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University of Groningen
Stereoselective synthesis of glycerol-based lipidsFodran, Peter
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1
Stereoselective Synthesis of Glycerol-based
Lipids
Peter Fodran
The work described in this thesis was carried out at the Stratingh Institute for
Chemistry, University of Groningen, The Netherlands.
This work was financially supported by the Zernike Institute for Advanced Materials.
Printed by: Ipskamp Drukkers, Enschede
Cover design: Peter Fodran
3
Stereoselective Synthesis of
Glycerol-based Lipids
PhD thesis
to obtain the degree of PhD at the University of Groningen on the authority of the
Rector Magnificus Prof. E. Sterken and in accordance with
the decision by the College of Deans
This thesis will be defended in public on
Friday 13th March 2015 at 12:45
By
Peter Fodran
born on 12th June 1985
in Bratislava, Slovak Republic
Supervisor Prof. A. J. Minnaard Assessment committee Prof. M. H. Clausen Prof. F. J. Dekker Prof. J. G. Roelfes
5
How come it is not possible? People are flying to the moon and you want to tell me
that you cannot fix this (vacuum cleaner, stove, hairdryer…)?
(Iveta Fodranová)
7
CONTENTS
Chapter 1 An Introduction to Phospholipids 1 Introduction 2 Nomenclature 4 Biosynthesis of fatty acids, sphingolipids, triacylglycerols, and glycerophospholipids 6
Biosynthesis of fatty acids 6 Biosynthesis of sphingolipids 8 Biosynthesis of triacylglycerols and glycerophospholipids 10
Biosynthesis of non-archaeal ether based lipids 15 Outline of this thesis 16 References and footnotes 18
Chapter 2 Synthesis of Methyl-branched Fatty Acids 21 Introduction 22
Tuberculostearic acid 25 Results and discussion 27
(R)-Tuberculostearic acid 27 Caspofungin fatty acid 28
Conclusions 32 Experimental part 32 References and footnotes 42
Chapter 3 Catalytic Synthesis of Enantiopure Mixed Diacylglycerols 45 Introduction 46 Results and discussion 49
Synthesis of enantiopure phospholipids 49 Enantiopurity does not decrease during the ring opening 51 Synthesis of the platelet-activating factor (PAF) 52
Conclusions 53 Experimental section 53 References and footnotes 61
Chapter 4 Enantiopure Triacylglycerols in Three Steps 63 Introduction 64
Reported syntheses of triacylglycerols 64 Results and discussion 68
Co[(R,R)-salen] catalyzed ring opening of glycidyl esters 68 Towards the automated synthesis of triacylglycerols 70
Conclusions 75 Experimental part 75 References and footnotes 93
Chapter 5 A Methyl Matters 95 Introduction 96 Results and discussion 101
Chemical synthesis of the phospholipids 101 Formation of the liposomes and proteoliposomes 102 Molecular dynamics study of the bilayers 104 Calcein efflux assay 106
Conclusions and outlook 110 Experimental part 111 References and footnotes 121
Chapter 6 Synthesis of a Cyclooctyne–based Lipidation Probe 123 Introduction 124
Design of a lipophilic lipidation probe 127 Results and discussion 128 Conclusions and outlook 131 Experimental part 132 References 140
Chapter 7 A Missing Link in Archaeal Lipid Biosynthesis; a Contribution from Organic Synthesis 143
Introduction 144 Biosynthesis of archaeal membrane lipids 145 Archaeal lipids as taxanomic markes 149
Results and discussion 149 Synthesis of 2,3-bis-O-(geranylgeranyl)-sn-glycero-1-phosphate 149 Identification of CDP-archaeol synthase 153 Catalytic alcoholysis of benzylglycidol as a key step in the synthesis of cyclo-
archaeol and -glucosyl-cyclo-archaeol 154 Conclusion 157 Experimental part 157 References and footnotes 167
Summary 171 Samenvatting 175 Acknowledgements 180
1
Chapter 1 An Introduction to Phospholipids
Abstract: Phospholipids are compounds with enormous significance for Life. For a
long time, they were considered as passive building blocks of the membranes.
However with the discovery of the phospholipid signalling, understanding of their
roles changed. In the first part, this chapter introduces the reader to the
nomenclature of phospholipids. The second part of this chapter briefly summarizes
the biosynthetic pathways of various phospholipid classes and presents some of their
functions in living organisms. The last part of this chapter presents an outline of this
thesis.
Chapter 1
2
1
Introduction
“Lipids” is a loosely defined term for substances of biological origin, soluble
in non-polar solvents.1 Chemically, lipids can be divided into non-saponifiable and
saponifiable lipids. Steroids, prostaglandins and fat soluble vitamins comprise the
class of non-saponifiable lipids. Glycerolipids, phospholipids, sphingolipids and
waxes constitute the class of saponifiable lipids. An intriguing difference between
these classes is that while non-saponifiable lipids act mostly as single molecules,
saponifiable lipids mainly act as a collective. This can be illustrated by the following
examples. Retinal (vitamin A) is a non-saponifiable lipid. Its molecular properties
allow light-induced cis/trans isomerization which is essential for vision (Figure 1-I).
Figure 1. ( I ) Cis/trans isomerization as a principle of vision; ( II ) organization of a lipid
raft in a liquid ordered membrane; ( III ) examples of a saponifiable lipids which act as single
molecules.
An Introduction to Phospholipids
3
1
A mixture of saturated phospholipids, cholesterol and sphingolipids is a collective
(Figure 1-II) in the liquid ordered phase. In the membrane this collective forms an
organized lipid raft2 which is essential for signal transduction.3 As this classification
to saponifiable and non-saponifiable lipids is historical, it is easy to find exceptions.
For example, lipid 1 (Figure 1-III) is a typical membrane lipid surrounded by billions
of similar lipids (slightly) differing in the length of the fatty acids and the number of
the double bonds. However, in the entire collective of the membrane lipids, 1 is also
the lipid involved in inflammation processes.4 An example of a non-saponifiable lipid
fulfilling the role of a saponifiable lipid is the archaeal membrane lipid 2. The ether
bonds make 2 resistant to saponification, but the function which 2 fulfils is typical
for saponifiable lipids.
The main challenge in the studies of saponifiable lipids is their variability.
Terms like glycero-, phospho- and spingolipids frequently account for 10s to 1000s
related, but different molecular species. This variability is determined by the modular
structure of these lipids, described in Figure 2. In Nature, 40 common fatty acids
occur which differ in their chain length and the degree of unsaturation (Figure 2-I).
Triacylglycerols (Figure 2-II) are esters of glycerol and fatty acids. Given that the 3
hydroxyl groups can be esterified with any of the 40 fatty acids, the estimated number
of possible triacylglycerols approaches 64 000 (403). In the case of glycero-
phospholipids (Figure 2-II), one position of the glycerol is already occupied by any
of the 6 common phosphorus headgroups. The remaining 2 positions can again be
esterified by any of the 40 fatty acids resulting in up to 9 600 (6 x 402) different
species. Spingolipids can display even greater variability (Figure 2-III). The primary
hydroxyl group can carry either a phosphorous headgroup or a glycan core resulting
in more than 100 000 different species. The current knowledge of lipids is far away
from understanding the biological significance of this variability, but it has been
established that subtle deviations in the fatty acid composition of lipids can be linked
to heart5 and neurodegenerative diseases6 or metabolic syndrome.7
This chapter briefly introduces 3 topics that are important for this thesis. In
the first part, the reader is introduced to the nomenclature of the lipids. The second
part offers a brief overview of the biosynthesis of fatty acids, triacylglycerols and
phospholipids together with some of their biological properties. The last part of the
chapter presents the outline of this thesis.
Chapter 1
4
1
Figure 2. Variability of saponifiable lipids. ( I ) 40 common fatty acids; ( II ) variability in
glycerol based lipids; ( III ) variability in sphingolipids.
Nomenclature
Lipid research covers multiple fields of chemistry, biology and medicine.
Therefore it is not a surprise that a unified and universally applied nomenclature is
lacking. Despite the nomenclature for organic compounds is rigorously defined by
IUPAC8, this is happily ignored in lipid research. The following table (Table 1)
An Introduction to Phospholipids
5
1
summarizes all the fatty acids that are mentioned in this thesis in all the common
nomenclatures.
Table 1. Names and symbols for fatty acids in this thesis.
Numerical
symbol
Structure
H3C-(hydrocarbon)-CO2H
Systematic name
(acid)
Trivial name
(acid)
4:0 -(CH2)2- butanoic butyric
6:0 -(CH2)4- hexanoic caproic
8:0 -(CH2)6- octanoic caprylic
10:0 -(CH2)8- decanoic capric
12:0 -(CH2)10- dodecanoic lauric
14:0 -(CH2)12- tetradecanoic myristic
16:0 -(CH2)14- hexadecanoic palmitic
18:0 -(CH2)16- octadecanoic stearic
18:1(11) -(CH2)7-CH=CH-(CH2)9- Z-9-octadecenoic oleic
18:2(9,12) -(CH2)5-(CH2CH=CH)2-(CH2)7- Z,Z-octadeca-9,12-
dienoic
linoleic
20:4(5,8,11,14) -(CH2)4-(CH2CH=CH)4-(CH2)3 Z,Z,Z,Z-eicosa-
5,8,11,14-tetraenoic
arachidonic
The description of the stereochemistry of the glycerol-based lipids might be
confusing. Given that glycerol is a prochiral compound, its substitution can lead to
a pair of enantiomers. Chemically, these are easily described using the Cahn-Ingold-
Prelog system (CIP). Although unambiguous, this nomenclature can obscure
biosynthetic relationships, for example in the case of triacylglycerols. Triacylglycerols
are biosynthesized by acylation of a diacylglycerol (Figure 3). An example below
(Figure 3-I) shows that depending on the length of the introduced fatty acid, the
corresponding triacylglycerols might have opposite configurational prefixes in CIP
system. In order to clearly present biological relationships, Hirschmann9 introduced
a stereospecific numbering (sn) system. This is based on the Fischer projection of the
substituted glycerol, placed in such a way that the secondary hydroxyl group points
to the left. The carbon on top is then designated as the sn-1 position and carbon on
the bottom sn-3. The advantage of the Hirschmann system is that a formal inversion
of the configuration is not possible. For comparison, the acylation of diacylglycerol
that was confusing in CIP (Figure 3-I) is now defined as an acylation on the sn-3
position (Figure 3-II).
Chapter 1
6
1
Figure 3. Comparison of 2 different systems for the stereochemical description of
glycerol-based lipids. ( I ) the CIP system commonly used in organic chemistry; ( II )
Hirschmanns system used in biology and biochemistry.
Biosynthesis of fatty acids, sphingolipids, triacylglycerols, and glycerophospholipids
Biosynthesis of fatty acids
Fatty acids are the building blocks of lipids. Their de novo synthesis is one of
the key metabolic pathways in living organisms. Chemically, this process is a
decarboxylative malonic ester synthesis of acyl coenzyme A with malonyl coenzyme
A (6) (Figure 4) followed by a deoxygenation. The synthesis of fatty acids starts with
a covalent attachment of acetyl coenzyme A 3 to the acyl carrying protein (ACP).
Malonyl coenzyme A (6) enters the cycle after covalent attachment to the acyl carrier
protein (ACP). The condensation of 4 and 6 results in thioaceto acetate 7 and
liberation of CO2.
An Introduction to Phospholipids
7
1
Figure 4. De novo biosynthesis of fatty acids.
The ß-keto group is first reduced to alcohol 8 by NADPH/H+ (Figure 4), which is
subsequently dehydrated to α,ß-unsaturated 9. The conjugated double bond of 9 is
reduced by NADPH/H+ and the resulting 10 can enter the second cycle.
Alternatively, 10 (or its higher homologue) can either be hydrolysed to the
corresponding fatty acid 11 or transthioesterified to acyl coenzyme A 12. All steps in
the fatty acid synthesis are catalyzed by a fatty acid synthase (FAS). In Nature, there
are 2 types of FAS. FAS type 1 is present in animals and fungi and FAS type 2 is
found in bacteria and plants. The difference between the 2 types is that FAS type 1
is a single enzyme with 7 distinct domains and FAS type 2 is an assembly of 7
separable enzymes. A notable exception is the CMN group of bacterial species
(Corynebacterium, Mycobacterium, and Nocardia), which possesses both types of FAS.10
Desaturation of fatty acids
The fatty acid synthases tightly cooperate with desaturases that introduce
double bonds in the fatty acid chain. The most common desaturation is the
conversion of stearic acid to oleic acid by abstraction of 9-pro-R and 10-pro-R
Chapter 1
8
1
hydrogens. In human metabolism, this is catalyzed by 3 membrane-bound proteins
(Figure 5). The necessary electrons come from the electron transport chain, which
begins by reduction of reductase bound FAD (E-FAD) by NADH.
Figure 5. Δ9 desaturation of fatty acids.
The electrons are further transferred to cytochrome b5 and finally to the non-heme
Fe of the desaturase. Iron in its Fe2+ state can interact with O2 and oxidize 13 to 14.
The resulting oleoyl coenzyme A (14) can be further elongated or desaturated.11,12
Once the fatty acid has the desired length and unsaturation(s), it can enter
other metabolic pathways. This can be, for example, further modification of the fatty
acid (i.e. methylation as in Mycobacterium tuberculosis, see Chapter 2) or conversion into
sphingolipids, triacylglycerols and glycerophospholipids.
Biosynthesis of sphingolipids
The biosynthesis of fatty acids is tightly connected to the biosynthesis of
sphingolipids (derivatives of sphingosine (21)) via palmitoyl coenzyme A (15). The
biosynthesis of 21 (Figure 6) starts with a decarboxylative condensation of 15 and
serine (16).
An Introduction to Phospholipids
9
1
Figure 6. Biosynthesis of sphingosine.
The resulting 17 is reduced to aminoalcohol 18. The nitrogen reacts with a fatty acid
coenzyme A and 19 is desaturated resulting in ceramide 20, which after hydrolysis of
the amide affords 21. Sphingosine (21) can be further modified (Figure 7) to
sphinholipids like cerebrosides 22, sphingomyelines 23 or gangliosides 24.
Figure 7. Examples of sphingolipids.
Chapter 1
10
1
Sphingolipids are responsible for diverse physiological functions. As the
membrane building blocks they are located at the outer leaflet of the phospholipid
bilayer.13 As signalling molecules14 sphingolipids are an important link between
overproduction of lipids and obesity.15
Biosynthesis of triacylglycerols and glycerophospholipids
The biosynthesis of triacylglycerols and glycerophospholipids is closely
related. Both pathways start with (R)-glycerol-1-phosphate (sn-glycerol-3-phosphate)
and share the same intermediates until the phosphatidic acid stage where the
pathways divide. First the biosynthesis of triacylglycerols is discussed.
Biosynthesis of triacylglycerols
The dominant route producing more than 90%16,17,18 of the triacylglycerols is
called the Kennedy pathway.19 In the endoplasmic reticulum, (R)-glycerol-1-
phosphate (sn-glycerol-3-phosphate) 25 (Figure 8) is esterified with a fatty acid
coenzyme A to form lysophosphatidic acid 26. In the next step, 27 is esterified with
a second fatty acid coenzyme A.
Figure 8. The Kennedy pathway.
The phosphate in the phosphatidic acid 27 is hydrolysed and the resulting
diacylglycerol 28 is esterified with a third fatty acid coenzyme A to afford the
An Introduction to Phospholipids
11
1
triacylglycerol 29. Subsequently, triacylglycerols can be stored in a specialized
organelle (a lipid droplet20) where they serve as energy reserve and precursors of
other lipid products.
Figure 9. ( I ) accumulation of triacylglycerols and fatty acids between the membrane
leaflets; ( II ) budding of a lipid droplet; ( III ) mature lipid droplet.
The mechanism of formation of the lipid droplets is poorly understood, but
a generally accepted theory states that these are formed by budding of the
endoplasmic reticulum (Figure 9)21 as a response to an elevated triacylglycerol
synthesis.22 Initially, the synthetized triacylglycerols concentrate between the leaflets
of the membrane (Figure 9-I). With the increasing amount of triacylglycerols, the
bud grows collecting more and more triacylglycerols (Figure 9-II). Finally, the lipid
droplet forms (Figure 9-III) as an independent organelle that can move into the
cytosol and interact with other organelles. Alternative mechanisms for the formation
of lipid droplets have been proposed by Ploegh23 and Walter and Farase.24
The content of the lipid droplets can be utilized when needed. The main
mechanism of utilization of the stored triacylglycerols and sterol esters is lipolysis.
Adipose triglyceride lipase and hormone sensitive lipase are moved to the surface of
the lipid droplet. The first enzyme hydrolyses at the sn-2 position of the
triacylglycerol. Hormone sensitive lipase further hydrolyses the 1,3-diacylglycerol to
a monoacylglycerol. The hydrolysis of the final fatty acid occurs in the cytosol and is
catalyzed by a monoacylglycerol lipase.25 The products of the hydrolysis of
triacylglycerols from the lipid droplets might be utilized for example in the
biosynthesis of phospholipids.
Chapter 1
12
1
As it was already mentioned, the biosynthesis of phospholipids is closely
related to the biosynthesis of triacylglycerols. The phosphatidic acid can be converted
to any of the 6 common phospholipids by 2 mechanisms. The first mechanism
involves cytidine triphosphate (CTP) activation of the phosphatidic acid leading to
phosphatidylinositols (PI), phosphatidylglycerols (PG) and cardiolipins. The second
mechanism utilizes CTP activation of the headgroup precursors leading to
phosphatidylcholines (PC), phosphatidylethanolamines (PE) and
phosphatidylserines (PS). The biosyntheses are presented in this order.
The activation of phosphatidic acid 27 (Figure 10) by CTP results in the cytidine
diphosphate (CDP) activated diacylglycerol 30 and liberation of diphosphate (PPi)
CDP activated 30 can react with inositol (31) resulting in phosphatidylinositol 32 and
cytidine monophosphate (CMP).
Figure 10. Biosynthesis of phospholipids via CTP activation of 27.
An Introduction to Phospholipids
13
1
Alternatively, the CDP activated 30 (Figure 10) can react with 25, which after
hydrolysis of phosphate results in phosphatidylglycerol 34 type lipid. 34 can react
with an additional molecule of CDP-diacylglycerol, affording cardiolipin 35.26
The phosphatidyl inositol 32, phosphatidyl glycerol 34 and cardiolipin 35
families of lipids fulfil various important functions in the entire cellular life. For
example the PI lipids anchor membrane proteins to the outer leaflet of the
membrane (via protein lipidation, see chapter 6). Another important role of the PI
lipids is in signal transduction in the plant and the animal kingdom via the action of
a specific phospholipase C.27 By this mechanism, the PI lipids influence the activity
of dozens of enzymes belonging to the protein kinase C family, thus controlling key
cellular functions like differentiation, proliferation, metabolism and apoptosis. The
PG lipids serve as precursors for the cardiolipins. Cardiolipins are mainly found in
the mitochondrial membrane, where they bind and regulate the activity of various
proteins.28 Abnormalities in the cardiolipin metabolism can be linked to a variety of
diseases, including Barth syndrome29, Parkinson, Alzheimer30 and Tengier disease.31
The mentioned functions of the families of lipids (PI, PG and cardiolipins) form
only a fraction of what has been reported.
The phosphatidic acid 27 can be transformed into PC, PE and PS
phospholipids via the second mechanism involving activation of the headgroup
precursor by CTP. This pathway starts with hydrolysis of 27 to diacylglycerol 28
(Figure 11-I). The CDP-phosphorylating agents 38 and 39 are synthetized in the
cytosol by the same mechanism (Figure 11-II). The corresponding alcohols 36 are
phosphorylated with ATP resulting in phosphates 37. These react with CTP leading
to the phosphorylating agents 38 and 39, which are further transported to the
endoplasmic reticulum, where they phosphorylate diacylglycerol 28. Phosphorylation
of 28 with CDP-ethanolamine 38 results in phosphatidylethanolamine type lipid 40
and phosphorylation of 27 with 39 results in phosphatidylcholine lipid type 41. Both
40 and 41 can be further converted to phosphatidylserine 42 type lipids. And finally,
42 can be converted back to 40 by decarboxylation.
PC, PE and PS lipids are the main building blocks of biological membranes.
PC is the most common lipid in animals and plants where it constitutes up to 50%
of all phospholipids. In bacteria, PC lipids are scarcer. Due to their molecular shape,
PC, PE and PS lipids have their preferred location in the membranes. PC lipids are
mainly located in the outer leaflet while PE and PS are located in the inner leaflet.
Distribution of the lipids between the leaflets is tightly regulated by enzymes –
flippases. However, in some events the distribution of the membrane lipids is altered.
Chapter 1
14
1
For example during apoptosis, PS lipids are moved to the outer leaflet, where they
are recognized by macrophages. By this mechanism the apoptic cell is removed
without triggering an inflamation.32
Figure 11. ( I ) Biosynthesis of phospholipids via CTP activation of the headgroup
precursors; ( II ) biosynthesis of the CDP activated headgroup precursors 38 and 39.
An Introduction to Phospholipids
15
1
Biosynthesis of non-archaeal ether based lipids
Plasmologens are ether analogues of the PE and PC lipids. Despite being
structurally related, their biosynthesis requires a specific pathway (Figure 12-I).
Figure 12. ( I ) Biosynthesis of plasmalogens; ( II ) mechanism of the substitution of acyl
for a long chain alcohol as the key step in the biosynthesis of plasmalogens.
Chapter 1
16
1
The biosynthesis starts in the peroxisome, by acylation of dihydroxyacetone
phosphate (43). In the second step the carboxylate is substituted by a long-chain
alcohol resulting in 45. The mechanism of this step was elucidated by Brown and
Snyder (Figure 12-II)33. In the active site of the alkylglycerone phosphate synthase,
44 tautomerizes to 46, which after protonation leads to 47. The resulting carbocation
is attacked by a nucleophilic centre Nu of the protein (probably an amino group in
the active site) resulting in departure of the carboxylate. In a subsequent step 49
reacts with a long-chain alcohol. 50 undergoes an E1 type elimination leading to 51
which finally tautomerizes to ketone 45. Reduction of 45 (Figure 12-I) results in 53
which is acylated in the endoplasmic reticulum. From 57 on, the biosynthesis is
similar to the synthesis of PC or PE lipids. First the phosphate is hydrolyzed and
resulting 55 is phosphorylated by CDP activated choline or ethanolamine. In case of
the choline headgroup, the biosynthesis stops at this point. The lipids with
ethanolamine headgroup can be further desaturated to 58.
The biological functions of plasmalogens are still not fully understood.
Structurally, they help to maintain physical properties of the membranes.34 Broniec
et al.35 reported that the analogues of 56 act as scavengers of reactive oxygen
suggesting that they play a role in oxidative stress. An important plasmalogen is the
platelet activating factor (Figure 12-I), which is an extremely potent signalling
molecule triggering the platelet aggregation and immunological responses at pM
concentrations (10-11 M).36 An efficient synthesis of PAF is described in Chapter 3.
Outline of this thesis
Lipids play vital roles in many processes essential for life. This is illustrated
by a lipid membrane, which is a complex mixture of (phospho)lipids with various
chain lengths and degree of unsaturation. In this complex mixture, every single
component has an irreplaceable role. Of course, lipids are in principle accessible from
their natural sources, but their isolation and purification (from other lipids) is tedious
and often virtually impossible. A convincing example in this connection starts with
the impressive contribution of R. J. Anderson in 1927,37 who was the first to isolate
and describe tuberculostearic acid 59. For his studies, he needed 2 200 culture flasks
with a volume of 200 cm3. Only in 2010, 83 years later, it was established beyond
reasonable doubt that 59 is part of phospholipid 60 in M. tuberculosis.38 From 1 g of a
total lipid extract of M. tuberculosis, the authors isolated 50 μg of pure lipid 60, and
determined its structure by independent synthesis. For further illustration, 1 g of the
total lipid extract corresponds roughly to 20 g of bacteria.
An Introduction to Phospholipids
17
1
Biology has a lot to gain from the availability of pure, well-defined, natural
and unnatural lipids in sufficient amounts, and organic chemistry can fulfil this need.
This is realized and illustrated in this thesis. In 7 chapters, novel, efficient, and
stereoselective approaches are described for the synthesis of ester-based and ether-
based phospholipids and triacylglycerols.
Chapter 2 describes the catalytic asymmetric synthesis of methyl-branched
fatty acids (59 in Figure 13). The approach is based on conjugate addition of
methylmagnesium bromide to α,ß-unsaturated thioesters and subsequent chain
elongation to the desired length by Wittig reaction with a functionalized ylide. This
modular approach is applied in the synthesis of the fatty acid chain of caspofungin,
which allowed a study in the group of Prof. R. M. J. Liskamp (Molecular Medicinal
Chemistry, University of Utrecht) on the influence of the stereochemistry of this
fatty acid on its antifungal properties.
Figure 13. Examples of a fatty acid and lipid isolated from natural sources.
The theme of Chapter 3 is the transformation of fatty acids into
phospholipids. Here, the Jacobsen Co(II) salen complexes play an important role,
granting the regiospecific opening of protected glycidol with fatty acids. The chapter
further describes a migration-free deprotection of the resulting silylated
diacylglycerols, solving a long-standing problem in this field. It allows the synthesis
of various glycerophospholipids. A small modification of the catalyst opens a
convenient access to mixed ether/ester lipids represented by platelet activating
factor.
Chapter 4 is an extension of this methodology to the synthesis of enantiopure
triacylglycerols in just 3 synthetic steps. This allows the preparation of a small (>15)
Chapter 1
18
1
library of triacylglycerols, as a prelude to the determination of the composition of
(cow) milk fat, a piece de resistance in diary research.
Chapter 5 describes the influence of phospholipids on the function of
mechanosensitive channels of large conductance (MscL). In particular, the role of
methyl-branched lipid 60 on the MscL from the same species is studied and related
to their non-branched analogues.
Chapter 6 describes the synthesis of a fatty acid equipped with a strained
cyclooctyne. This “clickable fatty acid” is a promising tool for further studies in
chemical biology.
Chapter 7, composed of 2 parts, is dedicated to the synthesis of ether-based
Archaea lipids. In this chapter, the introduction is dedicated to the metabolism of
the unique Archaea lipids. Part one describes the synthesis of an intermediate in
Archaea lipid biosynthesis. This lipid has been used in the department of Molecular
Microbiology (GBB, Prof. A. J. M. Driessen) for the identification of CDP-archaeol
synthase, the missing link in this biosynthesis. The second part describes the
application of the aforementioned Co(II) salen complexes in a total synthesis of
cyclo-archaeol.
References and footnotes
(1) Moss, G. P.; Smith, P. A. S.; Tavernier, D. Pure Appl. Chem. 1995, 67, 1307.
(2) Pike, L. J. J. Lipid Res. 2003, 44, 655.
(3) Simons, K.; Toomre, D. Nat. Rev. Mol. Cell Biol. 2000, 1, 31.
(4) Fernandis, A. Z.; Wenk, M. R. Curr. Opin. Lipidol. 2007, 18, 121.
(5) Beilin, L. J.; Burke, V.; Puddey, I. B.; Mori, T. A.; Hodgson, J. M. Clin. Exp.
Pharmacol. Physiol. 2001, 28, 1078.
(6) Han, X. Front. Biosci. 2007, 12, 2601.
(7) Carpentier, Y. A.; Portois, L.; Malaisse, W. J. Am. J. Clin. Nutr. 2006, 83, S1499.
(8) Eur. J. Biochem. 1977, 79, 11.
(9) Hirschmann, H. J. Biol. Chem. 1960, 235, 2762.
(10) Gebhardt, H.; Meniche, X.; Tropis, M.; Krämer, R.; Daffé, M.; Morbach, S.
Microbiology 2007, 153, 1424.
(11) Nakamura, M. T.; Nara, T. Y. Annu. Rev. Nutr. 2004, 24, 345.
(12) Qiu, X. Prostaglandins Leukot. Essent. Fatty Acids 2003, 68, 181.
(13) Berridge, M. J. Nature 1993, 361, 315.
(14) Spiegel, S.; Milstien, S. J. Biol. Chem. 2002, 277, 25851.
(15) Summers, S. A. Prog. Lipid Res. 2006, 45, 42.
(16) Lehner, R.; Kuksis, A. J. Biol. Chem. 1993, 268, 8781.
An Introduction to Phospholipids
19
1
(17) For other biosynthetic pathways see ref. 18 and 19.
(18) (a) Cagliari, A.; Margis, R.; Dos, S. M. F.; Turchetto-Zolet, A. C.; Loss, G.; Margis-
Pinheiro, M. Int. J. Plant Biol. 2011, 2, 40 (b) Coleman, R. A.; Lee, D. P. Prog. Lipid Res. 2004,
43, 134 (c) Karantonis, H. C.; Nomikos, T.; Demopoulos, C. A. Curr. Drug Targets 2009, 10,
302 (d) Lehner, R.; Kuksis, A. Prog. Lipid Res. 1996, 35, 169 (e) Lehner, R.; Kuksis, A. Prog.
Lipid Res. 1996, 35, 169 (f) Sorger, D.; Daum, G. Appl. Microbiol. Biotechnol. 2003, 61, 289 (g)
Sorger, D.; Daum, G. Appl. Microbiol. Biotechnol. 2003, 61, 289 (h) Yen, C.-L. E.; Stone, S. J.;
Koliwad, S.; Harris, C.; Farese, R. V., Jr. J. Lipid Res. 2008, 49, 2283 (i) Coleman, R. A.;
Mashek, D. G. Chem. Rev. 2011, 111, 6359.
(19) Weiss, S. B.; Kennedy, E. P. J. Am. Chem. Soc. 1956, 78, 3550.
(20) the alternative terms describing same organelle are: lipid bodies, oil bodies and
adiposomes
(21) Martin, S.; Parton, R. G. Nat. Rev. Mol. Cell Biol. 2006, 7, 373.
(22) Pol, A.; Martin, S.; Fernandez, M. A.; Ferguson, C.; Carozzi, A.; Luetterforst, R.;
Enrich, C.; Parton, R. G. Mol. Biol. Cell 2004, 15, 99.
(23) Ploegh, H. L. Nature 2007, 448, 435.
(24) Walther, T. C.; Farese Jr, R. V. Biochim. Biophys. Acta. - Mol. Cell Biol. L. 2009, 1791,
459.
(25) Guo, Y.; Cordes, K. R.; Farese, R. V.; Walther, T. C. J. Cell Sci. 2009, 122, 749.
(26) the biosynthesis of cardiolipins differs in prokaryotic and eucaryotic cells. The
depicted sequence corresponds to the prokaryotic cells.
(27) Irvine, R. F. Curr. Opin. Cell Biol. 1992, 4, 212.
(28) Haines, T. H. Biochim. Biophys Acta - Biomembranes 2009, 1788, 1997.
(29) Xu, Y.; Malhotra, A.; Ren, M.; Schlame, M. J. Biol. Chem. 2006, 281, 39217.
(30) Ruggiero, F. M.; Cafagna, F.; Petruzzella, V.; Gadaleta, M. N.; Quagliariello, E. J.
Neurochem. 1992, 59, 487.
(31) Oram, J. F. Biochim. Biophys. Acta - Mol. Cell Biol. L. 2000, 1529, 321.
(32) Verhoven, B.; Schlegel, R. A.; Williamson, P. J. Exp. Med. 1995, 182, 1597.
(33) Brown, A. J.; Snyder, F. J. Biol. Chem. 1983, 258, 4184.
(34) Farooqui, A. A.; Horrocks, L. A.; Farooqui, T. Chem. Phys. Lipids 2000, 106, 1.
(35) Broniec, A.; Klosinski, R.; Pawlak, A.; Wrona-Krol, M.; Thompson, D.; Sarna, T.
Free Radic. Biol. Med. 2011, 50, 892.
(36) Prescott, S. M.; Zimmerman, G. A.; Stafforini, D. M.; McIntyre, T. M. Annu. Rev.
Biochem. 2000, 69, 419.
(37) Anderson, R. J. J. Biol. Chem. 1927, 74, 525.
(38) ter Horst, B.; Seshadri, C.; Sweet, L.; Young, D. C.; Feringa, B. L.; Moody, D. B.;
Minnaard, A. J. J. Lipid Res. 2010, 51, 1017.
Chapter 1
20
1
21
Chapter 2 Synthesis of Methyl-branched Fatty Acids
Abstract: Branched-chain fatty acids are common in yeast and bacteria, where they
fulfil diverse functions. Their isolation from natural sources is lengthy and tedious.
This chapter presents an efficient and modular synthesis of methyl branched fatty
acids.
Parts of this chapter have been published:
Mulder, M. P. C.; Fodran P.; Kemmink, J.; Breukink, E. J.; Kruijtzer, J. A. W.;
Minnaard, A. J.; Liskamp, R. M. J. Org. Biomol. Chem. 2012, 10, 7491.
Fodran, P.; Minnaard, A. J. Org. Biomol. Chem. 2013, 11, 6919.
Chapter 2
22
2
Introduction
Methyl-branched fatty acids (a subset of branched-chain fatty acids) are
found at many places in Nature,1 although they are much less abundant than their
straight-chain congeners. They can be divided into several categories, but for the
purpose of this chapter a straightforward division based on the position of the
methyl-branch in the linear chain is sufficient. The most common pattern is a mono-
methyl branch, but also poly-methyl branched fatty acids occur.1b
Fatty acids can branch next to the carboxylic acid group - at the beginning of
the chain. Mycocerosic acids (Figure 1) 1 which are components of the Mycobacterium
tuberculosis cell wall are a prominent example. The biosynthesis2 of 1 is analogous to
the biosynthesis of fatty acids (Chapter 1). The long-chain acyl coenzyme A (2) is
initially extended by methylmalonyl coenzyme A, then decarboxylated and finally
deoxygenated. Additional cycle(s) lead to a multi-methyl branched 1. Remarkably,
the same M. tuberculosis produces the structurally very similar phthioceranic acid (3)
which has the opposite configuration of the methyl substituents.
Figure 1. Biosynthesis of fatty acids branched at the beginning of the chain.
Based on the abovementioned division, fatty acids can also branch in the
middle of the chain. Probably the best known example from this group is (R)-
tuberculostearic acid (4) (Figure 2-I). 4 is an important cytoplasmic membrane
component of M. tuberculosis. The biosynthesis3 (Figure 2-II) involves methylation of
the 10th carbon of oleolate 5 by S-adenosylmethionine (SAM). A subsequent Wagner-
Meerwein rearrangement of 6 results in a thermodynamically more stable tertiary
carbocation 7, which after deprotonation affords non-Zaitsev olefin 8. The final step
is the reduction of the methylene in 8 to the (R)-tuberculostearic acid carrying
phospholipid 9.
Synthesis of Methyl-branched Fatty Acids
23
2
Figure 2. ( I ) Structure of (R)-tuberculostearic acid (9); ( II ) biosynthesis of 9.
A third class of branched fatty acids carries the methyl-branch at the terminus of the
chain4 (Figure 3). They are biosynthetized in the same manner as linear fatty acids.
Figure 3. Biosynthesis of fatty acids branched at the terminus of the chain.
Chapter 2
24
2
The only difference is the primer, which determines the position of the
branch and the odd/even number of carbons in the chain. These primers, isobutyryl-
(10) isovarelyl- (11) and (S)-2-methylbutyryl-coenzyme A (12) are products of the
catabolism of L-valine, L-leucine and L-isoleucine respectively. Important to note is
that while iso-branched fatty acids 13 and 14 are achiral, the anteiso-branched acids 15
are chiral and enantiopure.
A particular example of a fatty acid branched at the end of the chain is 16
(Figure 4-I). 16 forms a lipophilic portion of lipopeptide pneumocandin B0 (17),
which is found in the fungus Zalerion arboricola. Chemical modification of 17
(Figure 4-II), leads to the better known caspofungin (18).5 18 together with
anidulafungin and micafungin6 are approved drugs of the enchinocandin class of
antifungals.
Figure 4. ( I ) An example of a fatty acid branched at the terminus of the chain; ( II ) antifugal
agents 17 and 18 originating from Zalerion arboricola.
Enchinocandins are modern non-competitive 1,3-ß-glucan synthase inhibitors, used
for the treatment of candidiasis and aspergillosis (also in immunocompromised
patients). Their advantages are low toxicity and a high antifungal activity. Their
pharmacological properties are dependent on the fatty acid component.6
A common denominator of all above-mentioned branched acids is their
relevance in medicine. For example, O’Sullivan et al. 7 reported that detection of
mycocerosic acid (1) in sputum can be used for the rapid detection of tuberculosis.
Synthesis of Methyl-branched Fatty Acids
25
2
Similarly, French et al.8 developed a method for diagnostics of tuberculous
meningitis based on the presence of tuberculostearic acid (4) in the cerebrospinal
fluid. The importance of branched fatty acids for medicine is not limited to
tuberculosis research, but extents also to cardiac9 and pediatric10 studies. A
procedure allowing an efficient synthesis of methyl-branched fatty acids, with the
methyl branch at any desired position in the chain would be a great aid in further
research on the role of methyl branching in fatty acids. To develop such a method,
(R)-tuberculostearic acid (4) served as prototype branched fatty acid.
Tuberculostearic acid
Since its first description in 1927, tuberculostearic acid has been synthetized
by multiple researches either in racemic or enantiopure form.11 Furthermore, the
natural enantiomer has been prepared starting from the chiral pool, using a chiral
auxiliary, and recently, also by enantioselective catalysis.11f The approach using
enantioselective catalysis was reported by Ter Horst et al.11f and utilizes an
enantioselective conjugate addition to an α,ß-unsaturated thioester as a key-step for
the introduction of the methyl branch (Scheme 1). The authors prepared thioester
22 by cross-metathesis of 19 (prepared from commercially available 10-undecenoic
acid) and 20 (prepared in 3 steps).
Scheme 1. Synthesis of 4 according to Ter Horst et al.11f
Chapter 2
26
2
Conjugate addition to 22 afforded 23 in an excellent 91% yield and a good 95:5 e.r.
(Scheme 1). A chemoselective reduction of the thioester in the presence of an oxo-
ester afforded the aldehyde and subsequent Wittig reaction afforded 25 in 92% yield
over 2 steps. Finally, hydrogenation using organocatalytically12 generated diimide and
subsequent alkaline hydrolysis of the isopropyl ester afforded (R)-tuberculostearic
acid 4 in 91% yield (over 2 steps). Although at that time the highest yielding synthesis,
it takes 7 linear steps starting from commercially available compounds.
A retrosynthetic analysis (Figure 5) revealed that the synthesis of 4 can in
principle be carried out in 5 linear steps. A disconnection between the 7th and 8th
carbon of 4 gives 27 and 28. Phosphonium salt 27 can be prepared in one step from
commercially available 7-bromoheptanoic acid.13
Figure 5. Retrosynthetic analysis of 4.
Aldehyde 28 can be obtained by enantioselective conjugate addition to α,ß-
unsaturated undecenoate14 followed by a reduction. From a broad spectrum of
suitable enoates,15 α,ß-unsaturated thioesters remain the best option as these afford
products of the conjugate addition in high yields and optical purity. Furthermore,
thioesters offer a better control of the reduction step compared to oxo-esters,16
therefore 29 can be envisioned as a suitable substrate. Finally, 29 is available via a
Wittig or Horner-Wadsworth-Emmons reaction of commercially available nonanal
(30). Alternatively, in analogy to Ter Horst, 29 can be prepared via cross-metathesis
of 20 and decene (31).17 On first sight, the cross-metathesis might seem more
Synthesis of Methyl-branched Fatty Acids
27
2
appealing especially from an atom economy point of view. This is however just
appearance as 20 is prepared by a Wittig reaction with paraformaldehyde.
The versatility of this approach will be illustrated with the synthesis of
tuberculostearic acid (4) and the caspofungin side chain 16. Its flexibility becomes
clear in particular because 16 (Figure 6) bears the methyl groups at different positions
of the chain compared to 4 and their absolute stereochemistry is opposite, this
underscores the need for a catalytic enantioselective approach.
Figure 6. Retrosynthetic analysis of 16.
Another aspect of this chapter is a study of the influence of 16 on the antifungal
activity of caspofungin and its derivatives.
Results and discussion
(R)-Tuberculostearic acid
According to the retrosynthetic analysis in Figure 5, (R)-tuberculostearic acid
(4) was prepared in 5 linear steps. Unsaturated thioester 29 (Scheme 2) was prepared
by treatment of nonanal (30) with an excess of stabilized Wittig reagent 3318 in the
presence of LiCl (20 mol%) using “on water“ conditions. The olefination proceeds
smoothly under these conditions resulting in an excellent E : Z ratio (>>95 : 5).
Claridge et al.19 reported similar selectivity in a related Horner-Wadsworth-Emmons
reaction using MeMgBr as a base. The on water procedure is advantageous as it does
not require inert atmosphere or exclusion of water. After the olefination, the trace
amount of undesired (Z)-29 (Scheme 2) was removed by flash chromatography and
(E)-29 was isolated in 92% yield. Next, 29 was subjected to a CuBr.(34)14,20 catalyzed
conjugate addition of MeMgBr. This afforded the branched product 35 in 94% yield
and 95 : 5 e.r. Thioester 35 was reduced with DIBAL to afford 28, which could be
subjected to Wittig reaction without any purification. Although multiple authors have
applied acid-functionalized Wittig reagents in their syntheses,21 their reported
Chapter 2
28
2
conditions resulted in low yields of 36. After optimization, a combination of an
excess of 27 in combination with LiHMDS, were the best conditions affording 36 in
79% yield over 2 steps.
Reagents and conditions: a) 33 (1.4 equiv), LiCl (20 mol%), water, RT, 18 h; b) CuBr.SMe2 (1.5 mol%), 34 (1.65 mol%), MeMgBr (1.2 equiv), tBuOMe, –78 °C, 3 h addition of 29 followed by stirring for 16 h; c) DIBAL (1.3 equiv), CH2Cl2, -78 °C, 2 h; d) 27 (1.4 equiv), LiHMDS (2.8 equiv), THF, 0 °C – 21 °C, 3 h; e) 26 (5 mol%), NH2NH2.H2O (21.0 equiv), EtOH, O2 (balloon), RT, 24 h.
Scheme 2. Second generation synthesis of tuberculostearic acid.
To avoid racemization of the homoallylic methyl-branched stereocenter in 36, the
double bond was reduced with diimide, produced by controlled oxidation of
hydrazine by O2 in the presence of 26.12 In this way, (R)-tuberculostearic acid (4) was
prepared in 63% overall yield, the shortest and highest yielding route to date.
Caspofungin fatty acid
The relative and absolute configuration of 16 (Scheme 3) was reported by
Leonard et al.22 The authors initially applied the Enders auxiliary method23 to prepare
ent-16 in 8 steps as a 90:10 (syn : anti) ratio of diastereomers. Later, using prolinol as
a chiral auxiliary,24 they synthetized the correct enantiomer as an 80 : 20 (syn : anti)
mixture of diastereomers also in an 8 step sequence.
The same strategy as in (R)-tuberculostearic acid (Scheme 2) could be applied
to the synthesis of caspofungin fatty acid 16 and its monomethyl analogue 46
Synthesis of Methyl-branched Fatty Acids
29
2
(Scheme 3). Commercially available (E)-pent-2-enoic acid 37 was converted into
thioester 32 using Steglich conditions.
Reagents and conditions: a) EtSH (2.0 equiv), DCC (1.1 equiv), DMAP (10 mol%), CH2Cl2, 0 - 21 °C, 3 h; b) MeMgBr (1.2 equiv), CuBr.Me2S (1.2 mol%), 25 (1.3 mol%), tBuOMe, –78 °C, 3 h addition of 32 followed by stirring for 16 h; c) DIBAL (1.2 equiv), CH2Cl2, –50 °C, 1 h; d) 40 (1.5 equiv), nBuLi (1.1 equiv), THF, 0-21 °C, 16 h; e) MeMgBr (1.3 equiv), CuBr.25 (1.3 mol%), tBuOMe, –78 °C, 3 h addition of 41 followed by stirring for 16 h; f) DIBAL, (1.2 equiv), CH2Cl2, -50 °C, 1 h; g) 27 (1.75 equiv), LiHMDS (2.0 equiv), THF, 0 °C – 21 °C, 3 h; h) NH2NH2.H2O (25.0 equiv), 26 (10 mol%), EtOH, O2 (balloon), 21 °C; i) 44 (1.7 equiv), LiHMDS (2.0 equiv), THF, 0 °C – 21 °C, 3 h; j) NH2NH2.H2O (30.0 equiv), 26 (10 mol%), O2, (balloon), EtOH, 21 °C.
Scheme 3. Synthesis of 15 and monomethyl analogue 46.
Distillation of the crude mixture under reduced pressure afforded pure 32 in
95%. Thioester 32 underwent CuBr.(S,Rp)-Josiphos (CuBr.25) catalyzed
enantioselective conjugate addition of MeMgBr smoothly, affording the desired 38
in 92% yield with a 98.5 : 1.5 e.r. This branched thioester was a key precursor in the
Chapter 2
30
2
synthesis of both 16 and 46. To achieve the caspofungin fatty acid 16, thioester 38
was reduced with DIBAL. After work-up, the aldehyde 39 was treated with Horner-
Wadworth-Emmons reagent 40 in basic conditions to afford 41 in 70% yield over 2
steps. After separation of the minor Z stereoisomer 41 was subjected to the second
conjugate addition resulting in a 42 in a 20 : 1 diastereomeric ratio. Flash
chromatography afforded diastereomerically pure 42 in 78% yield. This was reduced
with DIBAL and the corresponding aldehyde underwent Wittig reaction with 27 to
extend the chain to the desired length. Acid 43 was isolated in 47% over 2 steps. The
double bond in 43 was reduced with diimide, in situ formed by catalytic oxidation of
hydrazine. The caspofungin fatty acid 16 was isolated in 81% yield.
The synthesis of the mono methyl analogue 46 (Scheme 3) was performed in
a similar manner. The key thioester 38 was reduced by DIBAL and extended using
44. These 2 steps afforded 45 in 60% yield. The reduction of the resulting double
bond by in situ generated diimide resulted in monomethyl branched 46 in 80% yield.
The lower yields compared to the synthesis of tuberculostearic acid (Scheme
2) are caused by the volatility of the intermediates. Compared to the previously
published approach22 the present synthesis of the caspofungin side chain is higher
yielding (20% vs. 9% by Leonard et al.). The biggest advantage arises from the
iterative protocol, which yields 42 as 20:1 ratio of epimers, which are fully separable.
In the synthesis published by Leonard et al. the mismatching pair of chiral enolate
and chiral alkylating agent results in only a 4 : 1 ratio of epimers. The authors needed
2 recrystallizations of the fatty acid (as the corresponding cinchonidine salt) to
improve the diastereometic ratio to 97:3.
Influence of different branched fatty acids on the antifungal activity of caspofungine analogues
The following part of this research has been conducted by Dr. Mulder, from
the group of Prof. Liskamp, University of Utrecht.
The core of caspofungin (18) (Figure 7) consists of 6 amino acids. Given that
some of these amino acids are not commercially available, the effect of the branched
fatty acids on the antifungal activity of caspofungin was studied on the simplified
hexapeptide. In the simplified analogue (red in Figure 7) 3,4-dihydroxy
homotyrosine, 3-hydroxy ornithine and its diaminoethyl analogue were substituted
for homotyrosine and ornithine.
Synthesis of Methyl-branched Fatty Acids
31
2
Figure 7. Caspofungine and its analogues used in the study.
Caspofungine 18 and the analogues 47, 48 and 49 were then tested against a
panel of common Candida species in a broth microdilution assay (Table 1). The
results are expressed as minimum inhibitory concentration (MIC) values – the
minimum concentration of a compound which completely inhibits visible growth.
Table 1. Antifungal activity of caspofungin and its analogues.
species
derivative
C. albicans
CBS9975
(μg.ml-1)
C. dubliniensis
CBS7987
(μg.ml-1)
C. tropicalis
CBS94
(μg.ml-1)
C. glabrata
CBS138
(μg.ml-1)
C. krusei
CBS573
(μg.ml-1)
C. parapsilosis
CBS604
(μg.ml-1)
16 0.023 0.014 0.006 0.027 0.006 0.281
47 0.117 0.07 0.094 0.469 2.25 >4.25
48 0.188 0.047 0.188 0.625 1.875 >4.25
49 0.203 0.047 0.063 0.438 0.813 >4.25
The simplification of the core (derivative 16 vs. 47), leads to a decreased activity. In
the case of C. albicans and C. dubliniensis the decrease is only 5-fold. The simplification
has a larger impact on the activity against C. tropicalis and C. glabrata where the
decrease is in the order of a magnitude (15- and 19-fold). More pronounced effects
were observed in the case of C. krusei where 47 was less active in the order of 2
magnitudes. Analogue 47 was inactive against C. parapsilosis. To conclude on the
influence of the methyl groups, the dimethyl-47, monomethyl-48 and desmethyl-49
Chapter 2
32
2
demonstrated roughly the same activity against all the studied Candida species. This
result is partially in agreement with the study reported by Fujie6 on micafungin
derivatives. The substitution of the fatty acid did not have a major influence on the
antifungal activity, but was used to improve hemolysis, which is the most common
side effect of treatment by enchinocandins.
Conclusions
This chapter presents a novel synthetic approach to methyl-branched fatty
acids. The combination of an enantioselective conjugate addition of MeMgBr to
linear aliphatic unsaturated thioesters, in combination with a Wittig reaction with an
acid functionalized phosphonium salt, places the methyl group at the desired position
in the chain. These reactions were applied in the synthesis of (R)-tuberculostearic
acid, the caspofungin fatty acid, and its analogues. (R)-Tuberculostearic acid was
prepared in only 5 steps with an overall yield of 63%. The fatty acid residue of
caspofungin 16 was prepared in 8 steps with an overall yield of 20%. Its monomethyl
analogue 46 was prepared in 5 steps with 42% overall yield. Given that the methyl
substituents of these 2 fatty acids have the opposite configuration, enantioselective
catalysis is the approach of choice. In the case of the caspofungin fatty acid residue,
also the influence of methyl branching on antifungal activity was studied where no
obvious relationship was observed.
Experimental part
Solvents for chemical reactions were dried according to the standard procedures.
Solvents for flash chromatography were used without further purification. All
reagents were used without further purification unless noted otherwise. All the
reactions were performed using Schlenk techniques unless noted otherwise.
Glassware was dried by heating (150 °C) for at least 2 h and subsequent cooling
under vacuum before use. Reactions were monitored by GC/MS (GC, HP6890: MS
HP5973) equipped with an HP1 column (Agilent Technologies, Palo Alto, CA) or
by TLC on silica coated aluminium foils (60 Å, 0.25 mm coating thickness). TLCs
were visualized by the following stains: iodine stain, Seebach’s stain (2.5 g
phosphomolybdic acid 1.0 g Ce(SO4)2 and 6.0 ml conc H2SO4 sequentially added to
94 ml H2O, bromcresol green stain (40.0 mg of bromocresol green dissolved in 100
ml EtOH, 0.1M NaOH solution added until blue) or Dittmer stain (phospholipid
stain, Preparation: solution A: 4.0 g MoO3 in 100 ml of hot concentrated H2SO4;
solution B: dissolve 180 mg Mo (metallic molybdenum) in 50 ml of hot solution A,
stock solution: after cooling, mix 50 ml solution B with 50 ml solution A for
Synthesis of Methyl-branched Fatty Acids
33
2
phospholipids). Flash column chromatography was performed on 230-430 mesh
silica gel.1H-, 13C-, and 31P-NMR spectroscopy was performed on Varian VXR300 or
AMX400 spectrometers. Chemical shifts were determined relative to the residual
solvent peaks (CHCl3, δ = 7.26 ppm for 1H NMR, δ = 77.16 ppm for 13C NMR).
The reported shifts are in ppm. The ( - ) sign in the 13C NMR reports stands for
negative phase in APT (Attached Proton test). Optical rotations were measured on
a Schmidt+Haensch polarimeter (Polartronic MH8) with a 10 cm cell. The mass
spectra were recorded on an Thermoscientific LTQ OrbitrapXL spectrometer.
Synthesis of (R)-tuberculostearic acid (Scheme 2)
(E)-S-ethyl undec-2-enethioate (29)
To a suspension of 33 (5.10 g, 14.0 mmol, 1.4 equiv.) in water (10 ml),
lithium chloride (84.8 mg, 2.00 mmol, 20 mol%) and nonanal (1.70 ml, 10.0 mmol)
were added. The mixture was subsequently stirred for 18 h in an opened flask.
Subsequently, the water was evaporated, the residue was dissolved in CH2Cl2 (30 ml)
and adsorbed on silica. After evaporation, the residue was placed on top of the
column (dry loading) and chromatographed using 5% toluene in pentane.
The desired (E)-S-ethyl undec-2-enethioate25 (2.11 g, 92%) was obtained as a
colourless thick liquid.
1H NMR (400 MHz, CDCl3, δ): 6.88 (dt, J = 15.5, 7.0 Hz, 1H), 6.08 (d, J = 15.5 Hz,
1H), 2.93 (q, J = 7.4 Hz, 2H), 2.17 (ddd, J = 14.7, 7.3, 1.5 Hz, 2H), 1.43 (m, 3H),
1.26 (m, 12H), 0.87 (t, J = 6.9 Hz, 3H).
13C NMR (101 MHz, CDCl3, δ): 190.11, 145.41 ( - ), 128.63 ( - ), 32.14, 31.80, 29.3,
29.14, 27.97, 22.99, 22.62, 14.80 ( - ), 14.05 ( - ) (1 signal overlapping)
HRMS-ESI+ (m/z): [M + H]+ calculated for C13H25OS, 229.162; found 229.162.
(R)-S-ethyl 3-methylundecanethioate (35)
(R,SFe)-Josiphos.EtOH adduct (49.0 mg, 82.5 μmol, 1.65 mol%) and
CuBr.Me2S (15.0 mg, 75.0 μmol, 1.50 mol%) were stirred in freshly distilled tBuOMe
(45 ml) until homogeneous (approx. 20 min). The mixture was cooled to –78 °C
(cryostat) and a solution of MeMgBr in Et2O (Acros Organics, 3 M, 2.0 ml,
Chapter 2
34
2
7.50 mmol, 1.2 equiv) was added dropwise. After stirring for 10 min, a solution of
26 (1.14 g, 5.0 mmol) in tBuOMe (5 ml) was added over 3 h by syringe pump. After
complete addition, the mixture was stirred for an additional 16 h. Then, EtOH
(5.0 ml) was added and the flask was removed from the cooling bath. An aqueous
solution of NH4Cl (1 M, 20 ml) was added and the mixture was stirred for 20 min at
rt. The resulting solution was transferred to a separatory funnel and the aqueous layer
was diluted with water (30 ml). Layers were separated, the aqueous layer was
extracted with Et2O (3x15 ml), and the combined organic layers were washed with
brine (50 ml), dried over MgSO4 and evaporated. The crude residue (a yellow-orange
liquid) was purified by column chromatography (SiO2, 2% Et2O in pentane) to afford
35 (1.15 g, 94%) as a thick colourless liquid.
1H NMR (400 MHz, CDCl3, δ): 2.87 (q, J = 7.4 Hz, 3H), 2.52 (dd, J = 14.4, 6.0 Hz,
1H), 2.33 (dd, J = 14.4, 8.1 Hz, 1H), 2.00 (m, 1H), 1.25 (m, 17H), 0.92 (d, J = 6.7
Hz, 3H), 0.87 (t, J = 6.8 Hz, 3H).
13C NMR (101 MHz, CDCl3, δ): 199.32, 51.40, 36.61, 31.86, 31.06, 29.69 ( - ), 29.55,
29.27, 26.82, 23.23 , 22.65, 19.51 ( - ), 14.78 ( - ), 14.08 ( - ).
HRMS-ESI+ (m/z): [M + H]+ calculated for C14H29OS, 245.194; found, 245.195.
[α]D +3.3° (c = 1.02, CHCl3)
The enantiomeric excess was determined on the
corresponding carbamate, obtained by LiAlH4 reduction, treatment with phosgene
in toluene (5 equiv) and treatment with (S)-(−)-1-(1-naphthyl)ethylamine (1 equiv).
The retention times were compared to a racemic sample.
Chiracel OD-H, flow=1 ml/min, tminor=11.4 min, tmajor=12.4 min, e.r=95:5.
(R)-3-methylundecanal (28)
This compound was used without purification. One time, isolation was
performed in order to confirm its absolute configuration.
To a solution of (R)-S-ethyl 3-methyl undecathioate 35 (978 mg, 4.00 mmol) in
anhydrous CH2Cl2 (23 ml), cooled to –78 ºC (EtOH/dry ice), a solution of DIBAL
Synthesis of Methyl-branched Fatty Acids
35
2
in CH2Cl2 (1 M, 5.2 ml, 5.2 mmol, 1.3 equiv) was added. The mixture was stirred
until complete consumption of starting material (approx. 2 h, TLC). The solution
was poured into saturated Rochelle salt and stirred until phases separated (overnight).
The aqueous layer was extracted with CH2Cl2 (2x30 ml), the combined organic layers
were washed with brine, dried over MgSO4 and carefully evaporated. The residual
colourless liquid was purified by column chromatography (2% Et2O in pentane) to
afford 737 mg (94%) of a 28 as pleasantly smelling colourless liquid.
1H NMR (400 MHz, CDCl3, δ): 9.89 – 9.64 (m, 1H), 2.39 (ddd, J = 16.0, 5.7, 2.1 Hz,
1H), 2.22 (ddd, J = 16.0, 7.8, 2.6 Hz, 1H), 2.04 (d, J = 6.7 Hz, 1H), 1.28 (m, 14H),
0.96 (d, J = 6.7 Hz, 3H), 0.88 (t, J = 6.9 Hz, 3H).
13C NMR (101 MHz, CDCl3, δ): 180.15, 41.58, 36.64, 31.87, 30.12 ( - ), 29.69, 29.55,
29.27, 26.86, 22.65, 19.66 ( - ), 14.07 ( - ).
HRMS-ESI+ (m/z): [M + H]+ calculated for C12H25O, 185.191; found, 185.191.
[α]D + 10.3 (c = 0.25, hexanes).
Value of the optical rotation matches with a value previously reported.26
(R)-10-methyloctadec-7-enoic acid (33).
To a vigorously stirred suspension of 7-
(bromotriphenylphosphoranyl)heptanoic acid 27 (2.51 g, 5.3 mmol, 1.4 equiv) in
THF (4.0 ml) at 21 °C, a solution of LiHMDS (Sigma-Aldrich, 1 M, 10.6 ml, 10.6
mmol, 2.8 equiv) was added dropwise. After 30 min of stirring, (R)-3-
methylundecanal 28 (700 mg, 3.8 mmol) in a small amount of THF (1.00 ml) was
added dropwise over 5 min. The resulting reaction mixture was stirred until the
solution remained pale yellow (3 h), then HCl (1 M, aqueous) was added until the
pH reached 1. The mixture was transferred to a separatory funnel, the organic layer
was separated, and the aqueous layer was extracted with Et2O (2 x 25 ml). The
combined organic layers were washed with brine, dried over MgSO4 and evaporated.
The residual thick colourless liquid was purified by column chromatography (20%
Et2O in pentane 1% formic acid) to afford 890 mg (79%) of E and Z isomers of 36
as colourless liquid.
Chapter 2
36
2
1H NMR (400 MHz, CDCl3, δ): 5.38 (m, 2H), 2.35 (t, J = 7.5 Hz, 2H), 2.02 (m, 3H),
1.84 (m, 3H), 1.65 (dd, J = 9.9, 5.0 Hz, 1H), 1.27 (m, 2H), 0.86 (dt, J = 14.7,
7.3 Hz, 17H), 0.86 (dt, J = 14.7, 7.3 Hz, 6H).
13C NMR (101 MHz, CDCl3, δ): 173.49, 130.11, 128.81, 36.70, 34.52, 33.40, 31.91,
29.96 ( - ), 29.66, 29.34, 29.30, 28.72, 27.17, 27.07, 24.58, 22.67, 19.58 ( - ), 14.10 ( - ).
(R)-Tuberculostearic acid (4)
To a solution of (E)- and (Z)-(R)-10-
methyloctadec-7-enoic acid 33 (741 mg, 2.5 mmol) and hydrazine hydrate (2.5 ml, 53
mmol, 21 equiv) in an O2 atmosphere, riboflavin catalyst (47.5 mg, 0.13 mmol,
5.0 mol%) was added in one portion. The mixture turned from red to yellow and the
reaction was stirred for 24 h at ambient temperature (21 °C). After complete
conversion of the starting material, the solution was acidified with concentrated HCl
to pH = 1 and extracted with Et2O (3x50 ml). The combined organic layers were
dried over MgSO4 and evaporated. The residual yellow thick liquid was purified by
column chromatography (20% Et2O in pentane) to afford 724 mg of (R)-
tuberculostearic acid 23 (97%) as colourless liquid.
1H NMR (400 MHz, CDCl3, δ): 2.35 (t, J = 7.5 Hz, 2H), 1.63 (dt, J = 15.0, 7.5 Hz,
2H), 1.42 – 1.00 (m, 27H), 0.88 (t, J = 6.9 Hz, 3H), 0.83 (d, J = 6.5 Hz, 3H).
13C NMR (101 MHz, CDCl3, δ): 180.82, 37.08, 37.05, 34.06, 32.73 ( - ), 31.91, 30.02,
29.92, 29.68, 29.45, 29.35, 29.23, 29.05, 27.07, 27.02, 24.65, 22.67, 19.69 ( - ), 14.09
( - ).
HRMS-ESI+ (m/z): [M + H]+ calculated for C19H38O, 299.295; found, 299.295.
[α]D –0.2 (c = 3.1, CHCl3).
Caspofungin fatty acids (Scheme 3)
(E)-S-ethyl pent-2-enethioate (32)
To a cooled solution (0 °C) of (E)-pentenoic acid 37 (5.0 g, 50.0 mmol),
DCC (11.4 g, 55.0 mmol, 1.1 equiv) and DMAP (611 mg, 5.0 mmol, 10 mol%) in
pentane (500 ml), neat EtSH (7.2 ml, 0.1 mol, 2.0 equiv) was added dropwise. The
Synthesis of Methyl-branched Fatty Acids
37
2
mixture was allowed to slowly reach room temperature and stirred for 16 h. After
filtration through a short silica pad, volatiles were evaporated. The crude residue was
distilled on Kugelrohr and afforded product 32 as a colourless liquid (6.85 g; 95%).
1H NMR (400 MHz, CDCl3, δ): 6.90 (m, 1H), 6.06 (dd, J = 15.5, 1.5 Hz, 1H), 2.90
(m, 2H), 2.18 (qd, J = 7.2, 3.7 Hz, 2H), 1.15 (m, 3H), 0.99 (s, 3H).
13C NMR (101 MHz, CDCl3, δ): 190.03, 146.46 ( - ), 127.79 ( - ), 25.18, 22.95,
14.78 ( - ), 12.05 ( - ).
Spectral data correspond to literature.[27a]
(S)-S-ethyl 3-methylpentanethioate (38)
(S,RFe)-Josiphos.EtOH adduct (43.2 mg, 67 µmol, 1.3 mol%) and
CuBr.Me2S (12.8 mg, 62 µmol, 1.2 mol%) were stirred in freshly distilled tBuOMe
(56 ml) until the mixture remained homogeneous (typically 10-30 min). Then the
mixture was cooled to –78 °C and after 10 min a solution of MeMgBr in Et2O
(2.7 ml, 8.1 mmol, 1.6 equiv) was added dropwise during 10 min. After 15 min of
stirring a solution of thioester 10 (721 mg, 5.0 mmol) in tBuOMe (6.0 ml) was added
over 3 h by a syringe pump. The reaction mixture was stirred for an additional 16 h
at –78 °C, quenched with EtOH (5.0 ml) and allowed to reach ambient temperature.
Then a solution of NH4Cl (1 M, 50 ml) was added. The organic layer was separated
and the aqueous layer extracted with Et2O (3 20.0 ml). The combined organic layers
were dried over MgSO4 and carefully evaporated (the product is volatile). The
residual yellow liquid was purified by flash chromatography (20% Et2O in pentane)
to afford (S)-S-ethyl 3-methylpentanethioate (38) (800 mg, 80%) as a colourless
liquid.
1H NMR (400 MHz, CDCl3, δ): 2.87 (q, J = 7.4 Hz, 2H), 2.53 (dd, J = 14.4, 6.1 Hz,
1H), 2.34 (dd, J = 14.4, 8.1 Hz, 1H), 1.95 (m, 1H), 1.37 (m, 1H), 1.24 (t, J = 7.4 Hz,
4H), 0.9 (m, 6H).
13C NMR (101 MHz, CDCl3, δ): 199.36, 51.01, 32.63 ( - ), 29.23, 23.24, 19.02 ( - ),
14.78 ( - ), 11.21 ( - ).NMR spectra contain traces of solvents (≈5%). Spectral data
correspond to literature.20]
HRMS-ESI+ (m/z): [M + H]+ calculated for C8H16OS, 161.099; found 161.099.
Chapter 2
38
2
The enantiomeric ratio was determined on the corresponding methyl ester by chiral
stationary phase gas chromatography on a Chiraldex G-TA column (30 m x
0.25 mm), 60 ºC, retention times: 5.94 (R) / 6.05 (S) min: 98.5:1.5 (e.r.), 97% ee (as
reported in literature).20
[α]D +8.4 (c = 1.0 in CHCl3).
The absolute configuration was determined on the alcohol obtained by reduction
with LiAlH4.
[α]D +7.4 (c = 0.95 in CHCl3).
Literature[44] reports the opposite enantiomer with [α]D –8.5 (c = 1 in CHCl3).
(S,E)-S-ethyl 5-methylhept-2-enethioate (41)
A solution of (S)-S-ethyl 3-methylpentanethioate 38 (801 mg;
5.0 mmol) in CH2Cl2 (50 ml) was cooled to –55 °C and then a solution of DIBAL
(1 M in CH2Cl2, 6.0 ml, 6.0 mmol, 1.2 equiv) was added. The mixture was stirred
until complete conversion of starting material (ca 1.5 h). Subsequently, the mixture
was poured into a saturated Rochelles salt (potassium sodium tartrate) solution and
stirred until the phases separated (mostly within 2 h). Layers were separated and the
aqueous layer was extracted with CH2Cl2 (315 ml). The combined layers were dried
and carefully evaporated (the product is volatile) until the weight of the residue
corresponded to quantitative yield (501 mg).
To a cooled solution (0 °C) of 39 (1.8 g, 7.5 mmol, 1.5 equiv) in THF (25 ml) a
solution of n-BuLi (1.6 M in hexanes, 3.4 ml, 5.5 mmol, 1.1 equiv) was added
dropwise. The reaction mixture was stirred for 20 min at 0 °C. Then (S)-3-
methylpentanal from the previous step (ca 5.0 mmol) in a small amount of THF
(0.3 ml) was added. The reaction mixture was stirred overnight (16 h). The reaction
was quenched with water (10 ml). Layers were separated and the aqueous layer
extracted with Et2O (3 15 ml). The combined organic layers were dried over
MgSO4 and carefully evaporated (the product is volatile). The residue was purified
by flash chromatography on SiO2 (0.4% tBuOMe in pentane) and affored (S,E)-S-
ethyl 5-methylhept-2-enethioate 41 (651 mg, 70 %) as a colourless liquid.
1H NMR (400 MHz, CDCl3, δ): δ 6.85 (ddd, J = 8.6, 7.6, 1.2 Hz,), 6.08 (dd, J = 15.5,
1.3 Hz, 1H), 2.94 (m, 2H), 2.17 (ddd, J = 7.2, 6.5, 3.5 Hz, 1H), 2.00 (m, 1H), 1.53
Synthesis of Methyl-branched Fatty Acids
39
2
(dq, J = 13.3, 6.9 Hz, 1H), 1.36 (m, 2H), 1.26 (ddd, J = 7.4, 4.9, 1.2 Hz, 3H), 0.86 (m,
6H).
13C NMR (101 MHz, CDCl3, δ): 189.95, 144.27 ( - ), 129.69 ( - ), 39.21, 34.19 ( - ),
29.18, 22.99, 19.14 ( - ), 14.79 ( - ), 11.34 ( - ) + 2 peaks at 65.81 and 15.24 as Et2O
residues.
HRMS-ESI+ (m/z): [M + H]+ calculated for C10H18OS, 187.115; found, 187.115.
[α]D +8.2 (c = 1.8 in CHCl3);
(3S,5S)-S-ethyl 3,5-dimethylheptanethioate (42)
(S,RFe)-Josiphos-CuBr complex (29.1 mg, 1.30 mol%) was dissolved
in freshly distilled tBuOMe (24.0 ml) until the mixture remained homogeneous
(typically 10-30 minutes). Then the mixture was cooled to –78°C and after 10 min
a solution of MeMgBr in Et2O (3 M in Et2O, 1.20 ml, 1.30 equiv) was added
dropwise. After 15 min of stirring, a solution of thioester 41 (490 mg, 2.63 mmol) in tBuOMe (2.60 ml) was added over 3 h by a syringe pump. The mixture was stirred
for an additional 16 h at –78°C. The reaction was quenched by addition of EtOH
(2.00 ml) and the mixture was allowed to reach ambient temperature. Then an
aqueous solution of NH4Cl (1 M, 30 ml) was added, the organic layer separated and
the aqueous layer extracted with Et2O (3 20 ml). The combined organic layers were
dried over MgSO4 and carefully evaporated (the product is volatile). The residual
yellow liquid was purified by flash chromatography (0.4% tBuOMe in pentane) to
afford (3S,5S)-S-ethyl 3,5-dimethylheptanethioate 41 (418 mg, 78%) as a colourless
liquid.
1H NMR (400 MHz, CDCl3, δ): 2.86 (q, J = 7.4 Hz, 2H), 2.52 (dd, J = 14.4, 5.4 Hz,
1H), 2.28 (dd, J = 14.4, 8.5 Hz, 1H), 1.43 – 1.27 (m, 2H), 1.24 (t, J = 7.4 Hz, 4H),
1.04 (m, 3H), 0.92 (d, J = 6.6 Hz, 3H), 0.85 (m, 6H).
13C NMR (101 MHz, CDCl3, δ): 199.33, 51.29, 44.07, 31.53 ( - ), 29.00 ( - ), 28.66,
23.23, 20.15 ( - ), 19.48 ( - ), 14.79 ( - ), 11.07 ( - ).
HRMS-ESI+ (m/z): [M + H]+ calculated for C11H22OS, 203.147; found, 203.150.
[α]D +4.2 (c = 1.4 in CHCl3).
Chapter 2
40
2
(10S,12S)-10,12-dimethyltetradec-7-enoic acid (43)
A solution of (S)-S-ethyl 3-methylpentanethioate 41
(417 mg, 2.1 mmol) in CH2Cl2 (20 ml) was cooled to –55 °C and a solution of DIBAL
(1 M in CH2Cl2, 2.5 ml, 2.5 mmol, 1.2 equiv) was added. The mixture was stirred
until complete conversion of starting material (ca 1.5 h), then poured into saturated
aqueous Rochelles salt and stirred until phases separated (mostly within 2 h). Layers
were separated and the aqueous layer was extracted with CH2Cl2 (315 ml). The
combined layers were dried and carefully evaporated (the product is volatile) to a
weight corresponding to quantitative yield (294 mg).
To a stirred suspension of 7-(bromotriphenylphosphoranyl) heptanoic acid 27
(1.65 g, 3.8 mmol, 1.8 equiv) in THF (2.0 ml) at ambient temperature a solution of
LiHMDS (1 M in THF, 4.0 ml, 4.0 mmol, 2.0 equiv) was added dropwise. The
mixture was stirred until the suspension turned into a deep red solution. Then a
solution of (3S,5S)-3,5-dimethylheptanal (ca 2.1 mmol) in a small amount of THF
(300 µl) was added and the reaction mixture was stirred until complete consumption
of starting material (2 h). The mixture was acidified to pH=1 by dilute aq. HCl and
extracted with Et2O (3 20 ml). The combined organic layers were dried and
evaporated. The resulting thick liquid was purified by column chromatography (20%
Et2O in pentane) and afforded 43 (331 mg, 47%) as a colourless liquid (a mixture of
E and Z isomers).
1H NMR (400 MHz, CDCl3, δ): 5.37 (m, 2H), 2.35 (t, J = 7.5 Hz, 2H), 1.34 (m, 26H).
13C NMR (101MHz, CDCl3, δ): 131.22, 130.17, 129.03, 128.67, 44.23, 44.00, 39.77,
34.26, 34.03, 32.35, 31.61, 31.55, 30.70, 30.42, 29.29, 29.16, 28.72, 28.49, 27.08, 24.57,
24.51, 20.16, 20.06, 19.68, 11.14. – additional peaks observed due to the inseparable
E/Z mixture.
HRMS-ESI+ (m/z): [M + H]+ calculated for C16H32O2, 255.232; found, 255.235.
[α]D +12.2 (c = 1.8 in CHCl3).
(10R,12S)-10,12-dimethyltetradecanoic acid (16)
To a vigorously stirred solution of (10S,12S)-10,12-
dimethyltetradec-7-enoic acid 14 (310 mg, 1.2 mmol) and flavine catalyst
Synthesis of Methyl-branched Fatty Acids
41
2
(49.6 mg, 10 mol%) in EtOH (1.0 ml) under oxygen atmosphere, hydrazine hydrate
(1.6 ml, 31.7 mmol, 26 equiv) was added in one portion. Vigorous stirring continued
for 16 h. Then the reaction mixture was acidified to pH=1 by dilute aq. HCl and
extracted with Et2O (3 20 ml). The combined organic layers were dried and
evaporated. The residual red liquid was purified by column chromatography
(20% Et2O in pentane) and afforded 16 (524 mg, 81%) as a colourless thick liquid.
1H NMR (400 MHz, CDCl3, δ): 11.20 (bs, 1H), 2.34 (t, J = 7.5 Hz, 2H), 1.13 (m,
27H).
13C NMR (101MHz, CDCl3, δ): 180.15, 44.70, 36.85, 34.21, 31.56 ( - ), 30.00 ( - ),
29.93, 29.46, 29.24, 29.20, 29.07, 26.86, 24.73, 20.26 ( - ), 19.72 ( - ), 11.17 ( - ).
HRMS-ESI- (m/z): [M - H]+ calculated for C16H32O2, 255.232; found, 255.216.
[α]D +14.1 (c = 1.3 in CHCl3).
(S)-12-methyltetradec-9-enoic acid (45)
To a stirred solution of (S)-S-ethyl 3-
methylpentanethioate 38 (73.5 mg, 0.5 mmol) in CH2Cl2 (0.8 ml) at –50 °C, DIBAL
was added. After complete conversion of the thioester (2 h), the reaction mixture
was poured into a saturated solution of Rochelle salt. After clear layer separation, the
organic layer was separated and the aqueous layer extracted with Et2O (3 10 ml).
The combined organic layers were dried and carefully evaporated until the weight
corresponded to a quantitative yield (43.0 mg).
Then, to a stirred suspension of 9-(bromotriphenyl phosphoranyl)nonanoic acid 44
(425 mg, 0.9 mmol, 1.7 equiv.) in THF (0.8 ml) a solution of LiHMDS (1 M in THF;
1.0 ml, 1.0 mmol, 2.0 equiv) was added until the solution remained deep red. To this
solution, 3-methyl-pentanal in a small amount of THF (300 µL) was added. The
reaction mixture was stirred until complete conversion of the starting material (2 h)
and acidified to pH=1 by dilute aq. HCl. The resulting solution was extracted with
Et2O (3 20 ml) and the combined organic layers were dried and evaporated. The
resulting thick liquid was purified by column chromatography (20% Et2O in pentane)
and afforded 45 (65.9 mg, 60%) as a colourless liquid (as a mixture of E and Z
isomers).
Chapter 2
42
2
1H NMR (400 MHz, CDCl3, δ): 5.37 (dt, J = 6.0, 4.6 Hz, 2H), 2.35 (t, J = 7.5 Hz,
2H), 2.01 (d, J = 5.4 Hz, 2H), 1.63 (m, 2H), 1.30 (m, 14H), 0.88 (m, 6H).
13C NMR (101 MHz, CDCl3, δ): 177.89, 130.32 ( - ), 128.66 ( - ), 35.11, 34.13, 33.63,
29.69, 29.46, 29.19, 28.94, 28.88, 27.19, 24.64, 19.11 ( - ), 11.55 ( - ) (1 signal
overlapping)
HRMS-ESI- (m/z): [M - H]+ calculated for C15H29O2, 241.216; found, 241.217.
[α]D= +0.3 (c = 0.5 in CHCl3).
(S)-13- methylpentadecanoic acid (46)
To a vigorously stirred solution of (13S)-13-
methyltetradec-7-enoic acid 15 (60 mg, 250 μmol) and flavine catalyst 26 (10 mg,
25 µmol, 10 mol%) in EtOH (1.0 ml) under oxygen atmosphere, hydrazine hydrate
(375 µl, 7.5 mmol, 30 equiv.) was added in one portion. Vigorous stirring was
continued for 16 h and the mixture was acidified to pH=1 by dilute aq. HCl and
extracted with Et2O (3 20 ml). The combined organic layers were dried and
evaporated. The residual red liquid was purified by column chromatography (20%
Et2O in pentane) and afforded 46 (50.0 mg, 83%) as a colourless thick liquid.
1H NMR (400 MHz, CDCl3, δ): 2.34 (t, J = 7.5 Hz, 2H), 1.62 (m, 2H), 1.28 (m, 20H),
0.84(m, 6H).
13C NMR (101 MHz, CDCl3, δ): 220.66, 36.62, 34.38, 34.07, 29.99, 29.66, 29.58,
29.48, 29.42, 29.23, 29.05, 27.09, 24.66, 19.20 ( − ), 11.39 ( − ).
HRMS-ESI+ (m/z): [M + H]+ calculated for C15H31O2, 243.231; found, 243.233.
[α]D= +0.3 (c = 0.5 in CHCl3).
References and footnotes
(1) (a) Ohagan, D. Nat. Prod. Rep. 1993, 10, 593(b) Minnikin, D. E.; Kremer, L.; Dover,
L. G.; Besra, G. S. Chem. Biol. 2002, 9, 545(c) Rezanka, T.; Sigler, K. Prog. Lip. Res.
2009, 48, 206.
(2) Rainwater, D. L.; Kolattukudy, P. E. J. Biol. Chem. 1985, 260, 616.
Synthesis of Methyl-branched Fatty Acids
43
2
(3) Jauregui.G; Lenfant, M.; Toubiana, R.; Azerad, R.; Lederer, E. Chem. Commun. 1966,
855.
(4) Kaneda, T. Microbiol. Rev. 1991, 55, 288.
(5) Denning, D. W. The Lancet 2003, 362, 1142.
(6) Fujie, A. Pure Appl. Chem. 2007, 79, 603.
(7) O'Sullivan, D. M.; Nicoara, S. C.; Mutetwa, R.; Mungofa, S.; Lee, O. Y. C.; Minnikin,
D. E.; Bardwell, M. W.; Corbett, E. L.; McNerney, R.; Morgan, G. H. PLoS One
2012, 7.
(8) French, G. L.; Chan, C. Y.; Cheung, S. W.; Teoh, R.; Humphries, M. J.; O'Mahony,
G. The Lancet 1987, 330, 117.
(9) Knapp, F. F., Jr.; Ambrose, K. R.; Goodman, M. M. Eur. J. Nucl. Med. 1986, 12, S39.
(10) Ran-Ressler, R. R.; Devapatla, S.; Lawrence, P.; Brenna, J. T. Pediatr. Res. 2008, 64,
605.
(11) (a) Prout, F. S.; Cason, J.; Ingersoll, A. W. J. Am. Chem. Soc. 1947, 69, 1233 (b) Prout,
F. S.; Cason, J.; Ingersoll, A. W. J. Am. Chem. Soc. 1948, 70, 298 (c) Liu, X.; Stocker,
B. L.; Seeberger, P. H. J. Am. Chem. Soc. 2006, 128, 3638 (d) Dyer, B. S.; Jones, J. D.;
Ainge, G. D.; Denis, M.; Larsen, D. S.; Painter, G. F. J. Org. Chem. 2007, 72, 3282
(e) Roberts, I. O.; Baird, M. S. Chem. Phys. Lipids 2006, 142, 111 (f) ter Horst, B.;
Seshadri, C.; Sweet, L.; Young, D. C.; Feringa, B. L.; Moody, D. B.; Minnaard, A. J.
J. Lipid Res. 2010, 51, 1017.
(12) (a) Smit, C.; Fraaije, M. W.; Minnaard, A. J. J. Org. Chem. 2008, 73, 9482 (b) Teichert,
J. F.; den Hartog, T.; Hanstein, M.; Smit, C.; ter Horst, B.; Hernandez-Olmos, V.;
Feringa, B. L.; Minnaard, A. J. ACS Catal. 2011, 1, 309.
(13) Carballeira, N. M.; Cruz, H.; Hill, C. A.; De Voss, J. J.; Garson, M. J. Nat. Prod. 2001,
64, 1426.
(14) López, F.; Minnaard, A. J.; Feringa, B. L. Acc. Chem. Res. 2006, 40, 179.
(15) Harutyunyan, S. R.; den Hartog, T.; Geurts, K.; Minnaard, A. J.; Feringa, B. L. Chem.
Rev. 2008, 108, 2824.
(16) Fukuyama, T.; Tokuyama, H. Aldrichimica Acta 2004, 37, 87.
(17) van Zijl, A. W.; Minnaard, A. J.; Feringa, B. L. J. Org. Chem. 2008, 73, 5651.
(18) Keck, G. E.; Boden, E. P.; Mabury, S. A. J. Org. Chem. 1985, 50, 709.
(19) Claridge, T. D. W.; Davies, S. G.; Lee, J. A.; Nicholson, R. L.; Roberts, P. M.; Russell,
A. J.; Smith, A. D.; Toms, S. M. Org. Lett. 2008, 10, 5437.
(20) Des Mazery, R.; Pullez, M.; López, F.; Harutyunyan, S. R.; Minnaard, A. J.; Feringa,
B. L. J. Am. Chem. Soc. 2005, 127, 9966.
(21) (a) Corey, E. J.; Mascitti, V. J. Am. Chem. Soc. 2004, 126, 15664 (b) Corey, E. J.;
Mascitti, V. J. Am. Chem. Soc. 2006, 128, 3118 (c) Jakubowski, J. A.; Kohn, T. J.;
Mais, D. E.; Takeuchi, K.; True, T. A.; Wyss, V. L.; Mais, D. E.; True, T. A. Bioorg.
Med. Chem. Let. 1998, 8, 1943
(22) Leonard, W. R.; Belyk, K. M.; Bender, D. R.; Conlon, D. A.; Hughes, D. L.; Reider,
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Chapter 2
44
2
(24) White, J. D.; Johnson, A. T. J. Org. Chem. 1994, 59, 3347.
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45
Chapter 3 Catalytic Synthesis of Enantiopure Mixed Diacylglycerols
Abstract: Protected diacylglycerols are valuable precursors of phospholipids. A
catalytic one-pot synthesis of TBDMS-protected diacylglycerols has been developed,
starting from enantiopure glycidol. Subsequent migration-free deprotection leads to
stereo- and regiochemically pure diacylglycerols, which can be converted into the
desired phospholipids. Application of a more electrophilic catalyst allows synthesis
of mixed ether/ester lipids.
Parts of this chapter have been published: Fodran, P.; Minnaard, A. J. Org. Biomol.
Chem. 2013, 11, 6919.
Chapter 3
46
3
Introduction
Diacylglycerols and diacyl glycerophospholipids are compounds with an
enormous significance for living organisms.1 Besides being the major components of
cell membranes, phospholipids in particular are involved in numerous physiological
processes, and some of them even found therapeutic application.2 Natural
glycerophospholipids contain a phosphate head group and usually two different acyl
chains (Figure 1). A different chain length results in a different hydrophobic
thickness of the membrane formed from these lipids, which has a direct effect on
the functioning of membrane embedded proteins.3 The variability in head group and
acyl chains results in many different glycerophospholipid species (see Chapter 1).
While separation of phospholipids based on the head group is relatively
straightforward, separation based on the chain length is practically impossible.
Figure 1. Mixed diacyl glycerolphospholipids.
Despite their apparent structural simplicity, 1,2-diacylglycerols bearing two
different acyl residues are challenging synthetic targets. A commonly encountered
difficulty is the migration of the acyl group from the secondary to the primary
hydroxyl (Figure 2), which is accompanied by the release of steric strain.4
Figure 2. 1,2-to-1,3 acyl shift in diacylglycerols.
This acyl shift is catalyzed by traces of acids and bases. Furthermore, it also occurs
on chromatography stationary phases (silica gel, aluminium oxide) leading to
decreased yields and tedious purification.
Catalytic Synthesis of Enantiopure Mixed Diacylglycerols
47
3
Over the years, the limited access to diacylglycerols and phospholipids
bearing 2 different chains attracted the attention of organic chemists. Several
synthetic routes circumvent the acyl migration, usually by application of multiple
protection/deprotection steps. Starting from enantiopure 4-methoxybenzyl
protected glycerol, Martin et al.5 obtained mixed diacyl glycerolphospholipids in a
sequence of 8 steps and an overall 52% yield. Gras and Bonfanti6 used 5-hydroxy-
1,3-dioxan (formaldehyde protected glycerol) to synthetize selectively mixed 1,2-
diacylglycerols. A drawback of this approach is that the products are racemic.
Massing and Eibl7 applied protected D-mannitol in the synthesis of ether-based lipids
represented by platelet activating factor (PAF). Guanti et al.8 synthetized 1,2-
diacylglycerols by chemo-enzymatic methods. Starting from racemic solketal
(glycerol acetonide), the authors obtained 1,2-dipalmitoyl-sn-glycerol in 55% yield
using the Amano P protease.
Regioselective glycidol opening can be an attractive and atom economic
option for the synthesis of phospholipids. This strategy was pursued in several ways
(Scheme 1).
Scheme 1. Phospholipid syntheses with epoxide ring opening as the key step.
Chapter 3
48
3
Burgos et al.9 obtained monoacylglycerol 4 in 25% yield starting from glycidol 3 by
applying excess of stearic acid and Ti(OiPr)4. Besides the low yield (25%), the authors
reported a decreased enantiopurity of 4 compared to the starting material. Ali and
Bittman10 used BF3.OEt2 for ring-opening of tosyl glycidol 5 by a fatty acid
anhydride. Although the reaction afforded 6 in a good 76% yield, it is limited to 1,2-
diacylglycerols containing two identical acyl chains. Lindberg et al.11 achieved
regioselective acylation of epoxide 7 in conditions promoting SN2 reaction (good
nucleophile, polar aprotic solvent). Reaction with an excess (3 equiv) of palmitic acid
and cesium palmitate resulted in 8 in a good 65% yield. Stawinski and Stamatov12
described a 3 step procedure starting from 9. The epoxide 9 was first treated with an
excess of nBu4NI and the resulting iodohydrine was treated with an excess of acid
chloride to afford 10. The iodide, which prevented acyl migration, was substituted in
the last step with an excess of nBu4N salt of fatty acid affording 11 in an excellent
92% over 3 steps.
In the current era, in which sustainability is a frequently used term, a general,
efficient, and preferably catalytic approach to enantiopure mixed diacyl glycerols
would be highly desirable. A drawback of the development of such an approach is
the lack of catalysts that allow regioselective ring opening of terminal epoxides with
acids. Jacobsen et al.13 reported the application of a cobalt catalyst 13 in a
desymmetrization of meso-epoxide 12 (Scheme 2).
Scheme 2. Desymmetrization and kinetic resolution of epoxides using 13.
Catalytic Synthesis of Enantiopure Mixed Diacylglycerols
49
3
Although application of 13 to the kinetic resolution of terminal epoxides by fatty
acids was unsuccessful, in the case of hydrolytic kinetic resolutions of terminal
epoxides 15 the catalyst gives excellent results (Scheme 2).
The following part of this chapter describes the development of a catalytic
one-pot synthesis of protected diacylglycerols, and their further conversion into the
desired phospholipids bearing two different acyl groups. Furthermore, a slight
modification of the conditions allowed the synthesis of mixed ether/ester products,
which can be further converted into, for example, platelet activating factor.
Results and discussion
Synthesis of enantiopure phospholipids
It turned out that in solvent-free conditions, enantiopure TBDMS protected
glycidol (17) reacted smoothly with stearic acid in the presence of catalytic 13 and
stoichiometric iPr2NEt. These conditions led to monoacyl glycerol 18 as a single
regioisomer in quantitative yield as confirmed by 1H-NMR, 13C-NMR and GC-MS.
As 18 was the only product of the ring opening reaction it could be esterified in the
same pot using a second fatty acid, DCC and DMAP. After full conversion of 18,
the crude reaction mixture was transferred on a silica gel column without any workup
and chromatographed to afford 19 in 92% yield over two steps (Scheme 3). The
reaction could be readily scaled-up.
Reagents and conditions: (a) C17H35CO2H (1.0 equiv), iPr2NEt (1.0 equiv), 13 (1 mol%), 21 °C, 16 h; (b) C15H31CO2H (1.2 equiv), DCC (1.1 equiv), DMAP (10 mol%), heptane, 21 °C, 16 h.
Scheme 3 A two-step-one-pot synthesis of protected diacylglycerol 18.
Chapter 3
50
3
At 5 mmol scale, the protected diacylglycerol was isolated in 91% yield,
corresponding to 3.2 g of the desired product. The two-step-one-pot protocol also
allows synthesis of unsaturated protected diacylglycerols. When oleic acid was
applied for the ring opening and palmitic acid for the final esterification, the product
was isolated in 82% yield over 2 steps.
Acyl migration during deprotection of protected 1,2-diacylglycerols is a
common problem, which is enhanced by a difficult separation of the undesired
rearranged 1,3-diacylglycerol. Although conditions for migration-free deprotection
of benzylated and tritylated14 diacylglycerols have been reported,
Reagents and conditions: (a) BF3.CH3CN (1.1 equiv), CH2Cl2, 21 °C, 5 min; (b) 21 (1.2 equiv), 4,5-dicyanoimidazole (1.0 equiv), CH2Cl2, 15 min; (c) tBuOOH (3.0 equiv), 10 min; (d) Pd/C (5 mol%), MeOH / HCO2H (96 / 4), 21 °C.
Scheme 4. Conversion of a protected diacylglycerol into the corresponding phospholipid.
migration-free desilylation of 19 (Scheme 4) is a challenge for nearly 40 years.14-15
Examination of a wide variety of conventional deprotection conditions including
TBAF and TFA,16 led to substantial migration prior to full conversion. Conditions
that applied Lewis or Brønsted acids in only catalytic amounts17 gave similar results
at best. Finally, utilization of a small excess of BF3 (as its etherate or as its acetonitrile
complex) in CH2Cl2 at 0 °C, cleaved the TBDMS group with no detectable migration
(TLC, NMR18) within 5 min. The reaction was closely monitored (by TLC) and was
quenched immediately upon full conversion. To exclude any migration during the
work-up, the reaction was quenched with chilled phosphate buffer (1 M, pH = 7).
Catalytic Synthesis of Enantiopure Mixed Diacylglycerols
51
3
The 1,2-diacylglycerol 20 was isolated in quantitative yield and used without further
purification.
The target 1,2-diacyl phosphatidylethanolamine 23 was synthetized using the
phosphoramidite coupling/oxidation methodology19 which is widely used in solid-
phase DNA synthesis. The mild reaction conditions and short reaction times
minimize the possibility of acyl migration. Full conversion of 20 was achieved within
15 min using 4,5-dicyanoimidazole as the activator, 21 as the head group precursor
and tBuOOH as the oxidant. The reaction was very clean and 22 was obtained in
85% yield. Finally, the protecting groups were removed by hydrogenolysis using
catalytic Pd/C and HCO2H as hydrogen donor. This led, after purification over
silica-gel, to the desired phospholipid 23 in 72% yield starting from 19.
Enantiopurity does not decrease during the ring opening
There is a frequently overlooked pathway that might decrease the
enantiopurity of the diacylglycerol product. In an ideal case, the epoxide opening
step displays high regioselectivity on the C1 position of 17 (Figure 3), therefore the
optical purity of 24 does not erode. But, if the regioselectivity is insufficient, then an
SN220 attack on the C2 position of 17 results in 25, which after acyl migration gives
26. As a result, the optical purity of the ring-opened product is lower.
Figure 3. Decrease of enantiopurity as a result of insufficient regioselectivity in the ring opening.
Routinely, in the field of phospholipids, the optical purity is studied via analysis of
the corresponding Mosher’s esters.21 However, the reported 1H- and 19F-NMR shifts
are in some cases contradictory. 9-11,22 Furthermore, incomplete conversion of the
starting material in the esterification with Mosher’s acid chloride (leading to a kinetic
Chapter 3
52
3
resolution), together with an attempt to purify the diastereomeric products, might
lead to an undesired diastereomeric enrichment, thus leading to erroneous results.
To circumvent these issues, the optical purity in the cobalt catalyzed reactions was
studied by HPLC with a chiral stationary phase. To this end, the ring-opening
reaction was performed on the closely related TBDPS-glycidyl ether.23
Application of the identical reaction conditions as shown above (Scheme 3) led to
smooth ring opening, and the resulting protected monoacylglycerol was analyzed
without any further sample manipulation. HPLC analyses on a chiral stationary phase
showed no decrease of the optical purity during this step. It is therefore safe to
conclude that the reaction occurs regio-specifically on the terminal position of the
epoxide.
Synthesis of the platelet-activating factor (PAF)
The developed methodology was applied in the synthesis of platelet-
activating factor (33) (Scheme 5). This mixed ether/ester type phosphatidylcholine
lipid affects the aggregation of platelets, and has a function in processes like glycogen
degradation, reproduction, brain function and blood circulation. Up to date, several
syntheses of platelet-activating factor have been reported.7,24
Initially, the reaction of glycidol 17 with hexadecanol in the same conditions
as with carboxylic acids (Scheme 3) led only to a marginal conversion. However,
application of the more electrophilic 2725 led to full conversion (Scheme 5) of
hexadecanol in the presence of epoxide 17 in 3 days. The reaction proceeded in the
same clean manner as above, affording ring opened product 28 as a single
regioisomer. This was further converted to 29 with excess Ac2O in the presence of
DMAP and Et3N. 29 was further converted to 33 in 3 steps (Scheme 5). Desilylation
in the same conditions as in the case of diacylglycerols (CH3CN.BF3 complex)
resulted in a migration-free deprotection. The phosphatidylcholine head group was
installed by a known 2 step procedure.26 In the first step 30 was phosphorylated with
an excess of 31 in the presence of Et3N. After full conversion and removal of the
excess of the reagents, 31 was treated with Me3N in the presence of TMSOTf. The
crude reaction mixture was loaded on a low-surface silica gel column (see
experimental section) and chromatographed. This afforded 33 in 71% yield over 3
steps (46% starting from 17).
Catalytic Synthesis of Enantiopure Mixed Diacylglycerols
53
3
Reagents and conditions: (a) hexadecanol (0.55 equiv), 27 (2 mol%), THF, 21 °C, 3 d (b) Ac2O (2.5 equiv), Et3N (2.5 equiv), DMAP (10 mol%), CH2Cl2, 21 °C, 16 h; (c) BF3.CH3CN (1.1 equiv), CH2Cl2, 0 °C, 20 min; (d) 31 (4.0 equiv), iPr2NEt (4.0 equiv), CH2Cl2, 0 °C, 16 h; (e) Me3N (1.5 equiv), TMSOTf (2 equiv), CH2Cl2.
Scheme 5. Synthesis of platelet activating factor (33).
Conclusions
A one-pot synthesis of enantiopure mixed diacylglycerols was developed,
starting from TBDMS-protected glycidol. The longstanding problem of acyl
migration upon deprotection of silyl-protected diacylglycerols was solved with the
use of a TBDMS protecting group and a BF3-complex for the desilylation. The
protocol is experimentally straightforward and can be readily scaled-up to a multi-
gram scale. The overall synthesis of phospholipids was optimized and scaled-up. The
possibility of erosion of the optical purity during the ring-opening reaction was ruled
out. Application of the more electrophilic catalyst 27 allowed synthesis of mixed
ether/ester type phospholipids represented by platelet activating factor.
Experimental section
A two-step-one-pot synthesis of protected diacylglycerol (Scheme 3)
(R)-3-((tert-butyldimethylsilyl)oxy)-2-(palmitoyloxy)propyl stearate (19)
Chapter 3
54
3
Stearic acid (1.42 g, 5.0 mmol, 1.0 equiv) and 1313 (30 mg,
50 µmol, l mol%) were suspended in a small amount of ether (ca 1 ml) and stirred
in an oxygen atmosphere for 15 min at 21 °C. The solvent was evaporated, and
Hünigs base (873 µl, 5.0 mmol, 1.0 equiv) was added. After 5 min of stirring, (R)-
TBDMS-glycidyl ether (1.0 ml, 5 mmol) was added, and the mixture was stirred for
16 h.
After 1H-NMR showed (attenuation of the signals at 2.63 ppm and 2.77 ppm)
complete conversion of the glycidyl ether, all volatiles were evaporated in high
vacuum. To a solution of this intermediate (5.0 mmol) in heptane (10 ml), palmitic
acid (1.54 g, 6.0 mmol, 1.2 equiv) and DMAP (61.0 mg, 0.5 mmol, 5 mol%) were
added, the mixture was chilled to 0 °C, and DCC (1.24 g, 6.0 mmol, 1.2 equiv) was
added in one portion. The reaction mixture was stirred for 16 h and subsequently
directly placed on a SiO2 column and chromatographed using 9% Et2O in pentanes
to afford the desired product (3.24 g, 91%) as a white solid.
1H NMR (400 MHz, CDCl3, δ): 5.1 (m, 1H), 4.33 (dd, J = 11.8, 3.7 Hz, 1H), 4.15
(dd, J = 11.9, 6.3 Hz, 1H), 3.70 (m, 2H), 2.29 (td, J = 7.6, 2.1 Hz, 4H), 1.60 (m, 4H),
1.26 (broad s, 61H), 0.87 (m, 15H), 0.04 (s, 6H).
13C NMR (101 MHz, CDCl3, δ): 173.36, 173.01, 71.64( - ), 62.41, 61.43, 34.31, 34.13,
31.90, 29.68, 29.64, 29.61, 29.46, 29.34, 29.27, 29.11, 29.08, 25.72 ( - ), 24.92, 24.89,
22.66, 18.16, 14.07, -5.52 ( - ), -5.56 ( - ).
[α]D +7.1 (c = 2.3, CHCl3).
Melting point 45 °C
HRMS: (ESI+) calculated for C43H87O5Si [M+H] +: 710.624 found: 710.635.
Conversion of a protected diacylglycerol into the corresponding phospholipid (Scheme 4).
(S)-3-hydroxy-2-(palmitoyloxy)propyl stearate (20)
Catalytic Synthesis of Enantiopure Mixed Diacylglycerols
55
3
A solution of 19 (1.5 g, 2.1 mmol) in CH2Cl2 (20 ml) was immersed
in an ice bath (ice/water). Then CH3CN.BF3 (2.0 ml, 2.3 mmol, 1.1 equiv) was added,
and the resulting light yellow mixture was stirred for 5 min, while carefully monitored
by TLC. After full conversion, the reaction mixture was diluted with Et2O (100 ml)
and poured onto cooled phosphate buffer (pH = 7, 1 M, 25 ml). The organic layer
was separated and washed with saturated brine (50 ml), dried and evaporated to
dryness to afford 1-stearoyl-2-palmitoyl glycerol (1.25 g, 99%) as a white solid. The
compound was used directly without delay and further purification.
1H NMR (400 MHz, CDCl3, δ): 5.08 (m, 1H), 4.32 (dd, J = 11.9, 4.5 Hz, 1H), 4.23
(dd, J = 11.9, 5.7 Hz, 1H), 3.72 (d, J = 4.9 Hz, 2H), 2.31 (m, 4H), 1.61 (dd, J = 12.9,
6.8 Hz, 4H), 1.25 (s, 54H), 0.87 (t, J = 6.8 Hz, 6H).
13C NMR (101 MHz, CDCl3, δ): 173.75, 173.40, 72.09 ( - ), 62.00, 61.51, 34.27, 34.08,
31.90, 29.68, 29.64, 29.60, 29.46, 29.34, 29.25, 29.10, 29.07, 24.92, 24.87, 22.67, 14.09
( - ).
(2R)-3-(((benzyloxy)(2-(((benzyloxy)carbonyl)amino)ethoxy)phosphoryl)oxy)-2-(palmitoyloxy)propyl stearate (22)
To a stirred solution of (S)-3-hydroxy-2-
(palmitoyloxy)propyl stearate (1.25 g, 2.1 mmol) in CH2Cl2 (10 ml), phosphoramidite
21 (1.13 g, 2.5 mmol, 1.2 equiv) was added. The mixture was cooled to 0 °C and 1H-
imidazole-4,5-dicarbonitrile (323 mg, 2.7 mmol, 1.3 equiv) was added in one portion.
The reaction was stirred until complete conversion of the starting diacylglycerol
(monitored by TLC - typically 30 min). Subsequently, the mixture was cooled to –20
°C, and tBuOOH (ca 5 M in decane, 800 µl, 4.4 mmol, 2.1 equiv.) was added,
followed by stirring for 30 min. The reaction was then diluted with 10 ml of CH2Cl2
and poured into aqueous NaHCO3 (1 M, 200 ml). The organic layer was washed with
aqueous HCl (1 M, 200 ml), brine, dried and evaporated. The resulting crude yellow
oil was purified by column chromatography on SiO2 using 10% pentane in CHCl3 to
afford 22 (1.78 g, 85%) as a colorless thick liquid, together with an co-eluting
impurity.
Chapter 3
56
3
1H NMR (400 MHz, CDCl3, δ): 7.32 (m, 1H), 5.34 (broad s, 1H), 5.18 (dd, J = 9.6,
5.3 Hz, 1H), 5.07 (m, 4H), 4.27 (m, 1H), 4.09 (m, 4H), 3.42 (m, 2H), 2.28 (m, 4H),
1.57 (d, J = 7.0 Hz, 4H), 1.23 (m, 52H), 0.88 (t, J = 6.8 Hz, 6H), 0.83 (d, J = 6.5 Hz,
9H).
13C NMR (101 MHz, CDCl3, δ): 173.19, 172.81, 128.80( - ), 128.67 ( - ), 128.47 ( - ),
128.09 ( - ), 128.03 ( - ), 69.80, 66.78, 65.49, 61.56, 41.33, 37.09, 34.10, 33.97, 32.75
( - ), 31.91, 30.02, 29.97, 29.68, 29.64, 29.52, 29.47, 29.35, 29.28, 29.11, 29.06, 27.08,
24.81, 22.67, 19.69 ( - ), 14.10 ( - ).
31P NMR (162 MHz, CDCl3, δ): -0.81, -0.83.
HRMS (ESI+): calculated for C55H93O10NP [M+H]+: 958.653, found 958.653.
[α]D +7.1 (c = 2.3, CHCl3).
(R)-2-ammonioethyl (2-(palmitoyloxy)-3-(stearoyloxy)propyl) phosphate (23)
To a stirred solution of the lipid precursor 22 (1.78 g, 1.8
mmol) in MeOH/formic acid (96 / 4, 50 ml), Pd/C (Degussa Type E101 NE/W,
95.4 mg, 90 µmol, 5.0 mol%) was added. The mixture was stirred under hydrogen
atmosphere (balloon) until complete conversion of the starting material (typically 2 h,
according to TLC). Subsequently, the solution was diluted with CH2Cl2 (200 ml), and
SiO2 (10 g) was added, followed by evaporation of the volatiles. The SiO2 with
adsorbed phospholipid was transferred onto a short (20 g) SiO2 column, impurities
were eluted with Et2O (100 ml), followed by elution of the phospholipid with CHCl3
/MeOH / H2O (65 / 35 / 7), to afford 23 (1.09 g, 85%) as a white solid.
1H NMR (400 MHz, CDCl3/CD3OD/D2O 95/35/2, δ): 5.21 (d, J = 4.6 Hz, 1H),
4.38 (dd, J = 12.1, 2.9 Hz, 3H), 4.23 (d, J = 5.3 Hz, 1H), 4.15 (dd, J = 12.1, 7.3 Hz,
1H), 4.02 (t, J = 8.7 Hz, 2H), 3.95 (t, J = 5.9 Hz, 2H), 3.30 (dt, J = 3.2, 1.6 Hz, 1H),
3.19 – 3.10 (m, 2H), 2.29 (m, 4H), 1.58 (d, J = 6.6 Hz, 4H), 1.22 (broad s, J = 15.4
Hz, 54H), 0.86 (t, J = 6.8 Hz, 6H).
13C NMR (101 MHz, CDCl3/CD3OD/D2O, δ): 174.12, 173.77, 70.38 ( - ), 63.63,
62.68, 61.56, 34.15, 34.02, 31.83, 29.60, 29.47, 29.45, 29.25, 29.07, 29.03, 24.84, 24.77,
22.56, 13.84 ( - ).
Catalytic Synthesis of Enantiopure Mixed Diacylglycerols
57
3
31P NMR (162 MHz, CDCl3/CD3OD/D2O, δ): 0.10.
HRMS (ESI+): calculated for C39H79NO8P [M+H]+:720.554 found 720.559.
[α]D +6.5 (c = 1.0, toluene).
Melting point: 125 °C
benzyl (2-(((benzyloxy)(diisopropylamino)phosphanyl)oxy)ethyl)carbamate (21)
In a dry 50 ml round bottom flask (benzyloxy)bis(N,N-
diisopropylamino)-phosphine (1.51 g, 4.5 mmol, 1.5 equiv) and CBz-amino ethanol
(585 mg, 3.0 mmol) were dissolved in dry CH2Cl2 (6 ml). The solution was cooled to
0 °C in an ice/brine bath. To this solution, solid tetrazole (210 mg, 1 equiv) was
added in one portion. Stirring was continued at 0 °C until full conversion of the CBz-
amino ethanol (TLC, 2 h). The mixture was diluted with CH2Cl2 (to a final volume
of ca 30 ml), the organic layer was washed with saturated Na2CO3 (2 x 30 ml), dried
over MgSO4 and evaporated. The resulting thick residue was purified on silica using
pentane / ethyl acetate / Et3N in ration 95 / 5 / 5.
The reaction afforded 656 mg of the desired compound as a colorless liquid (51%).
1H NMR (400 MHz, CDCl3, δ): 7.30 (m, 10H), 5.18 (bs, 1H), 5.10 (s, 2H), 4.70 (m,
2H), 3.73 (dd, J = 12.0, 7.4 Hz, 1H), 3.63 (m, 2 H), 1.19 (d, J = 6.8 Hz, 12 H).
13C NMR (101 MHz, CDCl3, δ): 156.51( - ), 139.27( - ), 136.78( - ), 128.60, 128.42,
128.15, 127.51, 127.23, 66.73 ( - ), 65.66 ( - ), 65.48 ( - ), 62.84 ( - ), 62.68 ( - ), 43.14
(d C-P coupling), 24.76 (t).
31P NMR (162 MHz, CDCl3, δ): 149.1.
NMR data correspond to those published previously.19b
Studies on the regioselectivity of the ring-opening and the optical purity of the ring-opened products.
(rac)-tert-butyl(oxiran-2-ylmethoxy)diphenylsilane
Chapter 3
58
3
In an oven dried Schlenk flask, imidazole (1.58 g, 23.2 mmol, 2.2 equiv)
was dissolved in CH2Cl2 (5.0 ml). Neat TBDPSCl (3.4 ml, 13.2 mmol, 1.1 equiv) was
added whereupon the mixture turned into a thick suspension which was cooled to
0 °C. To this suspension (rac)-glycidol (0.8 ml, 12.0 mmol) was added. The mixture
was stirred for 17 h allowing to reach gradually to RT. Solids were filtered and washed
with CH2Cl2 (3 x 20 ml). The combined organic layers were dried and concentrated.
The crude residue was further purified on silica using 20% Et2O in pentane to yield
the desired product (3.75 g) in quantitative yield as a colourless oil.
1H NMR (400 MHz, CDCl3, δ): 7.68 (d, J = 7.8 Hz, 4H), 7.41 (m, J = 13.7, 6.8 Hz,
6H), 3.85 (dd, J = 11.9, 3.1 Hz, 1H), 3.71 (dd, J = 11.8, 4.8 Hz, 1H), 3.13 (m, J = 7.5,
3.8 Hz, 1H), 2.75 (t, J = 4.6 Hz, 1H), 2.61 (dd, J = 5.1, 2.7 Hz, 1H), 1.06 (s, 9H).
13C NMR (101 MHz, CDCl3, δ): 135.94, 135.88 ( − ), 133.58, 133.57, 130.08, 128.05,
128.04, 64.61 ( - ), 52.61, 44.77 ( - ), 27.08, 19.57( - ). 27
(R)-tert-butyl(oxiran-2-ylmethoxy)diphenylsilane
In an oven dried Schlenk flask, imidazole (295 mg, 4.4 mmol, 2.2 equiv)
was dissolved in CH2Cl2 (20.0 ml). Neat TBDPSCl (620 µl, 2.2 mmol, 1.1 equiv) was
added whereupon the mixture turned into a thick suspension which was cooled to
0 °C. To this suspension, (R)-glycidol (98% ee, 130 µl, 2.0 mmol) was added. The
mixture was stirred for 17 h allowing to reach gradually to rt. Solids were filtered and
washed with CH2Cl2 (3 x 20 ml), the combined organic layers were dried and
concentrated. The crude residue was further purified on silica using 20% Et2O in
pentane to yield the desired product in 86% yield (538 mg, colorless liquid).
1H NMR (400 MHz, CDCl3, δ): 7.68 (d, J = 7.8 Hz, 4H), 7.41 (m, J = 13.7, 6.8 Hz,
6H), 3.85 (dd, J = 11.9, 3.1 Hz, 1H), 3.71 (dd, J = 11.8, 4.8 Hz, 1H), 3.13 (m, J = 7.5,
3.8 Hz, 1H), 2.75 (t, J = 4.6 Hz, 1H), 2.61 (dd, J = 5.1, 2.7 Hz, 1H), 1.06 (s, 9H).
13C NMR (101 MHz, CDCl3, δ): 135.94, 135.88 ( - ), 133.58, 133.57, 130.08, 128.05,
128.04, 64.61 ( - ), 52.61, 44.77 ( - ), 27.08, 19.57( - ).
GC/MS: calculated for C15H15O2Si [M-tBu]: 255, found 255.
Spectral data in agreement with those previously published27.
[α]D =+2.5 (c = 2.0, CHCl3).
Catalytic Synthesis of Enantiopure Mixed Diacylglycerols
59
3
The epoxide ring opening:
Reaction for racemic TBDPS-glycidyl ether was performed at 1 mmol scale using
TBDPS-(rac)-glycidyl ether (312 mg, 1.0 mmol), butyric acid (91.8 mg, 1.0 mmol,
1.0 equiv), Hünigs base (175 µl, 1.0 mmol, 1.0 equiv) and Co[salen] catalyst (60.1
mg, 0.1 mmol, 10 mol%).
Reaction of enantiopure glycidyl ether was performed on 0.5 mmol scale using
glycidyl ether (156 mg, 0.5 mmol), butyric acid (46 µl, 0.5 mmol, 1.0 equiv), Hünigs
base (87 µl, 0.5 mmol, 1.0 equiv) and catalyst (6.0 mg, 10 μmol, 1 mol%). Reactions
were performed using following procedure:
A solution of Co[salen] (1.0 mol%) and butyric acid (1.0 equiv) in Et2O (1 ml) was
stirred under oxygen atmosphere (balloon) for 15 min. A change in color from bright
red to red-brown was observed. The solvent was evaporated, and to the resulting
brown mixture, Hünigs base (1.0 equiv) was added, and after 5 min of stirring (R)-
TBDPS-glycidyl ether (1.0 equiv) was added. The resulting mixture was stirred for
16 h after which 1H-NMR showed complete conversion of the glycidyl ether
(attenuation of the signals at 2.63 ppm and 2.77 ppm). Subsequently, volatiles were
evaporated using high vacuum. Crude residue was analyzed on HPLC.
1H NMR (400 MHz, CDCl3, δ): 7.65 (d, J = 7.7 Hz, 4H), 7.42 (m, 6H), 4.19 (m, 1H),
3.94 (m, 1H), 3.83 (m, 1H), 3.69 (m, 1H), 1.62 (dq, J = 15.0, 7.6 Hz, 2H), 1.06 (s, 9H),
0.93 (t, J = 7.3 Hz, 3H)
13C NMR (101 MHz, CDCl3, δ): 179.15, 135.51, 129.90, 127.81, 70.04, 64.90, 64.40,
35.99, 26.80, 26.72, 19.23, 18.37, 13.65.
HPLC (racemate, c = 1 mg/ml): CHIRACEL® OD-H 98:2 flow: 1 ml.min-1 t1=9.87
min t2=11.24 min.
HPLC (enantioenriched, c = 1 mg/ml): tmajor=10.03 min tminor= absent.
MS: calculated for C19H23O4Si [M-tBu]:343, found 343.
Synthesis of the platelet-activating factor (scheme 5).
(R)-1-((tert-butyldimethylsilyl)oxy)-3-(hexadecyloxy)propan-2-yl acetate (29)
Chapter 3
60
3
In a dry Schlenk flask, hexadecanol (242 mg, 1.0 mmol) was
dissolved in THF (0.4 ml). To this solution, (R)-TBDMS-glycidyl ether (390 µl, 1.9
mmol, 1.9 equiv) and Co[salen]OTs catalyst (20 mg, 25μmol, 2.5 mol%) were added.
The mixture was stirred for 3 days at rt (progress monitored by GC) until full
conversion of hexadecanol. The reaction mixture was diluted with anhydrous Et2O
(1.0 ml), cooled with an ice bath and subsequently DMAP (12.2 mg, 0.1 mmol,
10 mol%), Ac2O (240 µl, 2.0 mmol, 2.0 equiv) and Et3N (350 µl, 2.0 mmol, 2.0 equiv)
were added. The mixture was stirred for 16 h. Et2O was evaporated, the crude was
suspended in pentane, transferred onto a silica column and chromatographed with
5% Et2O in pentane to afford 321 mg of 29 (68% over 2 steps) as colorless liquid.
1H NMR (400 MHz, CDCl3, δ): 5.00 (m, 1H), 3.74 (m, 2H), 3.57 (m, 2H), 3.42 (m,
2H), 2.07 (s, 3H), 1.55 (s, 4H), 1.25 (s, 27H), 0.88 (s, 12H), 0.05 (s, 6H).
Shift at 1.55 ppm overlaps with water from the CDCl3.
13C NMR (101 MHz, CDCl3, δ): 170.62, 73.38 ( - ), 71.72, 68.96, 61.75, 32.06, 29.84,
29.82, 29.80, 29.76, 29.70, 29.60, 29.50, 26.20, 25.91 ( - ), 25.88, 22.83, 21.29 ( - ),
18.35, 14.25 ( - ), -5.31 ( - ).
HRMS (ESI+): calculated for C27H57O4Si [M+H]+: 473.401, found 473.402.
[α]D = +4.0 (c = 1.0, CHCl3).
Platelet-activating factor (33)
In a dry Schlenk flask, PAF precursor 29 (200 mg, 0.42
mmol) was dissolved in dry CH2Cl2 (4.2 ml). This solution was cooled to 0 °C in an
ice/water bath and treated with BF3.CH3CN (240 µl, 0.46 mmol, 1.1 equiv). The
reaction was closely monitored in the conversion of 29 (TLC), and after 20 min full
conversion was observed. The reaction was quenched by adding cooled phosphate
buffer (pH = 7, 1 M). The mixture was diluted with Et2O (20 ml), the organic layer
was washed with water (2 x 10 ml), and brine, dried and evaporated. The crude
residue was dried under high vacuum for 30 min and used without further
purification (1H NMR spectrum indicated no acyl migration).
Catalytic Synthesis of Enantiopure Mixed Diacylglycerols
61
3
1H NMR (400 MHz, CDCl3, δ): 4.99 (m, 1H), 3.82 (m, 2H), 3.61 (m, 2H), 3.45 (m,
2H), 2.11 (t, 3H), 1.55 (m, 30H), 0.88 (t, J = 6.7 Hz, 3H).
The residue was subsequently dissolved in THF (4.2 ml) and 2-chloro-1,3,2-
dioxaphospholan-2-oxide (154 µl, 1.7 mmol, 4.0 equiv) and iPr2NEt (300 µl,
1.7 mmol, 4.0 equiv) were added. The mixture was stirred overnight (ca 16 h). When
the reaction reached full conversion, the mixture was diluted with Et2O (25 ml). The
organic phase was washed with water, and brine, dried over MgSO4 and evaporated
to dryness. The crude was subsequently dissolved in CH2Cl2 (1.5 ml), and cooled in
an ice/water bath. The solution was treated with TMSOTf (140 µl, 0.84 mmol, 2.0
equiv). The addition was accompanied with a color change, first to brown then to
red). To this solution Me3N was added (55 µl, 0.63 mmol, 1.5 equiv). Given that
Me3N is a gas at RT, a syringe wrapped in cotton previously dipped in acetone/liquid
N2 was used. The reaction was monitored by TLC (disappearance of the spot with
Rf = 0.21 in Et2O). Upon full conversion all volatiles were evaporated. The crude
residue was transferred onto a silica column and carefully chromatographed on 150
Å Davisil silica gel using a gradient of 1% to 20% MeOH in CHCl3.
149 mg of PAF was obtained as a white waxy solid (71% over 3 steps). NMR spectra
of PAF were not indicative due to extensive peak-broadening.
HRMS (ESI+): calculated for C26H54NO7P [M+H]+: 524.362, found 524.366.
[α]D = +3.5 (c = 1.0, CHCl3).
Melting point: 240 °C (decomposition).
References and footnotes
(1) (a) Simons, K.; Vaz, W. L. C. Annu. Rev. Biophys. Biomol. Struct. 2004, 33, 269 (b)
Simons, K.; Toomre, D. Nat. Rev. Mol. Cell Biol. 2000, 1, 31 (c) Cevc, G. Phospholipids
handbook; Marcel Dekker, Inc.: New York, 1993.
(2) Mintzer, M. A.; Simanek, E. E. Chem. Rev. 2008, 109, 259.
(3) Perozo, E.; Kloda, A.; Cortes, D. M.; Martinac, B. Nat. Struct. Mol. Biol. 2002, 9, 696.
(4) (a) Kodali, D. R.; Tercyak, A.; Fahey, D. A.; Small, D. M. Chem. Phys. Lipids 1990,
52, 163 (b) Crossley, A.; Freeman, I. P.; Hudson, B. J. F.; Pierce, J. H. J. Chem. Soc.,
Perkin Trans. 1959, 760.
(5) Martin, S. F.; Josey, J. A.; Wong, Y.-L.; Dean, D. W. J. Org. Chem. 1994, 59, 4805.
(6) Gras, J.-L.; Bonfanti, J.-F. Synlett 2000, 248.
(7) Massing, U.; Eibl, H. Chem. Phys. Lipids 1995, 76, 211.
Chapter 3
62
3
(8) Guanti, G.; Banfi, L.; Basso, A.; Bevilacqua, E.; Bondanza, L.; Riva, R. Tetrahedron:
Asymmetry 2004, 15, 2889.
(9) Burgos, C. E.; Ayer, D. E.; Johnson, R. A. J. Org. Chem. 1987, 52, 4973.
(10) Ali, S.; Bittman, R. J. Org. Chem. 1988, 53, 5547.
(11) Lindberg, J.; Ekeroth, J.; Konradsson, P. J. Org. Chem. 2001, 67, 194.
(12) (a) Stamatov, S. D.; Stawinski, J. Org. Biomol. Chem. 2007, 5, 3787 (b) Stamatov, S.
D.; Kullberg, M.; Stawinski, J. Tetrahedron Lett. 2005, 46, 6855.
(13) Jacobsen, E. N.; Kakiuchi, F.; Konsler, R. G.; Larrow, J. F.; Tokunaga, M.
Tetrahedron Lett. 1997, 38, 773.
(14) Kodali, D. R.; Duclos Jr, R. I. Chem. Phys. Lipids 1992, 61, 169.
(15) (a) Dodd, G. H.; Golding, B. T.; Ioannou, P. V. J. Chem. Soc., Chem. Commun. 1975,
249 (b) De Medeiros, E. F.; Herbert, J. M.; Taylor, R. J. K. J. Chem. Soc., Perkin Trans.
1991, 2725 (c) Buchnea, D. Lipids 1974, 9, 55.
(16) Wuts, P. G. M.; Greene, T. W. Greene's Protective Groups in Organic Synthesis, 4th Edition;
John Wiley & Sons: New Jersey, 2007.
(17) Pedersen, P. J.; Adolph, S. K.; Subramanian, A. K.; Arouri, A.; Andresen, T. L.;
Mouritsen, O. G.; Madsen, R.; Madsen, M. W.; Peters, G. N. H.; Clausen, M. H. J.
Med. Chem. 2010, 53, 3782.
(18) No 1,3-diacylglycerol is visible in the 1H-NMR spectrum. For a smooth
deprotection dry conditions are required.
(19) (a) Hayakawa, Y.; Kawai, R.; Hirata, A.; Sugimoto, J.-i.; Kataoka, M.; Sakakura, A.;
Hirose, M.; Noyori, R. J. Am. Chem. Soc. 2001, 123, 8165 (b) Rzepecki, P. W.;
Prestwich, G. D. J. Org. Chem. 2002, 67, 5454.
(20) Alternatively, an SN1 reaction might be considered. However, this also leads to lower
optical purity of the product.
(21) (a) Dale, J. A.; Mosher, H. S. J. Am. Chem. Soc. 1973, 95, 512 (b) Dale, J. A.; Dull, D.
L.; Mosher, H. S. J. Org. Chem. 1969, 34, 2543.
(22) (a) Guivisdalsky, P. N.; Bittman, R. J. Org. Chem. 1989, 54, 4637 (b) Guivisdalsky, P.
N.; Bittman, R. J. Org. Chem. 1989, 54, 4643.
(23) The TBDPS group was chosen because it is chromophoric.
(24) (a) Nakamura, N.; Miyazaki, H.; Ohkawa, N.; Oshima, T.; Koike, H. Tetrahedron Lett.
1990, 31, 699 (b) Guivisdalsky, P. N.; Bittman, R. J. Org. Chem. 1989, 54, 4643 (c)
Kertscher, H. P.; Ostermann, G. Pharmazie 1986, 41, 596 (d) Tsuri, T.; Kamata, S.
Tetrahedron Lett. 1985, 26, 5195 (e) Marx, M. H.; Wiley, R. A. Tetrahedron Lett. 1985,
26, 1379 (f) Ohno, M.; Fujita, K.; Nakai, H.; Kobayashi, S.; Inoue, K.; Nojima, S.
Chem. Pharm. Bull. 1985, 33, 572.
(25) (a) Ferrer, C.; Fodran, P.; Barroso, S.; Gibson, R.; Hopmans, E. C.; Sinninghe
Damsté, J.; Schouten, S.; Minnaard, A. J. Org. Biomol. Chem. 2013, 11, 2482 (b)
Venkatasubbaiah, K.; Zhu, X.; Kays, E.; Hardcastle, K. I.; Jones, C. W. ACS Catal.
2011, 1, 489.
(26) Gadek, T. R. Tetrahedron Lett. 1989, 30, 915.
(27) Pospíšil, J.; Markó, I. E. Tetrahedron Lett. 2006, 47, 5933.
63
Chapter 4 Enantiopure Triacylglycerols in Three Steps
Abstract: With the development of chromatography and spectrometry, the analysis
of complex mixtures of closely related triacylglycerols has come within reach in a
number of laboratories. Next to the sophisticated instrumentation, this “lipidomics”
heavily relies on the analytical standards of triacylglycerols. However, these are
limited and expensive and their synthesis requires multiple steps, protecting groups,
and the use of an excess of reagents. This chapter presents an efficient, 3 step
synthesis of enantiopure triacylglycerols, which can be used as analytical standards in
the analysis of complex triacylglycerol mixtures like milk fat.
Fodran, P.; Das, N.; Eisink, N.; Welleman, I.; Kloek, W.; Minnaard, A. J. manuscript in preparation.
Chapter 4
64
4
Introduction
If a di- or triacylglycerol bears 2 different acyl chains on the sn-1 and the sn-3
position, the compound is chiral, which means it has a non-superimposable mirror
image (enantiomer). This fact is frequently overlooked in research connected to di-
and triacylglycerols, although it was noted already in 1939.1,2 In fact, their
stereochemistry is rarely considered. On the contrary, racemic versus enantiopure
triacylglycerols show for example different crystallization behavior – and, translated
to daily life, this means that butter, margarine or chocolate leave different sensations
depending on their stereochemical composition.3 Another area where the
stereochemistry of triacylglycerols is frequently overlooked is the analysis of fats and
oils. During the past 70 years, a lot of effort has been invested into the analysis of
milk fat. These efforts invariably involved chromatographic separation of the
triacylglycerols and their subsequent identification, and has met with moderate
success. Milk fat is typically composed of triacylglycerols containing 9 different fatty
acids, which results in 93=729 possible isomers. From this number, 92=81
triacylglycerols are achiral, the remaining 648 are chiral. Because the fatty acids are
similar in structure, that is, only differ in their chain length and possible unsaturation,
most triacylglycerols are extremely difficult to separate by chromatography.
Furthermore, they show only negligible optical rotations (are cryptochiral).4 This
severely complicates the analysis of oils and fats and in particular milk fat. An
unexplored approach in this connection is the unambiguous synthesis of
triacylglycerols and their use as reference compounds in HPLC-MS or GC-MS.
This chapter describes the synthesis of some of the most abundant
triacylglycerols of milk fat in a collaboration with the Dutch diary company
FrieslandCampina.
Reported syntheses of triacylglycerols
Chemical modification of triacylglycerols to alter the properties of edible fats
was first described in the 1920s and established as an industrial process in the 1940s
in Germany.5 To improve the spreadability and baking properties, lard (pig fat) was
treated with sodium methoxide. On the molecular level (Figure 1), this results in a
random intra- and intermolecular redistribution of the fatty acid residues on glycerol.
Today, this process is known as fat randomization 6 and plays an important role in the
food industry.7
Enantiopure Triacylglycerols in Three Steps
65
4
Figure 1. Triacylglycerols formed during fat randomization.6a
A more defined triacylglycerol composition can be achieved by lipases. Betapol, which is a human breast milk mimic, contains 47% of fat mainly as 1,3-dipalmitoyl-2-oleyl-sn-glycerol (3). This triacylglycerol has beneficial effects on infants like softer stool and better absorption of fatty acids and calcium.
Scheme 1. Industrial production of Betapol.
The industrial production of the fat component of Betapol is based on enzymatic
interesterification of tripalmitoyl glycerol (1) (Scheme 1)8 with oleic acid. (2).
Although enzymes are powerful tools in modifying triacylglycerols, they give
enantioenriched products only in a very limited number of cases.9 Another limitation
of the enzymatic synthesis of triacylglycerols is that different fatty acid chain lengths
often require different lipases.
Chemical synthesis can provide enantiopure triglycerides. Kristinsson and
Haraldsson10,11 reported a 6-step approach (Scheme 2). (S)-Solketal 4 (from mannitol)
Chapter 4
66
4
was first benzylated and then hydrolyzed in acidic aqueous ethanol affording 5 in
87% yield. An sn-3 selective acylation of 5 by immobilized Candida antarctica lipase
(CAL) and vinyl stearate, followed by hydrogenolysis with Pd/C gave
monoacylglycerol 6 in 85% yield over 2 steps. A second esterification with the same
enzyme and vinyl capriate afforded 7 in 85% yield. Finally, esterification of 7 with
eicosapentaenoic acid (EPA) led to triacylglycerol 8 in 91% yield. A careful choice of
protecting groups and the use of a selective lipase circumvents acyl migration
(Chapter 3).
Scheme 2. Synthesis of enantiopure triacylglycerols according to Kristinsson and Haraldsson.
Stamatov and Stawinski12 presented a different strategy. Heating glycidyl ether 9
(Scheme 3-I) with an excess of trifluoroacetic anhydride and Bu4NI afforded the
iodohydrin, which after esterification in the same pot yielded 11 in 90%. Substitution
of iodide 10 by acetate yielded 11 in 92% yield. The TBDMSO- group in 11 was
directly converted into the ester, yielding triacylglycerol 12 in 90% yield. Based on
their NMR studies, the authors proposed a mechanism for the direct conversion of
11 to 12 (Figure 3-II). Treatment of palmitic anhydride with TMSBr yields 13 and
acid bromide 14. The silyl substituted oxygen of 11 attacks 14 resulting in 15.
Subsequently, bromide can attack 15 at silicon, thus affording 12 and TBDMSBr. In
case the authors applied 16 as starting material (Scheme 3-III), the silyl substitution
could be omitted, thus affording 17 in only 3 steps. In this approach, the application
of iodohydrines and direct substitution of TBDMSO- group for an ester circumvents
the acyl migration.
Enantiopure Triacylglycerols in Three Steps
67
4
Scheme 3. ( I ) Synthesis of triacylglycerols according to Stamatov and Stawinski; ( II ) proposed mechanism of direct substitution of 11 to 12; ( III ) in case glycidyl esters are applied, the synthesis is one step shorter.
Despite both approaches avoid acyl migration and yield the triacylglycerols in high
yields, they have some drawbacks. The shorter approach presented by Stamatov and
Stawinski uses significant excess of the reagents (3 to 5 equiv), what is not
economical in case of expensive (i.e. polyunsaturated) fatty acids. The approach
presented by Kristinsson and Haraldsson uses nearly stoichiometric (1.1 – 1.25
equiv) conditions, but requires 2 protecting groups and 6 steps.
The previous chapter (chapter 3) showed that 1 mol% of (R,R)-N,N′-bis(3,5-
di-tert-butylsalicylidene)-1,2-cyclohexanediaminocobalt(II) catalyzes the ring opening
of glycidyl ethers with fatty acids under basic conditions. If glycidyl esters would
exhibit the same reactivity, they can afford triacylglycerols by the same procedure in
3 steps and without utilization of a large excess of reagents or protecting groups.
Chapter 4
68
4
Results and discussion
Co[(R,R)-salen] catalyzed ring opening of glycidyl esters
Glycidyl esters 16a-g were prepared under mild Steglich conditions (Scheme
4). In the presence of DCC and catalytic amounts of DMAP, (S)-glycidol 15 was
conveniently esterified with a small excess of fatty acid (1.2 equiv).
Reagents and conditions: a) RCO2H (1.2 equiv), DCC (1.1 equiv), DMAP (10 mol%), pentane, 0 °C, 4 h.
Scheme 4. Esterification of (S)-glycidol.
Homologous glycidyl esters 16a-f (Table 1, entries 1-6) were obtained in high yields
(82-92%). In addition to the saturated derivatives, unsaturated glycidyl oleate 16g
was prepared in 81% yield (Table 1, entry 7).
Table 1. Prepared glycidyl esters.
Entry Fatty acid R: Yield
1 butyric acid C3H7-16a 86%
2 caproic acid C5H11-16b 92%
3 capric acid C9H19-16c 86%
4 myristic acid C13H27-16d 82%
5 palmitic acid C15H31-16e 92%
6 stearic acid C17H35-16f 88%
7 oleic acid C17H33 [18:1 cis-9]-16g 81%
In an initial experiment (Scheme 5), neat glycidyl myristate 16d was treated
with a stoichiometric amount of stearic acid in the presence of 17 (1 mol%) and
Hünigs base. These conditions resulted in quantitative formation of the 1,3-
diacylglycerol. Progress of this reaction (Scheme 5) was monitored by 1H NMR by
following the attenuation of the signals corresponding to the protons of the primary
epoxide carbon (δ = 2.63 and 2.83 ppm). As the reaction afforded ring-opened
product 18 exclusively, and after dilution with heptane the crude residue was
esterified in the same flask with stearic acid, DCC and DMAP. After full conversion
of 18, the reaction mixture was transferred directly onto a silica column and
chromatographed to afford analytically pure triacylglycerol 19 in 83% yield.
Enantiopure Triacylglycerols in Three Steps
69
4
Reagents and conditions: a) stearic acid (1.0 equiv), Hünigs base (1.0 equiv), 17 (1.0 mol%), neat, 20 h, RT; b) stearic acid (1.2 equiv), DCC (1.1 equiv), DMAP (5.0 mol%) heptane, 20 h, RT.
Scheme 5. Triacylglycerols, prepared according to the described method.
The scope of this two-step-one-pot procedure was further investigated with
various acids for ring opening and final esterification (Table 2). The synthetized
triacylglycerols were obtained in yields in the range of 79-92% over two steps. The
obtained yields are similar or higher than those described by Kristinsson and
Haraldsson, and by Stamatov and Stawinski.
Table 2. Synthetized triacylglycerols.
entry
-label
starting glycidyl ester
(sn-3 position)
Fatty acid used in ring opening
(sn-1 position)
Fatty acid in final esterification
(sn-2 position)
Yield
1-19a glycidyl butyrate 16a myristic acid oleic acid 86%
2-19b glycidyl butyrate 16a stearic acid palmitic acid 79%
3-19c glycidyl butyrate 16a oleic acid stearic acid 86%
4-19d glycidyl butyrate 16a oleic acid oleic acid 83%
5-19e glycidyl butyrate 16a linoleic acid myristic acid 85%
6-19f glycidyl caproate 16b oleic acid palmitic acid 82%
7-19g glycidyl caprate 16c oleic acid palmitic acid 88%
8-19h glycidyl palmitate 16e myristic acid stearic acid 80%
9-19i glycidyl palmitate 16e stearic acid myristic acid 79%
10-19j glycidyl palmitate 16e oleic acid oleic acid 92%
11-19k glycidyl stearate 16f butyric acid palmitic acid 82%
12-19l glycidyl stearate 16f myristic acid palmitic acid 80%
13-19m glycidyl stearate 16f oleic acid oleic acid 80%
14-19n glycidyl oleate 16g palmitic acid stearic acid 80%
Chapter 4
70
4
Furthermore, the fatty acids are used in stoichiometric or nearly stoichiometric
amounts. Compared to the enzymatic synthesis of triacylglycerols, the developed
reaction conditions tolerate different chain lengths in all three positions (Table 2). A
pair of isomeric triacylglycerols (entry 8 vs. 9) was prepared in very similar yields. The
unsaturated oleic acid is tolerated on all 3 (sn-1, sn-2, sn-3) positions (entries 1, 3, 4,
6, 7, 10, 13, 14). The double bonds of the polyunsaturated linoleic acid (18:2 cis,cis-
9,12, entry 5) remained intact. And finally, these conditions allow the synthesis of a
pair of enantiomers (entry 2 and 11) both from (S)-glycidol 16, if the fatty acids are
introduced in reversed order.
17 was initially developed for the kinetic resolution of various terminal
epoxides including glycidyl esters and ethers. This raised the question whether 17
could also catalyze the kinetic resolution of glycidyl esters with fatty acids.
Reagents and conditions: a) ent-17 (5.0 mol%), Hünigs base (0.5 equiv), palmitic acid (0.5 equiv), THF, 0 °C, 18 h.
Scheme 6. Kinetic resolution of 20.
When racemic 20 (Scheme 6) was reacted with palmitic acid in the presence of ent-
17 (5.0 mol%) and Hünigs base, the reaction went to full conversion (in palmitic
acid). After tedious work-up and purification by flash chromatography, recovered
stearate 22 showed only marginal enantioenrichment (55 : 45 e.r.).
Towards the automated synthesis of triacylglycerols
In case triacylglycerols are required as analytical standard in the analysis of
(milk) fat, the reported 15 triacylglycerols (Table 2) correspond to only 2% of the
required standards.13 The time required for the synthesis of the remaining 98% of
the triacylglycerols may be reduced by the development of an automated protocol.
Commercially available liquid-handling platforms for parallel synthesis can perform
up to 96 reactions in the same batch, but the options for work-up and purification
are limited. In order to utilize this equipment to maximum extent, the two-step-one-
pot protocol has to be modified in such a way that pure product is obtained by
filtration. The two-step-one-pot protocol consists of ring-opening and esterification.
Various esterification methods leave the second step with multiple options. On the
Enantiopure Triacylglycerols in Three Steps
71
4
contrary, the first step is limited to 17 and Hünigs base. Hünigs base can be simply
evaporated. Then the removal of 15, which is soluble in a variety of organic solvents,
is the main challenge.
The wide application and excellent results in the numerous transformations
catalyzed by salen complexes made these catalysts attractive candidates for
immobilization.14 After covalent attachment to a support, the catalyst can be readily
removed from the reaction and recycled. Jacobsen et al.14a reported the synthesis and
application of modified salen ligand 27, which was immobilized on polystyrene beads
and converted into the corresponding cobalt catalyst.
Scheme 7. ( I ) Synthesis of modified salen ligand 27; ( II ) synthesis of aldehyde 26 as reported by Jacobsen et al.
The immobilization had no influence on the rate of the hydrolytic kinetic resolution
of terminal epoxides and the diol was obtained with similar enantiopurity after 4
times recycling of the catalyst. The authors prepared ligand 27 by condensation of
the aldehydes 25 and 26 with diamine 24 (Scheme 7). While diamine 24 and aldehyde
25 are commercially available, aldehyde 26 is not, but the same authors14a reported
its synthesis. First, diol 28 was monosilylated with TIPS-Cl on the sterically less
hindered 4-hydroxyl group. Then, the TIPS protected substrate was formylated with
paraformaldehyde in the presence of SnCl4. The final deprotection with TBAF
affords aldehyde 26. Even though Jacobsen et al.14a described the synthesis of
aldehyde 26, alternative routes avoiding toxic SnCl4 were explored. Formylation
according to Vilsmeier and Haack, Duff, and Hofsløkken and Skattebøl15,16 were
considered as suitable options (Scheme 8). The latter had already been reported17 for
the synthesis of 25 in a one-pot synthesis of the salen ligand, though not for the
Chapter 4
72
4
formylation of 28. These reactions were studied on benzyl protected substrate 29
(Scheme 8), which was obtained by benzylation of 28 followed by separation from
its bis-benzylated analogue.
Reagents and conditions: a) POCl3, DMF, CH2Cl2 b) hexamine (2.0 equiv), AcOH, reflux 4 h; c) MgCl2 (2.0 equiv), paraformaldehyde (3.0 equiv), Et3N (2.0 equiv), MeCN, reflux, 20 h; d) 1,4-cyclohexadiene (10 equiv), Pd/C (10 mol%), EtOH, 21 °C, 24 h.
Scheme 8. ( I ) Exploring the formylation of 29, ( II ) the mechanism of the MgCl2 mediated
formylation and formation of the side-product 32.
When 29 reacted with POCl3 and DMF (Scheme 8-I), only undesired O-formylated
30 was formed. An attempt to convert 30 into the desired C-formylated 31 by Fries
Enantiopure Triacylglycerols in Three Steps
73
4
rearrangement18 in the presence of BCl3 resulted only in decomposition of the
starting material. On the other hand, Duff reaction of 30 gave the desired aldehyde
31 in a moderate 52% yield. Finally, the formylation according to Hofsløkken and
Skattebøl15 using an excess of paraformaldehyde and anhydrous MgCl2 in basic
conditions was the best alternative affording 31 in a good 72% yield. Occasionally,
traces of methoxymethylated product 32 were formed as a side product. The
mechanism of this formylation is depicted in scheme (Scheme 8-II). Under basic
conditions, phenol 29 is deprotonated affording phenoxymagnesium chloride (33).
33 reacts with formaldehyde (from paraformaldehyde) resulting in intermediate 34.
This can subsequently undergo 2 different pathways.This can subsequently undergo
2 different pathways. Oxidation by another formaldehyde molecule (in blue) results
in the formylated 35, which after hydrolysis, affords the desired phenol 31.
Alternatively, 34 might form quinone methide intermediate 36 (depicted in red). 36
can undergo a Michael addition with methanol(ate), affording the undesired 32.
Despite 31 and 32 were inseparable by column chromatography, 32 could be readily
removed by trituration with cold pentanes. Finally, 31 was debenzylated (Scheme 8-
I). From the explored hydrogenolysis conditions a combination of Pd/C and 1,4-
cyclohexadiene19 was the best, affording the desired aldehyde 26 in 95% yield.
With all the building blocks in hand, the modified Jacobsen ligand 27 was
prepared according to the previously published procedure.14a Condensation of
aldehydes (Scheme 7-I, see above) 25 and 26 with (1R,2R)-cyclohexane-1,2-diamine
Figure 2. Mixture of the three ligands used for immobilization to poly-4-hydroxystyrene.
(24) resulted in a statistical mixture of ligands 27, 37, and 38 in a ratio of 6 : 1 : 920
(Figure 2). Given that 37 was present in a small amount (6%) and 38 does not react
in the immobilization step14a, the mixture was used without any purification.
Finally, 27 was immobilized on polystyrene beads. Poly-4-hydroxystyrene
beads (Scheme 9) were treated with 4-nitrophenyl chloroformate. After washing, the
incorporation of the 4-nitrophenyl carbonate was confirmed by IR spectroscopy,
Chapter 4
74
4
which showed 2 new bands corresponding to a carbonate stretch vibration (υ = 1765
cm-1) and an asymmetric stretch21 vibration of the nitro group (υ= 1528 cm-1).
Reagents and conditions: a) 4-nitrophenyl chloroformate (2.0 equiv), DMAP (50 mol%), CH2Cl2, b) 27, 37, 38, Hünigs base (1.0 equiv), DMAP (50 mol%), c) Co(OAc)2.4H2O, MeOH/toluene, air.
Scheme 9. Immobilization of the ligand on poly-4-hydroxy styrene support.
The functionalized polymer 39 was treated with the mixture of the ligands 27, 37,
38. After washing, the band corresponding to the asymmetric stretch vibration of
the nitro group (υ = 1528 cm-1) had disappeared and the band corresponding to the
carbonate stretch vibration had shifted to lower frequency (υ = 1745 cm-1).
Furthermore, a new band corresponding to the imine stretch vibration of the ligand
(υ = 1630 cm-1) appeared in the IR spectrum. Immobilized ligand 40 was treated with
Co(OAc)2.4H2O and exposed to air. In the IR spectrum, the band corresponding to
the imine (υ = 1630 cm-1) had disappeared. The elemental analysis indicated
incorporation of 0.83 mmol of Co3+[salen]OAc per gram of resin, based on the
nitrogen content.
The immobilized cobalt catalyst 41 was applied in the triacylglycerol synthesis
(Scheme 10). Glycidyl butyrate 16a afforded the corresponding diacylglycerol 36 in
the presence of oleic acid, Hünigs base and the immobilized catalyst (10 mol%) after
6 days. As envisioned, the salen catalyst was removed by filtration and the
diacylglycerol was esterified with palmitic acid under Steglich conditions to afford
19c in 80% yield. The yield of the reaction with the immobilized catalyst 41 is
comparable to the reaction with free catalyst 17. In the latter case, the same
Enantiopure Triacylglycerols in Three Steps
75
4
triacylglycerol was obtained in 86% yield. While Jacobsen et al. 14a reported no
influence of immobilization on the rate of hydrolytic kinetic resolution, in the case
of ring opening with carboxylic acids the effect was significant. Typically, the glycidyl
ester was converted to product in 16 h with non-immobilized catalyst but needed 6
days to be fully converted with the immobilized catalyst.
Reagents and conditions: a) oleic acid (1 equiv), Hünigs base (1 equiv), 35 (10 mol%) 6 days, then filtration b) palmitic acid (1.2 equiv), DCC (1.2 equiv), DMAP (5 mol%), heptane.
Scheme 10. Synthesis of triacylglycerol 19c with immobilized Co[salen] catalyst 41.
Conclusions
This chapter presents a novel access to triacylglycerols. The products are
obtained in a straightforward two-step-one-pot procedure in 79-92% yields. Double
bonds tolerate the mild conditions and no side products are formed. Furthermore,
this chapter presents a step towards the robot-operated synthesis of triacylglycerols.
For this purpose, an immobilized catalyst 35 was synthetized according to a literature
procedure. A synthesis of one of the building blocks for this catalyst was improved.
The immobilized catalyst was also applied in a synthesis of a triacylglycerol. Despite
a significantly longer reaction time, the desired triacylglycerol was isolated in 80%
yield. With these tools now in hand, the entire identification of the triacylglycerols in
milk fat has come a significant step closer
Experimental part
The configuration of triacylglycerols is presented using the Hirschmann’s nomenclature22 (see introduction).
General procedure for the synthesis of glycidyl esters 17a-17g (Scheme 4; Table 1)
A dried round bottom flask was charged with the fatty acid (12.0 mmol, 1.2 equiv),
and DMAP (122 mg, 10 mol%). These were dissolved in heptane (100 ml) and cooled
to 0 °C (ice bath). After stirring for 10 min, (S)-glycidol (670 μl, 10.0 mmol) and
DCC (2.24 g, 11.0 mmol, 1.1 equiv) were added, and the resulting mixture was stirred
for 4 h. A precipitate (dicyclohexyl urea) formed within 5 min. After full conversion
Chapter 4
76
4
of (S)-glycidol (TLC, anisaldehyde stain), the white precipitate was filtered off and
washed with pentane. The combined filtrates were concentrated in vacuo and the
resulting crude residue was purified by column chromatography on silica gel using
20% Et2O in pentanes to afford the desired glycidyl ester.
Glycidyl butyrate (17a)
Following the general procedure for the synthesis of glycidyl esters
with butyric acid. The reaction afforded 1.61 g of glycidyl butyrate as
pale yellow liquid (86% yield)
1H NMR (400 MHz, CDCl3, δ): 4.30 (dd, J = 12.3, 3.0 Hz, 1H), 3.81 (dd, J = 12.3,
6.3 Hz, 1H), 3.10 (m, 1H), 2.74 (dd, J = 11.6, 7.2 Hz, 1H), 2.54 (dd, J = 4.9, 2.6 Hz,
1H), 2.23 (t, J = 7.6 Hz, 2H), 1.65 – 1.49 (m, 2H), 0.85 (t, J = 7.4 Hz, 3H).
13C NMR (101 MHz, CDCl3, δ): 173.07, 64.59, 49.20 ( - ), 44.42, 35.73, 18.21, 13.45
( - ).
HRMS-ESI+ (m/z): [M + H]+ calculated for C7H13O3 , 145.085; found: 145.087.
Anal. Calcd for C7H12O3: C, 58.32; H, 8.39. Found: C 58.45; H 8.53.
[α]D –24.5° (c = 1.0, CHCl3).
Glycidyl caproate (17b)
Following the general procedure for the synthesis of glycidyl esters
with caproic acid. The reaction afforded 1.58 g of glycidyl caproate as pale yellow
liquid (92% yield).
1H NMR (400 MHz, CDCl3, δ): 4.34 (dd, J = 12.3, 3.1 Hz, 1H), 3.84 (dd, J = 12.3,
3.2 Hz, 1H), 3.19 – 3.04 (m, 1H), 2.76 (dd, J = 8.2, 4.3 Hz, 1H), 2.57 (dd, J = 7.8, 2.9
Hz, 1H), 2.28 (t, J = 7.6 Hz, 2H), 1.43 (m, 6H), 0.83 (t, J = 6.9 Hz, 3H).
13C NMR (101 MHz, CDCl3, δ): 173.31, 64.64, 49.25 ( - ), 44.46, 33.89, 31.16, 24.45,
22.19, 13.76 ( - ).
HRMS-ESI+ (m/z): [M + H]+ calculated for C9H17O3 , 173.117; found, 173.117.
Enantiopure Triacylglycerols in Three Steps
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4
Anal. Calcd for C9H16O3: C, 62.77; H, 9.36. Found: C, 63.04; H 9.51.
[α]D –27.0° (c = 1.0, CHCl3).
Glycidyl caprate (17c)
Following the general procedure for synthesis of glycidyl esters with
capric acid. Reaction afforded 2.17 g of glycidyl caprate as colourless liquid (86%
yield).
1H NMR (400 MHz, CDCl3, δ): 4.37 (dd, J = 12.3, 3.1 Hz, 1H), 3.88 (dd, J = 12.3,
6.3 Hz, 1H), 3.15 (m, 1H), 2.80 (dd, J = 5.8, 3.3 Hz, 1H), 2.60 (dd, J = 4.9, 2.6 Hz,
1H), 2.31 (t, J = 7.6 Hz, 2H), 1.43 (m, 14H), 0.84 (t, J = 6.8 Hz, 3H).
13C NMR (101 MHz, CDCl3, δ): 173.42, 64.68, 49.31 ( - ), 44.56, 34.00, 31.80, 29.34,
29.24, 29.19, 29.05 24.82, 22.60, 14.03 ( - ).
HRMS-ESI+ (m/z): [M + H]+ calculated for C13H25O3, 229.179; found, 229.179.
Anal. Calcd for C13H24O3: C, 68.38; H, 10.59. Found: C, 68.58; H, 10.70.
[α]D –19.8° (c = 1.0, CHCl3).
Glycidyl myristate (17d)
Following the general procedure for synthesis of the glycidyl esters
with myristic acid. The reaction afforded 2.40 g of glycidyl myristate as white solid
(82% yield).
1H NMR (400 MHz, CDCl3, δ): 4.41 (dd, J = 12.3, 3.1 Hz, 1H), 3.91 (dd, J = 12.3,
6.3 Hz, 1H), 3.20 (ddd, J = 6.4, 4.1, 3.0 Hz, 1H), 2.84 (dd, J = 4.9, 4.2 Hz, 1H), 2.64
(dd, J = 4.9, 2.6 Hz, 1H), 2.35 (t, J = 7.6 Hz, 2H), 1.45 (m, 22H), 0.88 (t, J = 6.9 Hz,
3H).
13C NMR (101 MHz, CDCl3, δ): 173.68, 64.87, 49.53 ( - ), 44.80, 34.21, 32.05, 29.81,
29.78, 29.73, 29.58, 29.49, 29.38, 29.26, 25.01, 22.82, 14.25 ( - ). (2 signals are
overlapping)
Chapter 4
78
4
HRMS-ESI+ (m/z): [M + H]+ calculated for C17H33O3, 285.242; found, 285.242.
Anal. Calcd for C13H24O3: C, 71.79; H, 11.34. Found: C, 71.52; H, 11.19.
[α]D –18.6° (c = 1.0, CHCl3).
Melting point 41 °C
Glycidyl palmitate (17e)
Following the general procedure for synthesis of the glycidyl esters
with palmitic acid. The reaction afforded 3.03 g of glycidyl palmitate as white solid
(92% yield)
1H NMR (400 MHz, CDCl3). δ 4.41 (dd, J = 12.3, 3.1 Hz, 1H), 3.91 (dd, J = 12.3,
6.3 Hz, 1H), 3.19 (m, J = 6.3, 4.1, 3.0 Hz, 1H), 2.84 (dd, J = 4.8, 4.2 Hz, 1H), 2.64
(dd, J = 4.9, 2.6 Hz, 2H), 2.35 (t, J = 7.6 Hz, 2H), 1.41 (m, 26H), 0.88 (t, J = 6.8 Hz,
3H).
13C NMR (101 MHz, CDCl3, δ): 173.70, 64.89, 49.55 ( - ), 44.82, 34.24, 32.08, 29.85,
29.84, 29.83, 29.81, 29.75, 29.60, 29.51, 29.40, 29.28, 25.04, 22.85, 14.28 ( - ). (2
signals are overlapping)
HRMS-ESI+ (m/z): [M + H]+ calculated for C19H37O3, 313.273; found, 313.273.
Anal. Calcd for C19H36O3: C, 73.03; H 11.61. Found: C, 73.21; H, 11.65.
[α]D –9.4° (c = 1.0, CHCl3).
Melting point: 48 °C
Glycidyl stearate (17f)
Following the general procedure for synthesis of the glycidyl esters
with stearic acid. The reaction afforded 3.41 g of glycidyl stearate as white solid (88%
yield).
Enantiopure Triacylglycerols in Three Steps
79
4
1H NMR (400 MHz, CDCl3, δ): 4.41 (dd, J = 12.3, 3.1 Hz, 1H), 3.91 (dd, J = 12.3,
6.3 Hz, 1H), 3.20 (ddd, J = 6.4, 4.1, 3.0 Hz, 1H), 2.84 (dd, J = 4.9, 4.2 Hz, 1H), 2.64
(dd, J = 4.9, 2.6 Hz, 1H), 2.35 (t, J = 7.6 Hz, 2H), 1.70 – 1.20 (m, 30H), 0.88 (t,
J = 6.9 Hz, 3H).
13C NMR (101 MHz, CDCl3, δ): 173.71, 64.89, 49.55 ( - ), 44.82, 34.22, 32.08, 29.85,
29.82, 29.81, 29.80, 29.75, 29.60, 29.52, 29.40, 29.27, 25.02, 22.85, 14.28 ( - ).
HRMS-APCI+ (m/z): [M + H]+ calculated for C21H41O3, 341.305; found, 341.305.
Anal. Calcd for C21H36O3: C, 74.07; H, 11.84. Found: C, 74.46; H, 11.65.
[α]D –11.2° (c = 1.0, CHCl3)
Melting point 56 °C
Glycidyl oleate (17g)
Following the general procedure for synthesis of glycidyl esters with
stearic acid. Reaction afforded 3.41 g of glycidyl oleate as white
solid (81% yield)
1H NMR (400 MHz, CDCl3, δ): 5.32 (m, 2H), 4.40 (dd, J = 12.3, 3.1 Hz, 1H), 3.90
(dd, J = 12.3, 6.3 Hz, 1H), 3.19 (ddd, J = 6.4, 4.1, 2.9 Hz, 1H), 2.82 (m, 1H), 2.63
(dd, J = 4.9, 2.6 Hz, 1H), 2.34 (t, J = 7.6 Hz, 2H), 1.99 (m, 4H), 1.61 (dd, J = 14.7,
7.3 Hz, 2H), 1.27 (m, 16H), 0.87 (t, J = 6.8 Hz, 3H).
13C NMR (101 MHz, CDCl3, δ): 173.54, 130.05 ( - ), 129.78 ( - ), 64.83, 49.44 ( - ),
44.70, 34.11, 31.99, 29.84, 29.76, 29.61, 29.40, 29.23, 29.16, 27.29, 27.23, 24.93, 22.77,
14.20 ( - ).
HRMS-ESI+ (m/z): [M + H]+ calculated for C21H39O3, 339.289; found, 339.289.
Anal. Calcd for C21H39O3: C, 73.03; H, 11.84. Found: C, 73.46; H, 11.65.
α = +2.0° (c = 1.0, CHCl3).
Chapter 4
80
4
Synthesis of triacylglycerols (Scheme 5, Table 2):
A roundbottom flask was charged with cobalt catalyst 15 catalyst (6.2 mg, 1.0 mol%)
and fatty acid (1.0 mmol, 1.0 equiv). A small amount of Et2O was added to dissolve
the compounds (ca 2 ml). This solution was stirred for ca 15 min exposed to air.
After the solution turned from red to brown (due to oxidation of Co2+ to Co3+), Et2O
was evaporated. To this residue, DIPEA (170 µl, 1.0 mmol, 1.0 eq) and glycidyl ester
(1.0 mmol) were added in this order and the reaction was stirred overnight (ca 16 h)
at RT (21 °C). Conversion was monitored by 1H NMR spectroscopy of the crude
reaction mixture aliquots (attenuation of the signals at 2.64 ppm and 2.84 ppm). After
full conversion (reaction mixture turns back to red), the crude residue was dissolved
in n-heptane (10 ml), the second fatty acid (1.2 equiv), DMAP (12.2 mg, 10 mol%)
and DCC (227 mg, 1.1 mmol, 1.1 equiv) were added in this order. The reaction
mixture was stirred overnight (ca 20 h) at RT (21 °C). After full conversion of the
intermediate 1,2-diacylglycerol (TLC), the crude mixture was transferred directly
onto a silica column and chromatographed with 9% Et2O in pentane to afford the
desired triacylglycerol.
NOTE: The measured optical rotations for triacylglycerols were below the precision
of the instrument (<0.005°)
1-myristoyl-2-oleoyl-3-butyryl- sn-glycerol (19a)
Following the general procedure for synthesis of triacylglycerols,
Myristic acid was used for ring opening of glycidyl butyrate and
oleic acid was used for the final esterification. Reaction afforded
544 mg of desired product as pale yellow liquid (86%)
1H NMR (400 MHz, CDCl3, δ): 5.35 (m, 2H), 5.25 (m, 1H), 4.23 (dd, J = 11.9, 4.3
Hz, 2H), 4.08 (dd, J = 11.9, 6.0 Hz, 2H), 2.30 (m, 6H), 2.00 (m, 4H), 1.61 (m, 6H),
1.26 (m, 40H), 0.94 (t, J = 7.4, 3H), 0.88 (t, J = 6.8 Hz, 6H).
13C NMR (101 MHz, CDCl3, δ): 173.30, 173.12, 172.92, 130.08 ( - ), 129.79 ( - ), 68.98
( - ), 62.18, 36.01, 34.31, 34.14, 34.13, 32.04, 32.02, 29.88, 29.81, 29.78, 29.74, 29.64,
29.60, 29.48, 29.43, 29.40, 29.28, 29.22, 29.19, 29.18, 27.33, 27.27, 25.01, 24.97, 24.95,
22.80, 18.45, 14.21 ( - ), 13.71 ( - ). (4 signals are overlapping)
HRMS-ESI+ (m/z): [M + H]+ calculated for C39H73O6, 637.538; found, 637.540.
Anal. Calcd for C39H72O6: C, 73.54; H, 11.39. Found: C, 73.67; H, 11.52.
Enantiopure Triacylglycerols in Three Steps
81
4
1-stearoyl-2-palmitoyl-3-butyryl-sn-glycerol (19b)
Following the general procedure for synthesis of
triacylglycerols, stearic acid was used for ring opening of
glycidyl butyrate and palmitic acid was used for the final
esterification. The reaction afforded 523 mg of desired product as white solid (79%)
1H NMR (400 MHz, CDCl3, δ): 5.27 (m, 1H), 4.29 (dd, J = 11.9, 3.3 Hz, 2H), 4.15
(dd, J = 11.9, 4.4 Hz, 2H), 2.31 (m, 6H), 1.65 (m, 6H), 1.26 (m, 52H), 0.94 (t,
J = 7.4 Hz, 3H), 0.88 (t, J = 6.8 Hz, 6H).
13C NMR (101 MHz, CDCl3, δ): 173.28, 173.07, 172.86, 68.85 ( - ), 62.07, 36.04,
35.89, 34.20, 34.03, 31.91, 29.68, 29.66, 29.64, 29.62, 29.61, 29.59, 29.53, 29.52, 29.47,
29.45, 29.34, 29.27, 29.25, 29.24, 29.10, 29.09, 29.06, 24.88, 24.84, 22.67, 18.35, 18.32,
14.10 ( - ), 13.59 ( - ), 13.53( - ) (6 signals are overlapping).
HRMS-ESI+ (m/z): [M + H]+ calculated for C41H79O6, 667.578; found, 667.579.
Anal. Calcd for C41H78O6: C, 73.82; H, 11.79. Found: C, 74.02; H, 11.91.
Melting point 52 – 56 °C
1-oleoyl-2-palmitoyl-3-butyryl-sn-glycerol (19c)
Following the general procedure for synthesis of
triacylglycerols, oleic acid was used for ring opening of
glycidyl butyrate and palmitic acid was used for the final
esterification. Reaction afforded 575 mg of desired product as
white solid (86%)
1H NMR (400 MHz, CDCl3, δ): 5.34 (m, 2H), 5.30 (m, 1H), 4.28 (dd, J = 11.9, 4.2
Hz, 2H), 4.13 (dd, J = 11.9, 6.0 Hz, 2H), 2.28 (m, 6H), 2.01 (m, 4H), 1.62 (td, J =
11.4, 5.7 Hz, 6H), 1.26 (m, 44H), 0.93 (t, J = 7.4 Hz, 3H), 0.86 (t, J = 6.8 Hz, 6H).
13C NMR (101 MHz, CDCl3, δ): 173.15, 172.97, 172.78, 129.94 ( - ), 129.64 ( - ), 68.84
( - ), 62.03, 36.02, 35.86, 34.16, 33.98, 31.90, 29.73, 29.67, 29.50, 29.46, 29.33, 29.29,
29.25, 29.14, 29.08, 29.05, 27.18, 27.13, 24.86, 24.81, 22.66, 18.30, 14.07 ( - ),
13.57 ( - ) ppm. (11 signals are overlapping)
HRMS-ESI+ (m/z): [M + H]+ calculated for C41H77O6, 665.571; found, 665.570.
Chapter 4
82
4
Anal. Calcd for C41H76O6: C, 74.05; H, 11.52. Found: C, 74.31; H, 11.63.
1-oleoyl-2-oleoyl-3-butyryl-sn-glycerol (19d)
Following the general procedure for synthesis of triacylglycerols,
oleic acid was used for ring opening of glycidyl butyrate and oleic
acid was used for the final esterification. Reaction afforded
575 mg of desired product as pale yellow liquid (83%).
1H NMR (400 MHz, CDCl3, δ): 5.35 (m, 4H), 5.26 (m, 1H), 4.29 (dd, J = 11.9, 4.2
Hz, 2H), 4.14 (dd, J = 11.9, 5.9 Hz, 2H), 2.31 (m, 6H), 2.00 (m, 8H), 1.62 (m, 6H),
1.27 (m, 40H), 0.93 (t, J = 7.4 Hz, 3H), 0.87 (t, J = 6.7 Hz, 6H).
13C NMR (101 MHz, CDCl3, δ): 173.18, 172.99, 172.77, 129.97 ( - ), 129.95 ( - ),
129.68 ( - ), 129.66 ( - ), 68.85 ( - ), 62.05, 35.84, 34.19, 33.99, 31.81, 29.74, 29.63,
29.61, 29.58, 29.50, 29.41, 29.36, 29.25, 29.21, 29.16, 29.05, 29.03, 27.19, 27.14, 27.11,
27.09, 24.80, 22.65, 18.31, 14.07 ( - ), 13.58 ( - ) (12 signals are overlapping).
HRMS-ESI+ (m/z): [M + H]+ calculated for C43H79O6 , 691.587; found, 691.587.
Anal. Calcd for C43H78O6: C, 74.73; H, 11.38. Found: C, 74.78; H, 11.46.
1-oleoyl-2-oleoyl-3-butyryl-sn-glycerol (19e)
Following the general procedure for synthesis of
triacylglycerols, linoleic acid was used for ring opening of
glycidyl butyrate and myristic acid was used for the final
esterification. Reaction afforded 541 mg of desired product
as pale yellow liquid (85%)
1H NMR (400 MHz, CDCl3, δ): 5.36 (m, 4H), 5.23 (m, 1H), 4.29 (dd, J = 11.9, 3.2
Hz, 2H), 4.14 (dd, J = 11.9, 6.0 Hz, 2H), 2.76 (t, J = 6.4 Hz, 2H), 2.30 (m, 6H), 2.04
(q, J = 6.7 Hz, 4H), 1.65 (m, 6H), 1.27 (m, 34H), 0.94 (t, J = 7.4, 3H), 0.87 (t,
J = 3.4 Hz, 6H).
13C NMR (101 MHz, CDCl3, δ): 173.18, 173.01, 172.81, 130.15, 129.95 ( - ), 128.02
( - ), 127.86 ( - ), 68.84 ( - ), 62.04, 36.03, 35.88, 34.18, 34.01, 33.89, 31.50, 29.65,
29.62, 29.60, 29.58, 29.45, 29.33, 29.25, 29.23, 29.16, 29.09, 29.05, 27.17, 25.60, 24.87,
24.83, 22.66, 22.54, 22.31, 18.35, 14.08 ( - ), 13.58 ( - ) (3 signals are overlapping).
Enantiopure Triacylglycerols in Three Steps
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4
HRMS-ESI+ (m/z): [M + H]+ calculated for C39H71O6, 635.523; found, 635.524.
Anal. Calcd for C39H70O6: C, 73.77; H, 11.11. Found: C, 74.07; H, 11.28.
1-oleoyl-2-palmitoyl-3-caproyl-sn-glycerol (19f)
Following the general procedure for synthesis of
triacylglycerols, oleic acid was used for ring opening of glycidyl
caproate and palmitic acid was used for the final esterification.
Reaction afforded 565 mg of desired product as pale yellow
liquid (82%).
1H NMR (400 MHz, CDCl3, δ): 5.32 (m, 2H), 5.24 (m, 1H), 4.27 (dd, J = 11.9, 4.3
Hz, 2H), 4.11 (dd, J = 11.9, 6.0 Hz, 2H), 2.28 (t, J = 7.5 Hz, 6H), 2.00 (m, 4H), 1.60
(m, 6H), 1.27 (m, 48H), 0.85 (t, J = 6.9 Hz, 9H).
13C NMR (101 MHz, CDCl3, δ): 173.19, 173.17, 172.79, 129.95 ( - ), 129.65 ( - ), 68.84
( - ), 62.06, 34.18, 33.99, 33.97, 31.90, 31.21, 29.74, 29.67, 29.63, 29.50, 29.47, 29.34,
29.29, 29.26, 29.15, 29.08, 29.06, 29.00, 27.19, 27.14, 24.87, 24.81, 24.50, 22.66, 22.26,
14.08 ( - ), 13.85 ( - ) (10 signals are overlapping).
HRMS-ESI+ (m/z): [M + H]+ calculated for C43H81O6, 693.599; found, 693.602.
Anal. Calcd for C43H80O6: C, 74.52; H, 11.63. Found: C, 74.17; H, 11.70.
1-oleoyl-2-palmitoyl-3-capryl-sn-glycerol (19g)
Following the general procedure for synthesis of
triacylglycerols, oleic acid was used for ring opening of glycidyl
caprate and palmitic acid was used for the final esterification.
Reaction afforded 652 mg of desired product as pale yellow
liquid (88%)
1H NMR (400 MHz, CDCl3, δ): 5.35 (m, 2H), 5.26 (m, 1H), 4.29 (dd, J = 11.9, 4.3
Hz, 2H), 4.14 (dd, J = 11.9, 6.0 Hz, 2H), 2.30 (t, J = 7.6 Hz, 6H), 2.01 (m, 4H), 1.61
(m, 6H), 1.27 (m, 56H), 0.87 (t, J = 6.8 Hz, 9H).
13C NMR (101 MHz, CDCl3, δ): 173.09, 173.07, 172.70, 129.90 ( - ), 129.61 ( - ),
68.83, 62.02, 34.14, 33.98, 33.96, 31.89, 31.87, 31.83, 29.73, 29.67, 29.63, 29.60, 29.58,
Chapter 4
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4
29.49, 29.44, 29.33, 29.29, 29.26, 29.23, 29.13, 29.07, 29.04, 27.17, 27.12, 24.86, 24.82,
24.80, 22.64, 22.62, 14.05 ( - ) (12 signals are overlapping).
HRMS-ESI+ (m/z): [M + H]+ calculated for C47H89O6, 749.662; found, 749.665.
Anal. Calcd for C47H88O6: C, 75.96; H, 11.12. Found: C, 75.56; H, 11.93.
1-myristoyl-2-stearoyl-3-palmitoyl-sn-glycerol (19h)
Following the general procedure for synthesis of
triacylglycerols, myristic acid was used for ring opening of
glycidyl palmitate and stearic acid was used for the final
esterification. Reaction afforded 730 mg of desired product as white solid (80%).
NOTE: small amount of THF (ca 0.5 ml) is necessary to dissolve the starting
materials
1H NMR (400 MHz, CDCl3, δ): 5.25 (m, 1H), 4.29 (dd, J = 11.9, 4.3, 2H), 4.14 (dd,
J = 11.9, 6.0, 2H), 2.29 (m, 6H), 1.63 (m, 6H), 1.27 (m, 72H), 0.88 (t, J = 6.8, 9H).
13C NMR (101 MHz, CDCl3, δ): 173.28, 172.86, 68.84 ( - ), 62.08, 34.21, 34.04, 31.91,
29.69, 29.67, 29.65, 29.61, 29.50, 29.47, 29.43, 29.35, 29.28, 29.26, 29.10, 29.07, 24.89,
24.85, 22.67, 14.10 ( - ) (28 signals overlapping).
HRMS-ESI+ (m/z): [M + H]+ calculated for C51H99O6, 807.743; found, 807.743.
Anal. Calcd for C51H98O6: C, 75.87; H, 12.24. Found: C, 76.19; H, 12.34.
Melting point 58 – 59 °C
1-stearoyl-2-myristoyl-3-palmitoyl-sn-glycerol (19i)
Following the general procedure for synthesis of
triacylglycerols, stearic acid was used for ring opening of
glycidyl palmitate and myristic acid was used for the final
esterification. Reaction afforded 720 mg of desired product as white solid (79%)
NOTE: small amount of THF (ca 0.5 ml) is necessary for dissolving the starting
materials
Enantiopure Triacylglycerols in Three Steps
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4
1H NMR (400 MHz, CDCl3, δ): 5.26 (m, 1H), 4.29 (dd, J = 11.9, 4.3, 2H), 4.14 (dd,
J = 11.9, 6.0, 2H), 2.31 (td, J = 7.6, 2.4, 6H), 1.62 (m, 6H), 1.26 (m, 72H), 0.88 (t, J
= 6.8, 9H).
13C NMR (101 MHz, CDCl3, δ): 173.28, 173.28, 172.86, 68.85 ( - ), 62.08, 34.21,
34.04, 31.91, 29.69, 29.65, 29.61, 29.49, 29.47, 29.35, 29.28, 29.26, 29.11, 29.07, 24.89,
24.85, 22.68, 14.10 ( - ) (29 signals are overlapping).
HRMS-ESI+ (m/z): [M + H]+ calculated for C51H99O6, 807.743; found, 807.743.
Anal. Calcd for C51H98O6: C, 75.87; H, 12.24. Found: C, 76.09; H, 12.25.
Melting point 58 – 59 °C
1-oleoyl-2-oleoyl-3-palmitoyl-sn-glycerol (19j)
Following the general procedure for synthesis of
triacylglycerols, oleic acid was used for ring opening of glycidyl
palmitate and myristic acid was used for the final esterification.
Reaction afforded 791 mg of desired product as pale yellow
liquid (92%)
1H NMR (400 MHz, CDCl3, δ): 5.34 (m, 4H), 5.25 (m, 1H), 4.28 (dd, J = 11.9, 4.3
Hz, 2H), 4.13 (dd, J = 11.9, 4.3 Hz, 2H), 2.29 (td, J = 7.6, 2.2 Hz, 6H), 2.01 (m, 8H),
1.60 (m, 6H), 1.26 (m, 64H), 0.86 (t, J = 6.8 Hz, 9H).
13C NMR (101 MHz, CDCl3, δ): 173.13, 173.10, 172.70, 129.94 ( - ), 129.92 ( - ),
129.63 ( - ), 129.60 ( - ), 68.85 ( - ), 62.03, 29.74, 29.64, 29.51, 29.45, 29.32, 29.27,
29.17, 29.09, 29.05, 27.15, 25.59, 24.83, 22.65, 22.31, 14.06 ( - ), 14.02 ( - ) (31 signals
overlapping).
HRMS-ESI+ (m/z): [M + H]+ calculated for C55H103O6, 859.774; found: 859.776
Anal. Calcd for C55H102O6: C, 76.87; H, 11.96. Found: C, 76.99; H, 12.04.
1-butyryl-2-palmitoyl-3-stearoyl-sn-glycerol (19k)
Following the general procedure for synthesis of
triacylglycerols, butyric acid was used for ring opening of
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86
4
glycidyl stearate and palmitic acid was used for the final esterification. Reaction
afforded 546 mg of desired product as white solid (82%)
NOTE: small amount of THF (ca 0.5 ml) is necessary for dissolving the starting
materials
1H NMR (400 MHz, CDCl3, δ): 5.27 (m, 1H), 4.30 (dd, J = 11.9, 4.3, 2H), 4.15 (dd,
J = 11.9, 6.0, 1.3, 2H), 2.34 (m, 6H), 1.64 (m, 6H), 1.25 (m, 54H), 0.95 (t, J = 6.1,
3H), 0.88 (t, J = 6.8, 6H).
13C NMR (101 MHz, CDCl3, δ): 173.28, 173.07, 172.86, 68.85 ( - ), 62.07, 36.04,
35.89, 34.20, 34.03, 31.91, 29.68, 29.66, 29.64, 29.62, 29.61, 29.59, 29.53, 29.52, 29.47,
29.45, 29.34, 29.27, 29.25, 29.24, 29.10, 29.09, 29.06, 24.88, 24.84, 22.67, 18.35, 18.32,
14.10 ( - ), 13.59 ( - ), 13.53 ( - ) (6 signals are overlapping).
HRMS-ESI+ (m/z): [M + H]+ calculated for C41H78O6, 667.578; found, 667.579.
Anal. Calcd for C41H78O6: C, 73.82; H, 11.79. Found: C, 74.00; H, 11.91.
Melting point 52 – 62 °C
1-myristoyl-2-palmitoyl-3-stearoyl-sn-glycerol (19l)
Following the general procedure for synthesis of
triacylglycerols, myristic acid was used for ring opening of
glycidyl stearate and palmitic acid was used for the final
esterification. Reaction afforded 546 mg of desired product as white solid (80%)
NOTE: small amount of THF (ca 0.5 ml) is necessary for dissolving the starting
materials
1H NMR (400 MHz, CDCl3, δ): 5.28 (m, 1H), 4.25 (dd, J = 11.9, 4.3, 2H), 4.14 (dd,
J = 11.9, 6.0, 2H), 2.32 (td, J = 7.6, 2.4, 6H), 1.60 (m, 6H), 1.28 (m, 72H), 0.88 (t, J
= 6.8, 9H).
13C NMR (101 MHz, CDCl3, δ): 173.56, 173.30, 172.88, 68.86 ( - ), 62.10, 49.40,
44.67, 34.23, 34.09, 34.06, 31.93, 29.71, 29.67, 29.65, 29.63, 29.60, 29.51, 29.49, 29.45,
29.37, 29.30, 29.28, 29.25, 29.13, 29.09, 24.92, 24.87, 22.70, 14.12 ( - ) (22 signals
overlapping)
Enantiopure Triacylglycerols in Three Steps
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4
HRMS-ACPI (m/z): [M + Na]+ calculated for C51H98O6Na, 829.725; found, 829.725.
Anal. Calcd for C51H98O6: C, 75.87; H, 12.24. Found: C, 75.57; H, 11.93.
Melting point 38 °C
1-oleoyl-2-oleoyl-3-stearoyl-sn-glycerol (19m)
Following the general procedure for synthesis of
triacylglycerols, oleic acid was used for ring opening of glycidyl
stearate and oleic acid was used for the final esterification.
Reaction afforded 680 mg of desired product as pale yellow
liquid (80%)
1H NMR (400 MHz, CDCl3, δ): 5.34 (m, 4H), 5.24 (m, 1H), 4.27 (dd, J = 11.9, 4.3
Hz, 2H), 4.12 (dd, J = 11.9, 4.3 Hz, 2H), 2.29 (m, 6H), 2.00 (m, 8H), 1.59 (m, 6H),
1.26 (m, 68H), 0.85 (t, J = 6.8 Hz, 9H).
13C NMR (101 MHz, CDCl3, δ): 173.13, 173.10, 172.70, 129.94 ( - ), 129.92 ( - ),
129.63 ( - ), 129.60 ( - ), 68.85 ( - ), 62.03, 29.74, 29.64, 29.51, 29.48, 29.45, 29.34,
29.26, 29.15, 29.08, 29.05, 27.15, 25.67, 24.85, 22.71, 22.33, 14.08 ( - ), 14.04 ( - )
(31 signals overlapping).
HRMS-ESI+ (m/z): [M + H]+ calculated for C57H107O6, 887.806; found, 887.808.
Anal. Calcd for C57H106O6: C, 77.14; H, 12.04. Found: C, 76.75; H, 11.14.
1-palmitoyl-2-stearoyl-3-oleoyl-sn-glycerol (19n)
Following the general procedure for synthesis of
triacylglycerols, palmitic acid was used for ring opening of
glycidyl oleate and stearic acid was used for the final
esterification. Reaction afforded 681 mg of desired product as
white solid (80%)
1H NMR (400 MHz, CDCl3, δ): 5.34 (m, 2H), 5.25 (m, 1H), 4.29 (dd, J = 11.9, 4.3,
2H), 4.14 (dd, J = 11.9, 6.0, 2H), 2.31 (t, J = 7.6, , 6H), 2.00 (m, 4H), 1.61 (s, 6H),
1.25 (m, 72H), 0.88 (t, J = 6.8, 9H).
Chapter 4
88
4
13C NMR (101 MHz, CDCl3, δ): 173.24, 173.21, 172.83, 129.97 ( - ), 129.69 ( - ), 68.84
( - ), 62.08, 34.20, 34.03, 34.01, 31.91, 31.89, 29.75, 29.69, 29.65, 29.63, 29.61, 29.51,
29.48, 29.47, 29.35, 29.30, 29.28, 29.26, 29.16, 27.20, 27.15, 24.89, 24.85, 22.67, 22.66,
22.32, 14.09 ( - ), 14.03( - ) (21 signals are overlapping)
HRMS-ESI+ (m/z): [M + H]+ calculated for C55H105O6, 861.790; found: 861.790.
Anal. Calcd for C57H104O6: C, 77.69; H, 12.17. Found: C, 76.75; H, 11.97.
Melting point 38 °C
Synthesis of the modified Jacobsen ligand (Scheme 8-I)
2-(tert-butyl)-4-benzyloxyphenol (29)
A roundbottom flask was charged with tert-butyl hydroquinone (7.51 g,
45 mmol) and potassium iodide (375 mg, 5.0 mol%). Solids were dissolved
in CH3CN (150 ml), and the remaining compounds were added in the
following order: benzyl bromide (6.5 ml, 54 mmol, 1.2 equiv) and K2CO3
(7.5 g, 54 mmol, 1.2 equiv). The reaction mixture was immersed into a preheated oil
bath (80 °C) and the mixture was refluxed for 3 h. The oil bath was removed and
reaction mixture was allowed to cool to RT. Once cooled down, the acetonitrile was
evaporate in vacuo. The crude residue was suspended in CH2Cl2 and filtered. The
filtrate was concentrated in vacuo and the crude residue was further purified by flash
chromatography on silica gel using 10% Et2O in pentane. Fractions with Rf = 0.36
in 10% Et2O in pentanes were collected and evaporated to afford 5.7 g of the desired
product monobenzylated 29 (50%) as a brown solid.
5.48 g of the bisprotected derivative was also isolated (35%).
1H NMR (400 MHz, CDCl3, δ): 7.48 (d, J = 7.3 Hz, 2H), 7.42 (t, J = 7.3 Hz, 2H),
7.36 (t, J = 7.1 Hz, 1H), 7.00 (d, J = 2.9 Hz, 1H), 6.70 (dd, J = 8.5, 2.8 Hz, 1H), 6.59
(d, J = 8.5 Hz, 1H), 5.03 (s, 2H), 4.83 (bs, 1H), 1.44 (s, 9H).
13C NMR (101 MHz, CDCl3, δ): 152.61, 148.56, 137.64, 137.40, 128.58 ( - ), 127.94
( - ), 127.68 ( - ), 116.82 ( - ), 115.25 ( - ), 111.78 ( - ), 70.84, 34.75, 29.53 ( - ), ppm.
HRMS-ESI+ (m/z): [M + H]+ calculated for C17H20O2, 257.153; found, 257.153.
Melting point: 82 – 83 °C
Enantiopure Triacylglycerols in Three Steps
89
4
Bisprotected derivative
1H NMR (400 MHz, CDCl3, δ): 7.48 (t, J = 5.3 Hz, 4H), 7.41 (t, J = 7.2 Hz, 4H),
7.35 (d, J = 6.9 Hz, 2H), 7.04 (d, J = 2.9 Hz, 1H), 6.87 (d, J = 8.8 Hz, 1H), 6.77 (dd,
J = 8.7, 2.9 Hz, 1H), 5.09 (s, 2H), 5.03 (s, 2H), 1.43 (s, 9H).
13C NMR (101 MHz, CDCl3, δ): 152.61, 152.02, 139.96, 137.65, 137.39, 129.03,
128.55, 128.50, 127.88, 127.61, 127.21, 115.37, 113.00, 110.89, 70.58, 70.55, 35.04,
29.76.
Melting point 73 – 75 °C
5-(benzyloxy)-3-(tert-butyl)-2-hydroxybenzaldehyde (31), conditions a.
A roundbottom flask was charged with the benzyl derivative 29 (256 mg,
1.0 mmol) and hexamine (280 mg, 2.0 mmol, 2.0 equiv). These compounds were
dissolved in glacial acetic acid (10 ml) and immersed into preheated oil bath (120 °C).
After heating for 4 h, TLC indicated full conversion of the starting material (29). The
flask was removed from the oil bath and allowed to cool to RT. The reaction mixture
was then poured into water and extracted with Et2O (3 x 30 ml). Combined organic
layers were washed with water and brine, dried over MgSO4 and evaporated to
dryness. The crude residue was purified by flash chromatography on silica gel using
10% Et2O in pentanes.
Reaction afforded 168 mg of the desired aldehyde 31 as a brown solid (53%)
1H NMR (400 MHz, CDCl3, δ): 11.44 (s, 1H), 9.73 (s, 1H), 7.36 (t, J = 5.9 Hz, 2H),
7.32 (d, J = 7.3 Hz, 1H), 7.27 (dd, J = 6.1 Hz, 2H), 7.17 (d, J = 3.7 Hz, 1H), 6.81 (d,
J = 3.0 Hz, 1H), 4.97 (s, 2H), 1.33 (s, 9H).
13C NMR (101 MHz, CDCl3, δ): 196.58 ( - ), 156.33, 151.14, 140.19, 136.67, 128.66
( - ), 128.55, 128.15 ( - ), 127.57 ( - ), 119.80 ( - ), 113.26 ( - ), 70.82, 35.02, 29.11( - )
ppm.
Chapter 4
90
4
HRMS-ESI+ (m/z): [M + H]+ calculated for C18H21O3, 285.148; found, 285.149.
Melting point: 55 – 56 °C
5-(benzyloxy)-3-(tert-butyl)-2-hydroxybenzaldehyde (31), conditions c.
A dried three-necked 250 ml flask equipped with a reflux condenser and connected
to a nitrogen line was charged with phenol 29 (5.12 g, 20 mmol), anhydrous MgCl2
(3.8 g, 40 mmol, 2.0 equiv) and paraformaldehyde (loading based on monomeric
formaldehyde, 1.8 g, 60 mmol, 3.0 equiv, dried overnight over P2O5 and high
vacuum) under a positive stream of nitrogen. Solids were dissolved in dry THF (150
ml) and Et3N (5.5 ml, 40 mmol, 2.0 equiv) was added. The resulting mixture was
immersed into a preheated oil bath (72 °C) and refluxed for 20 h. The oil bath was
removed and the reaction mixture was allowed to cool to RT. Aqueous HCl (ca 1 M,
150 ml) was carefully added. The aqueous layer was further extracted with Et2O
(3 x 100 ml), washed with brine, dried over Mg2SO4 and evaporated. The crude
residue was chromatographed using 5% Et2O in pentanes to afford 4.5 g of the
desired compound as yellow-brown solid (80%) containing 8% of methoxymetylated
derivative based on 1H NMR.
NOTE: 32 (methoxymethylated derivative) which occasionally forms has the same
Rf as the desired product 31, but it can be identified in 1H-NMR based on the
following set of signals: (400 MHz, CDCl3) 6.93 (d, J = 3.0 Hz, 1H), 6.53 (d, J = 3.0
Hz, 1H), 4.61 (s, 2H), 3.44 (s, 3H). Trituration with cold pentanes is sufficient to
remove contaminant 32 from the desired aldehyde 31.
1H NMR (400 MHz, CDCl3, δ): 11.44 (s, 1H), 9.73 (s, 1H), 7.36 (m, 5H), 7.17 (d, J =
3.0 Hz, 1H), 6.80 (d, J = 3.0 Hz, 1H), 4.96 (s, 2H), 1.33 (s, 9H).
13C NMR (101 MHz, CDCl3, δ): 196.58 ( - ), 156.33, 151.14, 140.19, 136.67, 128.66
( - ), 128.55, 128.15 ( - ), 127.57 ( - ), 119.80 ( - ), 113.26 ( - ), 70.82, 35.02, 29.11( - ).
Melting point 55 – 56 °C
3-(tert-butyl)-2,5-dihydroxybenzaldehyde (20)
An oven-dried roundbottom flask was charged with aldehyde 31 (2.5 g,
8.8 mmol). This was dissolved in EtOH (150 ml) and Pd/C (Degussa type
E101 NE/W, 10 mol%, 930 mg) and cyclohexa-1,4-diene (8.3 ml, 10
equiv) were added. The mixture was stirred for 24 h until full conversion
Enantiopure Triacylglycerols in Three Steps
91
4
of 31 was achieved (TLC). Reaction mixture was filtered over a pad of celite. The
filtrate was evaporated to dryness and the crude residue was purified by flash
chromatograph using 30% AcOEt in pentanes to afford 1.62 g of desired aldehyde
20 as yellow solid (95%).
1H NMR (400 MHz, CDCl3, δ): 11.38 (s, 1H), 9.79 (s, 1H), 7.10 (d, J = 3.0 Hz, 1H),
6.83 (d, J = 3.1 Hz, 1H), 4.71 (bs, 1H), 1.41 (s, 9H).
13C NMR (101 MHz, CDCl3, δ): 196.54 ( - ), 155.90, 147.60, 140.09, 123.34 ( - ),
120.08, 115.39 ( - ), 34.94, 29.06 ( - ).
HRMS-ESI+ (m/z): [M + H]+ calculated for C11H15O3, 195.100; found, 195.101.
Anal. Calcd for C11H14O3: C, 68.02; H, 7.27. Found: C, 68.26; H, 7.34.
Melting range: 140 – 141 °C
Immobilization of ligand 24 on solid support (scheme 7)
Salen ligands 27, 37, 38
A roundbottom flask was charged with 3-tert-butyl-2,5-dihydroxybenzaldehyde 26
(620 mg, 3.2 mmol) and 3,5-di-tert-butylsalicylaldehyde 25 (2.25 g, 9.6 mmol,
3.0 equiv). These solids were dissolved in CH2Cl2 (30 ml), and (1R,2R)-cyclohexane-
1,2-diamine 24 (730 mg, 6.4 mmol, 2.0 equiv) was added. The reaction mixture was
stirred for 24 h at RT. The solvent was removed under reduced pressure to afford a
mixture of salen ligands 27, 37, and 38 in statistical ratio of 6:1:9 as a yellow solid.
This mixture was used without further purification in the next reaction.
Resin bounded [R,R]-Co(Salen) catalyst 41
Chapter 4
92
4
A roundbottom flask was charged with poly-4-
hydroxystyrene resin (Advanced ChemTech, 1% cross-
linked, 90 µm, 3.6 mmol/g, 36 mg what is considered as
0.13 μmol). Beads were further suspended in CH2Cl2
(2.5 ml) and 4-nitrophenyl chloroformate (400 mg, 2.0
mmol, 15 equiv) and DMAP (61 mg, 0.46 mmol, 3.5 equiv) were added in this order.
The mixture was stirred for 1 h at RT. The solids were filtered using a glass filter
(por 4) and washed with CH2Cl2. The resulting white beads were dried for 1 h under
high vacuum (oil pump).The IR spectrum showed a band at 1765 cm-1 corresponding
to the carbonate stretch vibration and a prominent band at 1528 cm-1 corresponding
to the asymmetric stretch vibration of the nitro group. The white beads were further
suspended in DMF (2.5 ml) and the mixture of salen ligands 27, 37 and 38 followed
by DIPEA (180 μl, 1.0 mmol, 8.0 equiv) and DMAP (61 mg, 0.46 mmol, 3.5 equiv)
were added. The mixture was stirred for 1.5 h. An immediate color change to yellow
attributed to p-nitro-phenolate was observed. After 1.5 h of stirring, the resin was
filtered using a glass filter (por 4) and washed with solvents in the following order:
DMF, CH2Cl2, MeOH and CH2Cl2. The resulting yellow beads were subjected to IR
analysis. In the IR spectrum the band corresponding to carbonate shifted towards a
lower wave number (1745 cm-1) and the band corresponding to asymmetric stretch
vibration of the nitro group disappeared. Furthermore, a new band corresponding
to the stretch vibration of an imine appeared in the IR spectrum (1630 cm-1). The
resulting yellow beads were dissolved in MeOH / toluene (1 / 1, 2.5 ml) and
Co(OAc)2.4H2O (249 mg, 1.0 mmol, 8.0 equiv). After a short period of time, the
beads turned dark red. After 1.5 h of stirring, the beads were filtered using a glass
filter (por 4) and washed with solvents in the following order: MeOH, CH2Cl2 /
toluene 9 / 1, AcOH, CH2Cl2, MeOH and CH2Cl2 to yield dark red/brown beads.
The nitrogen content N = 2.33% of the resin corresponds to 0.83 mmol of ligand
per 1 g of the resin. Assuming the quantitative incorporation of the Co2+ the final
loading of the Co3+[R,R-(SALEN)] is 0.83 mmol/g.
Anal. Calcd found: N, 2.33; C, 66.56; H, 6.37.
Ring opening using immobilized catalyst (scheme 10)
To a 10 ml round-bottom flask, resin bound Co[R,R-Salen]
catalyst 35 (144 mg of the resin corresponds to 10 mol% of
the Co catalyst was added. This was suspended in small
amount of CH2Cl2 (0.5 ml) and oleic acid (320 µl, 1.0 mmol,
Enantiopure Triacylglycerols in Three Steps
93
4
1.0 equiv), followed by glycidyl butyrate, 1 (102 µl, 1.0 mmol) and Hünigs base (170
µl, 1.0 mmol, 1.0 equiv) were added in this order. The reaction was followed by 1H
NMR. After 6 days, full conversion of glycidyl butyrate was achieved. The resin
bound catalyst was removed by filtration and the filtrate was diluted with n-heptane
(10 ml). To this mixture, myristic acid (274 mg, 1.2 mmol, 1.2 equiv) and DMAP (6.0
mg, 50 μmol, 5 mol%) followed by DCC (227 mg, 1.1 mmol, 1.1 equiv) were added,
and the resulting reaction was stirred for 20 h. The crude residue was directly
transferred onto a silica column and chromatographed using 10% Et2O in pentane
to afford 411 mg of the desired product as a yellowish liquid (80% yield).
Spectral data corresponded to those reported in table 2, entry 4.
References and footnotes
(1) Baer, E.; Fischer, H. O. L. J. Biol. Chem. 1939, 128, 475.
(2) The band corresponding to the symmetric stretching vibration of the nitro group
was overlapping with other bands.
(3) Craven, R. J.; Lencki, R. W. Cryst. Growth Des. 2011, 11, 1723.
(4) Schlenk, W., Jr. J. Am. Oil Chem. Soc. 1965, 42, 945.
(5) Patent, O. G. G. a. W. N. G., 1920.
(6) (a) Sreenivasan, B. J. Am. Oil Chem. Soc. 1978, 55, 796 (b) Konishi, H.; Neff, W.;
Mounts, T. J. Am. Oil Chem. Soc. 1993, 70, 411 (c) Eckey, E. W. Ind. Eng. Chem. 1948,
40, 1183.
(7) (a) Jandacek, R. J.; Webb, M. R. Chem. Phys. Lipids 1978, 22, 163 (b) Smith, R. E.;
Finley, J. W.; Leveille, G. A. J. Agric. Food Chem. 1994, 42, 432.
(8) (a) Xu, X. Eur. J. Lipid Sci. Technol. 2000, 102, 287 (b) Mu, H.; Porsgaard, T. Prog.
Lipid Res. 2005, 44, 430 (c) Lee, K. T.; Akoh, C. C. Food Rev. Inter. 1998, 14, 17.
(9) Chandler, I.; Quinlan, P.; McNeill, G. J. Am. Oil Chem. Soc. 1998, 75, 1513.
(10) Kristinsson, B.; Haraldsson, G. G. Synlett 2008, 2178.
(11) (a) Halldorsson, A.; Magnusson, C. D.; Haraldsson, G. G. Tetrahedron 2003, 59, 9101
(b) Halldorsson, A.; Magnusson, C. D.; Haraldsson, G. G. Tetrahedron Lett. 2001, 42,
7675 (c) Haraldsson, G. G. In Biocatalysis and Bioenergy; John Wiley & Sons, Inc.,
2008.
(12) (a) Stamatov, S. D.; Stawinski, J. Synlett 2006, 2251 (b) Stamatov, S. D.; Stawinski, J.
Bioorg. Med. Chem. Lett. 2006, 16, 3388 (c) Stamatov, S. D.; Stawinski, J. Tetrahedron
Lett. 2006, 47, 2543 (d) Stamatov, S. D.; Stawinski, J. Synlett 2007, 439 (e) Stamatov,
S. D.; Stawinski, J. Org. Biomol. Chem. 2007, 5, 3787 (f) Stamatov, S. D.; Stawinski, J.
Eur. J. Org. Chem. 2008, 2635 (g) Stamatov, S. D.; Stawinski, J. Org. Biomol. Chem.
2010, 8, 463.
(13) 9 common fatty acids in milk fat correspond to 729 different possible triacylglcerols.
(21/729) x 100 = 2.8%
Chapter 4
94
4
(14) (a) Annis, D. A.; Jacobsen, E. N. J. Am. Chem. Soc. 1999, 121, 4147 (b) Baleizão, C.;
Garcia, H. Chem. Rev. 2006, 106, 3987.
(15) Hofsløkken, N. U.; Skattebøl, L. Acta Chem. Scand. 1999, 53, 258.
(16) Hansen, T. V.; Skattebøl, L. Org. Synth. 2005, Vol. 82, p. 64-68; 2009, Coll. Vol. 11,
p. 267-271. Addendum: Org. Synth. 2012, 89, 220-229.
(17) Hansen, T. V.; Skattebøl, L. Tetrahedron Lett. 2005, 46, 3829.
(18) Bagno, A.; Kantlehner, W.; Kress, R.; Saielli, G. Z. Naturforsch. B 2004, 59, 386.
(19) The other explored hydrogen sources were ammonium formate and H2 (balloon).
(20) the ratio of 25 : 26 is 1 : 3, translated to % this means the mixture consists of 25%
of 25 and 75% of 26. Therefore ligand 37 will correspond to (0.25 x 0.25) x 100 =
6.25%, ligand 38 (0.75 x 0.75) x 100 = 56.3% and the desired C2 symmetrical ligand
27 will be present in 2 x (0.25 x 0.75) x 100 = 37.5%
(21) The band corresponding to the symmetric vibration of the nitro group was
overlapping with other bands.
(22) Hirschmann, H. J. Biol. Chem. 1960, 235, 2762.
95
Pag
e95
Chapter 5 A Methyl Matters
Abstract: Methyl branched fatty acids and their corresponding phospholipids are
found as components of bacterial membranes. Despite their occurrence, it is
challenging to isolate a reasonable amount of these lipids and use them for studies.
Therefore, these amounts have been obtained by chemical synthesis.
This work has been carried out in collaboration with Mac Donald José, and Dr. A.
Koçer (Department of Medical Physiology, UMCG), and Dr. H. I. Ingólfsson, Dr.
A. de Vries and Prof. Dr. S.-J. Marrink (Department of Molecular Dynamics, GBB,
University of Groningen).
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Introduction
Methyl-branched fatty acids, introduced in Chapter 2, are components of
phospholipids that occur in a number of specific bacterial species. The influence of
the methyl branch on the physical properties of membranes composed of these lipids
is a topic of discussion. Currently, it is accepted that the main function of the methyl
group is to increase the fluidity of the membranes, acting as a chemically stable
analogue of a double bond. However, methyl branching can have more roles, for
example, the corresponding membranes might be more stable and less permeable.
In this context, the study of Elferink et al.1 is relevant. The authors compared the
stability and the proton permeability of liposomes from linear, branched and archaeal
(that is; multimethyl-branched) lipids, which were obtained as lipid extracts from
Escherichia coli, Bacillus stearothermophilus (Figure 1) and Sulfolobus acidocaldarius (Figure
2).
Figure 1. Typical lipid and fatty acid composition of E. coli grown at 25 °C2; ( II ) typical
lipid and fatty acid composition of B. Stearothermophilus grown at 55 °C.3
The composition of the lipids differs in the headgroups, chain lengths, unsaturation
and branching. The study of Elferink et al.1 showed that the liposomes of B.
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stearothermophilus lipids were more stable and less proton permeable than the
liposomes of E. coli lipids.
Figure 2. Typical lipid composition of S. acidocaldarius.4
The liposomes composed of archaeal lipids showed even greater stability. From the
compared lipid mixtures it is difficult to conclude, if methyl branching has any effect
on the stability of the liposomes.
Due to their difficult accessibility, branched-chain lipids are studied often in
silico.5 Recently, Lim and Klauda6 published a computational study of the influence
of branched lipids (Figure 3) from Chlamidia trachomatis on the properties of the
corresponding bilayer. One of their conclusion was that the branched lipids form
stiffer membranes. However, from their publication it is not clear whether the
authors considered the stereochemistry of the lipids.
Figure 3. Membrane-forming lipids studied by Lim and Klauda by molecular dynamics
simulation.
Stereochemistry of the branched lipids might have an impact not only on the
properties of the bilayers (an ensemble - or macroscopic effect), but also on lipid-
protein interactions in a more molecular effect. Substitution of the glycerol with a
chiral, branched fatty acid introduces an additional stereogenic center to the
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molecule. The resulting lipid then comes in 4 stereoisomers. One might expect, that
the protein (which is chiral and enantiopure) will interact with each stereoisomer in
a different way.
In this context it is interesting to study the interaction of the
mechanosensitive protein channels with lipids. Mechanosensitive channels regulate
the pressure inside the cell by opening when this gets to life-threating levels. High
turgor pressure stretches the lipid membrane, what triggers a conformational change
in the protein that opens the channel’s pore. Through the opened pore an unselective
efflux takes place. When the pressure is below the life-threating value, the channel
closes. One of the best understood mechanosensitive channels is the
mechanosensitive channel of large conductance7 (MscL) from Mycobacterium
tuberculosis.
The crystal structure of M. tuberculosis MscL revealed that it is a
homopentamer (Figure 4-I, 4-II) with a simple topology.
Figure 4. Schematic representations of the MscL channel from the M. tuberculosis crystal
structure. ( I ) side view of the MscL pentamer; ( II ) top view from the periplasmic side, (
III ) individual MscL subunit with the positions of the two transmembrane helices, TM1 and
TM2.
The cytoplasmic N-terminus is followed by a transmembrane helix (TM1), which is
connected to the second transmembrane helix (TM2), followed by the cytoplasmic
C-terminus (Figure 4-III). The MscL is embedded in the inner membrane of the M.
tuberculosis cell wall, which is largely composed by lipids bearing the methyl branched
fatty acid tuberculostearic acid.
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There are several techniques to study the MscLs. Probably, the most widely
used technique is patch-clamp.8 As this is experimentally difficult, other, more
convenient techniques have been developed. One of the most versatile is a calcein
efflux essay.9 The idea of the calcein efflux essay is the preparation of liposomes filled
with calcein (Figure 5-I), which is a self-quenching fluorescent dye.
Figure 5 ( I ) Principle of the calcein efflux essay; ( II ) release triggered by MTSET; ( III )
release triggered by LPC; ( IV ) chemical structures of the compounds used in the calcein
efflux essay.
Subsequently, the MscL is reconstituted into these dye-filled liposomes. The
efflux of the dye is triggered either by introduction of a charge into the pore of the
MscL or by deformation of the bilayer, which triggers a change in the MscL
conformation or leads to the opening of the pore. To trigger the MscL by charge
(Figure 5-II), the proteoliposomes with a reconstituted mutated MscL are treated
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with 2-(trimethylammonium)ethyl methanethiosulfonate (MTSET). MTSET labels
the cysteine residues of TM1, which are in the middle of the pore. In the same
manner, the charge is introduced onto the remaining 4 MscL subunits. The
introduced charge leads to mutual repulsion of the subunits, thus forcing the pore to
open. Through the opened pore, the calcein can efflux, which is recorded as an
increase in the fluorescence. Triggering of the MscL through membrane induced
deformation is achieved by addition of lysophosphatidylcholine (LPC). LPC (Figure
5-III) has a conical shape and can insert into the outer leaflet of the bilayer. The
insertion leads to the deformation of the membrane. This deformation leads to a
change in the conformation of the MscL, which again leads to the opening of the
pore. The effluxed calcein again increases the fluorescence. At the final stage of the
calcein efflux assay, the amount of the effluxed dye is determined after bursting of
the liposomes induced with a detergent.
Until now, there is no study that directly compares branched and non-
branched lipids in their influence on an MscL, although the best studied MscL
originates from M. tuberculosis. Recently, Ter Horst et al. showed that 1 (Figure 6) is
the most abundant lipid in M. tuberculosis by comparison of the MS/MS spectra of
synthetic and natural 1. The authors needed 1 g of lipid extract (corresponding to 20
g of bacteria) for the isolation of 50 μg of natural 1.
Figure 6. The most abundant membrane lipid in M. tuberculosis.
Extrapolating, for 30 mg of 1, what is an acceptable amount to study MscL
incorporation and function in liposomes, one would need 600 g of the extract,
corresponding to 12 kg of M. tuberculosis.
The previous chapters (chapter 2 and 3) showed the efficient synthesis of
branched fatty acids and their phospholipids. These methods can be applied to the
synthesis of 1 providing sufficient amounts of the lipid for further studies.
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Results and discussion
Chemical synthesis of the phospholipids
The phospholipids used in this study were prepared according to the
synthetic routes developed in the previous chapters (Chapter 2, Chapter 3).
Figure 7. ( I ) (R)-tuberculostearic acid and its enantiomer; ( II ) branched lipids used in this
study; ( III ) linear chain lipids used in this study.
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Commercially available nonanal was converted into (R)-tuberculostearic acid in 5
steps. Application of the opposite enantiomer of the ligand, used for the introduction
of the methyl branch, led to the non-natural (S)-tuberculostearic acid (Figure 7-I).
Both fatty acids were obtained in comparable yields and in the same optical purity.
The fatty acids could be further converted into the corresponding
phospholipids (Figure 7-II). As Okuyuma10 and Ter Horst11 independently reported,
the phospholipids of M. tuberculosis have the sn-1 position mainly substituted by (R)-
tuberculostearic acid and the sn-2 position by (saturated) palmitic acid. Therefore,
lipid 1 (Figure 1-II) was synthetized and used for the study. To facilitate the
formation of the liposomes, a PC analogue 2 was also prepared. The non-natural (S)-
tuberculostearic acid was converted into the lipid 3. 3 is a chain epimer of 1. Lipids
1 and 3 allow a controlled study of the influence of the configuration of the methyl
branch on the properties of the liposomes. The chemical synthesis afforded 32.6 mg
of lipid 1, 73.5 mg of lipid 2 and 50.0 mg of lipid 3.
The methyl branched lipids were compared to their desmethyl analogues 4
and 5 (Figure 1-III). Lipid 4 is commercially available, and the synthesis of lipid 5 is
described in the previous chapter (Chapter 3).
Formation of the liposomes and proteoliposomes
The initial experiments with lipids 1 and 3 showed that these do not form
liposomes. Therefore, the lipids were studied in two-component liposomal
formulations which were prepared as a 1/1 (w/w) ratio of the PE and PC
phospholipids. This resulted in the following mixtures:
1:1 mixture of compounds 1 and 2, designated as RR
1:1 mixture of compounds 3 and 2, designated as SR
1:1 mixture of compounds 1 and 4, designated as R0
1:1 mixture of compounds 3 and 4, designated as S0
1:1 mixture of compounds 5 and 4, designated as 00
The lipid mixtures are designated based on the presence and configuration of the
methyl branch in the lipid. The first letter stands for the configuration of the PE
component and the second letter for the configuration of the PC component.
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Formation of the liposomes already revealed different behavior of the
branched and non-branched lipids. The mixtures consisting only of branched lipids
(RR, SR) smoothly afforded liposomes at room temperature. From the “half-
branched” mixtures R0 and S0, only S0 afforded liposomes, after heating above the
transition temperature of the PC component. At the same conditions, R0 did not
yield any liposomes. The lipids 4 and 5 displayed low solubility in organic solvents,
what together with their high transition temperatures resulted in failure to form
liposomes from the 00 formulation.
The SR and S0 liposomes were subsequently studied by cryo-electron
microscopy (Figure 8). This revealed, that while the SR mixture yields round, well-
shaped liposomes, the S0 mixture yields edgy, uneven liposomes, probably due to
phase separation in the membrane.
Figure 8. ( I ) uneven liposomes from the SR mixture, ( II ) well defined liposomes from
the S0 mixture (courtesy of Dr. M. Stuart).
The MscL proteins from E. coli and M. tuberculosis were reconstituted into liposomes
based on the branched (RR, SR) and half-branches (S0) mixtures. The efficiency of
the reconstitution was estimated using SDS-PAGE (Figure 9). This showed, that
roughly the same amount of E. coli and M. tuberculosis protein was reconstituted in
the SR and RR liposomes (lanes 2-5). However, when the proteins were
reconstituted into the S0 mixture, the MscL from M. tuberculosis reconstituted with
greater efficiency (lane 13 and 14), than the MscL from E. coli. The proteins were
reconstituted also in liposomes based on soy-extract (20%) (lanes 6-9 and 11-12),
which was used as a control experiments in the calcein efflux assays.
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Figure 9. SDS-PAGE of the reconstituted proteoliposomes; ( I ) reconstitution of the E.
coli and M. tuberculosis proteins in the RR and SR -based liposomes; ( II ) reconstitution of
the E. coli and M. tuberculosis proteins in S0-based liposomes.
Molecular dynamics study of the bilayers
This part of the chapter briefly summarizes results obtained by Dr. Helgi I. Ingólfsson and Dr. Alex de Vries, department of Molecular Dynamics.
Simulation of a phospholipid bilayer using molecular dynamics (MD) is a
powerful technique. One of the big advantages is its predictive power. Out of the 5
studied liposomal formulations, only 3 afforded liposomes. However, thanks to the
MD simulations all 5 formulations could be compared. In the case of the fully
branched formulations SR and RR, the bilayer resides in a liquid phase at 25 °C
(Table 1).12 In the case of S0 and R0, where only 50% of the lipids are branched, the
bilayer resides in a gel phase.13 The lipid mixture 00, which is composed only from
linear lipids is also in the gel phase. The phase in which lipids reside is closely related
to the area per lipid. Table 1 further shows, that the removal of the methyl branches
leads to tighter packing of the membrane. The mixtures SR and RR (0.634 ± 0.012
and 0.640 ± 0.012 nm2 area per lipid) show very similar packing. The removal of a
portion of methyl branched lipids as in the mixtures S0 and R0 leads to tighter
packing compared to the SR and RR bilayers.
Table 1. Summary of the molecular dynamics study.
Lipid mixture Phase Area per lipid / nm2 Phosphate distance / Å
SR Liquid 0.634 ± 0.012 39.2 ± 0.7
RR Liquid 0.640 ± 0.012 38.8 ± 0.6
S0 Gel 0.544 ± 0.021 44.0 ± 1.3
R0 Gel 0.496 ± 0.009 47.1 ± 0.7
00 Gel 0.475 ± 0.003 47.0 ± 0.3
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Interesting is the difference between the S0 and R0 mixtures. Despite being
diastereomeric, the stereogenic centers are distant. However, mixture S0 shows a
higher area per lipid (0.544 ± 0.021 nm2) compared to R0 (0.496 ± 0.009 nm2). The
subsequent removal of a methyl-branch as in mixture 00 leads to an even tighter
packing (0.475 ± 0.003 nm2). The MD simulations also provided information about
the thickness of the bilayers (expressed as the transbilayer distance of the phosphate
head-groups). The thickness of the membrane in the SR and RR bilayers is
comparable (39.2 ± 0.7 Å and 38.8 ± 0.6 Å). The bilayers composed of the
diastereomers S0 and R0 again were notably different. The bilayer corresponding to
R0 was thicker than the one of S0 (47.1 ± 0.7 Å and 44.0 ± 1.3 Å). In comparison
to the previous mixtures, the 00 mixture formed the thickest bilayer (47.0 ± 0.3 Å).
The simulations of the studied lipid bilayers have been summarized in an illustrative
fashion. The residues in pink correspond to the PE components and the blue
residues correspond to PC. In the case of the branched SR and RR mixtures (Figure
10), the fatty acid residues are unorganized and randomly ordered.
Figure 10. Simulated bilayers of SR and RR lipid mixtures.
However, the mixtures S0 and R0 (Figure 11) are considerably more ordered. The
aggregation of the PC (blue) and PE (pink) residues in the mixture S0 might explain
the formation of the observed edgy uneven liposomes, as observed by cryo-electron
microscopy (Figure 8-II). Comparison of the diastereomers S0 a R0 suggests that
despite the fact that the stereogenic centers (of the methyl-branch and the head
group) are distant, they have an impact on the macroscopic properties of the bilayer.
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Figure 11. Simulated bilayers of S0 and R0 mixtures.
Simulation of the bilayer from the linear lipid mixture 00 (Figure 12) showed that
this is highly organized.
Figure 12. Simulated bilayer of the 00 mixture.
Calcein efflux assay
This part of the research has been conducted by Mac Donald José and Dr. Armagan Koçer, department of medical physiology, UMCG.
In the calcein efflux assay, MscLs from 2 different species were studied. The
MscL from M. tuberculosis and the one from from E. coli. In Nature, the membranes
in which these MscLs are embedded differ. The M. tuberculosis channel is embedded
in a membrane consisting of methyl-branched lipids and the E. coli channel is
embedded in lipids from linear and unsaturated fatty acids. The first studied trigger
in the calcein efflux essay was MTSET. The increase of fluorescence was plotted
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against time (Figure 13). After the fluorescence did not increase anymore, the total
amount of released calcein was determined after bursting of the liposomes. The
measured data points were fitted by an exponential function. The initial rate and the
total amount of effluxed calcein are the studied parameters. These are summarized
in Table 2.
Figure 13. Graphs of the calcein efflux essay triggered by MTSET.
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Table 2. Summary of the calcein efflux essay triggered by MTSET.
M. tuberculosis MscL E. coli MscL
Lipid mixture Release Initial rate (s-1) Release Initial rate (s-1)
Soy extract 94.0 ± 1.0% 1.13 ± 0.5 98 ± 1.0% 1.15 ± 0.03
SR 94 ± 1.0% 1.13 ± 0.04 79 ± 3.0% 0.92 ± 0.3
RR 95 ± 1.0% 0.78 ± 0.1 85 ± 7.0% 0.53 ± 0.06
S0 90 ± 1.0% 1.2 ± 0.6 22 ± 3.0% 0.06 ± 0.01
As the graph (figure 13-I) and Table 2 show, the M. tuberculosis MscL is not much
affected by the different lipid mixtures. The release of the calcein is the same in all 4
cases and the rate is only slightly slower in the case of the RR lipid mixture. However,
the difference in the rate is not significant. The E. coli MscL shows comparable
release in soy extract, SR and RR mixture. The release in S0 mixture is dramatically
lower (22 ± 3.0%). Furthermore, E. coli MscL shows only negligible difference in the
rates in soy extract and SR mixture. However, the rate of the RR mixture is 2 times
slower compared to the soy extract. The rate in the S0 mixture is again significantly
lower (0.06 ± 0.01 s-1). Both the lower release and rate of E. Coli MscL in the S0
mixture are probably caused by much lower reconstitution efficiency as shown on
SDS-PAGE above (Figure 7, lane 14).
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Release triggered by LPC
The second studied trigger was LPC. The experimental set up and the output
are very similar to the previous MTSET-experiment. Following figure (Figure 14)
summarizes the results in graphs.
Figure 14. Graphs of the calcein efflux essay triggered by LPC.
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The releases and rates are summarized in Table 3.
Table 3. Summary of the calcein efflux assay triggered by LPC.
M. tuberculosis MscL E. coli MscL
Lipid mixture Release Initial rate (s-1) Release Initial rate (s-1)
Soy extract 82 ± 1.0% 1.6 ± 0.1 59 ± 4.0% 1.10 ± 0.01
SR 81 ± 1.0% 1.8 ± 0.1 34 ± 1.0% 1.14 ± 0.01
RR 90 ± 1.0% 1.0 ±0.2 19 ± 1.0% 1.09 ± 0.01
S0 70 ± 1.0% 0.4 ± 0.1 29 ± 6.0% 1.09 ± 0.01
Using LPC as the trigger, the M. tuberculosis MscL shows similar
characteristics as in the MTSET experiments. The release is comparable (though not
identical, see the RR mixture) in all studied liposomes. The lower release and rate of
the M. tuberculosis MscL in the S0 mixture can be attributed to a hydrophobic
mismatch; the hydrophobic region of the M. tuberculosis MscL spans 35 Å. The
phosphate distances calculated by MD simulations of the mixture SR and RR is
comparable to this; 39.2 ± 0.7 Å and 38.8 ± 0.6 Å, respectively. The S0 mixture,
however, forms bilayers of 44.0 ± 1.3 Å.
Another aspect which has to be considered in the case of liposomes based
on the S0 mixture is the concentration of liposomes. The concentration of added
LPC, which was kept constant in all the experiments, leads in the case of the S0
mixture to a greater ratio of the trigger to liposomes. The E. coli MscL shows
different, deviating behavior in the calcein efflux assay when LPC is used as the
trigger (Figure 11-II). Only the control experiment with soy extract shows the same
kinetics. The apparent linear dependence of the release against time in the SR, RR
and S0 mixtures suggest, that in these mixtures the release follows a different kinetics.
The total release is in all cases lower than the corresponding M. tuberculosis MscL
experiments.
Conclusions and outlook
The methyl-branched phospholipids were synthetized according to procedures
described in the previous chapters. Both natural (R)-tuberculostearic and non-natural
(S)-tuberculostearic acid were prepared in the same optical purity and comparable
yields. Both fatty acids were further converted to a set of epimeric PE lipids. The
natural (R)-tuberculostearic acid was also converted to a PC lipid. All of the lipids
were obtained in reproducible yields and in sufficient amounts (up to 100 mg) to
conduct further studies, which showed that these lipids do not form single
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component liposomes. Therefore, the liposomes were formed as 2 component
PE/PC 1/1 (w/w) mixtures. The mixtures consisting of lipids with branched fatty
acids formed regular round liposomes. From the mixtures of linear and branched
lipids, only 1 mixture afforded liposomes. These, however had uneven edgy shapes.
The mixtures were studied by molecular dynamics simulations, which provided
insight into the organization and thickness of the bilayers. MscLs from 2 different
species, M. tuberculosis and E. coli were reconstituted in these liposomes. The function
of the MscLs was further studied in a calcein efflux assay. While the M. tuberculosis
MscL showed similar behavior with both studied triggers, the E. coli MscL did not
perform well in the lipid mixture consisting of methyl branched lipids.
As such, the use of natural (synthetic) lipids in this study more closely mimics
the natural environment of in particular the M. tuberculosis MscL. Despite the studied
lipids in this chapter are natural, they were not the best choice for a comparison of
the properties of branched and linear lipids. The low solubility and high transition
temperature of the linear lipids are the probable causes for their reluctance to form
liposomes. In future research, this can be addressed by studies of shorter analogues.
For linear lipids this would lower the transition temperature and increase the
solubility. Furthermore, in future research, the MscLs should be studied by patch-
clamp as this gives more insight into the properties of the bilayer.
Experimental part
Note: The synthesis of (R)-tuberculostearic acid is described in previous chapters (Chapter 2)
(S)-S-ethyl 3-methylundecanethioate
A dried Schlenk flask was charged with CuBr.SMe2 (Sigma-Aldrich, 24.7 mg, 2
mol%) and (R,S)-Josiphos.EtOH adduct (Sigma-Aldrich, 92.2 mg, 2.4 mol%). The
flask was evacuated in three cycles and MTBE (54 ml) was added. The resulting
solution was stirred for 30 min at rt (21 °C) before cooling to −78 °C. After stirring
for 10 min at −78 °C a solution of MeMgBr (Sigma-Aldrich, 3 ml, 1.5 equiv) was
added dropwise during 10 min accompanied by formation of a voluminous yellow
precipitate. To this suspension, (E)-S-ethyl undec-2-enethioate (1.37 g, 6 mmol) was
added as a solution in MTBE (6 ml) during 3 h. The reaction was stirred for an
additional 16 h and then poured into an NH4Cl/ice mixture. The organic layer was
separated, and the aqueous layer was extracted with Et2O (3 x 20 ml). The combined
organic layers were dried over MgSO4 and evaporated. The residual orange liquid
was chromatographed on silica gel using 3% of Et2O in pentane.
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The reaction afforded the desired (S)-S-ethyl 3-methylundecanethioate as colorless
liquid (1.38 g, 95%)
1H NMR (400 MHz, CDCl3) δ 2.87 (q, J = 7.4 Hz, 3H), 2.52 (dd, J = 14.4, 6.0 Hz,
1H), 2.33 (dd, J = 14.4, 8.1 Hz, 1H), 2.00 (broad s, J = 5.9 Hz, 1H), 1.33 – 1.17 (m,
17H), 0.92 (d, J = 6.7 Hz, 3H), 0.87 (t, J = 6.8 Hz, 3H).
13C NMR: (101 MHz, CDCl3) δ 199.32, 51.40, 36.61, 31.86, 31.06, 29.69 ( - ), 29.55,
29.27, 26.82, 23.23 , 22.65, 19.51 ( -), 14.78 ( - ), 14.08 ( - ).
HRMS-ESI+ (m/z): [M + H]+ calculated for C14H29OS: 245.193 found 245.193.
[α]D +3.32° (c = 1.00, CHCl3).
The enantiomeric excess was determined on the corresponding carbamate, obtained
by LiAlH4 reduction, treatment with phosgene and subsequent treatment with (S)-1-
(1-naphthyl)ethylamine.
Chiracel OD-H, flow=1 ml/min, tminor = 11.4 min, tmajor = 12.4 min, e.r. = 95 : 5.
(S)-3-methylundecanal
A solution of (S)-S-ethyl 3-methylundecanethioate (1.2 g, 4.9 mmol) in CH2Cl2 (30
ml) in a 100 ml roundbottom flask was immersed into a – 40 °C bath. After 5 min
of stirring, a solution of DIBAL-H (Sigma-Aldrich, 1 M in CH2Cl2, 1.2 equiv) was
introduced slowly via the wall of the flask to cool down the reagent. The reaction
was stirred for 1 h (full conversion, TLC) before Rochelle salt (saturated aqueous
solution, 10 ml) was added to stop the reaction. The flask was removed from the
cooling bath and stirred at rt (21 °C) until the layers fully separated. The organic layer
was diluted with additional CH2Cl2 (50 ml), and filtered over a Whatman® 1PS phase
separator filter paper. The filtrate was carefully evaporated (maximum vacuum 100
mbar).
The reaction afforded 915 mg (100%) of the desired aldehyde as a pleasantly smelling
colorless liquid. This was used without further purification in subsequent step.
(S)-10-methyloctadec-7-enoic acid (E/Z mixture)
A dry Schlenk flask was charged with 7-(bromotriphenylphosphoranyl)-heptanoic
acid (Wittig reagent) under a stream of nitrogen. After degassing in three cycles,
LiHMDS (Sigma-Aldrich, 1 M solution in THF, 2.2 equiv) was added dropwise
(addition time ca 3 min, the Wittig reagent is treated without previous suspending or
dissolving). The obtained suspension was stirred for 15 min at rt (21 °C) until it
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turned into a cloudy (unreacted phosphonium salt) deep red solution. This was
further cooled to 0 °C by immersion into an ice/water bath. After 5 min of stirring,
(S)-3-methylundecanal (915 mg, 4.9 mmol) in THF (1 ml added to rinse the flask)
was added dropwise over 15 min. After addition of ca 30% of the aldehyde,
triphenylphosphine oxide started to precipitate. The reaction was stirred for 2 h at 0
°C, and then for 30 min at rt (21 °C). Subsequently it was cooled again by immersing
into the ice/water bath and water was added (5 ml). The pH of the solution was
adjusted to 1 by careful addition of concentrated HCl. The reaction mixture was
transferred into a separatory funnel and extracted with CH2Cl2 (3 x 20 ml), the
combined organic layers were dried over MgSO4 and evaporated with a small amount
of silica gel (10 g, column chromatography using solid loading). The silica gel with
the adsorbed compound was transferred onto a silica gel column and was further
chromatographed with 5% of Et2O in pentane (to elute unreacted aldehyde). After
complete elution of the aldehyde the polarity of the eluent was increased to 60%
Et2O in pentane.
The reaction afforded a mixture of E and Z (S)-10-methyloctadec-7-enoic acid (1.04
g, 71% over 2 steps) and 150 mg of unreacted aldehyde (yield based on recovered
starting material 83% over two steps).
1H NMR (400 MHz, CDCl3) δ 5.53 – 5.23 (m, 2H), 2.35 (t, J = 7.5 Hz, 2H), 2.12 –
1.92 (m, 2H), 1.65 (dd, J = 9.9, 5.0 Hz, 1H), 1.53 – 1.01 (m, 23H), 0.86 (dt, J = 14.7,
7.3 Hz, 6H).
13C NMR (101 MHz, CDCl3) δ 173.49, 130.11, 128.81, 36.70, 34.52, 33.40, 31.91,
29.96 ( - ), 29.66, 29.34, 29.30, 28.72, 27.17, 27.07, 24.58, 22.67, 19.58 ( - ), 14.10 ( - ).
(S)-Tuberculostearic acid
In a 500 ml roundbottom flask, (S)-10-methyloctadec-7-enoic acid (940 mg, 3 mmol)
was dissolved in MeOH (20 ml). To this solution, Pt/C (Sigma-Aldrich, 10% loading,
589 mg, 5 mol%) was added with caution (PYROPHORIC). The flask was capped
with a H2 balloon and vigorously stirred for 20 h at rt (21 °C). The MeOH was
evaporated and the black oily residue was transferred directly onto a silica column.
The desired compound was eluted with 60% Et2O in pentane.
1H NMR (400 MHz, CDCl3) δ 2.35 (t, J = 7.5 Hz, 2H), 1.63 (dt, J = 15.0, 7.5 Hz,
2H), 1.42 – 1.00 (m, 27H), 0.88 (t, J = 6.9 Hz, 3H), 0.83 (d, J = 6.5 Hz, 3H).
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13C NMR (101 MHz, CDCl3) δ 180.82, 37.08, 37.05, 34.06, 32.73 ( - ), 31.91, 30.02,
29.92, 29.68, 29.45, 29.35, 29.23, 29.05, 27.07, 27.02, 24.65, 22.67, 19.69 ( - ), 14.09
( - ).
HRMS-ESI+ (m/z): [M + H]+ calculated for C19H38O2, 299.295; found, 299.295.
[α]D +0.2 (c = 3.0, CHCl3).
Synthesis of the 3-protected-1,2-diacylglycerols
(S)-(R)-3-((tert-butyldimethylsilyl)oxy)-2-(stearoyloxy)propyl tuberculostearate
A dry flask was charged with (R,R)-(−)-N,N′-Bis(3,5-di-tert-butylsalicylidene)-1,2-
cyclohexanediaminocobalt(II) complex (12 mg, 2 mol%) and (R)-TBSA (298 mg, 1
mmol) and guarded with a CaCl2 tube. The mixture was stirred at rt (21 °C) for 30
min (change of color from brick red to brown). Hünigs base (175 µl, 1 equiv) was
added, followed by tert-butyldimethylsilyl (R)-glycidyl ether (Sigma-Aldrich, 210 µl,
1 equiv). The mixture was stirred under a nitrogen atmosphere until full conversion
of the epoxide (followed by 1H NMR). All volatiles were removed under vacuum.
The brownish liquid residue was dissolved in heptane (5 ml), and palmitic acid (332
mg, 1.3 equiv) and DMAP (12 mg, 0.1 equiv) were added sequentially. To the
brownish solution, dicyclohexylcarbodiimide (247 mg, 1.2 equiv) was added in one
portion. The solution turned into a suspension in less than 1 min. The mixture was
further stirred at rt (21 °C) for 16 h (the reaction is complete after ca 4 h). After full
conversion of the alcohol (TLC, NMR), the reaction mixture was directly transferred
onto a silicagel column and chromatographed using 5% Et2O in pentane.
The reaction afforded 658.8 mg of desired product as colorless liquid (89% yield)
1H NMR (400 MHz, CDCl3) δ 5.15 – 4.97 (m, 1H), 4.34 (dd, J = 11.8, 3.7 Hz, 1H),
4.16 (dd, J = 11.8, 6.3 Hz, 1H), 3.71 (m, 2H), 2.33 – 2.27 (m, 4H), 1.60 (d, J = 6.8
Hz, 4H), 1.27 (d, J = 11.1 Hz, 48H), 0.88 (s, 16H), 0.83 (d, J = 6.5 Hz, 4H), 0.05 (s,
6H).
13C NMR (101 MHz, CDCl3) δ 173.57, 173.22, 71.82 ( - ), 62.59, 61.61, 37.25, 34.50,
34.31, 32.91 ( - ), 32.08, 30.20, 30.14, 29.86, 29.82, 29.79, 29.68, 29.64, 29.52, 29.47,
29.45, 29.30, 29.26, 27.25, 27.23, 25.90 ( - ), 25.10, 25.07, 22.84, 19.85( - ), 18.35,
14.26 ( - ), -5.33 ( - ), -5.37 ( - ).
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HRMS-ESI+ (m/z): [M + H]+ calculated for C44H89O5Si: 725.647, found: 725.649
[α]D = 5.1 (c = 2.0, CHCl3)
(R)-(R)-3-((tert-butyldimethylsilyl)oxy)-2-(palmitoyloxy)propyl tuberculostearate
The reaction afforded 632 mg of the desired product (86%) as a colorless liquid.
1H NMR (400 MHz, CDCl3) δ 5.15 – 4.97 (m, 1H), 4.34 (dd, J = 11.8, 3.7 Hz, 1H),
4.16 (dd, J = 11.8, 6.3 Hz, 1H), 3.71 (m, 2H), 2.33 – 2.27 (m, 4H), 1.60 (d, J = 6.8
Hz, 4H), 1.27 (d, J = 11.1 Hz, 48H), 0.88 (s, 16H), 0.83 (d, J = 6.5 Hz, 4H), 0.05
(s, 6H).
13C NMR (101 MHz, CDCl3) δ 173.57, 173.22, 71.82 ( - ), 62.59, 61.61, 37.25, 34.50,
34.31, 32.91 ( - ), 32.08, 30.20, 30.14, 29.86, 29.82, 29.79, 29.68, 29.64, 29.52, 29.47,
29.45, 29.30, 29.26, 27.25, 27.23, 25.90 ( - ), 25.10, 25.07, 22.84, 19.85 ( - ), 18.35,
14.26 ( - ), -5.33 ( - ), -5.37 ( - ).
HRMS-ESI+ (m/z): [M + H]+ calculated for C44H89O5Si: 725.647, found: 725.649.
[α]D = +4.1 (c = 2.3, CHCl3)
Major lipid precursor
To a solution of (R,R)-3-((tert-butyldimethylsilyl)oxy)-2-(palmitoyloxy)propyl 10-
methyloctadecanoate (81.3 mg, 112 µmol) in CH2Cl2 (1 ml), CH3CN●BF3 (100 µl,
123 µmol, 1.1 eq) was added and the resulting light yellow reaction mixture was
stirred for 5 min, carefully monitored by TLC. Upon full conversion, the reaction
mixture was diluted with Et2O (15 ml) and poured onto chilled sodium phosphate
buffer (1 M, 5 ml). The organic layer was separated and washed with saturated brine
(5 ml), dried and evaporated to dryness to afford (R, S)-3-hydroxy-2-
(palmitoyloxy)propyl 10-methyloctadecanoate (65.6 mg, 96%) as a colorless oil. The
compound was used directly without delay and further purification.
Chapter 5
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1H NMR (400 MHz, CDCl3) δ 5.09 (m, 1H), 4.32 (m, 1H), 4.26 (s, 1H), 3.75 (s, 1H),
2.34 (dd, J = 15.8, 8.1 Hz, 4H), 2.01 (s, 1H), 1.63 (s, 4H), 1.54 (dd, J = 8.2, 4.9 Hz,
2H), 1.27 (d, J = 8.1 Hz, 47H), 0.94 – 0.79 (m, 9H).
To a stirred solution of 1-(R)-TBSA-2-palmitoyl glycerol (63.6 mg, 104 µmol) in
CH2Cl2 (0.5 ml), phosphoramidite 15 (54.0 mg, 125 µmol, 1.2 eq) was added. The
mixture was cooled to 0 °C and 1H-imidazole-4,5-dicarbonitrile (15.4 mg, 0.13 µmol,
1.3 eq) was added in one portion. The reaction was stirred until complete conversion
of the starting diacylglycerol (monitored by TLC - typically 30 min). Subsequently,
the mixture was cooled to –20 °C and tBuOOH (ca 5 M in decane, 38 µl, 0.208
mmol) was added, followed by stirring for 30 min. Then the reaction was diluted
with 10 ml of CH2Cl2 and poured into aqueous NaHCO3 (1 M, 10 ml). The organic
layer was washed with aqueous HCl (1 M, 10 ml), brine, dried and evaporated.
The resulting crude yellow oil was purified by column chromatography on SiO2
(CHCl3 : pentane 9 : 1) to afford the desired product (85 mg, 85%) as a colorless thick
liquid, together with a co-eluting impurity.
1H NMR (400 MHz, CDCl3) δ 7.47 – 7.19 (m, 1H), 5.34 (broad s, 1H), 5.18 (dd, J =
9.6, 5.3 Hz, 1H), 5.11 – 5.02 (m, 4H), 4.31 – 4.22 (m, 1H), 4.17 – 4.01 (m, 4H), 3.42
(m, 2H), 2.32 – 2.24 (m, 4H), 1.57 (d, J = 7.0 Hz, 4H), 1.46 – 0.99 (m, 52H), 0.88 (t,
J = 6.8 Hz, 6H), 0.83 (d, J = 6.5 Hz, 9H).
13C NMR (101 MHz, CDCl3) δ 173.19, 172.81, 128.80( - ), 128.67( - ), 128.47 ( - ),
128.09 ( - ), 128.03 ( - ), 69.80, 66.78, 65.49, 61.56, 41.33, 37.09, 34.10, 33.97, 32.75 (
- ), 31.91, 30.02, 29.97, 29.68, 29.64, 29.52, 29.47, 29.35, 29.28, 29.11, 29.06, 27.08,
24.81, 22.67, 19.69 ( - ), 14.10 ( - ).
31P NMR (162 MHz, CDCl3) δ 0.81, 0.83.
HRMS-ESI+ (m/z): [M + H]+ calculated for C55H93O10NP: 958.653, found 958.653.
[α]D = +7.1 (c = 2.3, CHCl3)
Epimer of the major TBSA lipid
Starting from 103 mg of protected diacylglycerol, the reaction afforded 120 mg (93%)
of the desired product.
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1H NMR (400 MHz, CDCl3) δ 7.47 – 7.19 (m, 10H), 5.34 (broad s, 1H), 5.18 (dd,
J = 9.6, 5.3 Hz, 2H), 5.11 – 5.02 (m, 4H), 4.31 – 4.22 (m, 1H), 4.17 – 4.01 (m, 4H),
3.42 (m, 2H), 2.32 – 2.24 (m, 4H), 1.57 (d, J = 7.0 Hz, 4H), 1.46 – 0.99 (m, 52H),
0.88 (t, J = 6.8 Hz, 6H), 0.83 (d, J = 6.5 Hz, 9H).
13C NMR (101 MHz, CDCl3) δ 173.19, 172.81, 128.80 ( - ), 128.67 ( - ), 128.47 ( - ),
128.09 ( - ), 128.03 ( - ), 69.80, 66.78, 65.49, 61.56, 41.33, 37.09, 34.10, 33.97,
32.75( - ), 31.91, 30.02, 29.97, 29.68, 29.64, 29.52, 29.47, 29.35, 29.28, 29.11, 29.06,
27.08, 24.81, 22.67, 19.69 ( - ), 14.10 ( - ).
31P NMR (162 MHz, CDCl3) δ 0.81, 0.83.
HRMS-APCI+ (m/z): [M + Na]+ calculated for C55H92NO10PNa: 980.635 found
980.635.
Synthesis of the phospholipids
Phospholipid 1
To a stirred solution of the TBSA lipid precursor (50 mg, 52 µmol) in MeOH/formic
acid (2 ml, 96/4), Pd/C (Sigma-Aldrich, Degussa Type E101 NE/W, 3 mg, 2.6 µmol,
5 mol%) was added. The suspension was stirred under a hydrogen atmosphere
(balloon) until complete conversion of the starting material (typically 2 h, according
to TLC). Subsequently, the solution was diluted with CH2Cl2 (10 ml) and SiO2 (2 g)
was added followed by evaporation of the volatiles. The SiO2 with adsorbed
phospholipid was transferred onto a short (5 g) SiO2 column (Sigma-Aldrich, Silica
gel, high-purity grade (Davisil Grade 633), pore size 60 Å, 200-425 mesh particle
size), impurities were eluted with Et2O (100 ml) followed by elution of the
phospholipid with CHCl3/MeOH/H2O (65 /35/7) to afford the M. tuberculosis
phospholipid 1 (32.6 mg, 0.044 mmol, 87%) as a white sticky solid.
1H NMR (400 MHz, CDCl3/CD3OD/D2O, v/v, 95/35/2) δ 5.19 (s, 1H), 5.07 (s,
1H), 4.36 (d, J = 11.3 Hz, 1H), 4.21 – 3.80 (m, 4H), 3.64 (s, 4H), 3.37 (s, 1H), 3.09
(s, 1H), 2.27 (dd, J = 15.5, 8.1 Hz, 4H), 1.56 (s, 4H), 1.48 – 0.98 (m, 44H), 0.98 –
0.77 (m, 9H).
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5
13C NMR (101 MHz, CDCl3/CD3OD/D2O v/v 95/35/2 ) δ 173.42, 173.14, 128.41
( - ), 127.97 ( - ), 37.14, 34.24, 34.06, 32.79 ( - ), 31.92, 30.05, 29.75, 29.70, 29.42,
29.37, 29.25, 27.15, 27.11, 24.94, 24.87, 22.68, 19.65 ( - ), 14.10 ( - ). 31P NMR (162 MHz, CDCl3) δ +0.29
HRMS-ESI+ (m/z): [M + H]+ calculated for C40H81NO8P: 734.570, found 730.569.
[α]D = +7.0 (c = 0.3, CHCl3).
Spectral data correspond to those previously published17
Phospholipid 2
Starting from 72 mg of the precursor reaction afforded 50 mg (91%) of the desired
compound.
1H NMR (400 MHz, CDCl3/CD3OD/D2O, v/v, 95/35/2) δ 5.24 (d, J = 4.2 Hz,
1H), 4.40 (dd, J = 12.1, 2.9 Hz, 2H), 4.17 (dd, J = 12.1, 7.1 Hz, 1H), 4.08 (t, J = 8.4
Hz, 1H), 3.99 (t, J = 6.0 Hz, 2H), 3.24 – 3.13 (m, 2H), 2.33 (dd, J = 15.7, 8.2 Hz,
4H), 1.66 – 1.56 (m, J = 5.7 Hz, 4H), 1.27 (bs, 50H), 0.89 (t, J = 6.8 Hz, 6H), 0.84
(d, J = 6.5 Hz, 3H)
13C NMR (101 MHz, CDCl3/CD3OD/D2O, v/v, 95/35/2) δ 173.90, 173.55, 77.48,
77.16, 76.84, 62.50, 61.59, 40.17, 36.92, 34.04, 33.90, 32.57 ( - ), 31.73, 29.83, 29.51,
29.49, 29.46, 29.39, 29.37, 29.16, 28.98, 28.94, 26.89, 24.73, 24.67, 22.47, 19.41 ( - ),
13.78 ( - ).
31P NMR (162 MHz, CDCl3/CD3OD/D2O, v/v, 95/35/2) δ 0.65.
HRMS-ESI+ (m/z): [M + H]+ calculated for C40H81NO8P: 734.569, found 734.569.
Phospholipid 3
To a solution of (R)-(R)-3-((tert-butyldimethylsilyl)oxy)-2-(palmitoyloxy)propyl 10-
methyloctadecanoate (200 mg, 280 µmol) in CH2Cl2 (2.8 ml), CH3CN●BF3 (220 µl,
1.1 eq) was added and the resulting light yellow reaction mixture was stirred for 5
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5
min carefully monitored by TLC. Upon full conversion, the reaction mixture was
diluted with Et2O (30 ml) and poured onto chilled phosphate buffer (1 M, 10 ml).
The organic layer was separated and washed with saturated brine (10 ml), dried and
evaporated to dryness to afford (R)-(S)-3-hydroxy-2-(palmitoyloxy)propyl 10-
methyloctadecanoate (151 mg, 90%) as a colorless oil. The compound was used
directly without delay and further purification.
1H NMR (400 MHz, CDCl3) δ 5.09 (m, 1H), 4.32 (m, 1H), 4.26 (s, 1H), 3.75 (s, 1H),
2.34 (dd, J = 15.8, 8.1 Hz, 4H), 2.01 (s, 1H), 1.63 (s, 4H), 1.54 (dd, J = 8.2, 4.9 Hz,
2H), 1.27 (d, J = 8.1 Hz, 47H), 0.94 – 0.79 (m, 9H)
2-Chloro-2-oxo-1.3.2-dioxaphospholane (distilled under reduced pressure at 150 °C,
and 3.6 mbar, before use, 80 μl, 3 equiv) was added to a chilled solution of
diacylglycerol. To this solution, DMAP (103 mg, 3 equiv) was added. The reaction
was allowed to reach rt and stirred for 24 h at rt (21 °C). The reaction was diluted
with Et2O (20 ml), and the organic layer was washed with water and brine, dried and
evaporated. The crude residue (ca 170 mg) was dissolved in CH2Cl2 (2 ml) and cooled
to 0 °C with an ice bath. To this solution, NMe3 (125 μl, 4 equiv) was added using a
plastic syringe wrapped in cotton previously dipped in acetone/liquid N2.
Subsequently, TMSOTf (100 μl, 2 equiv) was added. The mixture was stirred until
full conversion of the starting material. All volatiles were evaporated and the crude
residue was purified by column chromatography on silica gel (Davisil high purity
silica gel Grade 633, pore size 150 Å, 200-425 mesh particle size) carefully using a
gradient from 1% MeOH to 30% MeOH in CHCl3. The reaction afforded 73.5 mg
of desired product (34% over three steps).
1H-NMR was inconclusive because of strong significant signal-broadening.
13C NMR (101 MHz, CDCl3/CD3OD/D2O, v/v, 95/35/2) δ 173.54, 173.18, 70.14,
70.04, 69.95, 63.12, 62.37, 58.87, 58.82, 55.07, 55.03, 53.72 ( - ), 51.15 ( - ), 36.68,
33.85, 33.70, 32.33 ( - ), 31.50, 29.60, 29.28, 29.26, 29.23, 29.16, 29.13, 28.93, 28.75,
28.70, 26.66, 24.54, 24.47, 22.25, 19.22 ( - ), 13.60 ( - ).
31P NMR (162 MHz, CDCl3/CD3OD/D2O, v/v, 95/35/2) δ 2.94.
HRMS-ESI+ (m/z): [M + H]+ calculated for
This part of the research was conducted by Mac Donald José
Preparation of large unilamellar vesicles (LUV) or liposomes
Chapter 5
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5
20% total soy extract (Avanti polar lipids) was dissolved in lipid buffer (10 mM
sodium phosphate and 150 mM NaCl, pH 8) to 20 mg/ml. The dissolved lipid was
then passed through an alternate five cycles of freezing in liquid nitrogen and thawing
in a water bath set at 50 oC. The sample was stored at -20 oC until use.
For the synthetic lipids, both pure lipids and lipid mixtures were dissolved first in
chloroform and dried using a rotatory evaporator under vacuum for 1.5 h. After
drying, the lipids were dissolved (10 mg/ml) in lipid buffer (10 mM sodium
phosphate and 150 mM NaCl, pH 8) and passed through five cycles of freeze and
thaw just like soy lipid extract. The liposomes were composed as follows:
Lipid mixture SR: 1:1 mixture of compounds 2 and 3 (18:(10-(S)Me)16:0 PE 18:(10-
(R)Me)16:0 PC)
Lipid mixture RR: 1:1 mixture of compounds 1 and 2 (18:(10-(R)Me)16:0 PE and
18:(10-(R)Me)16:0 PC)
Lipid mixture S0: 1:1 mixture of compounds 3 and 4 (18:(10-(S)Me)16:0 PE and
18:0 16:0 PC)
Soy extract (control-100%)
Proteoliposomes reconstitution
The lipids were then extruded through a polycarbonate filter of 400 nm by passing
to and fro eleven times. Two aliquots of the extruded lipids (300 l) were destabilized
by addition of Triton X-100 for five min in a waterbath set to 50 oC (the amount of
detergent for destabilization was different for each lipid and was found by prior
titration). To one portion of lipid sample MscL cysteine mutant protein from M.
tuberculosis (A20C) was added and to another portion of the same lipid MscL cysteine
mutant protein from E. coli (G22C)was added to a protein to lipid ratio of 1 to 50
(wt/wt). A control sample was prepared which contained no protein for monitoring
unspecific release from the liposomes. This was followed by incubation at 50 oC for
30 min. After incubation, one volume of calcein (200 mM calcein, 10 mM sodium
phosphate pH 8) was added to each portion. About 200 mg (wet weight) of biobeads
(SM-2-Absorbents) were added to each tube to remove the detergent during
overnight incubation at 4 oC. The excess calcein was removed from the
proteoliposomes by passing the samples through a Sephadex G50 size exclusion
column using efflux buffer (150 mM NaCl, 10 mM sodium phosphate, 1 mM EDTA
pH 8).
Calcein efflux assay
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The activity of the protein in the calcein filled proteoliposomes was determined by
following the increase in fluorescence at an excitation wavelength of 495 nm and an
emission wavelength of 515 nm. Into 2.2 ml of efflux buffer (150 mM NaCl, 10 mM
sodium phosphate, 1 mM EDTA pH 8) 2-5 l proteoliposomes were added and
allowed to equilibrate for about 50 s before adding the trigger. Two triggers were
used, MTSET to a final concentration of 1 mM and LPC (lysophosphatidyl choline)
to a final concentration of 4.5 M. The release was followed for about 12 min at
which point complete release was observed. Another portion of detergent (Triton)
to a final concentration of about 0.5% was added to destroy all the proteoliposome
to obtain maximum calcein release.
References and footnotes
(1) Elferink, M. G. L.; de Wit, J. G.; Driessen, A. J. M.; Konings, W. N. Biochim. Biophys.
Acta - Biomembranes 1994, 1193, 247.
(2) Marr, A. G.; Ingraham, J. L. J. Bacteriol. 1962, 84, 1260.
(3) (a) Bezbaruah, R. L.; Pillai, K. R.; Gogoi, B. K.; Baruah, J. N. Antonie van Leeuwenhoek
1988, 54, 37(b) Jurado, A. S.; Pinheiro, T. J. T.; Madeira, V. M. C. Arch. Biochem.
Biophys. 1991, 289, 167.
(4) Langworthy, T. A. J. Bacteriol. 1977, 130, 1326.
(5) Gabriel, J. L.; Chong, P. L. G. Chem. Phys. Lipids 2000, 105, 193.
(6) Lim, J. B.; Klauda, J. B. Biochim. Biophys. Acta - Biomembranes 2011, 1808, 323.
(7) Chang, G.; Spencer, R. H.; Lee, A. T.; Barclay, M. T.; Rees, D. C. Science 1998, 282,
2220.
(8) Hamill, O. P.; Marty, A.; Neher, E.; Sakmann, B.; Sigworth, F. J. Pflugers Archiv - EJP
1981, 391, 85.
(9) Kocer, A.; Walko, M.; Feringa, B. L. Nat. Protoc. 2007, 2, 1426.
(10) Okuyama, H.; Kankura, T.; Nojima, S. J. Biochem. 1967, 61, 732.
(11) ter Horst, B.; Seshadri, C.; Sweet, L.; Young, D. C.; Feringa, B. L.; Moody, D. B.;
Minnaard, A. J. J. Lipid Res. 2010, 51, 1017.
(12) This is partially supported by DSC measurment where no transition was observed
between -10 and 80 °C.
(13) transition temperature at 43 °C
Chapter 5
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123
Chapter 6 Synthesis of a Cyclooctyne–based Lipidation Probe
Abstract: Protein lipidation is an important, relatively unexplored posttranslational
modification. Usually, the protein lipidation is studied with radioactive or fluorescent
radioactive probes. With the development of bioorthogonal chemical reporters,
azides and alkynes fatty acid analogues became important tools for the lipidation
studies. However, a probe which could be used in living cells is still lacking. This
chapter describes the design and synthesis of a cyclooctyne–based fatty acid that can
be potentially applied in the study of the protein lipidations.
Chapter 6
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6
Introduction
Previous chapter (Chapter 5) discussed the influence of lipids on the function
of the membrane-embedded proteins via lipid-protein interactions. Lipids can
control the function also in a different way. Protein lipidation is a posttranslational
modification in which hydrophobic groups are attached to the protein, thus altering
its activity and subcellular location. Depending on the lipidation pattern, modified
proteins can be associated with the inner or the outer membrane leaflet.
Proteins modified with a glycosylphosphatidylinositol-anchor (GPI-anchor,
Figure 1) are localized at the outer leaflet of the membrane towards the extracellular
space.
Figure 1. GPI-anchor modification.
This very complex anchor consists of a lipophilic portion, a glycan core and
a phosphoethanolamine linkage. The phosphoethanolamine linkage attaches to the
C-terminus of the protein. GPI-anchor modified proteins fulfil diverse biological
roles. Usually, they are signal transduction enzymes, antigens or receptors.1
Synthesis of a Cyclooctyne-based Lipidation Probe
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The lipidation patterns for anchoring to the inner leaflet of the membrane
are less complex (Figure 2). Typically, these are acyl residues bound to the N-
terminus of the protein (N-myristoylation or N-palmitoylation), prenyl residues
bound to the sulfur of a terminal cysteine (S-farnesylation and S-geranylgeranylation)
or a phosphatidylethanolamine residue bound to the C-terminus of the protein (C-
phosphatidylethanolaminylation).
Figure 2. Lipidation patterns for anchoring to the intracellular leaflet of the membrane.
Another common lipidation is acylation of the sulphur of cysteine (S-palmitoylation).
In comparison to the other lipidation patterns, the S-palmitoylation is specific,
because it is reversible, thus more challenging to study.
Chapter 6
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6
Lipidations play a central role in many physiological processes and diseases.2
For example, Fearson et al.3 reported that inhibitors of N-myristoyltransferase of
Trypanosoma brucei are promising pharmaceutical leads for treatment of the African
sleeping sickness. S-farnesylation of lamin A protein plays an important role in the
Hutchinson-Gilford progeria syndrome.4 Inhibitors of the corresponding farnesyl
transferase5 improved the disease symptoms and survival in mice. Proteins modified
by C-phosphatidylethanolamination are important in autophagy, cellular homeostasis
and infection resistance.6 The cycle of S-palmitoylation, depalmitoylation and
repalmitoylation is vital for the localization and signalling activity of Ras proteins.7
Inhibition of the corresponding palmitoylase reduced the growth of human tumour
cells.8
The importance of lipidated proteins motivated the development of many
tools and procedures for their study.9 Traditionally, palmitoylation has been studied
by radioactively-labelled palmitic acid analogues 1 and 2 (Figure 3).10
Figure 3. Radioactively-labelled palmitic acid analogues.
Although this method is very sensitive and the change in the molecular
properties are minimal, 1 and 2 are radioactive, which can be seen as a disadvantage.
To address the radioactivity issue, Dursina et al. 11 developed the fluorescently–
labelled prenyl analogue 3.11 The authors also applied 3 for the development of
farnesyl and geranylgeranyl transferase inhibitors.
Figure 4 . Fluorescently–labelled prenyl analogue.
The development of bio-orthogonal chemical reporters brought new
opportunities for the lipidation studies. Hang et al. 12 successfully applied 4 and 5
(Figure 5) in the studies of the dynamic S-palmitoylation and protein turnover in a
cell lysate.
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Figure 5. Bio-orthogonal probes used to study protein lipidation.
Logical progress in the field would be the utilization of a copper-free
cycloaddition to monitor the lipidation in the living cells. However, this tool is still
lacking. An unique example of application of the copper-free cycloaddition in a lipid
related study was reported by Schultz and Neef.13 The authors developed and applied
6 (Figure 6) to visualize membrane lipids in living cells.
Figure 6. An analogue of a diacylphosphoglycerol to visualize membrane lipids in living cells.
One of the reasons for the slow development of strain-promoted probes in
the lipid research is the general trend towards reactive and hydrophilic probes14 for
labelling of proteins, whereas for lipid studies hydrophobic probes are required.
Design of a lipophilic lipidation probe
The design of a suitable lipophilic lipidation probe can be based on the
recently reported15 bicyclononanes 7 or 8 (Figure 7).
Figure 7. Strained cyclooctynes.
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6
These are readily available (4 steps) and undergo a fast, strain promoted dipolar
cycloaddition. Even though cyclooctynes 9, 10 and 11 display higher rate of dipolar
cycloaddition, their synthesis is longer and their structure significantly deviates from
the hydrocarbon nature of fatty acids. The characteristics cyclooctynes 7-11 are
summarized in the following table (Table 1).
Table 1. Comparison of the rate and synthesis of cyclooctynes 7-11.
Entry Cyclooctyne number of steps Yield Ratea
(10-3 M-1s-1)
115 7 4 15% 110
215 8 4 35% 140
316 9 6 11% 240
417 10 9 41% 410
518 11 6 18% 960 arate constant determined in reaction with benzyl azide as a model compound.
Results and discussion
12 (Figure 8) can be a suitable lipophilic lipidation probe. The bicyclononyne
can be attached to a fatty acid by a Wittig reaction, thus giving 12 (Figure 8) as the
desired compound. Given that the strained triple bond is the most sensitive
functionality in 12 (Figure 8), this can be introduced in the final step using a double
Figure 8. Retrosynthesis of 12.
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HBr elimination. Dibrominated 13 can be prepared by a Wittig reaction and double
bond reduction sequence. Synthesis of functionalized ylide 14 from 15 was described
in a previous chapter (Chapter 2). The aldehyde 16 can be easily obtained from
bicyclononenol 17 via bromination and oxidation. And finally, 17 is available via
cyclopropanation of 18 and subsequent reduction.15
The synthesis towards 12 (Scheme 1) started with the cyclopropanation of 18
according to previously described conditions.15 Purification by chromatography
afforded 20 and 21 in 30% and 51% yield, respectively.
Reagents and conditions: a) ethyl diazoacetate (0.13 equiv) added over 12 h, Rh2(OAc)4 (2.0 mol%), CH2Cl2, 24 h, 21 °C; b) LiAlH4 (1.0 equiv), 0 °C, then 10 min at 21 °C, Et2O; c) pyridinium bromide perbromide (1.2 equiv), CH2Cl2, 1 h, 21 °C; d) TPAP (5.0 mol%), NMO (1.5 equiv), 3 Å molecular sieves, CH2Cl2, 1 h.
Scheme 1. Attempted synthesis of the lipidation probe.
The synthesis continued with reduction of the exo ester 21 with LiAlH4, yielding
alcohol 17 in quantitative yield. The subsequent bromination with Br2 according to
the described conditions15 resulted in decomposition of 21. A slight excess of
pyridinium bromide perbromide was an excellent alternative for Br2 affording the
desired 22 in 95% yield. Oxidation of 22 with TPAP/NMO afforded the aldehyde
16 in 65% yield. Despite a substantial effort, the planned Wittig reaction did not
afford the desired 23, but resulted in the decomposition of the starting material.
The unsuccessful Wittig reaction forced a new design of the lipidation probe,
in which the bicyclononyl moiety is bound to the linker via an ether bond (24 in
Figure 9). The strained triple bond is available (again) via a debromination of 25.
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Figure 9. Retrosynthetic analysis of a fatty acid probe with an ether linkage.
Acid 25 can be synthetized from alcohol 26 by oxidation, which in turn, can be
obtained by bromination and deprotection of 27. Finally, 27 is available from 17 and
28 via a Williamson ether synthesis.
The synthesis of the ether-linked clickable fatty acids was straightforward
(Scheme 2). The steps of the synthesis were carried out with both the endo and the
exo stereo-isomer (separately), but for clarity only the exo-isomer is discussed. The
yields for the endo stereoisomer are showed in brackets. Alkylation of 17 in basic
conditions afforded 30 in 65% yield (endo-30 in 48%) based on recovered 17. Despite
a substantial number of experiments, the competing elimination of 29 could not be
fully suppressed. Ether 30 was further converted into dibromide 26 in 2 steps. First,
the tetrahydropyranyl (THP) group was removed by refluxing in methanol in
presence of Amberlite (acidic form). Amberlite was removed by filtration, and the
intermediate alcohol was brominated using pyridinium bromide perbromide.
Dibromide 26 was isolated in 86% yield over 2 steps after flash chromatography
(endo-26 in 95% yield). Subsequent oxidation of 26 to acid 25 was carried out with
TPAP/NMO to the corresponding aldehyde followed by treatment with oxone in
DMF, affording 25 in 80% yield (endo-25 in 71%). Finally, 25 was debrominated by
refluxing in THF in the presence of excess KOtBu. The lipidation probe exo-24 was
obtained in 99% yield (endo-24 71%).
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Reagents and conditions: a) NaH (1.2 equiv) DMF 2 h, then 29 (1.2 equiv), 16 h, 0- 21 °C, b) Amberlite (100 mg/1 mmol), MeOH, 4 h, reflux c ) TPAP (5 mol%), NMO (2.0 equiv), CH2Cl2, 4 h, 0 °C then Oxone (3.0 equiv), DMF, 16 h, 21 °C; d) KOtBu (3.3 equiv), THF, 2 h, reflux.
Scheme 2. Synthesis of clickable fatty acid 24.
The reactivity of the clickable fatty acid 24 was explored in a dipolar
cycloaddition in an aqueous solution (Scheme 3). 24 underwent full conversion with
a stoichiometric amount of 31 in phosphate buffer (100 mM NaHPO4, pH 7.4) at 37
°C, in 20 min. Triazole 32 was isolated in 99% yield.
Reagents and conditions: a) 31 (1.0 equiv), sodium phosphate buffer (100 mM, pH = 7.4), 20 min, 37 °C.
Scheme 3. Strain promoted dipolar cycloaddition of 24.
Conclusions and outlook
A cyclooctyne based fatty acid is accessible in 6 steps from 1,5-cycloocta-
diene. The lipidation probe exo-24 undergoes smooth and fast dipolar cycloaddition
with an azide, resulting in a fluorescent adduct.
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Preliminary study showed, that 24 is metabolised by eukaryotic cells.
However, a further study is required in order to determine if 24 is incorporated to
the lipids or proteins.
Experimental part
Synthesis of bicyclononanols (scheme 1).
Exo- and endo- ethyl bicyclo[6.1.0]non-4-ene-9-carboxylate (20 and 21)
Prepared as a mixture according to Van Delft et al. with a modification in
the reagent addition time and the eluent for flash chromatography.
To the stirred mixture of freshly distilled cycloocta-1,5-diene (120 ml, 0.96 mol.
8.0 equiv) and Rh2(OAc)4 (106 mg, 0.2 mmol, 0.2 mol%) in CH2Cl2 (120 ml) ethyl
diazoacetate (15 ml, ca 0.12 mmol) was added dropwise over 12 h. After the addition
was complete, the mixture was stirred for 24 h at RT (21 °C). The crude reaction
mixture was filtered over a short silica pad. The resulting filtrate was fractionally
distilled to remove CH2Cl2 (800 mbar, 40 °C), and the unreacted 1,5-cyclooctadiene
(20 mbar, 70 °C) on a rotatory evaporator. The distillation residue was purified by
flash chromatography over silica gel using 2% diisopropyl ether in pentanes.
The reaction afforded 7.2 g (30%) of exo-isomer 11, 10.0 g (43%) of endo-isomer 12
and 2.2 g (10%) of a mixed fraction that was further separated by a second column
chromatography using the same eluent to afford 2 g of 12 (8%)
exo-ethyl bicyclo[6.1.0]non-4-ene-9-carboxylate (20)
1H NMR (400 MHz, CDCl3, δ): 5.62 (m, 2H), 4.08 (q, J = 7.1, 2H), 2.29 (m, 2H),
2.18 (m, 2H), 2.16 (m, 2H), 1.49 (m, 4H), 1.23 (t, J = 7.1, 3H), 1.17 (t, J = 4.5, 1H).
13C NMR (100 MHz, CDCl3, δ): 174.6, 130.1( - ), 60.4, 28.4, 28.11, 27.95, 26.87 ( - ),
14.52 ( - ).
endo-Ethyl bicycle[6.1.0]non-4-ene-9-carboxylate (21)
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1H NMR (400 MHz, CDCl3, δ): 5.60 (m, 2H), 4.10 (q, J = 7.1, 2H), 2.49 (m, 2H), 2.19 (m, 2H), 2.04 (m, 2H), 1.81 (m, 2H), 1.68 (m, 1H), 1.39 (m, 2H), 1.25 (t, J = 7.1, 3H).
13C NMR (100 MHz, CDCl3, δ): 172.47, 129.63 ( - ), 59.90, 27.27, 24.37,
22.85, 22.13 ( - ), 14.60 ( - ).
The NMR data are in agreement with previously reported.15
exo-bicyclo[6.1.0]non-4-en-9-ylmethanol (17)
20 (9.65 g, 50 mmol) was dissolved in Et2O (300 ml). The solution was
cooled on an ice bath, and LiAlH4 (920 mg, 50 mmol, 1.0 equiv) was added
in small portions (GAS EVOLUTION) over 10 min. After complete
addition, the cooling bath was removed, and the resulting grey suspension
was stirred for 15 min at RT (21 °C). The reaction was monitored by TLC. As soon
as the starting material was consumed, the reaction mixture was immersed again into
an ice bath. Water (1 ml) was added with great caution (GAS EVOLUTION). After
stirring for 10 min, aqueous NaOH (15%, 3 ml) was added. The grey suspension
turned into a white suspension to which water (1 ml) was added. The mixture was
stirred for 15 min and then filtered over a Celite pad.
After evaporation of the volatiles, 7.35 g (99 %) of alcohol 13 was obtained.
1H NMR (400 MHz, CDCl3, δ): 5.63 (m, 2H), 3.71 (d, J = 7.6, 2H), 2.36 (m, 2H),
2.10 (m, 2H), 1.98 (m, 2H), 1.58 (m, 2H), 1.31 (s, 1H), 1.13 (m, 1H), 1.00 (m, 2H).
13C NMR (100 MHz, CDCl3, δ): 130.22 ( - ), 60.73, 28.17, 24.38, 21.25 ( - ), 19.49
( - ).
Anal. Calcd for C10H16O: C, 78.90; H, 10.59. Found C, 78.70; H, 10.77.
endo-bicyclo[6.1.0]non-4-en-9-ylmethanol.
The same procedure as for the exo isomer was employed. Endo-12 (6.84 g,
35 mmol) was dissolved in Et2O (200 ml) and treated with LiAlH4 (650 mg, 35 mmol,
1.0 equiv).
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The reaction afforded 5.31 g (99%) of alcohol 14.
1H NMR (400 MHz, CDCl3, δ): 5.62 (m, 2H), 3.45 (d, J = 6.9, 2H), 2.26 (m, 2H),
2.15 (m, 2H), 2.5 (m, 2H), 1.40 (m, 3H), 0.76 (m, 2H), 0.64 (m, 1H).
13C NMR (100 MHz, CDCl3, δ): 130.65 ( - ), 67.69, 29.51, 29.34, 27.57 ( - ), 22.60 ( - ).
Anal. Calcd for C10H16O: C, 78.90; H, 10.59. Found: C, 78.60; H, 10.78.
Synthesis of lipid probe 24 (scheme 2)
2-((9-bromononyl)oxy)tetrahydro-2H-pyran (29)
In a 250 ml flask, 9-bromo nonanol (10.4 g, 46 mmol) was
dissolved in CH2Cl2 (46 ml). To this solution, 3.4-dihydro-2H-pyran (13 ml, 0.14 mol,
3 equiv) and pyridinium p-toluene sulphonate (1.17 g, 4.6 mmol, 10 mol%) were
added. The resulting suspension was immersed into a pre-heated oil bath (65 °C) and
refluxed until full conversion of the starting alcohol as monitored by TLC
(typically 3 h). The flask was removed from the bath and allowed to cool down. All
volatiles were evaporated using a rotatory evaporator. The resulting slurry was
dissolved in Et2O (150 ml), washed with water, brine, dried and evaporated. The
crude liquid residue was purified by column chromatography using 5% Et2O in
pentane.
The reaction afforded 14.0 g (>99%) of the desired product as a colorless liquid.
1H NMR (400 MHz, CDCl3, δ): 4.57 (m, 1H), 3.87 (m, 1H), 3.73 (m, 1H), 3.50
(m, 1H), 3.38 (m, 3H), 1.84 (m, 3H), 1.71 (m, 1H), 1.56 (m, 6H), 1.35 (m, 10H).
13C NMR (100 MHz, CDCl3, δ): 99.02 ( - ), 77.48, 77.16, 76.84, 67.80, 62.53, 34.20,
32.97, 30.94, 29.88, 29.51, 28.85, 28.30, 26.34, 25.66, 19.87.
NMR data correspond to those previously published.19
Endo-(Z)-2-((9-(bicyclo[6.1.0]non-4-en-9-ylmethoxy)nonyl)oxy)tetrahydro-2H-
pyran (30)
A dry Schlenk flask was charged with NaH (60% in oil,
672 mg, 17 mmol, 1.2 equiv). The mineral oil was
removed by washing with pentane (4 x 15 ml), and the resulting white powder was
Synthesis of a Cyclooctyne-based Lipidation Probe
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suspended in DMF (dry, 70 ml). This suspension was immersed to an ice bath
(ice/brine) and 17 (2.12 g, 14 mmol) was added dropwise (GAS EVOLUTION).
The mixture was removed from the ice bath and stirred at RT (21 °C) until the gas
evolution ceased (2 h). The mixture was again cooled in an ice bath, and 29 (5.14 g,
17 mmol, 1.2 equiv) was added. The mixture was allowed to slowly reach RT and
stirred overnight (16 h). The obtained solution was diluted with Et2O (75 ml),
transferred into a separatory funnel and washed with water, brine, dried and
evaporated. The resulting liquid was purified using the Reveleris® X2 Flash
Chromatography System with the following gradient: 10 column volumes 5% Et2O
in pentane followed by 3 column volumes of 60% Et2O in pentane.
The reaction afforded 2.38 g (yield based on 65% of recovered starting material) of
desired 30 as colourless liquid and 406 mg of the starting alcohol.
1H NMR (400 MHz, CDCl3, δ): 5.60 (m, 2H), 4.54 (m, 1H), 3.85 (m, 1H), 3.71 (m,
1H), 3.40 (m, 6H), 2.32 (m, 2H), 2.07 (m, 2H), 1.94 (m, 2H), 1.76 (m, 1H), 1.68 (m,
1H), 1.54 (m, 10H), 1.30 (m, 10H), 1.09 (m, 1H), 0,96 (m, 2H).
13C NMR (400 MHz, CDCl3, δ): 129.90 ( - ), 99.00 ( - ), 71.12, 68.04, 67.84, 62.51,
30.94, 29.96, 29.91, 29.71, 29.59, 27.90, 26.38, 26.35, 25.66, 24.06, 19.87, 18.87 ( - ),
18.12 ( - ).
IR (cm-1): 2927, 2853, 1462, 1352, 1106, 1078, 1032.
Anal. Calcd for C24H42O3: C, 76.14; H, 11.18. Found: C, 76.35; H, 11.37.
HRMS-APCI (m/z): [M + Na]+ calculated for C24H43NaO3, 401.303; found, 401.301.
Exo-(Z)-2-((9-(bicyclo[6.1.0]non-4-en-9-ylmethoxy)nonyl)oxy)tetrahydro-2H-pyran
The same procedure as for the synthesis of 30 was
employed, starting from endo-17 (1.23 g, 7.9 mmol).
The reaction afforded 963 mg (48% yield based on the recovered starting material)
of desired endo-17 as colorless liquid and 406 mg of the starting alcohol.
1H NMR (400 MHz, CDCl3, δ): 5.61 (m, 2H), 4.55 (m, 1H), 3.85 (m, 1H), 3.70 (m,
1H), 3.48 (m, 1H), 3.36 (m, 3H), 3.27 (d, J = 6.8, 2H), 2.27 (m, 2H), 2.15 (m, 2H),
Chapter 6
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6
2.02 (m, 2H), 1.81 (m, 1H), 1.70 (m, 1H), 1.53 (m, 8H), 1.32 (m, 12H), 0.73 (m, 2H),
0.55 (m, 1H).
13C NMR (100 MHz, CDCl3, δ): 130.18 ( - ), 98.83 ( - ), 74.88, 70.41, 67.68, 62.34,
30.78, 29.75, 29.74, 29.53, 29.44, 29.42, 28.96, 27.12, 26.22, 26.16, 25.89, 25.49, 22.31
( - ), 19.70 ( - ).
IR (cm-1): 3008, 2927, 2854, 1454, 1102, 1032.
HRMS-APCI (m/z): [M + Na]+ calculated for C24H43NaO3, 401.303; found, 401.301.
Anal. Calcd for C24H42O3: C, 76.14; H, 11.18. Found: C, 76.29; H, 11.36.
Endo- 9-((4,5-dibromobicyclo[6.1.0]nonan-9-yl)methoxy)nonan-1-ol.
30 (2.38 g, 6.3 mmol) was dissolved in MeOH (63
ml), and Amberlite IR 120 in its H+ form (630 mg,
100 mg/1 mmol of the starting material) was
added. The mixture was immersed into a pre-heated oil bath and refluxed for 4 h.
After full conversion (TLC), the reaction mixture was allowed to cool to RT. All
volatiles were evaporated, and the crude residue was suspended in Et2O (100 ml).
The precipitate was removed by filtration, and the filtrate was evaporated to dryness.
The crude residue was used without further purification in the subsequent reaction.
The crude product from the previous step was dissolved in CH2Cl2 (63 ml) and
pyridinium tribromide (3.02 g, 9.5 mmol, 1.5 equiv) was added in one portion. The
mixture was stirred until full conversion of the starting material (4 h). The conversion
was determined by 1H NMR of reaction mixture aliquots. After full conversion, the
reaction mixture was transferred into a separatory funnel, washed with water, brine,
dried and evaporated to dryness. The crude residue was purified by flash
chromatography over silica using 50% Et2O in pentanes.
The reaction afforded 2.47 g (86%) of the desired product as a colourless liquid.
1H NMR (400 MHz, CDCl3, δ): 4.81 (m, 2H), 3.41 (t, J = 6.7, 2H), 3.33 (d, J = 6.9,
2H), 2.63 (m, 2H), 2.34 (t, J = 7.5, 2H), 2.25 (m, 1H), 2.08 (m, 3H), 1.60 (m, 4H),
1.44 (m, 1H), 1.31 (m, 9H), 0.86 (m, 2H), 0.61 (m, 1H).
Synthesis of a Cyclooctyne-based Lipidation Probe
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13C NMR (100 MHz, CDCl3, δ): 179.31, 74.55, 70.70, 56.54 ( - ), 53.61 ( - ), 35.18,
35.08, 34.10, 29.87, 29.46, 29.39, 29.20, 26.31, 25.45( - ), 24.86, 24.56, 23.80, 22.93
( - ), 20.23 ( - ).
Anal. Calcd for C19H34Br2O2: C, 50.23; H, 7.53. Found: C, 50.49; H, 7.56.
endo-9-((4,5-dibromobicyclo[6.1.0]nonan-9-yl)methoxy)nonan-1-ol.
The same procedure as for the synthesis of 26 was
employed. Starting from endo-26 (963 mg, 2.6 mmol).
The reaction afforded 1.10 g (95%) of endo-26 as colourless liquid.
1H NMR (400 MHz, CDCl3, δ): 4.82 (m, 2H), 3.64 (t, J = 6.6, 2H), 3.50 (d, J = 7.2,
2H), 3.42 (t, J = 6.7, 2H), 2.67 (m, 2H), 2.21 (m, 2H), 1.91 (m, 2H), 1.57 (m, 5H),
1.45 (m, 2H), 1.32 (m, 10H), 1.19 (m, 2H), 1.07 (m, 1H).
13C NMR (100 MHz, CDCl3, δ): 71.10, 67.37, 63.32, 56.70 ( - ), 53.67( - ), 35.23,
33.02, 30.03, 29.78, 29.63, 29.60, 26.42, 25.95, 20.21, 20.07( - ), 19.41( - ), 19.28,
17.11( - ).
Anal. Calcd for C19H34Br2O2: C, 50.23; H, 7.53. Found: C, 50.52; H, 7.69.
Exo-9-((4,5-dibromobicyclo[6.1.0]nonan-9-yl)methoxy)nonanoic acid (25)
Dibromide 26 (2.03 g, 4.4 mmol) was dissolved in
CH2Cl2 (44 ml). This solution was cooled to 0 °C (brine/ice) and stirred for 5 min.
Then TPAP (77.3 mg, 0.2 mmol, 5.0 mol%) followed by NMO (1.03 g, 8.8 mmol,
2 equiv) were added. The mixture was stirred for 4 h. After full conversion of the
starting material, all volatiles were evaporated, and the crude black residue was used
in the next step.
The crude product from the previous step was dissolved in DMF (44 ml). Oxone (4
g, 13.2 mmol, 3.0 equiv) was added, and the mixture was stirred overnight (16 h) at
RT (21 °C). The reaction mixture was diluted with Et2O (200 ml), transferred into a
separatory funnel and washed with water and brine. The organic layer was dried and
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6
evaporated. The crude residue was purified by a Reveleris® X2 Flash
Chromatography System with the following gradient: from 20% Et2O to 60% Et2O.
The reaction afforded 1.64 g of the desired product (80% over 2 steps) as colourless
thick liquid.
1H NMR (400 MHz, CDCl3, δ): 4.82 (dd, J = 12.5, 7.0 Hz, 2H), 3.41 (t, J = 6.8 Hz,
2H), 3.32 (d, J = 6.9 Hz, 2H), 2.64 (m, 2H), 2.34 (t, J = 7.5 Hz, 1H), 2.23 (m, 1H),
2.09 (dd, J = 15.8, 9.9 Hz, 3H), 1.59 (m, 4H), 1.37 (m, 10H), 0.85 (ddd, J = 20.4,
16.1, 7.8 Hz, 2 H), 0.61 (m, 1H).
13C NMR (100 MHz, CDCl3, δ): 179.69, 74.45, 70.61, 56.47 ( - ), 53.53 ( - ), 35.10,
35.00, 34.13, 29.81, 29.40, 29.33, 29.13, 26.24, 25.38 ( - ), 24.80, 24.47, 23.72, 22.83
( - ), 20.12 ( - ).
Anal. Calcd for C19H32Br2O3: C, 48.73; H, 6.89. Found: C, 48.90; H, 6.98.
Endo-9-((4,5-dibromobicyclo[6.1.0]nonan-9-yl)methoxy)nonanoic acid
The same procedure as for the synthesis of 25 was
employed, starting from endo-26 (1.32 g, 2.9 mmol).
The reaction afforded 970 mg (71%) of endo-25 as a colourless thick liquid.
1H NMR (400 MHz, CDCl3, δ): δ 4.88 – 4.77 (m, 2H), 3.50 (d, J = 7.1 Hz, 2H), 3.41
(t, J = 6.7 Hz, 2H), 2.77 – 2.57 (m, 2H), 2.34 (t, J = 7.5 Hz, 2H), 2.31 – 2.20 (m, 1H),
2.14 (dt, J = 15.3, 5.1 Hz, 1H), 1.98 – 1.83 (m, 2H), 1.69 – 1.45 (m, 6H), 1.31 (s, 8H),
1.23 – 1.01 (m, 3H).
13C NMR (100 MHz, CDCl3, δ): 179.63, 70.82, 67.15, 56.44, 53.41, 35.01 ( - ), 34.97
( - ), 33.98, 29.72, 29.24, 29.19, 28.99, 26.11, 24.65, 19.95, 19.83, 19.13, 19.02 ( - ),
16.87 ( - ).
Anal. Calcd for C19H32Br2O3: C, 48.73; H, 6.89. Found: C, 49.04; H, 6.97.
Exo-9-(bicyclo[6.1.0]non-4-yn-9-ylmethoxy)nonanoic acid (24)
Synthesis of a Cyclooctyne-based Lipidation Probe
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25 (960 mg, 2.0 mmol) was dissolved in THF. The
solution was cooled in an ice bath, and KOtBu (Sigma-Aldrich 1 M solution, 0.75 ml,
6.6 mmol, 3.3 equiv) was added. After complete addition, the reaction mixture was
immersed in a pre-heated oil bath, and the reaction was refluxed for 2 h. After
cooling to RT, the reaction mixture was quenched with saturated aqueous NH4Cl
solution. The mixture was transferred to a separatory funnel and extracted with
CH2Cl2. The combined organic layers were washed with brine, dried and evaporated.
The reaction afforded 630 mg of 24 (99%).
24 could be further chromatographed on silica using a Reveleris® X2 Flash
Chromatography System with following gradient: from 20% Et2O to 60% Et2O.
The chromatography usually resulted in a decreased yield (ca 50%).
1H NMR (400 MHz, CDCl3, δ): 3.47 (d, J = 7.7, 2H), 3.40 (t, J = 6.7, 2H), 2.26 (m,
6H), 1.59 (m, 6H), 1.31 (m, 10H), 0.88 (m, 3H).
13C NMR (100 MHz, CDCl3, δ): 179,44, 99.18, 71.10, 67.76, 34.13, 29.93, 29.45,
29.40, 29.37, 29.21, 26.35, 24.87, 21.72, 20.04 ( - ), 18.89( - ).
HRMS-ESI (m/z): [M + H]+ calculated for C19H31O3, 307.226; found, 307.225.
Endo-9-(bicyclo[6.1.0]non-4-yn-9-ylmethoxy)nonanoic acid
The same procedure as for the synthesis of 24 was
employed, starting from endo-25 (870 mg, 2.6 mmol).
The reaction afforded 570 mg (99 %) of desired endo-22 as a colourless liquid.
1H NMR (400 MHz, CDCl3, δ): 3.42 (t, J = 6.7, 2H), 3.36 (d, J = 6.4, 2H), 2.28 (m,
6H), 1.60 (m, 4H), 1.33 (m, 12H), 0.63 (m, 3H).
13C NMR (100 MHz, CDCl3, δ): 179.44, 99.12, 75.01, 70.71, 34.08, 33.61, 29.94,
29.48, 29.40, 29.21, 26.34, 24.89, 24.67( - ), 22.98( - ), 21.74.
Chapter 6
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6
HRMS-ESI (m/z): [M + H]+ calculated for C19H31O3, 307.226; found, 307.225.
Strain promoted dipolar cycloadition (scheme 3)
Adduct 32
Round-bottom flask containing sodium
phosphate buffer of pH = 7.4 (100 mM, 5.0 ml) was warmed to 37 °C. In this
solution, 24 (16 mg, 50 μmol) was dissolved. To this solution, a solution of 3-azido-
7-(diethylamino)-2H-chromen-2-one (12.9 mg, 1 equiv) in DMSO (100 μl) was
added. The progress of the reaction was monitored by TLC. After 20 min, full
conversion was reached. Water was evaporated and the crude residue was purified
by flash chromatography using 1% MeOH in CH2Cl2.
The reaction afforded 28 mg of 32 (99%) as orange viscous liquid.
1H NMR (400 MHz, CDCl3, δ): δ 7.75 (s, 1H), 7.28 (s, 1H), 6.45 (d, J = 2.3 Hz, 1H),
6.41 (d, J = 2.5 Hz, 1H), 3.30 (m, 8H), 2.74 (m, 4H), 2.18 (t, J = 7.6 Hz, 2H), 1.20
(m, 22H), 0.66 (m, 3H).
13C NMR (100 MHz, CDCl3, δ): 176.01, 164.13, 158.10, 156.78, 141.89, 128.93,
128.13, 125.20, 116.05, 109.76, 97.17, 79.03, 74.02, 70.39, 44.94, 44.70, 37.72, 34.09,
32.37, 29.60, 29.19, 29.18, 29.16, 29.02, 24.84, 12.35, 12.33.
HRMS-ESI (m/z): [M + H]+ calculated for C32H44N4O5, 556.338; found, 556.335.
References
(1) (a) Ferguson, M. A. J. J. Cell Sci. 1999, 112, 2799(b) Paulick, M. G.; Bertozzi, C. R.
Biochemistry 2008, 47, 6991(c) Low, M. G. Biochim. Biophys. Acta 1989, 988, 427(d)
Ikezawa, H. Biol. and Pharm. Bull. 2002, 25, 409.
(2) Resh, M. D. Trends in Molecular Medicine 2012, 18, 206.
(3) Frearson, J. A.; Brand, S.; McElroy, S. P.; Cleghorn, L. A. T.; Smid, O.; Stojanovski,
L.; Price, H. P.; Guther, M. L. S.; Torrie, L. S.; Robinson, D. A.; Hallyburton, I.;
Mpamhanga, C. P.; Brannigan, J. A.; Wilkinson, A. J.; Hodgkinson, M.; Hui, R.; Qiu,
W.; Raimi, O. G.; van Aalten, D. M. F.; Brenk, R.; Gilbert, I. H.; Read, K. D.;
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Fairlamb, A. H.; Ferguson, M. A. J.; Smith, D. F.; Wyatt, P. G. Nature 2010, 464,
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Erdos, M. R.; Robbins, C. M.; Moses, T. Y.; Berglund, P.; Dutra, A.; Pak, E.; Durkin,
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Kuhlmann, J.; Brunsveld, L.; Chandra, A.; Ellinger, B.; Waldmann, H.; Bastiaens, P.
I. H. Cell 2010, 141, 458.
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J. D.; Smith, C. D. Mol. Cancer Ther. 2006, 5, 1647.
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A.; Kalinin, A.; Evstifeev, V.; Ciobanu, D.; Szedlacsek, S. E.; Waldmann, H.; Goody,
R. S.; Alexandrov, K. J. Am. Chem. Soc. 2006, 128, 2822.
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Acad. Sci 2010, 107, 8627.
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(14) Debets, M. F.; Prins, J. S.; Merkx, D.; van Berkel, S. S.; van Delft, F. L.; van Hest, J.
C. M.; Rutjes, F. P. J. T. Org. Biomol. Chem. 2014, 12, 5031.
(15) Dommerholt, J.; Temming, R.; Hendriks, L. J. A.; Rutjes, F. P. J. T.; Van Hest, J. C.
M.; Van Delft, F. L.; Schmidt, S.; Friedl, P.; Lefeber, D. J. Angew. Chem., Int. Ed.
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van Delft, F. L. Chem. Commun. 2010, 46, 97.
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Chapter 7 A Missing Link in Archaeal Lipid Biosynthesis; a Contribution from Organic Synthesis
Abstract: The Archaea form a separate domain of Life known to occupy extreme
ecological niches. Their extremophilicity is often associated with their characteristic
membrane lipids, which require different biosynthetic pathways than eukaryotes and
bacteria. Given that the in vivo identification of the involved enzymes is ambiguous,
their function needs to be confirmed by in vitro experiments. However, development
of the detection methods, assay conditions and isolation of the substrates are
challenges on their own. At least with the substrates, organic synthesis can help. This
chapter describes a chemical synthesis of an intermediate in the biosynthesis of
archaeal lipids. This intermediate was essential for an in vitro assay, which revealed
one of the missing links in biosynthesis of archaeal lipids. A second contribution of
this chapter is a total synthesis of cycloarcheol and its ß-glucosyl analogue, which are
important taxonomic tools in Archaea.
Parts of this chapter have been published.
Jain, S.; Caforio, A.; Fodran, P.; Lolkema, J. S.; Minnaard, A. J.; Driessen, A. J. M.
Chemistry & Biology, 2014, 21, 1392.
Ferrer, C.; Fodran, P.; Barroso, S.; Gibson, R.; Hopmans, E. C.; Sinninghe Damsté,
J.; Schouten, S.; Minnaard, A. J. Org. Biomol. Chem. 2013, 11, 2482.
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Introduction
Archaea form the third domain of life, comprising up to 20% of the biomass
on Earth. Since 1977, when the domain Archaea was first described,1 their
evolutionary origin has been a topic of intense debate. Recently, Forterre2 presented
a hypothesis in which the Archaea and the Eukarya evolved from a common
ancestor. He further hypothesized, that the ancestors of the Archaea escaped from
their proto-eukaryotic predators by invading ecological niches with harsh
environmental conditions. Surroundings like hydrothermal vents,3 geysers3-4 with
temperatures over 120 °C, highly acidic (pH = 0)5 or alkaline (pH >10)6 springs, or
lakes with salt7 concentrations 10 times higher than sea water required a lot of
adaptation. One of the features that distinguishes Archaea from Bacteria and
Eukaryota is their cell envelope. This lacks a general cell wall polymer, and contains
membrane phospholipids, which differ from bacterial and eukaryotic lipids in three
aspects (Figure 1-I). First, in Archaea the hydrophobic part comprises of two
terpenoid (mostly phytanyl) chains, while this part is composed of two fatty acid
residues in bacterial and eukaryotic lipids.
Figure 1. ( I ) Comparison of bacterial and eukaryotic lipids with archaeal lipids;
( II ) common archaeal lipid backbones.
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Second, in Archaea the phytanyl chains are linked to the glycerol via an ether bond,
while in Bacteria and Eukarya the fatty acids bind via an ester bond. Third, the
stereogenic centre in the glycerol moiety of archaeal phospholipids has the opposite
configuration compared to that in bacterial and eukaryotic lipids. Furthermore,
Archaea display a greater variability of the lipid backbones compared to Bacteria and
Eukarya. Structures like cycloarcheol (1) (Figure 1-II) and caldarcheol (2) are
common. Despite substantial investigations on archaeal membrane lipid
biosynthesis, several steps and their corresponding enzymes remain unknown.
The extremophilic nature of Archaea is often associated with their unique
lipids. Driessen et al.8 studied the stability of liposomes from lipid extracts of
Escherichia coli (mesophilic Bacteria), Bacillus stearothermophilus (thermophilic Bacteria)
and Sulfolobus acidocaldarius (thermophilic Archaea). The Archaea-derived liposomes
(archeosomes) showed significantly higher stability at all studied temperatures. In the
same study, the authors also reported that while the bacterial liposomes gradually
released about 50% of their content (fluorescent dye) over 62 days, the archeosomes
showed only 8-10% release over the same period of time. The higher stability of
archeosomes already found application in bioelectronics9, gene delivery10 and
vaccination.11 A bottleneck for their wider application is their limited availability.
Growing Archaea is technically more difficult than growing Bacteria or eukaryotic
cells. Furthermore, the yields of lipids are low. Typically, 1 g of lyophilized archaeal
cells affords only 0.11-54 mg of crude lipid extracts.12
Biosynthesis of archaeal membrane lipids
Complete genome sequencing13 of Archaeoglobus fulgidus allowed a better
understanding of the lipid metabolism in Archaea. Archaea species have a complete
set of genes encoding fatty acid metabolism,14 similar to the bacterial and eukaryotic
metabolism. Nevertheless, the fatty acids in Archaea are not used for the synthesis
of membrane lipids, but for posttranslational modification of proteins. As mentioned
above, the lipophilic portion of archaeal membrane lipids is exclusively terpenoid-
based. These terpenes are biosynthesized15 in a pathway that is similar to the bacterial
and eukaryotic pathways (Figure 2). Diphosphate 3 (coming from the mevalonic acid
pathway) is isomerised to diphosphate 4. A three- or fourfold extension of 4 affords
geranylgeranyl diphosphate (5) or farnesylgeranyl diphosphate (6), which
subsequently enters the lipid biosynthetic pathway.
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Figure 2. Biosynthesis of terpenes in Archaea.
The biosynthesis of the phospholipids starts with glycerol monophosphate 8
(Figure 3), which is obtained by the reduction of 7 (a product of glycolysis). Given
that the absolute configuration of glycerol phosphate (8) is opposite in Bacteria and
Eukarya, the corresponding reductase was considered unique for Archaea. Babinger
et al.16 recently showed that Bacillus subtilis produces a homologous enzyme. The
biosynthesis of the phospholipids continues with the attachment of the terpenoid
(here geranylgeranyl) chains to 8 (Figure 3). First, the primary hydroxyl group is
prenylated in the cytosol. The significantly more lipophilic 9 is transferred to the cell
membrane where the second prenylation takes place. Both prenylations proceed via
a same SN1 type mechanism. In the active site of the enzyme, the diphosphate of 5
is cleaved with the assistance of Mg2+. Subsequently, the resulting allylic carbocation
reacts with a nucleophilic hydroxyl group of the glycerol. With the bis-prenylated
glycerol 10, the biosynthesis of the archaeal phospholipids continues by attachment
of the polar head group, which is achieved in two steps (Figure 3). The first step is
an activation of 10 with cytidine triphosphate (CTP). Although an analogous step
takes place in all three domains of life, the corresponding archaeal enzyme has been
elusive until now. The identification of this enzyme is the topic of the first part of
this chapter. The second step of the attachment of the phosphorous headgroup is
the conversion of 11 to the final phospholipid 12, 13 or 14. With the headgroup
attached, 12, 13 or 14 need to undergo 1 or 2 more transformations, depending on
the species. First, the reduction of the double bonds – a transformation that is
common in all Archaea species. The reduction was studied by Nishimira and Eguchi,17
who purified and characterized the corresponding enzyme in Thermoplasma
acidophilum. This enzyme did not show any preference for a headgroup –
phospholipids 12, 13 and 14 were reduced in a similar rate.
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Figure 3. Biosynthesis of phospholipids in Archaea.
While for some of the Archaea, the biosynthesis ends with the reduction of the
prenyl chains (from 15 to 16 in Figure 4-I), some other members further modify the
chain by dimerization (17 in Figure 4-I), cyclization (18 in Figure 4-I), or dimerization
followed by cyclization (19 in Figure 4-I). The exact mechanism of these
transformations remains unknown. A study by Eguchi et al.18 suggests that the
terminal double bonds are crucial for the dimerization. These findings are however
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in contrast to the findings of Nemoto et al.,19 who reported that the dimerization
takes place at fully saturated precursors. Fitz and Arigoni20 studied an analogous
dimerization in Butyrvibrio fibrisolvens (a genus of Bacteria), which produces a
membrane spanning diabolic acid (21) (Figure 4-II).
Figure 4. ( I ) Final steps in the biosynthetic pathway of membrane-spanning Archaea-
lipids; ( II ) dimerization of palmitic acid in Butyrivibrio fibrisolvens.
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The authors showed that diabolic acid is synthetized by dimerization of a fully
saturated 20.
Archaeal lipids as taxanomic markes
The second part of this chapter is dedicated to the total synthesis of the
cycloarcheol lipid core 1 and its glycolipid analogue 22. The lipids in Archaea fulfil
also an important taxonomic function which highlights the necessity of their
unambiguous structural determination. A total synthesis of the lipid is one of the
options. Further comparison of the HPLC chromatograms and mass spectra of the
synthetic and natural samples gives a high confidence in determining its presence. A
total synthesis of cycloarcheol 1 (Figure 1) and its ß-glucosyl analogue 22 (Figure 5)
is interesting in this context. Cycloarcheol was detected for the first time in 198321 in
a deep sea hydrothermal vent.
Figure 5. ß-glucosyl analogue of 1.
Results and discussion
Synthesis of 2,3-bis-O-(geranylgeranyl)-sn-glycero-1-phosphate
Retrosynthetic analysis 10 (Figure 6) suggests its preparation by
phosphorylation of 2,3-bis-O-geranylgeranyl-sn-glycerol (23). 23 can be synthetized
from a suitably protected glycerol derivative 24 and geranylgeranyl halide 25 via a
Williamson ether synthesis. Another alternative would be ring-opening of protected
enantiopure glycidol 26 (more details in chapter 3) with geranylgeraniol (27),
followed by etherification of the formed secondary alcohol. Given that allyl halides
are excellent partners in Williamson’s reaction, this is the method of choice for the
construction of the unsaturated derivatives. This strategy was already recognized by
Morii, Nishihara and Koga.22In their synthesis, the authors used geranylgeranyl
bromide and enantiopure benzylglycerol. The benzyl group was subsequently
removed by Na/NH3 (liq). Phosphorylation with dimethyl chlorophosphate in basic
conditions and subsequent demethylation with TMSBr afforded 10 in <6% overall
yield. The authors explained their low overall yield by instability of the intermediate
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compounds. In an unrelated publication, Dannenmuller et al.23 reported the synthesis
and properties of archaeal membrane phospholipids analogues of 10.
Figure 6. Retrosynthetic analysis of 10.
The authors prepared bisgeranylgeranyl glycerol 23 via Wiliamson reaction of
geranylgeranyl chloride and dimethoxybenzyl protected glycerol. Application of this
protecting group is advantageous compared to the benzyl group because it can be
removed using mild oxidative conditions. The reported conditions were applied to
the synthesis with some minor modifications.
The glycerol derivative 29 was prepared in 2 steps from commercially
available (R)-solketal.24 The geranylgeranyl chloride as etherification partner was
prepared by treatment of geranylgeraniol with N-chlorosuccinimide and Me2S.25
Reaction of both 29 and geranylgeranyl chloride (Scheme 1) in the presence of dimsyl
sodium (sodium methylsulfinylmethylide) in DMSO afforded the desired diallylated
30 in 61% yield, together with monoallylated 31 in 16% yield. The yield of 30 is in
perfect agreement to that reported by Dannenmuller et al.23 (60%). Deprotection of
30 (Scheme 1) with DDQ in CH2Cl2/H2O (40/1) afforded 23 in 60% isolated yield,
again in a very good agreement with the literature (60%).23
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Reagents and conditions: a) NaDMSO (2.1 equiv), DMSO, then geranylgeranyl chloride (2.0 equiv.), RT, 16 h; b) DDQ (2.0 equiv), CH2Cl2, H2O, 0 °C
Scheme 1. Synthesis of bisgeranylgeranyl glycerol 13.
The phosphorylation (Scheme 2) of 23 turned out to be a challenging step.
Phosphorylation of 23 with POCl3 (Scheme 2) and subsequent hydrolysis in the
presence of AgNO326 resulted only in decomposition of the starting material. The
procedure reported by Morii, Nishihara and Koga22 (Scheme 2) afforded
dimethylphosphate 32, but all attempted demethylations resulted in its
decomposition. Phosphoramidites could be another viable option. First explored
reagent 33 (scheme 2) underwent phosphoramidite coupling with 23 in the presence
of tetrazole, and subsequent in situ oxidation with a solution of tBuOOH in decane
afforded bisprotected 34 in 54% yield. However, all the explored deprotection
methods of 34 resulted only in monodeprotected 35. Next, phosphoramidite 36 was
explored. The pKa value of the corresponding phosphate suggests a greater base-
lability.27 Synthesis of 36 was straightforward, but all attempts to purify the reagent
resulted in its decomposition. Finally, a reaction of 23 with an excess of crude 36 in
the presence of tetrazole and subsequent oxidation with tBuOOH, afforded
bisprotected 37 in 94% yield. Reaction of 37 with excess Et3N resulted again in
monodeprotected 38. Treatment of 38 with aqueous NaOH (1 M) resulted in the
removal of the second fluorenylmethyl group, affording 10 in 48% yield. After
further optimization, both protecting groups could be cleaved in 1 reaction. Stirring
37 in a 1 M aqueous NaOH in dioxane mixture followed by acidification and column
chromatography on 130 Å Davisil silica gel afforded 10 in 68% yield. Overall, 10 was
prepared in 4 steps and 23% overall yield, starting from 29.
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Reagents and conditions: a) OP(OMe)2Cl, Et3N CH2Cl2, 21 °C, 2h; b) 33, tetrazole 4h, 21 °C then tBuOOH (2.0 equiv), -10 °C, 15 min; c) 36 (3.0 equiv), tetrazole (3.0 equiv), 24 h, 21 °C, then tBuOOH (4.0 equiv) -10 °C, 1 h; d) Et3N (20 equiv), 21 °C, 18 h; e) 1 M aqueous NaOH; f) dioxane/1 M aqueous NaOH.
Scheme 2. Explored phosphorylations methods of 23.
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Identification of CDP-archaeol synthase
This paragraph summarizes the experiments of Dr. Samta Jain, and Dr.
Antonella Caforio, from the department of Molecular Microbiology of the
Rijksuniversiteit Groningen.
Based on the analogy between the biosynthesis of CDP-activated precursor
11 (Figure 3) in Archaea and Bacteria, bioinformatic analysis could identify a putative
CDP-archaeol synthase in Archaea. The sequence of bacterial phosphatidate
cytidylyltransferase28 (CDP- diacylglycerol synthase) served as input for an NCBI-
BLAST analysis. This resulted in a list of hypothetical proteins. Their sequences were
aligned to an averaged hydropathy (hydrophobicity) profile. The alignment revealed
common structural features of the hypothetical proteins – an extracellular N-
terminus and 5 transmembrane helices. Although the bacterial enzymes are longer
than the archaeal ones, the alignment of the family averaged hydropathy profile of
the two showed a common pattern at the C-terminal region. Furthermore, analysis
of the sequence of one of the protein loops revealed a consensus sequence between
the archaeal and bacterial enzyme. This putative enzyme could be the CDP-archaeol
synthase. The corresponding amino acid sequence was codon optimized for
expression in E. coli and the C-terminus of the protein was equipped with an
octahistidin tag. The enzyme was isolated after affinity chromatography. The
predicted function of the enzyme was confirmed in two assays. Synthetic 10, the
natural substrate for the enzyme, was incubated in the presence of Mg2+ salts and
cytidine triphosphate. LC/MS analysis of the reaction mixture confirmed the
presence of CDP-archaeol 11. When 10 was incubated with 2′-deoxycytidine 5′-
triphosphate under identical conditions, LC/MS analysis confirmed the presence of
deoxy-CDP-archaeol. Application of the other nucleosides did not lead to the
corresponding products. In the second assay, 10 was incubated with a radiolabelled
cytidine triphosphate ([5-T]CTP) under the same conditions. TLC analysis of the
reaction mixture showed only a single radioactive spot.
With the identified enzyme CDP-archaeol synthase, the archaeal lipid
biosynthesis could be reconstituted in vitro (Figure 7). After combining isopentenyl
diphosphate (3), dihydroxyacetone phosphate (7), farnesyl diphosphate 38, five
enzymes catalyzing the steps of the biosynthesis and NADH, LC/MS analysis
confirmed the presence of 11.
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Figure 7. In vitro reconstitution of the biosynthesis of 11.
Catalytic alcoholysis of benzylglycidol as a key step in the synthesis of cyclo-
archaeol and -glucosyl-cyclo-archaeol
The following part of the chapter summarizes research performed together
with Dr. Catalina Ferrer and Dr. Santiago Barroso.
Although bisgeranylgeranyl glycerol 10 and cycloarchaeol 1 (as their
corresponding phosphates) are part of the same biosynthetic route, the synthetic
challenges in 1 are considerably larger. Enzymatic reduction of the double bonds
introduces 8 new stereogenic centers, making the synthesis of the hydrocarbon chain
a challenge. A second, frequently underestimated hurdle is the construction of the
ether bonds. While in the case of reactive, unsaturated allylic derivatives (as in the
case of 10) the Wiliamson synthesis is straightforward, in the case of the saturated
alkylsulphonates or alkyl iodides, the competing elimination is a problem frequently
resulting in low yields of the etherification. At least a partial solution can be
alcoholysis of an enantiopure glycidyl ether catalysed by Jacobsen’s catalyst.
A two-fold conjugate addition (Scheme 3) on cyclo-octadienone (39),
followed by ozonolysis and esterification, afforded hydroxyl ester 40 with two
methyl-branched stereogenic centres. One portion of 40 was converted in 3 steps to
protected tetrazole 41, the second portion of ester 40 was oxidized to aldehyde 42.
41 and 42 were coupled in a Julia-Kocienski reaction. Hydrogenation using in situ
generated diimide using the aforementioned flavine catalyst, afforded alcohol 43. A
part of 43 was converted to iodide 44. The first ether bond was constructed (Scheme
4) by alcoholysis of (R)-benzylglycidol 45 with alcohol 43 using 8.0 mol% of the
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Co[R,R-(salen)]OTs, affording the desired ring-opened product 46 in 87% yield. The
second ether bond was constructed via Williamson reaction. After testing a series of
reaction conditions, best results were obtained when a mixture of 46 and the iodide
44 were treated with freshly ground KOH and catalytic nBu4NBr under solvent-free
conditions. In a slow reaction, this provided the desired product 47 with yields
varying from 35% to 55%. These values are in good agreement to the literature.29.
The contrast between the two applied etherification methods is noteworthy. While
the epoxide alcoholysis is a clean reaction with 87% yield, Wiliamson reaction affords
the ether in significantly lower 35 to 55% yield, together with side products comming
from the elimination.
Reagents and conditions: a) Me2Zn (3.0 equiv), Cu(OTf)2 (5.0 mol%), L-Phos (10 mol%), 39 added over 6 h, toluene, -25 °C, overnight; b) Me2Zn (1.5 equiv), Cu(OTf)2 (2.5 mol%), L-Phos (5.0 mol%), substrate added over 6 h, toluene, -25 °C, overnight then Et3N (3.5 equiv), TMSCl (5.0 equiv), 2 h c) crude TMS enol ether dissolved in MeOH, CH2Cl2, O3, -78 °C, then NaBH4; d) p-toluenesulfonic acid (5.0 mol%), MeOH, reflux, 24 h; e; TBDPSCl (1.6 equiv), 1H-imidazole (2.0 equiv), DMF, rt, 16 h; f) DIBAL (5.0 equiv), THF, -78 °C, 2 h; g) 1-phenyl-1H-tetrazole-5-thiol (2.0 equiv), PPh3 (1.5 equiv), DIAD (1.8 equiv), rt, overnight, then mCPBA (5.0 equiv), rt, overnight; h) TPAP (5.0 mol%), NMO (1.5 equiv), CH2Cl2, rt, overnight; i) LiHMDS (1.0 equiv), 41 (1.0 equiv), then 42 added, THF, -78 °C to rt, overnight; j) DIBAL (5.0 equiv), THF, -78 °C, 2 h; k) NH2NH2.H2O (20 equiv) added over 10 h, L-flav (2.0 equiv), EtOH, rt, 2 h; l) N,N,-dimethyl-N-(methansulfanylmethylene)ammonium iodide (1.5 equiv), 1H-imidazole (0.5 equiv), toluene, 85 °C, 16 h.
Scheme 3. Synthesis of methyl-branched precursors 43 and 44.
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However, the amounts of the building blocks were sufficient to complete the
synthesis of 1 (Scheme 4). The deprotection, oxidation and a Wittig reaction
sequence afforded bis-alkene 48, which was cyclized by ring closing metathesis. The
resulting double bond was reduced by hydrogenation over Pt/C catalyst because the
flavin generated diimide did not result in a full conversion.
Reagents and conditions: a) 43 (0.55 equiv), Co[R,R-(salen)]OTs (4.5 mol%), O2 (ballon), rt, 16 h, b) 44 (1.1 equiv), nBu4Br (0.5 equiv), KOH (2.7 equiv), 42 °C, 48 h; c) TBAF (4.0 equiv), THF, rt, overnight, d) Dess-Martin periodinane (2.5 equiv), CH2Cl2, rt, 1 h; e) Me3PPh3Br (4.5 equiv), KHMDS (4.2 equiv), THF, rt, 1 h; f) 2nd Grubbs catalyst (15 mol%), CH2Cl2 (0.002 M), reflux, 48 h; g) Pt/C (20 mol%), MeOH/CH2Cl2 (2/1), H2 (ballon), rt, 16 h; Pd/C (Degussa type E101 NE/W, 25 mol%), H2 (ballon), EtOAc, rt, 16h; i) 49 (3.5 equiv), AgOTf (3.5 equiv), tetramethylurea (4.5 equiv), toluene/CH2Cl2 (1/1), 0 °C; j NaOMe (30 equiv), MeOH, rt.
Scheme 4. Final steps of synthesis of 1 and 22.
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Final debenzylation afforded cycloarcheol 1. The Koenigs–Knorr glycosylation
followed by deprotection of the hydroxyl groups afforded the desired ß-glucosyl
derivative 22.
Detection of 1 and 22 in the deep sea samples
Both compounds were used to confirm their presence in hydrothermal vents.
The analyzed sample was collected from the Rainbow hydrothermal vent (36°14′N)
field located on the Mid-Atlantic Ridge. Samples were collected during a sampling
campaign in 2008 using the remotely operated vehicle Jason. The sample is
composed of material from the interior of a vent chimney collected at a depth of
2293 meters below the sea level. Analysis by GC-MS (in the case of 1) and
HPLC/ESI/MS (in the case of 22) showed that synthetic and natural compounds
co-eluted and that their mass spectra were identical. This suggest the presence of
methanogenic Archaea in the Rainbow.
Conclusion
The chemical synthesis of unsaturated archaeatidic acid has been important
in the identification of CDP-archaeol synthase, one of the missing links in the
biosynthesis of archaeal membrane lipids. The synthetically challenging step was the
phosphorylation of bisgeranylgeranyl-glycerol. This was achieved by the application
of bisfluorenylmethyl substituted phosphoramidite, in situ oxidation, and subsequent
deprotection under basic conditions.
In the second part of this chapter, a key step in the synthesis of cyclo-archaeol
is described. As in chapter 3, the catalytic regioselective ring opening of a protected
glycidol is successfully applied as an alternative for a Williamson ether synthesis with
a glycerol derivative. A versatile method for the subsequent alkylation of the
secondary hydroxyl group is still lacking, but the currently applied procedure is
acceptable. The synthesis of cycloarchaeol and ß-glucosyl cycloarchaeol allowed to
unambiguously establish their presence in a sample taken from a hydrothermal vent
field.
Experimental part
(S)-4-(((3,4-dimethoxybenzyl)oxy)methyl)-2,2-dimethyl-1,3-dioxolane
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A dry three-necked round-bottom flask equipped with a reflux condenser
was charged with (R)-1,2-isopropylidene glycerol (744 mg, 4.0 mmol) and nBu4Itetra-n-butylammonium iodide (147 mg, 0.4 mmol, 10 mol%,).
Solids were degassed in three cycles before dry THF (12 ml) was added.
To the obtained solution, KH (50% in paraffin, 370 mg, 4.6 mmol, 1.15 equiv) was
added in small portions. The mixture was stirred for 10 min before 4-(chloromethyl)-
1,2-dimethoxybenzene30 (860 mg, 4.6 mmol, 1.15 equiv) was added in one portion.
The so-obtained reaction mixture was immersed into a preheated oil bath (87 °C)
and refluxed for 16 h. After removal from the oil bath and cooling down to rt, solid
NH4Cl (1 g) was added. The mixture was stirred for 15 min, filtered and the collected
filtrate was evaporated to dryness. The yellow liquid residue was further purified by
flash chromatography using 50% Et2O in pentane. Fractions with an Rf = 0.37 (50%
Et2O in pentane) were collected and concentrated to afford 1.04 g of the desired
compound as colourless thick liquid (92%).
1H NMR (400 MHz, CDCl3, δ): 6.82 (m, 3H), 4.49 (m, 2H), 4.27 (m, 1H), 4.03 (dd,
J = 8.2, 6.5 Hz, 1H), 3.71 (dd, J = 8.2, 6.4 Hz, 1H), 3.51 (dd, J = 9.8, 5.8 Hz, 1H),
3.44 (dt, J = 12.3, 4.7 Hz, 1H), 1.40 (s, 3H), 1.34 (s, 3H).
13C NMR (100 MHz, CDCl3, δ): 149.20, 148.83, 130.66 ( - ), 120.51, 111.21, 111.02
( - ), 109.54 ( - ), 74.93 ( - ), 73.56, 70.96, 67.00, 56.06 ( - ), 55.99 ( - ), 26.95 ( - ), 25.54
( - ).
αD =+15.9 (c = 0.067, CHCl3).
Anal. Calcd for C15H22O5: C, 63.81; H, 7.85. Found: C, 63.51; H, 7.88%.
The spectroscopic data correspond to previously published24
(R)-3-((3,4-dimethoxybenzyl)oxy)propane-1,2-diol
The corresponding acetonide (950 mg, 3.4 mmol) was dissolved in
CH2Cl2/MeOH (10 ml/10 ml). Amberlite IR120 (acid form, 100 mg)
was added, and the mixture was stirred at RT until full conversion (36
h). The catalyst was filtered off and the filtrate was concentrated in vacuo.
The reaction afforded 815 mg of desired product (>99%) as colourless very thick
liquid.
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1H NMR (400 MHz, CDCl3, δ): 6.85 (m, 3H), 4.47 (s, 2H), 3.87 (m, 7H), 3.69 (dd, J
= 11.4, 3.9 Hz, 1H), 3.61 (dd, J = 11.4, 5.5 Hz, 1H), 3.57 – 3.48 (m, 2H), 2.31 (s,
2H).
13C NMR (100 MHz, CDCl3, δ): 149.02, 148.76, 130.18, 120.47, 111.16 ( - ), 110.94
( − ), 73.45, 71.44, 70.71 ( - ), 64.02, 55.89 ( - ), 55.86 ( - ).
The spectral data corresponds to previously reported.24
Geranylgeraniol prepared following the literature procedure
Farnesyl bromide
A dry flask was charged with farnesol (2.0 g, 8.9 mmol). Dry THF (30 ml)
was added, and resulting solution was immersed into a -47 °C bath (ethanol,
cryostat). After stirring for 10 min, freshly distilled MsCl (900 µl, 12 mmol, 1.3 equiv)
was added via syringe over 5 min. Subsequently, Et3N (2.5 ml, 18 mmol, 2.0 equiv)
was added over another 5 min. After complete addition, the mixture was stirred for
45 min at –47 °C. To the resulting suspension, a solution of LiBr (3.0 g, 36 mmol,
4.0 equiv) in dry THF (10 ml) was added dropwise over 5 min. After complete
addition, the reaction vessel was transferred to a 0 °C bath (ice/water) and stirred
for 1 h. The reaction mixture was poured into chilled saturated NaHCO3 solution.
The organic layer was separated, the aqueous layer was extracted with cold Et2O (a
mixture of Et2O with pieces of ice, 3 x 25 ml), the combined organic layers were
washed with cold water, brine, dried over MgSO4 and evaporated. The crude farnesyl
bromide was obtained as a yellow liquid and used without further manipulation.
The reaction afforded 1.91 g of the desired product as a light yellow oil (75% yield)
which was stored at -80 °C, and used within 1 day.
(6E,10E)-ethyl 7,11,15-trimethyl-3-oxohexadeca-6,10,14-trienoate
A dry Schlenk flask was charged with NaH (60%
dispersion, 880 mg, 22 mmol, 3.3 equiv). The
mineral oil was removed by three washings with pentane (3 x 10 ml). The resulting
white solid was dried in vacuum. Dry THF (16 ml) was added and the resulting white
suspension was cooled to 0 °C (ice/water bath). To this suspension, freshly distilled
ethyl acetoacetate (2.6 ml, 3.0 equiv) was added dropwise over 5 min. After complete
addition the suspension turned into a light yellow solution. To this solution was
added a solution of nBuLi in hexanes (2.5 M, 8.3 ml, 3.1 equiv) over 15 min. The
resulting orange solution was stirred for additional 15 min at 0 °C before a solution
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of farnesyl bromide (from the previous experiment 1.91 g, 6.7 mmol) in dry THF
(3.5 ml) was added dropwise over 5 min. The resulting solution was stirred at 0 °C
for 15 min, during which formation of a precipitate was observed. The reaction was
quenched by careful addition of chilled aqueous HCl (1 M, 10 ml,
EXOTHERMIC). The mixture was transferred into a separatory funnel, where the
organic layer was separated, the aqueous layer was extracted with Et2O (3 x 10 ml),
the combined organic layers were washed with brine, dried and evaporated.
The title compound was obtained after column chromatography using 10% Et2O in
pentane as 1.67 g of a light yellow liquid (55% starting from farnesol, lit 70%).
1H NMR (400 MHz, CDCl3) δ 12.09 (s, 0.2H), 5.08 (d, J = 6.5 Hz, 3H), 4.19 (dt, J
= 7.2, 5.3 Hz, 2H), 3.42 (s, 2H), 2.56 (t, J = 7.4 Hz, 2H), 2.38 – 2.17 (m, 3H), 2.05 -
1.98 (m, 8H), 1.68 (s, 3H), 1.64 – 1.58 (m, 9H), 1.28 (t, J = 7.1 Hz, 3H).
The spectral data corresponds to previously reported.31
Ethyl (2Z,6E,10E)-3-((diethoxyphosphoryl)oxy)-7,11,15-trimethylhexadeca-
2,6,10,14-tetraenoate
A dry Schlenk flask was charged with NaH (60%
dispersion, 228 mg, 1.15 equiv). The mineral oil was
removed by three successive washings with hexane (3 x 5 ml) and the resulting white
solid was suspended in dry Et2O (21 ml). The suspension was immersed in an
ice/water bath of 0 °C and a solution of (6E,10E)-ethyl 7,11,15-trimethyl-3-
oxohexadeca-6,10,14-trienoate (1.65 g, 5.0 mmol) was added as a solution in dry
Et2O (7 ml) over 15 min. After the addition was complete, the resulting light yellow
solution was stirred for 15 min at 0 °C and for 15 min at 21 RT. Then the solution
was again cooled in the ice bath and neat (EtO)2P(O)Cl (1.1 ml, 7.5 mmol, 1.5 equiv)
was added dropwise. The resulting reaction mixture was stirred for 15 min at 0 °C
and subsequently quenched by addition of saturated aqueous NH4Cl solution (15
ml). The organic layer was separated, and the aqueous layer was extracted with Et2O
(3 x 15 ml). The combined organic layers were washed with saturated aqueous
NaHCO3 (3 x 15 ml), brine (2 x 15 ml), dried over MgSO4 and the solvent was
removed in vacuo.
The resulting crude (2.08 g) was obtained as a yellow liquid and used without further
purification in the following step.
ethyl (2E,6E,10E)-3,7,11,15-tetramethylhexadeca-2,6,10,14-tetraenoate
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A dry Schlenk flask was charged with CuI (1.7 g,
8.9 mmol, 1.8 equiv) which was suspended in dry Et2O
(5.5 ml) and cooled to 0 °C. The resulting suspension was treated with MeLi (1.6 M
in Et2O, 11.0 ml, 18 mmol, 3.6 equiv). The suspension turned yellow after initial
addition of MeLi, after complete addition the CuI fully dissolved affording a nearly
colourless solution.
The reaction vessel with Me2CuLi was immersed in a cryostat at –78°C32 and
a solution of the phosphate (2.08 g, ca 5.0 mmol) in Et2O (dry, 7 ml) was added
dropwise via the cold wall of the Schlenk flask (in order to cool the solution of the
phosphate). After complete addition, the colour changed to orange/red and the
resulting solution was stirred at –78 °C. After 1 h the bath was allowed to warm to –
47 °C and the reaction mixture was stirred at –47 °C for 2 h. After this time TLC
showed full conversion of the phosphate and a new spot had appeared on TLC. MeI
(630 µl) was added to quench the unreacted cuprate. After stirring for 10 min, the
reaction mixture was carefully poured into a solution of NH4Cl (24 ml) and NH4OH
(6 ml) (can be exothermic with gas evolution). The mixture was stirred until all
solids dissolved. Layers were separated, the aqueous layer was extracted with Et2O
(3 x 20 ml) and the combined organic layers were washed with NH4OH (10%, 2 x
40 ml), brine (2 x 40 ml), dried and evaporated.
The reaction afforded 1.15 g of a yellow liquid which was used without further
purification.
Geranylgeraniol
The ethyl ester from the previous step (1.15 g, 4.5
mmol) was dissolved in toluene (p.a. grade, 17 ml).
This solution was cooled to –78 °C (N2/acetone bath) and a solution of DIBAL (1M
in hexane, 14.0 ml, 14 mmol, 3.0 equiv) in hexane (15 ml) was added dropwise. The
mixture was stirred until complete consumption of the starting material (TLC). The
reaction was quenched by careful addition of MeOH (3.0 ml, added over 10 min,
EXOTHERMIC, gas evolution). When gas evolution ceased, the mixture was
removed from the bath and stirred for 10 min at rt. The reaction mixture was poured
into saturated NH4Cl (50 ml)/HCl (50 ml) solution and stirred until clear separation
of the layers took place (ca 30 min). The aqueous layer was extracted with Et2O (3 x
50 ml). The combined organic layers were washed with water (2 x 50 ml) and brine
(2 x 50 ml), dried over MgSO4 and evaporated. The residual thick liquid was further
purified by flash chromatography on silica using 30% Et2O in pentane as the eluent.
The reaction afforded 857 mg of geranylgeraniol (60% over three steps) with >99%
double bond isomer purity according to GC analysis.
Chapter 7
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The spectral data corresponds to previously reported31
Geranylgeranyl chloride
N-chlorosuccinimide (1.2 g, 9.0 mmol, 1.3 equiv) was
suspended in dry CH2Cl2 (15 ml). The suspension was
cooled to –30 °C (acetone/liquid N2 bath) and after stirring for 5 min, dimethyl
sulfide (750 µl, 10 mmol, 1.5 equiv) was added. The reaction was stirred for 10 min
at –30 °C and 10 min at 0 °C. Then the solution was cooled to –40 °C. A solution
of geranylgeraniol (2.0 g, 6.9 mmol) in CH2Cl2 (dry, 5 ml) was added dropwise. The
resulting suspension was allowed to warm during 150 min to 0 °C, turning into a
cloudy solution at –15 °C. The reaction mixture was poured into pentane (150 ml),
The organic layer was washed with water (2 x 50 ml), brine (50 ml), dried over MgSO4
and evaporated.
The reaction afforded 1.94 g of geranylgeranyl chloride as a colourless liquid which
was used without further purification.
Synthesis of 2,3-bisgeranylgeranyl-sn-glycerol (23), (scheme 1)
1-((3,4-dimethoxybenzyl)oxy)- 2,3-bisgeranylgeranyl-sn-glycerol (30)
A dry Schlenk flask was charged
with NaH (60% dispersion in mineral oil, 265 mg, 6.6 mmol, 2.1 equiv). The mineral
oil was removed by washing with pentane (3 x 5 ml) and the white solid was dried in
high vacuum before suspending in DMSO (5 ml). The obtained suspension was
immersed in a preheated oil bath (70 °C) and stirred for 40 min during which the
suspension turned into a pale yellow solution. The flask was removed from the bath
and allowed to cool to rt (21 °C). To this solution, a solution of (R)-3-((3,4-
dimethoxybenzyl)oxy)propane-1,2-diol (595 mg, 3.2 mmol) in dry DMSO (5 ml) was
added carefully. After the complete addition, the reaction mixture was stirred for 1
h at rt (21 °C). Then a mixture of geranylgeranyl chloride (1.94 g from the previous
experiment, ca 6.3 mmol) in a small amount of DMSO (2 ml) was added. The
resulting solution was stirred for 16 h before pouring into saturated aqueous NH4Cl
solution (20 ml). The aqueous layer was extracted with Et2O (3 x 50 ml). The
combined organic layers were washed with brine, dried and evaporated. The crude
residue was further chromatographed using 30% Et2O in pentane to afford the
desired product and the product of the mono-alkylation.
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Dialkylated product 1.52 g (61%) as colourless liquid
1H NMR (400 MHz, CDCl3, δ): 6.97 – 6.75 (m, 3H), 5.35 (dt, J = 13.5, 6.6 Hz, 2H),
5.10 (d, J = 5.8 Hz, 6H), 4.49 (s, 2H), 4.16 (d, J = 6.7 Hz, 2H), 4.01 (d, J = 6.7 Hz,
2H), 3.85 (s, 3H), 3.89 (s, 3H), 3.68 (dt, J = 10.0, 5.1 Hz, 1H), 3.62 – 3.46 (m, 5H),
2.16 – 1.90 (m, 23H), 1.68 (s, 6H), 1.65 (s, 26), 1.59 (s, 16H)
13C NMR (100 MHz, CDCl3, δ): 149.06, 148.60, 140.20, 139.95, 135.43, 135.40,
135.06, 131.39, 131.10, 124.51 ( - ), 124.32 ( - ), 124.31 ( - ), 124.03 ( - ), 123.99 ( - ),
121.33 ( - ), 120.99 ( - ), 120.31 ( - ), 111.07 ( - ), 110.91 ( - ), 77.03( - ), 73.40, 70.35,
70.19, 68.02, 66.95, 56.03 ( - ), 55.92 ( - ), 39.86, 39.84, 39.78, 26.89, 26.78, 26.53,
26.50, 25.85( - ), 17.83 ( - ), 16.69( - ) , 16.66 ( - ), 16.15 ( - ).
αD =+5.2 (c = 1.0, CHCl3)
NMR data correspond to those previously published23.
Monoalkylated product 256 mg
(16%) as colourless liquid.
1H NMR (400 MHz, CDCl3, δ): 6.84 (m, 3H), 5.34 (d, J = 7.1 Hz, 2H), 5.10 (s, 3H),
4.48 (d, J = 5.0 Hz, 2H), 4.01 (m, 3H), 3.85 (s, 3H), 3.89 (s, 3H), 3.76 – 3.40 (m, 5H),
2.05 (m, 12H), 1.66 (d, J = 8.3 Hz, 6H), 1.59 (s, 9H).
13C NMR (100 MHz, CDCl3, δ): 149.11, 148.76, 148.73, 140.72, 140.67, 135.49,
135.45, 135.05, 131.36, 130.62, 124.47 ( - ), 124.26 ( - ), 123.88 ( - ), 123.85 ( - ),
120.79 ( - ), 120.59 ( - ), 120.48 ( - ), 120.38 ( - ), 111.16 ( - ), 111.02 ( - ), 110.95 ( - ),
110.93 ( - ), 77.61 ( - ), 73.53, 73.48, 71.34, 71.22, 69.93, 69.70 ( - ), 67.94, 66.67,
63.01, 56.01 ( - ), 55.93 ( - ), 55.92 ( - ), 39.83, 39.80, 39.72, 26.86, 26.72, 26.43, 25.82
( - ), 17.80 ( - ), 16.66 ( - ), 16.63 ( - ), 16.13 ( - ) 16.12 ( - ).
αD =+9.2 (c = 1.0, CHCl3).
HRMS-APCI (m/z): [M + Na]+ calculated for C32H50O5Na, 538.354; found, 538.355.
Chapter 7
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2,3-bisgeranylgeranyl-sn-glycerol (23)
30 (1.44 g, 1.8 mmol) was dissolved in
CH2Cl2 (30 ml). To this solution, H2O (0.75 ml) was added. The obtained biphasic
solution was cooled in an ice bath (0 °C) and DDQ (830 mg, 3.6 mmol, 2.0 equiv)
was added. The reaction was stirred for 4 h at 0 °C until TLC showed full conversion
of the starting material. The crude reaction mixture was filtered over a small silica
pad and washed with CH2Cl2 (200 ml). The washings were combined and evaporated.
The obtained yellow liquid was further purified by column chromatography (30%
Et2O in pentane).
The reaction afforded 705 mg of the desired product (60%) as yellow thick liquid
containing traces of unidentified co-eluting impurities.
1H NMR (400 MHz, CDCl3, δ): 5.35 (dt, J = 13.5, 6.7 Hz, 2H), 5.11 (t, J = 6.5 Hz,
6H), 4.26 – 4.06 (m, 2H), 4.02 (d, J = 6.7 Hz, 2H), 3.77 – 3.41 (m, 5H), 2.16 – 1.93
(m, 24H), 1.68 (s, 12H), 1.60 (s, 21H)
13C NMR (100 MHz, CDCl3, δ): 140.68, 135.49, 135.47, 135.07, 131.37, 124.52 ( - ),
124.32 ( - ), 123.9 ( - )3, 120.91 ( - ), 120.69 ( - ), 77.61 ( - ), 70.16, 68.08, 66.66, 63.21,
39.88, 39.86, 39.83, 39.75, 26.90, 26.77, 26.50, 26.46, 25.83 ( - ), 17.82 ( - ), 16.68 ( - ),
16.65 ( - ), 16.15 ( - ), 16.14 ( - ).
HRMS-APCI+ (m/z): [M + Na]+ calculated for C43H72O3Na, 659.536; found,
659.537.
Synthesis of 2,3-bis-O-(geranylgeranyl)-sn-glycero-phosphate (10) (Scheme 2)
bis((9H-fluoren-9-yl)methyl) diisopropylphosphoramidite
PCl3 (7 ml, 80 mmol) was dissolved in pentane (700 ml).
Via an addition funnel, a solution of diisopropylamine
(distilled from CaH2, 23 ml, 0.16 mol, 2.0 equiv) in
pentane (100 ml) was added dropwise over 15 min.
After this time, a significant amount of white precipitate had formed. The suspension
was stirred for 2 h. The mixture was transferred to a separatory funnel. The pentane
layer was washed with acetonitrile (the pentane layer stays on top, 5 x 100 ml of
acetonitrile, after the washing the pentane layer was fully transparent). Pentane was
subsequently evaporated. The reaction afforded 1,1-dichloro-N,N-
diisopropylphosphinamine (6 g, 38%) as colourless liquid
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1H NMR (400 MHz, CDCl3, δ): 4.02 – 3.83 (m, 1H), 1.28 (d, J = 6.8 Hz, 6H),
31P NMR (400 MHz, CDCl3, δ): 169.59.
From the obtained liquid, an aliquot was taken (2.0 g, 10 mmol). This was dissolved
in dry THF (40 ml) and DIPEA (3.5 ml, 20 mmol, 2.0 equiv) was added. The solution
was cooled in an ice bath and (9H-fluoren-9-yl)methanol (3.92 g, 20 mmol, 2.0 equiv)
in dry THF (10 ml) was added. The solution was stirred for 10 h during which a
white precipitate formed. The reaction was poured into aqueous phosphate buffer
(1 M, pH = 7) and extracted with ethyl acetate (4 x 50 ml). The combined organic
extracts were washed with the same phosphate buffer, brine, dried and evaporated.
The crude product was used without further purification due to its sensitivity.
31P NMR (162 MHz, CDCl3) δ 148.01.
bis((9H-fluoren-9-yl)methyl) ((S)-2,3-bisgeranylgeranyl)oxy)propyl) phosphate 37
(R)-2,3-bisgeranylgeranyl glycerol
(66.0 mg, 0.1 mmol)was dissolved
in CH2Cl2/CH3CN (0.5 ml/0.5
ml) and 36 (154 mg, 0.3 mmol,
3.0 equiv) was added. The mixture was cooled to 0 °C and tetrazole (21 mg, 0.3
mmol, 3.0 equiv) was added. The mixture was allowed to gradually warm to rt (21
°C) and stirred overnight. When full conversion of starting material was observed
(TLC), the mixture was cooled and a solution of tBuOOH (5 M in decane, 64μl, 0.3
mmol, 3.2 equiv) was added in one portion followed by stirring for 45 min. The
mixture was subsequently poured into aqueous phosphate buffer (1 M, pH = 7) and
extracted with Et2O (4 x 20 ml). The combined extracts were washed with brine,
dried and concentrated. The crude residue was purified by flash chromatography
using (50% Et2O in pentane). Fractions with an Rf = 0.4 (50% Et2O in pentane)
were collected to afford 102 mg (94%) of the desired compound.
1H NMR (400 MHz, CDCl3, δ): 7.71 (dd, J = 12.2, 4.3 Hz, 4H), 7.56 (m, 4H), 7.38
(m, 4H), 7.22 (m, 4H), 5.27 (q, J = 6.8 Hz, 2H), 5.09 (m, 6H), 4.28 (m, 4H), 4.14 (m,
3H), 3.97 (m, 5H), 3.59 (dd, J = 9.7, 4.9 Hz, 1H), 3.54 (m, 2H), 2.03 (m, 24H), 1.68
(s, 6H), 1.59 (dd, J = 11.2, 6.0 Hz, 24H)
13C NMR (100 MHz, CDCl3, δ): 147.46, 147.40, 145.60, 144.69, 144.56, 139.59,
139.57, 139.21, 135.52 ( - ), 132.10 ( - ), 131.36 ( - ), 129.48 ( - ), 129.43 ( - ), 128.66
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( - ), 128.47 ( - ), 128.09 ( - ), 124.89 ( - ), 124.86 ( - ), 124.25 ( - ), 124.21 ( - ), 80.44
( - ), 80.36 ( - ), 73.59, 73.53, 73.07, 72.22, 71.45, 71.40, 71.07, 52.24 ( - ), 52.16 ( - ),
44.00, 43.98, 43.87, 31.04, 30.93, 30.70, 30.65, 29.98 ( - ), 21.97 ( - ), 20.78 ( - ), 20.29
( - ).
HRMS-APCI (m/z): [M + Na]+ calculated for C71H93O6PNa, 1095.660; found
1095.660.
2,3-bis-O-(geranylgeranyl)-sn-glycero-phosphate (10)
Bisprotected phosphoric ester 37 (102
mg, 0.10 mmol) was dissolved in acetonitrile (5.0 ml). To this solution, Et3N (280
μmol, 2.0 mmol, 20 equiv) was added and the resulting mixture was stirred overnight.
All volatiles were evaporated and the crude mono deprotected phosphoric ester (as
assumed from the TLC) was suspended in aqueous NaOH (1 M, 5.0 ml) until full
conversion of the monoprotected ester was observed (TLC, 3 h). The mixture was
acidified with HCl (1 M) to pH = 1 and extracted with Et2O (3 x 20 ml). The
combined extracts were dried over MgSO4 and concentrated. The crude residue was
purified on a silica column using a carefully established gradient of 2%
MeOH/CHCl3 33% MeOH/CHCl3.
Reaction afforded 34.3 mg (48%) of the desired compound as a colourless liquid.
One pot procedure for the deprotection:
Bisprotected phosphoric ester (102 mg, 0.10 mmol) was dissolved in dioxane (1 ml).
To the stirred solution aqueous NaOH soulution (1 M, 1 ml) was added. The mixture
was stirred until full conversion of the starting material (3 h). The mixture was
acidified with HCl (1 M) to pH = 1 and extracted with Et2O (3 x 20 ml). The
combined extracts were dried over MgSO4 and concentrated. The crude residue was
purified on a 120 Å Davisil silica column using a carefully established gradient of 2%
MeOH/CHCl3 33% MeOH/CHCl3.
The reaction afforded 48.6 mg (68%) of the desired compound as a colourless oil
NOTE: The final product was stored in the freezer (–20 °C).
1H NMR (400 MHz, CDCl3, δ): 5.32 (m 2H), 5.09 (m, 6H), 4.15 (m, 2H), 4.00 (m,
4H), 3.57 (m, 3H), 2.01 (m, 24H), 1.77 – 1.49 (m, 30H).
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13C NMR (100 MHz, CDCl3, δ): (101 MHz, CDCl3, the spectrum shows
a considerable number of overlapping signals) δ 135.23, 134.85, 131.16, 124.38,
124.22, 123.88, 120.71, 39.75, 39.72, 26.76, 26.61, 25.67, 17.66, 16.53, 16.00, 15.97.
31P NMR (162 MHz, CDCl3) δ 1.37.
HRMS-ESI+ (m/z): [M + H]+ calculated for C43H72O6P, 715.507; found, 715.506.
Total synthesis of cycloarcheol 1 (Scheme 4)
(4S,9R,13R,17S,21S)-9,13,17,21,25,25-hexamethyl-1,24,24-triphenyl-2,6,23-trioxa-
24-silahexacosan-4-ol (46)
To a roundbottom flask containing
neat 13 (488 mg, 0.9 mmol) was
added neat benzyl-(S)-glycidol (250 μl, 1.6 mmol, 1.65 equiv) and Co[R,R-
(salen)]OTs (35 mg, 8.0 mol%). An atmosphere of dry oxygen was applied (balloon,
1 bar). The mixture was stirred for 16 h and then purified by silica gel
chromatography using 20% Et2O in hexane. The reaction afforded 533 mg of the
desired product as colourless liquid (85%)
1H NMR (400 MHz, CDCl3, δ): 7.69 – 7.65 (m, 4H), 7.45 – 7.27 (m, 11H), 4.57 (s,
2H), 3.98 (br s, 1H), 3.59 – 3.40 (m, 8H), 2.47 (br d, J = 3.1 Hz, 1H), 1.70 – 1.47 (m,
4H), 1.46 – 1.14 (m, 20H), 1.06 (s, 9H), 0.92 (d, J = 6.7 Hz, 3H), 0.88 (d, J = 6.6 Hz,
3H), 0.85 (d, J = 5.9 Hz, 3H), 0.83 (d, J = 6.0 Hz, 3H).
13C NMR (100 MHz, CDCl3, δ): 138.0, 135.6, 134.1, 129.4, 128.4, 127.7, 127.7, 127.5,
73.4, 71.8, 71.4, 70.0, 69.5, 68.9, 37.5, 37.4, 37.4, 36.6, 35.7, 33.5, 32.8, 32.8, 29.9,
26.9, 24.5, 24.4, 24.4, 19.8, 19.7, 19.3, 17.0.
HRMS-APCI (m/z): [M + Na]+ calculated for C46H72O4SiNa, 739.509; found:
739.509.
[]D -0.3 (c = 1.2, CHCl3).
References and footnotes
(1) Woese, C. R.; Fox, G. E. Proc. Nat. Acad. Sci. 1977, 74, 5088.
(2) Forterre, P. Archaea 2013, 2013, 18.
(3) Blöchl, E.; Rachel, R.; Burggraf, S.; Hafenbradl, D.; Jannasch, H. W.; Stetter, K. O.
Extremophiles 1997, 1, 14.
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(4) Barns, S. M.; Fundyga, R. E.; Jeffries, M. W.; Pace, N. R. Proc. Nat. Acad. Sci. 1994,
91, 1609.
(5) Schleper, C.; Puehler, G.; Holz, I.; Gambacorta, A.; Janekovic, D.; Santarius, U.;
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A Missing Link in Archaeal Lipid Biosynthesis; a Contribution from Organic Synthesis
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Chapter 7
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7
Summary
171
Summary
The idea that saponifiable phospholipids are only building blocks and energy
source of a cell has been refuted by the discovery of phospholipid signalling.
Currently, these lipids are widely studied, mainly because of their potential
undiscovered functions. One aspect that makes the study of phospholipids difficult,
is their limited availability in pure form. Biological membranes, which are the main
source of phospholipids, are typically composed of several tens to hundreds very
similar species. In this mixture, only a minute amount of a single species might have
a specific physiological property. To find, and to isolate this lipid reminds of the
phrase “search the needle in a haystack”. Despite organic chemistry cannot help
finding the needle directly, it offers a different solution; to prepare a new needle.
However, this requires efficient strategies that allow the synthesis of defined,
chemically pure lipids and their derivatives. This is what this thesis presents.
Chapter 2 describes a modular synthesis of branched fatty acids, which are
mainly lipid components of the membranes of various bacterial pathogens. The
described synthesis allows preparation of a fatty acid bearing the methyl-branch at
any position of the linear chain together with the desired absolute configuration.
Figure 1. Modular synthesis of branched fatty acids.
This modular approach was successfully applied in the synthesis of 3 different fatty
acids in the amounts relevant for further studies.
Despite the fact that glycerophospholipids seem relatively simple
compounds, their synthesis might be surprisingly complex. Chapter 3 is devoted to
the synthesis of these glycerophospholipids. A cobalt catalyst allowed the
regioselective ring opening of a silyl protected glycidol. The obtained protected
Summary
172
monoacylglycerol was further esterified in the same pot. The resulting silyl protected
diacylglycerol was deprotected without any notable migration, and subsequently
converted to a phospholipid.
Figure 2. One pot synthesis of protected diacylglycerols and the subsequent conversion to
a phospholipid.
Chapter 4 is an extension of this methodology to the synthesis of
triacylglycerols. When glycidyl esters were used as the starting material, the same
conditions afforded enantiopure triacylglycerols. This methodology was
demonstrated in the synthesis of 18 different triacylglycerols.
Figure 3. An efficient, 3 step synthesis of triacylglycerols.
Furthermore, this chapter described the first steps towards the automated synthesis
of triacylglycerols by a liquid-handling platform. The obtained triacylglycerols can be
applied as analytical standard in the analysis of triacylglycerols mixtures such as milk
fat.
The synthetic solutions described in the chapters 2 and 3 allowed the
synthesis of phospholipids bearing methyl branched fatty acids. These lipids were
Summary
173
prepared in amounts, which allowed initial studies of their properties as membrane
components.
Figure 4. Study of the influence of a methyl-branch on the organization of a bilayer.
The phospholipids were converted into 2-component liposomes. The bilayers of
these liposomes were studied by molecular dynamics simulations. Another studied
aspect of these lipids was their interaction with membrane proteins.
Chapter 6 is also dedicated to the influence of lipids on protein function, but from a
different perspective. Protein lipidation is a posttranslational modification, by which
lipids control the activity of proteins. The chapter describes the synthesis of a lipid
probe, which might be useful in protein lipidation studies in living cells.
Figure 5. A probe for the study of protein lipidation displays a rapid and clean dipolar
cycloaddition.
Summary
174
Chapter 7 is dedicated to (phospho)lipids found in Archaea. Archaea form a
domain of Life, which early in evolution strayed from the remaining 2 domains.
Figure 6. ( I ) Synthesis of a biosynthetic intermediate in archaeal lipid biosynthesis for the
identification of an involved enzyme; ( II ) Epoxide ring opening as a key step in the total
synthesis of cycloarchaeol.
The separated evolution granted, that the species from this domain
developed their own lipid biosynthetic pathways. Study of these pathways is
challenging because there are few assays and the corresponding biosynthetic
intermediates are not available. Chapter 7 presents a synthesis of one of these
intermediates, which was necessary for the identification of one of the missing
enzymes in archaeal lipid biosynthesis. Furthermore, chapter 7 describes a key step
in the total synthesis of cycloarchaeol – an archaeal lipid backbone.
Samenvatting
175
Samenvatting
Het idee dat verzeepbare fosfolipiden alleen functioneren als bouwstenen
en energiebron voor de cel werd weerlegd door de ontdekking van signaalfuncties
van fosfolipiden. Tegenwoordig worden deze lipiden uitgebreid bestudeerd, en
steeds worden nieuwe functies ontdekt. Een aspect wat de studie van fosfolipiden
bemoeilijkt, is hun gelimiteerde beschikbaarheid in zuivere vorm. Biologische
membranen, de hoofdbron van fosfolipiden, bestaan vaak uit honderden zeer
vergelijkbare lipiden. Daarin is soms maar een zeer geringe hoeveelheid van een
bepaald lipide met een specifieke fysiologische eigenschap aanwezig. Het vinden en
isoleren van dit lipide doet denken aan het spreekwoord “een speld in een hooiberg
zoeken”. Hoewel de organische chemie niet direct kan helpen naar de zoektocht
naar de speld, geeft het wel een andere oplossing; het maken van een zo’n speld.
Dit vereist wel een efficiënte strategie voor de synthese van gedefinieerde,
chemisch zuivere lipiden en hun derivaten. Dat is wat dit proefschrift beschrijft.
Hoofdstuk 2 beschrijft de modulaire synthese van vertakte vetzuren, vaak
hoofdbestanddelen van membranen van bacteriële pathogenen. De beschreven route
maakt het mogelijk om vetzuren te synthetiseren die methylsubstituenten hebben op
elke positie van de lineaire keten, in combinatie met de gewenste absolute
configuratie.
Figure 1. De modulaire synthese van vertakte vetzuren
Deze modulaire benadering is succesvol toegepast in de synthese van 3 verschillende
vetzuren in hoeveelheden die toereikend zijn voor verdere studies.
Samenvatting
176
Ondanks het feit dat glycerofosfolipiden een vrij eenvoudige structuur lijken
te hebben, is hun synthese verrassend complex. Hoofdstuk 3 is dan ook gewijd aan
de synthese van deze glycerofosfolipiden. Met behulp van een kobalt katalysator kan
een beschermd glycidol regioselectief worden geopend. Het verkregen
monoacylglycerol wordt nogmaals veresterd in dezelfde reactiekolf. Het resulterende
beschermde diacylglycerol wordt ontschermd zonder acylmigratie en vervolgens
omgezet naar het fosfolipide.
Figure 2. Eénpotssynthese van beschermde diacylglycerolen en de omzetting naar een
fosfolipide
Hoofdstuk 4 is een uitbreiding van deze methodologie tot de synthese van
triacylglycerolen. Door glycidyl-esters te gebruiken als startmateriaal, kunnen onder
dezelfde condities enantiozuivere triacylglycerolen worden geïsoleerd. Deze
methodologie is gedemonstreerd in de synthese van 18 verschillende
triacylglycerolen.
Figure 3. Een efficiënte, 3-stapssynthese van triacylglycerolen.
Samenvatting
177
Bovendien beschrijft dit hoofdstuk ook de eerste stap in de richting van de
geautomatiseerde synthese van triacylglycerolen met behulp van een pipetteer robot.
De verkregen triacylglycerolen kunnen gebruikt worden als standaarden in de analyse
van triacylglycerolmengsels zoals in melkvet.
Hoofdstuk 2 en 3 beschrijven een strategie voor de synthese van fosfolipiden
met methylvertakte vetzuren. Deze lipiden zijn gesynthetiseerd in hoeveelheden die
initiële studies toelieten naar hun eigenschappen als membraanlipiden.
Figure 4. De invloed van methylvertakkingen op de organisatie van de lipide bilaag, links
de moleculaire dynamicasimulaties.
De fosfolipiden werden gebruikt in liposomen. De bilaag van deze liposomen is
gesimuleerd met moleculaire dynamica. Het blijkt dat kanaalvormende
membraaneiwitten succesvol in deze liposomen kunnen worden gereconstitueerd.
Hoofdstuk 6 is ook gewijd aan de invloed van lipiden op de functie van eiwitten,
maar dan vanuit een ander perspectief. Eiwitlipidering is een posttranslationele
modificatie waarbij lipiden de activiteit van eiwitten sturen. Dit hoofdstuk beschrijft
Samenvatting
178
de synthese van een lipide-sonde, die gebruikt zou kunnen worden in levende cellen
voor de modificatie van eiwitten met lipiden om ze dusdanig te kunnen bestuderen.
Figure 5. Een sonde voor de studie van eiwitlipidering laat een schone en snelle
dipolaire cycloadditie zien.
Hoofstuk 7 is gewijd aan (fosfo)lipiden die gevonden worden in Archaea.
Archaea vormen één van de domeinen van het leven, die in een beginstadium van de
evolutie is afgesplitst van de andere twee domeinen.
Figure 6. ( I ) Synthese van een intermediair uit de biosynthese van archaiële lipiden in
verband met de identificatie van het betrokken enzym. ( II ) Epoxide ringopening als de
belangrijkste stap in de totaalsynthese van cycloarchaeol.
Samenvatting
179
De gescheiden evolutie van deze domeinen liet toe dat Archaea hun eigen
route hebben ontwikkeld voor lipidensynthese. Het bestuderen van deze
syntheseroute is uitdagend, omdat er maar enkele analyses bestaan en de bijhorende
biosynthese-intermediairen niet beschikbaar zijn. Hoofdstuk 7 beschrijft de synthese
van één van deze intermediairen, die leidde tot de identificatie van een enzym in de
lipide-biosynthese. Hoofdstuk 7 beschrijft ook een belangrijke stap in de
totaalsynthese van cycloachaeol, een membraanlipide uit Archaea.
Acknowledgements
180
Acknowledgements
Možno netypicky, ale v prvom rade sa chcem podakovať mojím rodičom. Tí
si so mnou užili viac než dosť počas môjho predchádzajúceho štúdia. Mama, ty si
ma naučila, že ak sa chcem niekam dostať, tak nemôžem byť čajová nula, ktorá sa
vzdá pri prvej, druhej, alebo dvadsiatej prekážke. Oco, nakoniec som sa predsa len
“potatil”. Zrejme ta to môžem poďakovať genetickej výbave, ktorú som po tebe
zdedil. Keď teba môže baviť labák a pokusy po viac ako 50 rokoch, prečo by
nemohol aj mňa...? Kuku (Tomáš)! Bez teba by som bol úplne iný človek! Dúfam, že
úspešne doštuduješ, potom úspešne skončíš PhD a potom ovládneme svet...muhaha!
Leti, existen un millón de razones por las que te estoy inmensamente
agradecido, dos de las cuales son: primero, te agradezco todo tu esfuerzo que has
puesto en la corrección de tanto mi tesis como de mis artículos. Segundo y
muchísimo más importante, te quiero agradecer la oportunidad que me has regalado
al elegir compartir tu vida conmigo. Estoy ansioso por conocer que nos va a regalar
el futuro juntos.
Adri, I would like to thank you for at least dozen of different things. First of
all, thank you for the opportunity to join your group and do PhD. under your
supervision. Then, thank you for your supervision, care, trust when you offered me
to work on Mycolic acid on everything you did for me. Dank je wel!
Niek and Manuel, where to start. Well, first of all, thank you for being my
paranymphs, friends and gym sparring partners. You deserve all my respect for all
the “gins at the bar”, “just 2 more repetitions”, “I know an awesome
exercise”...gentlemen, you are the real champions!
Selma, Nick, Jelle and Steven. With students like you I kind off won a lottery.
It was a pleasure to supervise a bunch of motivated and enthusiastic students. Thank
you!
I would also like to thank to the group of people joining for the Friday
dinners at Papa Joe’s, Klein Moghul, Tel Aviv and other places we visited. Paul, Ilse,
Mattia, Mannathan, Francesco, Guilloume, Melanie, Hylke and all those, which
I forgot (sorry). Thank you for your company.
Thanks also goes to all the members of Stratingh institute, which I had the
pleasure to meet.