ACTAUNIVERSITATIS

UPSALIENSISUPPSALA

2015

Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Science and Technology 1329

Light-Triggered ConformationalSwitches for Modulation ofMolecular Recognition

Applications for Peptidomimetics andSupramolecular Systems

MAGNUS BLOM

ISSN 1651-6214ISBN 978-91-554-9431-5urn:nbn:se:uu:diva-267845

Dissertation presented at Uppsala University to be publicly examined in B41, BMC,Husargatan 3, Uppsala, Thursday, 28 January 2016 at 10:15 for the degree of Doctor ofPhilosophy. The examination will be conducted in English. Faculty examiner: ProfessorMorten Grötli (University of Gothenburg).

AbstractBlom, M. 2015. Light-Triggered Conformational Switches for Modulation of MolecularRecognition. Applications for Peptidomimetics and Supramolecular Systems. DigitalComprehensive Summaries of Uppsala Dissertations from the Faculty of Science andTechnology 1329. 61 pp. Uppsala: Acta Universitatis Upsaliensis. ISBN 978-91-554-9431-5.

The main focus of this thesis is on photochemical modulation of molecular recognitionin various host-guest systems. This involves the design, synthesis and integration of light-triggered conformational switches into peptidomimetic guests and molecular tweezer hosts.The impact of the switches on guest and host structures has been assessed by spectroscopicand computational conformational analysis. Effects of photochemical structure modulation onmolecular recognition in protein-ligand and supramolecular host-guest systems are discussed.

Phototriggerable peptidomimetic inhibitors of the enzyme M. tuberculosis ribonucleotidereductase (RNR) were obtained by incorporation of a stilbene based amino acid moiety intooligopeptides between 3-9 residues long (Paper I). Interstrand hydrogen bond probability inthe E and Z forms of the peptidomimetics was used as a tool for predicting conformationalpreferences. Considerable differences in inhibitory potency for the E and Z photoisomers weredemonstrated in a binding assay.

In order to advance the concept of photomodulable inhibitors, synthetic routes towards aminoacid derivatives based on the more rigid stiff-stilbene chromophore were developed (Paper II). The effect of E-Z isomerization on the conformational properties of peptidomimetic inhibitorsincorporating the stiff-stilbene chromophore was also assessed computationally (Paper III). Itwas indicated that inhibitors with the more rigid amino acid derivative should display largerconformational divergence between photoisomers than corresponding stilbene derivatives.

Bisporphyrin tweezers with enediyne and stiff-stilbene spacers have been synthesized, andthe conformational characteristics imposed by the spacers have been studied and comparedto a glycoluril linked tweezer. The effects of spacers on tweezer binding of diamine guestsand helicity induction by chiral guests have been investigated (Paper IV). Connectionsbetween spacer flexibility and host-guest binding strength have been established. The structuralproperties of the stiff-stilbene spaced tweezer made it particularly susceptible to helicityinduction by both monotopic and bitopic chiral guests. Finally, the possibility of photochemicalbite-size variation of tweezers with photoswitchable spacers has been assessed. Initial studieshave shown that photoisomerization of the tweezers is possible without photochemicaldecomposition. Conformational analyses indicate that isomerization should impact bindingcharacteristics of the tweezers to a significant extent (Paper V).

Keywords: Bisporphyrin tweezers, peptidomimetics, host-guest systems, conformationalanalysis, supramolecular chemistry, photochemistry

Magnus Blom, Department of Chemistry - BMC, Synthetical Organic Chemistry, Box 576,Uppsala University, SE-75123 Uppsala, Sweden.

© Magnus Blom 2015

ISSN 1651-6214ISBN 978-91-554-9431-5urn:nbn:se:uu:diva-267845 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-267845)

Happiness can be found, even in the darkest of times, if one only remembers to turn on the light.

-Steve Kloves

Till Lisa

List of Papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals.

I Karlsson, C., Blom, M., Johansson M., Jansson, M. A., Scifo,

E., Karlén, A., Govender, T., Gogoll, A. Phototriggerable pep-tidomimetics for the inhibition of Mycobacterium tuberculo-sis ribonucleotide reductase by targeting protein-protein binding. Org. Biomol. Chem. 2015, 13, 2612-2621.

II Blom, M., Huang, H., Gogoll, A. Synthesis and characteriza-tion of photoswitchable stiff-stilbene based amino acid de-rivatives. Manuscript.

III Blom, M., Huang, H., Gogoll, A. Photoswitchable pep-tidomimetics with a stiff-stilbene chromophore for inhibi-tion of Mycobacterium tuberculosis RNR. Preliminary Manu-script.

IV Blom, M., Norrehed, S., Andersson, C.-H., Huang, H., Light, M., E., Bergquist, J., Grennberg, H., Gogoll, A. Synthesis and properties of bis-porphyrin molecular tweezers: Effects of spacer flexibility on binding and supramolecular chirogene-sis. Accepted.

V Blom, M., Olsson, K. S., Norrehed, S., Andersson, C.-H., Grennberg, H., Gogoll, A. Photomodulable bis-porphyrin molecular tweezers as dynamic host systems for diamine guests. Preliminary Manuscript.

VI Appendix

Reprints were made with permission from the respective publishers.

Contribution report

The author wishes to clarify his contribution to the research presented in the present thesis I Contributed to the planning of the research problem and performed

control synthesis experiments for characterization of the heptapep-tide derivatives. Prepared samples for the binding studies of the short peptide analogues, and quantified the photochemical conversion of all peptidomimetics. Wrote the first draft of the paper and contribut-ed significantly to writing and revision of the final manuscript.

II Contributed significantly to the project layout. Planned, screened and designed synthetic routes to the stiff-stilbene amino acid derivatives. Performed a significant part of the synthetic work and characteriza-tion of new compounds. Carried out all photochemical studies. Wrote the first draft of the paper and contributed to writing of the fi-nal manuscript.

III Took part in the planning and formulation of the research problem. Planned and contributed to the conformational analyses. Performed the ligand epitope mapping and binding studies for all model com-pounds. Wrote the first draft of the manuscript.

IV Performed a significant part of the synthetic work and characteriza-tion of the tweezers. Took part in the interpretation of the conforma-tional analyses. Planned and carried out all UV-binding studies, and performed 1H NMR complexation studies with the stiff-stilbene tweezer. Contributed to the planning of the chiral recognition exper-iments, synthesized the chiral guest molecules and performed all the CD studies. Contributed significantly to writing of the final manu-script.

V Performed a significant part of the experimental work. Took part in the interpretation of the conformational analyses. Carried out all pho-tochemical experiments and related UV and 1H NMR studies. Con-tributed to writing the manuscript.

Contents

1. Svensk populärvetenskaplig sammanfattning ........................................... 11

2. Introduction ............................................................................................... 13 2.1 Light-induced alteration of chemical structure ................................... 13

2.1.1 Photochemistry ........................................................................... 13 2.1.2 Photomodulable biological systems ............................................ 15 2.1.3 Photomodulable conformational switches .................................. 16

2.2 Molecular recognition and host-guest systems ................................... 18 2.2.1 Peptides and peptidomimetics as guests ..................................... 18 2.2.2 Molecular tools as hosts .............................................................. 20

3. This thesis ................................................................................................. 22 3.1 Photomodulable peptidomimetics.................................................. 22 3.2 Photomodulable bis-porphyrin tweezers ....................................... 23 3.3 Objectives of the present study ...................................................... 23

4. Overview of employed methods ............................................................... 24 4.1 Solid phase peptide synthesis ............................................................. 24 4.2 Conformational analysis ..................................................................... 25

4.2.1 Circular Dichroism Spectroscopy ............................................... 25 4.2.2 X-ray diffraction crystallography ............................................... 25 4.2.3 Nuclear Magnetic Resonance Spectroscopy ............................... 26 4.2.4 Computational Methods .............................................................. 27

4.3 Determination of binding constants in protein-ligand interactions and supramolecular host-guest systems ................................................... 28

4.3.1Saturation transfer difference....................................................... 29 4.3.2 Fluorescence polarization ........................................................... 30

5. Synthesis of photomodulable conformational switches for host-guest systems .......................................................................................................... 31

5.1 Synthetic pathways to stiff-stilbene conformational switches ........... 32 5.2 Conclusions ........................................................................................ 35

6. Photoregulated inhibition of Mycobacterium Ribonucleotide Reductase (RNR) (Papers I and III) ............................................................................... 36

6.1 Design of photomodulable peptidomimetics based on the C-terminal of the R2 subunit of RNR .......................................................... 37

6.1.1 Design of more drug-like heptapeptide analogues ...................... 38 6.1.2 Synthesis of photomodulable peptidomimetics .......................... 40 6.1.3 Conformational analysis by NMR spectroscopy ........................ 41 6.1.4 Binding studies of peptidomimetics ........................................... 41

6.2 Conclusions ........................................................................................ 42

7. Photomodulable units as linkers for bisporphyrin molecular tweezer hosts (Papers IV and V) ................................................................................ 44

7.1 Impact of linkers on tweezer characteristics (Paper IV) .................... 45 7.1.1 Conformational analysis of linker and tweezer geometries ........ 46 7.1.2 Synthesis of bisporphyrin tweezers ............................................ 50 7.1.3 Photostability of bisporphyrin linkers ......................................... 50 7.1.4 Isomerization of tweezers ........................................................... 51 7.1.5 Binding studies with diamine guests (Paper IV and V) .............. 51 7.1.6 Chiral guest induced helicity ...................................................... 53

7.2 Conclusions ........................................................................................ 54

8. Outlook ..................................................................................................... 55

9. Acknowledgements ................................................................................... 56

10. References ............................................................................................... 58

Abbreviations

Boc tert-Butoxycarbonyl CD Circular Dichroism DCM Dichloromethane DHP Dihydrophenanthrene DIPEA N,N-diisopropylethylamine DMF Dimethylformamide DMSO Dimethylsulfoxide DNA Deoxyribonucleic Acid ECCD Excition Coupled Circular Dichroism Fmoc 9-Fluorenylmethyloxy carbonyl FP Fluorescence Polarization HBTU N,N,N’,N’-Tetramethyl-O-(1H-benzotriazol-1-yl)uronium

hexafluorophosphate HOMO Highest Occupied Molecular Orbital HPLC High Performance Liquid Chromatography LUMO Lowest Unoccupied Molecular Orbital MALDI Matrix Assisted Laser Desorption/Ionization MCMM Monte Carlo Molecular Mechanics MM Molecular Mechanics MS Mass Spectrometry Mtb Mycobacterium tuberculosis NMR Nuclear Magnetic Resonance NOESY Nuclear Overhauser Effect Spectroscopy Ppm Parts per million RNR Ribonucleotide Reductase SPPS Solid Phase Peptide Synthesis STD Saturation Transfer Difference TOCSY Total Correlation Spectroscopy

The natural amino acids

Aspartic acid (D)Asp

Glutamate (E)Glu

O–

OO

O–

N H 3+

O–

O

O–

O

N H 3+

Histidine (H)His

Arginine (R)Arg

Lysine (K)Lys

O–

O

N+H3

N H3+

O–

N H 2+

NH 2 NH

O

N H3+

O–

N +H

NH

O

N H3+

Glycine (G)Gly

Alanine (A)Ala

Valine (V)Val

Leucine (L)Leu

Isoleucine (I)Ile

O–

O

N H 3+

O–

O

N H 3+

O–

O

NH 3+

O–

O

NH3+

O–

O

H

NH3+

Methionine (M)Met

Phenylalanine (F)Phe

Tryptophan (W)Trp

Proline (P)Pro

O–

O

N H 2+

O–

O

NH

NH 3+

O–

O

NH3+

O–

O

S

NH 3+

Serine (S)Ser

Threonine (T)Thr

Cysteine (C)Cys

Tyrosine (Y)Tyr

Asparagine (N)Asn

Glutamine (Q)Gln

O–

OO

NH2

NH3+

O–

N H 2

O

O

N H3+

O–

O

OHNH3

+O–

O

SH

NH 3+

O–

OOH

N H 3+

O–

O

OH

N H 3+

1. Svensk populärvetenskaplig sammanfattning

Modulering av molekylär igenkänning I princip alla kroppens viktiga processer kontrolleras av interaktioner mellan proteiner och olika typer av bindande molekyler (ligander). Den här typen av interaktioner brukar gå under benämningen värd-gäst system där en (oftast) större molekyl binder mindre molekyler. Dessa processer kan ske på ett spe-cifikt sätt i kroppen för att proteiner och ligander ”känner igen varandra”, och den igenkänningen beror bland annat på kompatibiliteten mellan respek-tive molekyls tredimensionella struktur. Den här avhandlingen handlar om hur man kan påverka den molekylära igenkänningen i olika värd-gäst system genom att reglera värdens eller gästens struktur med hjälp av ljus. För att genomföra detta använder vi oss av små ljuskänsliga molekyler (switchar) som kan växla mellan ett öppet och ett stängt läge. Genom att bygga in en switch i våra värd- eller gästmolekyler, kan vi kontrollera deras tredimens-ionella strukturer genom belysning med ljus av olika våglängder.

Figur 1 Schematisk figur av ljusreglerad molekylär igenkänning i ett värd-gäst sy-stem där en switch ingår i gästmolekylens struktur. Belysning orsakar strukturför-ändring hos switchen som växlar från ett öppet läge (lila) till ett stängt läge (orange).

Det finns många vardagliga exempel där växelverkan mellan ljus och ma-teria leder till förändrad struktur och kemisk sammansättning. Uppbyggnad och nedbrytning av ozon i den övre atmosfären är en ljusreglerad process som är livsviktig för vår överlevnad. Fotosyntes är ett annat exempel på en

11

12

process där växter med hjälp av ljus omvandlar koldioxid i luften till bio-massa.

I den första delen av avhandlingen undersöker vi effekten av en inbyggd switch i kända peptider som binder till enzymet Ribonukleotid reduktas och hämmar dess aktivitet. Enzymet sköter bland annat uppbyggnad och reparat-ion av DNA och är identifierat som en målreceptor för antibakteriella läke-medel. Vi har visat att belysning av switchen kan förändra peptidernas struk-tur och på så sätt påverka hur bra den ”känns igen” av enzymet. Vi har också utvecklat en ny och bättre switch som leder till större strukturförändringar, och som är lättare att reglera med ljus av olika våglängder.

Figur 2 Representation av en molekylär pincett med en inbyggd ljuskänslig switch som genom belysning kan begränsa rörligheten hos en flexibel gästmolekyl.

Avhandlingens andra del handlar om hur vi framställer funktionella värdmolekyler (molekylära verktyg) som kan hjälpa till vid strukturanalys av små flexibla molekyler. En god kännedom om molekylers struktur är mycket viktigt, särskilt inom läkemedelsutveckling, där till exempel spegelbilder av samma kirala substans kan ha helt olika effekt i kontakt med kroppens prote-inreceptorer. I normala fall är sådana flexibla molekyler svåra att karaktäri-sera då deras rörlighet är allt för snabb för att hinna detekteras med vanliga spektroskopiska analysmetoder. Våra verktyg är utformade som små pincet-ter som kan fånga upp de rörliga molekylerna och begränsa deras rörlighet, så att detaljerade strukturanalyser kan genomföras. Med en inbyggd switch kan avståndet mellan pincettens sidoväggar varieras. På så sätt kan man ex-empelvis gynna igenkänning för gäster av viss storlek eller påverka den in-bundna gästens tredimensionella form. Beroende på vilken switch som byggs in, kan verktygens egenskaper påverkas. Exempelvis har vi utvecklat en molekylär pincett som kan binda en kiral molekyl och anpassa sin form efter dess spegelbild. Strukturförändringen hos verktyget ger då upphov till en tydlig signal som är relaterad till gästens struktur.

Ljus

2. Introduction

2.1 Light-induced alteration of chemical structure Interaction between matter and energy in the form of electromagnetic radia-tion is one of the fundamental processes that can modulate molecular struc-ture.

2.1.1 Photochemistry By absorbing a photon, a molecule can convert from its natural electronic ground state S0 to an exited state. An electronically excited molecule can then undergo reactions, which are not possible in the ground state. Many of the most essential processes in nature proceed via photochemically excited intermediates. The most recognizable example is photosynthesis, a light driven process of fundamental importance for our survival. Another example is the Chapman cycle of light mediated generation and breakdown of ozone in the upper atmosphere.1

Figure 1. Overview of the Chapman cycle of photo-initiated splitting of molecular oxygen into oxygen atoms (1) followed by generation of ozone (2) and its subse-quent photochemical breakdown (3).

There are many other light-driven processes in biological systems, involv-ing chromophoric proteins such as rhodopsin or various fluorescent pro-teins.2 Two photochemical processes of particular importance for humans are the pericyclic reaction of 7-dehydrocholesterol and subsequent thermal [1,7]-sigmatropic rearrangement into vitamin D3 (Figure 2),2-4 and the photoisom-erization of retinal involved in visual perception.

O2 2 O (1)UV

O2 + O O3 (2)UVO3 O2 + O (3)

13

Figure 2. Route for the biosynthesis of vitamin D3 (cholecalciferol) via photo trig-gered electrocyclic ring opening of 7-dehydrocholesterol and subsequent [1, 7]-sigmatropic rearrangement.

When a compound is in its low energy singlet ground state S0, its elec-trons have antiparallel spins. Absorption of a photon results in an electron transition to an excited state. There are many possible excited states, but the ones of most relevance in photochemistry are the first singlet and triplet states S1 and T1, where the electrons have antiparallel and parallel spins re-spectively (Figure 3).

Figure 3. Jablonski diagram of the electronic transitions between the singlet ground state S0 and the first singlet and triplet excited states S1 and T1 described as excita-tions from HOMO to LUMO. 1) Absorption; 2) Internal conversion (IC); 3) Fluo-rescence; 4) Inter system crossing (ISC); 5) ISC; 6) Phosphorescence. Solid lines represent radiative transitions whereas dashed lines depict non radiative transitions.

According to the Franck-Condon principle, electronic transitions occur without alteration of molecular structure or electronic spin state.5-6 Thus, a direct transition between S0 and T1 is prohibited because of the required in-version of electron spin. The triplet state T1 is instead attained via a spin inversion from the first singlet state S1 (intersystem crossing). Electronically excited molecules are high-energy species and thus they tend to quickly re-turn to lower energy states either by photochemical alterations (e.g. isomeri-zation) or radiative processes (fluorescence, phosphorescence) and non-radiative release of heat (Figure 3).

UV

OH

OHOH

CH3

CH3

S1

S0

T1

E

1 2 3

4

5 6

HOMO

LUMO

14

2.1.2 Photomodulable biological systems There are some cases where photomodulation has been used in vivo. Photo-therapy of jaundice or hyperbilirubinemia in new born infants is one such example. Jaundice is a condition caused by an over accumulation of biliru-bin, which is toxic in high concentrations.7 (Z,Z)-Bilirubin is formed enzy-matically by cleavage of the porphyrin ring of heme in red blood cells (Figure 4).8

Figure 4. Top left: Neonatal phototherapy of jaundice; Top right: Intramolecular H-bonding in (Z,Z) bilirubin; Bottom: Enzymatic cleavage of a heme group to form bilirubin, and subsequent photoisomerization.

Due to extensive intramolecular hydrogen bonding, (Z,Z)-bilirubin is very lipophilic, and it binds tightly to proteins like serum albumin. These attrib-utes in combination make it essentially unexcretable in the human body. Normally, conjugation with glucuronic acid bypasses this issue, but in in-fants the activity of the necessary enzyme is low. If left untreated, harmful levels of bilirubin can accumulate with increased risk of brain damage as a consequence. However, phototherapy with visible blue light can effectively isomerize the insoluble (Z,Z) isomer into the (E,Z) form which is more easily excreted.

15

2.1.3 Photomodulable conformational switches A way of accessing photochemical regulation of a property in a system is to incorporate a photo receptive molecule. Reversibly photoswitchable small molecules have found a wide field of potential applications particularly in the cross section between materials and life sciences.9-13 This type of com-pound has been known since the end of the 19th century and their photo-chemical characteristics have been studied for over 50 years. One of the earliest examples of the concept was the reversible change of color in dyes, which was observed during the mid-19th century.14 The term photochromism was later given to this phenomenon by Hirschberg in the 1950s.15 The vari-ous types of photoswitches are typically classified according to the type of chemical change that occurs upon irradiation. The most common types un-dergo either intramolecular ring opening/closing or isomerization reactions of double bonds. Herein we focus on switches belonging to the latter catego-ry. Our approach is to use conformational switches to alter steric bulk by means of photochemical isomerization of a central double bond. For this purpose we want to achieve maximal structural difference between pho-toisomers.

Figure 5. Chromophores used as photoswitches/triggers: I) X = CH, stilbene; (X = N, azobenzene; II) thioaurone (hemithioindigo); III) enediyne; IV) stiff-stilbene.

Some common examples from this class of switches are azobenzene, thi-oaurone and stilbene.16 Out of these, the flexible azobenzenes stand out as the by far most frequently used.16 The planar E-isomer is thermally more stable than the skewed Z-isomer, which will revert back to the more stable form spontaneously.17 The half-life of the Z-isomer depends on factors such as solvent, pH and double bond substituents and can vary from tens of milli-seconds to a few days at room temperature.18-21 The N=N double bond of azobenzenes is known to be chemically unstable towards reducing agents like thiols.22-23

Stilbenes are analogous to azobenzenes with the exception of the central C=C double bond. Despite their structural similarities, stilbenes display some key differences from azobenzenes. Stilbenes do not exhibit the same thermal instability of the Z-isomers as do azobenzenes. The chemical stabil-

I

II

III

IV

X = C, N

XX

XX

S

O

O

S

UV

UV/Vis

Vis

Vis

UV

UV

UV

UV/Vis

16

ity towards reducing agents is also superior to that of azobenzenes.24 A po-tential issue is the possibility of photochemical formation of dihydrophenan-threne (DHP) from the Z-isomer (Figure 6). However, the extent of this side reaction can be restricted, as DHP can revert back to the Z-isomer both pho-tochemically and thermally under non-oxidative conditions.

Figure 6. Possible side reactions during irradiation of stilbene and enediyne deriva-tives. Formation of DHP from Z-stilbene (top); photochemical or thermal formation of 1, 4 biradical intermediate via Bergman Cyclization (bottom).

Insertion of acetylene units between the olefin and the aryl rings of stil-bene yields a diaryl enediyne. Enediynes in general are mostly known for their potent anticancer and antibacterial properties which arise from their ability to form 1,4 biradical aromatic intermediates thermally or photochem-ically via Bergman cyclization (BC) (Figure 6).25

However, acyclic aryl substituted enediynes display a high degree of thermal and photochemical stability towards BC, and therefore their potential as photomodulable com-pounds has received some attention.26-28 Diaryl enediyne has similar photo-chemical characteristics as stilbene29and the Z-isomer is also thermally sta-ble. The large difference in end-to-end distance between photoisomers, makes it a promising candidate for a conformational switch.

Thioaurone or (hemithioindigo) based switches are more structurally rigid compared to azobenzene and stilbene derivatives. For thioaurones, it is the Z-isomer that is thermodynamically more stable, but owing to the unstrained nature of both isomers, the E-isomer of thioaurones is more thermally stable Z-azobenzenes.30-32 Photochemical stability is of thioaurones in organic sol-vents is also well documented.33,34 Nevertheless, their applicability as con-formational switches in peptidomimetics is limited by their propensity to degrade under standard peptide synthesis conditions.35-37

Annulating the stilbene motif with two cyclopentane rings yields a tetra-hydro-biindenylidene (stiff-stilbene), and results in significant changes of two important factors. Firstly, rotation of the aromatic rings is restricted, making the chromophore more rigid. Secondly, because of the tetra-substituted C=C bond, the UV-Vis absorption bands of both isomers are red shifted and more separated when compared to native stilbene.38,39 Stiff-

R

R.

.

R

R

R

R

DHP

HH

[H]UVor heat

280 nm

360 nmor heat

[O2]

17

stilbenes have previously been used in several molecular assemblies and display remarkable photochemical and thermal stability.

Table 1. Characteristics of photomodulable conformational switches.

Property Chromophore Azoben-

zene Stilbene Enediyne Thioaurone Stiff-

stilbene Typical λ (E→Z/ Z→E) Thermally reversible Chemical stability Photostability Divergent isomers

350/450 Yes +/- +

+/-

300/280 No +

+/- +/-

340-400 No +/- +/- +

480/400 Yes

- + +

300/360 No + + +

+ Suitable properties, +/- tolerable properties, - inferior properties

We have chosen to incorporate stilbene, enediyne and stiff-stilbene chromo-phores into our photomodulable systems because of their thermal and chem-ical stabilities.

2.2 Molecular recognition and host-guest systems Molecular recognition describes non covalent interactions between molecules, and includes host-guest chemistry ranging from relatively small synthetic assemblies to large protein-ligand complexes in biological systems. A host-guest system can essentially be any complex of a larger molecule (host) that binds smaller (guest) molecules. Research in the field of molecular recognition aims for better understanding of binding energies and selectivity in such systems.40 Supramolecular chemistry is a subdivision of this field and focuses on non-covalent interactions in synthetic molecular assemblies. The field has evolved from the studies of crown ether chemistry and self organization of molecular assemblies like micelles and mem-branes.41-43 In comparison to covalent bonds, individual non-covalent bind-ing interactions are usually weaker in strength. Some of the most common are ionic bonds (100-350 kJ/mol), metal-ligand (50-300 kJ/mol), ion-dipole (50-200 kJ/mol), dipole-dipole (5-50 kJ/mol), hydrogen bonds (2-50 kJ/mol), aromatic or π-π interactions (0-50 kJ/mol) and van der Waals interactions (<5 kJ/mol).44,45 However, several non-covalent binding interactions in tan-dem can result in significantly stronger binding forces.

2.2.1 Peptides and peptidomimetics as guests Peptides are essentially sequences consisting of amino acids that are coupled together by amide bonds. Their involvement as ligands (guests) in nearly all biological processes46 makes them interesting as models or lead compounds in the development of new drugs. Peptide chemistry had its earliest break-

18

through with the first reported synthesis of hippuric acid by Theodor Curtius in the late 19th century.47 His development of the azide coupling reaction was paramount for the field, and it remained in use for several decades.48

Figure 7. Synthesis of hippuric acid by Theodor Curtius in 1881, and synthesis of the first free dipeptide by Emil Fischer in 1901.

The hydrolytic ring opening of piperazine-2,5-dione by Emil Fischer in 1901 is regarded as the first synthesis of a peptide.49 He too contributed with pioneering developments in peptide chemistry procedures. One such exam-ple is the use of acid chlorides for amide bond formation.50At an early stage there was a realization of the correlation between the structure of peptides and their biological activity. One important example of this connection came from Vigneaud via the synthesis and structural characterization of the signal substance oxytocin, which was the first peptide sold as a commercial drug.51 Elucidation of the conformational behavior of peptides is essential in order to understand the different aspects of biological molecular recognition such as binding to receptors, antigens and enzymes. However, the majority of short peptides interconvert rapidly between populations of conformations. In some cases, the population of a group of similar conformations is energeti-cally favored, and this leads to folding into a more defined secondary struc-ture (Figure 8).

Figure 8. Examples of common peptide secondary structures: alpha helix (left) and beta sheet (right).

The applicability of natural peptides as orally administered drugs is lim-ited because of poor gastrointestinal uptake and permeability, as well as en-zymatic degradation. This has led to the development of peptidomimetics,

Glycyl glycine

HCl

Hippuric acid

O

OHNH

OO

OHNH2

O

Cl –O

O

OHNH

N+H3O

O

N H

NH

N3 +

19

which mimic the biological effect of natural peptides but have better phar-macological properties and longer metabolic half-life. When designing pep-tidomimetics, the amino acid residues deemed non-essential for the specific molecular recognition are replaced with non-peptidic units. These can help to improve the stability and induce conformational preference for a certain secondary structure.

2.2.2 Molecular tools as hosts Any supramolecular assembly that performs a specific task can be regarded as a supramolecular tool. The first “molecular tweezer” was reported in the late 70s and referred to a bis-caffeine derivative (host) capable of forming pincer like 1:1 sandwich complexes with aromatic guests.52 The predominant non-covalent intermolecular force holding this complex together is π-π inter-actions between the aromatic units of the host’s sidewalls and the guest.

Figure 9. The first reported “molecular tweezer”-type host-guest system by Whit-lock from 1978. 52

Generally, molecular tweezers consist of two binding sites (sidewalls) co-valently joined by a spacer unit (linker). Together, the spacer and the side-walls form a binding cavity with three open faces. Since their first report, a wide range of molecular tweezers have been described with various combi-nations of binding sites and linkers targeting different types of guest mole-cules.53 Porphyrins are a group of biologically abundant heterocyclic com-pounds consisting of four pyrrolic units connected by sp2 carbon bridges (meso-carbons). Generally, their complexes with different metals are used as components of supramolecular systems (Figure 10). Metallated porphyrins are strong electron acceptors capable of coordinating ligands with donor atoms like oxygen and nitrogen. For this reason, tweezers with metallated porphyrin sidewalls have been used extensively as hosts for ditopic binding of guest molecules of various sizes.54 Due to their versatility as hosts, bisporphyrin tweezers have been applied as tools for a variety of tasks such as size selective binding of guests55,56 and chiral recognition.57-59

Bis-caffeinemolecular tweezer

Aromatic guest

Linker

N

N

N

O

N

ON

N

N

O

N

OOH

O

O–

OH

20

Figure 10. Schematic representations of: I) a bis-porphyrin molecular tweezer with bound guest; II) a porphyrin unit indicating available functionalization sites.

The flexibility and size of the host cavity (bite size) depends highly on the structural properties of the linker. Consequently, characteristics such as guest size selectivity can be induced by alteration of the linker structure. Binding properties and conformational preferences of the tweezers can also be regu-lated by the choice of coordinated metal and by substitution on the porphyrin rings.

21

3. This thesis

Introduction of a photomodulable unit into a peptidomimetic would make it possible to affect its overall conformation by an external stimulus (light). This notion provides the chemist with a light switch for regulating biological processes.60-62 Integrating a photo-triggered conformational switch as a link-er opens the possibility of controlled variation of the host cavity in a tweezer by an external stimulus. Such a complex would offer even larger binding adaptability than conventional tweezers, and might even allow for light modulated guest binding and release including investigation of binding dy-namics.63

3.1 Photomodulable peptidomimetics Photoswitches have been used extensively to characterize biological pro-cesses such as peptide and protein folding. Although most examples involve the azobenzene chromophore there are a number of cases where other alter-natives are discussed. Previous studies from our group have covered pho-toswitchable β-hairpin mimetics containing the stilbene chromophore. Cyclic peptides converted between random coil and β-hairpin conformations upon irradiation with UV-light.64 The catalytic activity of a dimeric artificial hy-drolase was modulated through incorporation of a stilbene switch in the turn region between two α-helical segments (Figure 11).

Figure 11. Illustration of the photomoduation of the tertiary structure of an artificial hydrolase by isomerization of an incorporated stilbene based amino acid ( ).

Photoisomerization of the switch caused conformational changes affect-ing also the tertiary structure of the hydrolase and thus reduced its catalytic effect.37

22

3.2 Photomodulable bis-porphyrin tweezers Some examples of photoswitchable bis-porphyrin tweezers have been re-ported in the literature. However, these complexes employ rather flexible azobenzene65 or stilbene66 as the switchable unit. Even with the apparent flexibility of these tweezers, differences in binding constants for various guest molecules to the E or Z photoisomer, respectively have been observed. Incorporation of a more rigid conformational or higher structural discrepan-cy should likely result in even more pronounced differences in binding char-acteristics. Furthermore, binding dynamics for photoswitching in the pres-ence of bound guests have so far not been investigated.

3.3 Objectives of the present study

Photomodulable guests • Evaluation of the effect of photoisomerization of photomodulable

amino acids on the affinity of oligopeptides for Mycobacterium tu-berculosis RNR.

• Design and synthesis of a new stiff stilbene amino acid with im-proved photochemical properties and more pronounced structural divergence between E and Z isomers.

Photomodulable hosts • Design, structural characterization, and binding studies of novel

bisporphyrin tweezers with photomodulable stiff-stilbene and ene-diyne spacers.

• Explore the impact of photochemical bite size alteration in a bisporphyrin tweezer on its binding properties.

23

24

4. Overview of employed methods

4.1 Solid phase peptide synthesis Synthesis of peptides involves a series of successive reactions, which couple amino acids together into the desired sequence. If performed in solution phase, the process is dependent on isolation and purification of intermediates before coupling of each new amino acid. When R. B. Merrifield introduced peptide synthesis on solid phase (SPPS) in the early 1960s, it was a ground-breaking development, which simplified the synthetic procedure considera-bly (Figure 12).67 SPPS is performed on a solid, functionalized insoluble polymer (resin), offering a number of advantages as compared to traditional liquid phase methods. As the growing peptide chain is covalently linked to the resin during synthesis, time-consuming purification steps between cou-plings are superfluous. The method also allows for use of excess reagents and amino acid in order to maximize yields.

Figure 12. A schematic depiction of the standard procedures involved in Fmoc solid phase peptide synthesis (SPPS).

4.2 Conformational analysis Conformation refers to the three dimensional molecular structure of a com-pound, that may be varied by rotation about single bonds. Characterization of the structural preferences of peptides and supramolecular complexes often involves the application of a broad array of different spectroscopic and com-putational methods. For peptides with several interconverting conformers, spectroscopic analysis can sometimes be sufficient to determine overall ste-ric arrangements. However, the lifetime of one or more of these conformers needs to be long enough for detection on the spectroscopic time scale. The type of supramolecular complexes covered in this thesis, is generally more ordered and their conformations can be characterized by spectroscopic methods to a larger extent. This section provides a short depiction of the employed methods in this thesis.

4.2.1 Circular Dichroism Spectroscopy Circular dichroism is a highly sensitive chiroptical technique, which can be used to obtain information about the conformational behavior of chiral mol-ecules. For peptides it can be applicable as an indicator for secondary struc-tures since they have characteristic CD-signatures. For example, alpha heli-ces typically give a positive signal at 190 nm and two negative bands be-tween 208-222 nm whereas beta-hairpins result in positive bands at 195 nm and a negative band between 217-222 nm. Random coil structures give a negative signal response at 195 nm and a weak positive one at 210-222 nm.68,69 The fact that these characteristic signals appear at low UV-wavelengths is a significant drawback since high UV absorption in this re-gion is quite common among typical organic solvents.

Circular dichroism can also be used for stereochemical analysis of chiral guest molecules in bis-porphyrin supramolecular complexes (Paper IV). When a chiral substrate forms a complex with the tweezer, the porphyrin units may adopt an orientation resulting in helical chirality, thus producing a strong CD-signal. Importantly, when porphyrins constitute the chromophore, there is no problem using common organic solvents like DCM, since their characteristic absorption bands (Soret bands) appear around 400 nm.70

4.2.2 X-ray diffraction crystallography The most conclusive method for determination of molecular structure in terms of atom connectivity is X-ray crystallography. It relies on X-ray irra-diation of single crystals and subsequent analysis of the resulting diffraction pattern. Although accurate, the static solid state structures obtained by this technique do not necessarily represent solution state conformers of small flexible molecules such as peptides. Upon crystallization, peptide confor-mations are stabilized by intermolecular interaction forces, which are not

25

present to the same extent in solution phase.71 Obtaining single crystals of the sample is another issue that can hamper its applicability.

4.2.3 Nuclear Magnetic Resonance Spectroscopy Nuclear magnetic resonance (NMR) spectroscopy is by far the most versatile spectroscopic technique available for elucidation of chemical structure. This technique can obtain information about molecular structure on the atomic level by utilizing the magnetic properties of nuclei.72

Chemical shifts Chemical shift (δ) provides information about the electron density surround-ing a nucleus. This electron density is predominantly affected by the type of atoms that are bonded to or in the near vicinity of the observed nucleus. Hy-bridization, resonance and anisotropy effects amongst others also affect chemical shifts.72 In supramolecular bisporphyrin complexes the cyclic aro-matic sidewalls give rise to a strong anisotropy effect73 which drastically lowers the proton chemical shifts of the bound guest (Paper IV and V). Changes in chemical shift of the guest signals are directly related to their distance to the sidewalls, and can thus be used to provide information of guest orientation within the tweezer.

The amide proton chemical shifts (δNH) may provide insight regarding presence of intramolecular hydrogen bonding, which can be of significant relevance for the elucidation of overall peptide conformation.74 Intramolecu-lar hydrogen bonding amide protons typically are found in the δNH = 7-9 ppm region whereas signals at 6-7 ppm are indicative of hydrogen bonding with solvent. Amide proton chemical shifts also display a very prominent solvent dependence. Addition of a strong H-bond donor solvent like DMSO to a peptide solution in CHCl3 results in marginal changes (~0.2 ppm) for H-bonding amide proton shifts, whereas non-H-bonding protons exhibit a 10 times higher change in δNH.75,76 There is also a strong temperature depend-ence on δNH and therefore their temperature coefficients (ΔδNH/ΔT) can be used as an indicator for H-bonding (see Paper I). In polar aprotic solvents like DMSO, H-bonding amide protons display temperature coefficients <4.6 ppb/K while solvent exposed protons will give values >4.6 ppb/K.76,77

Scalar coupling constants Scalar coupling constants (J) are influenced by neighboring atoms, and are manifested as a splitting of NMR signals. By analyzing this signal splitting, one can obtain information about the chemical connectivity in a compound. The multiplicity of the splitting reveals the number of coupling nuclear spins, while the magnitude of the signal splitting is related to, e. g., their proximity and molecular geometry. Therefore, conformational parameters such as bond angles and distances between different nuclei can be derived

26

from the coupling constants. For example, the size of vicinal 3JNH, Hα cou-pling constants can be used to predict peptide conformation. The Karplus relation describes the relation between the (3JNH, Hα) and the dihedral angle θ, and is an empirical measure for determining conformational preferences.78,79 For example, alpha helices tend to have vicinal coupling constants of about 4 Hz, whereas random coils and beta sheets have values of 3JNH, Hα >7.5 Hz and 6-7 Hz, respectively.

Nuclear Overhauser effects Nuclear Overhauser Effects (NOE:s) can provide information about through space interactions between nuclei in close proximity to one another. Thus, they can be very useful when determining the three-dimensional structure of a molecule. The effect (NOE) arises from dipolar cross relaxation through space between two nuclei. Consequently the effect is highly distance de-pendent, and can usually be observed between protons within a distance of up to 3.5 Å.80 Apart from distance in space, the NOE is also affected by the degree of molecular motion in a molecule. However, it is important to re-member that NMR has a longer timescale than other spectroscopic methods. Therefore, the observed parameters are usually average values for different conformations in equilibrium.

4.2.4 Computational Methods Computational methods for the elucidation of conformational preferences can be a valuable compliment to experimental data obtained from spectro-scopic techniques. Simulations can offer a rationale of observed spectroscop-ic results and provide an extra dimension to any given experimental study. Calculations also open up the possibility for rational design and selections of compound candidates in a study (Papers I and IV). However, careful con-sideration has to be taken about the quality of the input data during evalua-tion of data from computational studies. This is to assure that the input data, e.g. the applied force field parameters, solvent models and basis sets are of sufficient quality and suitability to describe the studied system.

Molecular mechanics Molecular mechanics (MM) or force field methods is a commonly used technique for conformational analysis of organic molecules. Force field methods calculate the energy of a system as a function of the positions of the atomic nuclei in a molecule. In terms of computational cost, MM is a rela-tively economic method. This makes it possible to perform calculations on larger molecular systems with many atoms like peptides or supramolecular complexes, which can be inaccessible for more sophisticated methods (see papers I, III, IV and V).81 Simulations in solution phase with varied solvents are also readily available, which makes the output data more likely to reflect experimental conditions.

27

Monte Carlo conformational search A Monte Carlo (MC) conformational search in combination with molecular mechanics is commonly used for analysis of peptide conformations. Monte Carlo simulations generate configurations of a molecular system by making randomized changes of torsion angles at the different atomic positions. Each new arrangement is then energy minimized with MM, using predefined crite-ria for standard values of bond angles and lengths. Deviation from these criteria results in higher energies. Each new conformation within the preset energy limits is kept and those exceeding them are discarded. Every retained conformation serves as the starting point for the following iteration. This process is repeated until a pre-set number of iterations is reached or until no new conformations are obtained.81 The output, i.e. a series of conformations, is then analyzed in terms of energy-dependent probability via the Boltzmann equation. This may be combined with so called clustering where populations of similar conformations are grouped together.82

4.3 Determination of binding constants in protein-ligand interactions and supramolecular host-guest systems Binding interactions between two species A and B are quantified by the binding constant K, which is a thermodynamic property related to the free energy (G) of the binding process through the Gibbs equation. The free en-ergy can also be related to entropy (S) and enthalpy (H).

A + B 𝐾𝐾⇌ 𝐴𝐴𝐴𝐴

𝐾𝐾 = [𝐴𝐴𝐴𝐴]

[𝐴𝐴][𝐴𝐴]

𝛥𝛥𝛥𝛥 = −𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝐾𝐾

𝛥𝛥𝛥𝛥 = 𝛥𝛥𝐻𝐻 − 𝑅𝑅𝛥𝛥𝑇𝑇

There are many different procedures for the characterization of binding interactions, but it typically involves spectroscopic measurement of the change of a given parameter as a function of the concentration of either spe-cies A or B. Here follows a brief overview of the different techniques used in this thesis. The most frequently used method for obtaining binding constants for supramolecular systems is through a titration experiment by gradual ad-dition of the guest molecule to a solution of host. For these studies, changes

28

in the UV-absorption spectrum or 1H NMR chemical shifts were recorded as a function of host-guest ratio variation. Binding constants were then calcu-lated via non-linear regression analysis based on iterative fitting (Paper IV).83

4.3.1Saturation transfer difference Saturation transfer difference (STD) is an NMR based technique, which is very useful for screening the binding potency of ligands to proteins.84-85

Figure 13. Binding equilibria between protein and ligands.

The technique relies on the fact that magnetization perturbation (satura-tion) introduced into proteins spreads fast throughout the entire structure, including any bound ligands. When the ligand dissociates from the binding site, it retains some of the perturbation. If an NMR spectrum is recorded under these conditions, and compared to a spectrum without perturbation (an off resonance reference spectrum), the degree of retained saturation can be visualized and information about which protons are in closest contact with the protein (Figure 14).

Figure 14. A general scheme of the STD-NMR experiment involving a protein binding ligand (turquoise) and non-binders (orange) A) Off resonance spectrum; B) on resonance; C) difference spectrum.

This makes the technique especially useful for binding epitope mapping during the initial screening process when developing new ligands or inhibi-tors for proteins (Paper III). However, STD is limited to mid to weak affini-ty binding ligands in the 10-3-10-8 M range. If the ligand binds too tightly it will behave as a part of the protein, yielding no change in the difference

29

spectrum, whereas a too weak binder would not receive sufficient saturation and thus appear as a non-binder.

4.3.2 Fluorescence polarization Fluorescence polarization (FP) is a common and highly sensitive method used for studies of protein-ligand interactions.86 Generally, FP-methods are designed as competitive equilibrium binding assays using a fluorescence labelled probe. When the probe is bound to the enzyme, it produces a signif-icant fluorescence polarization signal. However, when the probe is displaced by a competitive ligand it results in diminished FP. By plotting the degree of probe displacement against the ligand concentration, binding constants can be calculated (Paper I).

30

5. Synthesis of photomodulable conformational switches for host-guest systems

To enable photomodulation of our host and guest systems there was a need to develop systematic methods to incorporate the photo responsive units into the peptides and tweezers. For this purpose, we designed stilbene and stiff-stilbene based amino acid derivatives 5.1 (Paper I) and 5.3 (Paper III), which could be integrated into peptides by means of standard SPPS method-ologies. Dicarboxylic linkers 5.2 and 5.4 were designed for coupling to amine functionalized porphyrin moieties (Paper IV) (Figure 15).

Figure 15. Conformational switches for peptidomimetics (left) and bisporphyrin tweezers (right).

Our group has previously reported the synthesis of the stilbene amino acid derivative by microwave assisted Heck couplings (Figure 16).35,64

31

Figure 16. Synthetic scheme for the formation of stilbene and enediyne backbones: a) Pd(OAc)2, (o-CH3C6H4)3P, Et3N, DMF, microwave, 120 °C, 30 min; b) 1,2-Z-dichloroethylene, Pd(PPh3)2Cl2, CuI, Et2NH, toluene, 0 °C, N2-atm, 48 h; c) NaOH, EtOH, reflux, 5 h.

The enediyne linker scaffold for bisporphyrin tweezer host was accessed via microwave assisted Sonogashira coupling which has been reported by Kosinki.87

5.1 Synthetic pathways to stiff-stilbene conformational switches

For the stiff-stilbene moieties, we opted for the reductive dimerization of carbonyl compounds, otherwise known as McMurry reaction. This reaction pathway makes use of low valent titanium as the active species, and this reagent can be generated in numerous ways.88 In our studies we have utilized the well-known system of TiCl4 and Zn (0), which has been reported for similar systems.38 The generally accepted mechanism of titanium mediated reductive carbonyl coupling involves formation of a titanium pinacolate intermediate via 1- or 2-electron transfer. This intermediate is then deoxy-genated to afford the final alkene species (Figure 17).89,90

Figure 17. Mechanism of reductive coupling of carbonyl compounds by 1- or 2 electron transfer.

32

This approach allowed for the use of highly accessible indanone carbox-ylic acid precursors.

Figure 18. Synthetic pathway for indanone carboxylic acids. 5.5b: i) AlCl3, NaCl, 180 °C, 1h, 42%; 5.6e: ii) dimethylcarbonate. H2SO4 (conc.), 80 °C, 17 h, 77 %. iii) AlCl3, CH3COCl, DCM, 0 °C – 60 °C, 4h, 73 % iv) 1) NaOH(aq), 95 °C, 10 min, HCl(aq). 2) sulfur, morpholine, reflux, 6 h, HCl(aq). 3) NaOH (50% in H2O), MeOH, reflux overnight, HCl (50% in H2O), 73 %. v) polyphosphoric acid, 110 °C, 50 min, 28 %.

Indanone derivatives with no (5.5b) or one (5.6e) methylene group sepa-rating the aromatic ring and the carboxyl group were synthesized via intra-molecular ring closing of their corresponding aryl propanoic acid derivatives (Figure 18).91 The moderate yields in the synthesis of 5.5b were predomi-nantly caused by complications during work up of the highly insoluble crude product. In the case of 5.6e, the Wilgerodt-Kindler reaction afforded a com-plex crude product mixture, which presented us with some challenges. For the reductive McMurry coupling, the indanone carboxylic acids 5.5b and 5.6e were first converted into their ethyl esters 5.5c and 5.6f (Figure 19). McMurry has previously discussed functional group compatibility under reductive coupling conditions in a review.88 Even though both carboxylic acids and esters were considered semi-compatible to these conditions, there are very few successful examples involving carboxylic acids found in the literature.

Figure 19. Outline for syntheses of stiff-stilbene core: i) HCl conc. (1 mol eq.) etha-nol, reflux 12 h; ii) TiCl4 (3 eq.), Zn powder (6 eq.), THF, reflux 12 h.

33

When we attempted to use 5.5b directly, only complex mixtures were ob-tained, with no product detectable by 1H NMR. The ethyl ester derivatives on the other hand survived even prolonged reaction times without decreased yields. For tweezer linker syntheses, the fraction of desired Z-isomer was enriched to (E:Z 1:1.1) by preparative irradiation of E-Z mixtures in DCM, with UV-light at 300 nm. Recrystallization from ethanol afforded pure E-isomer, and subsequent chromatographic separation of the mother liquor gave pure E and Z isomers (70% total yield). Hydrolysis of Z-5.5d with NaOH in ethanol gave the corresponding dicarboxylic acid 5.4 in 94% yield.

Figure 20. Synthetic route to stiff-stilbene tweezer linkers, and amino acid deriva-tives. i) NaOH, EtOH, reflux, 6 h, 94% ; ii) NaOH (1.2 eq.), TBAB (1 eq.), DCM:EtOH:H2O (1:2:0.3), reflux 50°C 12h, (E-5.5e, 67%); iii) (COCl)2 (10 eq.), DCM, r.t. 3 h (not isolated); iv) NaN3 (10 eq.), toluene:H2O (2:1), 0° C, 4 h (not isolated); v) tBuOH, toluene, reflux, 2h (E-5.3a, 45%).

A selective mono-hydrolysis of one of the ester groups was performed, in order to introduce the amide functionality. The poor solubility of diester E-5.5d in EtOH prompted us to use a three-phase solvent system. The starting material was first dissolved in a minimal amount of DCM and heated to re-flux before addition of preheated EtOH and the aqueous base. Addition of cold solvents to the DCM solution was avoided since it tended to cause pre-cipitation of the starting material. The proportion of water in the reaction mixture was kept at 9-10 %, and a phase transfer catalyst (TBAB or TEAB) was employed since this has been shown to facilitate selective mono-hydrolysis.92

The amide functionality was obtained via a Curtius rearrangement, con-verting the carboxylic acid into the corresponding carboxylic azide E-5.3g via treatment of acid chloride intermediate E-5.3f with NaN3. Upon heating, compound E-5.3g underwent thermal decomposition, forming an isocyanate, which in the presence of tBuOH rearranges into the Boc protected amide E-5.3a (Figure 21).

34

Figure 21. Mechanism for the Curtius rearrangement of the azide functionality of compound 5.3g into the Boc-protected amine 5.3h.

A one-pot procedure for the Curtius rearrangement, using DPPA as a more soluble source of azide was attempted; however, this only afforded the starting material 5e. The poor solubility of the monoacid in the reported sol-vent DME is a likely cause for this issue. An alternative route where the indanone ester and amide derivatives were prepared separately and then used in a subsequent McMurry cross coupling did not afford the desired product either. Although amides should be tolerated under reductive conditions88, McMurry hetero coupling reactions are known to have poor reproducibility and selectivity for cross coupling is highly dependent on the compatibility of two substrates. Thus, even though the sequential approach requires work up of the intermediates it provided reproducible and consistent results.

5.2 Conclusions A synthetic route to a novel conformational switch based on the stiff-stilbene chromophore has been developed. Reductive homo coupling of readily available indanone carboxylic acid starting precursors allows for facile varia-tion of substituent patterns. We have also demonstrated that introduction of the amino acid N-terminus is feasible via Curtius rearrangement. To expand this investigation, it would be interesting to further explore the possibilities of asymmetric formation of the stiff-stilbene core via other pathways like olefin metathesis.

35

6. Photoregulated inhibition of Mycobacterium Ribonucleotide Reductase (RNR) (Papers I and III)

Ribonucleotide reductase (RNR) is an enzyme composed of 2 dimeric subu-nits R1 and R2, and it is integral in the life cycle of the bacterium Mycobac-terium tuberculosis (Mtb). The enzyme catalyzes the reduction of ribonucle-otides into deoxy ribonucleotides and thus plays a central role in DNA syn-thesis and repair.93-95 Its catalytic function is dependent on the interplay be-tween the 2 R1 and R2 subunits that associate to form the active holoenzyme.

Figure 22. X-ray crystal structure of Salmonella typhimurium Ribonucleotide reduc-tase holoenzyme. The two monomers of the R1 subunit are colored red and gold and the two of the R2 subunit are colored dark and light blue.96

Designing an inhibitor for RNR can be done using several approaches. The most common one is to target the catalytically active site and thus block access to the natural substrate of the enzyme. An alternative, is to hinder the association of the two subunits and thus to disrupt formation of the active holoenzyme.97-101 For these studies we opted for the latter approach.

36

6.1 Design of photomodulable peptidomimetics based on the C-terminal of the R2 subunit of RNR To probe the possibility to photo regulate of the inhibitory potency of 6.1, (Figure 23) we attached the stilbene amino acid derivative 6.2 (Figure 24) (for synthesis see Figure 16 p. 32) directly onto the N-terminus of the pep-tide.

Figure 23. Structure of the Heptapeptide sequence 6.1 (Glu-Asp-Asp-Asp-Trp-Asp-Phe) derived from the native C-terminal of the R2 subunit of Mycobacterium tuber-culosis RNR.

We expected that the overall peptide conformation would be altered upon photoisomerization of the conformational switch, resulting in a change of affinity for the R2 binding site in the enzyme.

Figure 24. E-Z isomerization of phototriggerable stilbene 6.2 (left) and stiff-stilbene amino acid derivatives 6.3 and 6.4 (right).

In order to verify this assumption, a series of three heptapeptide deriva-tives 6.5 – 6.7 were designed with varying N-terminal functionalization of the stilbene moiety (Figure 25). Upon E-Z isomerization, mimetic 6.5 with its free charged ammonium group was anticipated to become more perturbed when compared to the natural heptapeptide by forming strong ionic interac-tions with the charged Asp-residues in the sequence. In the N-terminally acylated compound 6.6 only inter-strand hydrogen bonds are expected. Fi-nally, mimetic 6.7 with its bulky N-acylated Trp residue would exert larger sterical crowding in its Z-isomer compared to the more linear E-isomer. Be-cause of these N-terminal modifications, the Z-isomers were envisioned to be the less tight binders in all three cases.

O

O

O

ONH

O

O

NH

NH

NH

NH

NH

NH

NH2 COOH

COOHCOOH

COOH

COOH

COOH

37

Figure 25. Phototriggerable peptidomimetic analogues of the R2 C-terminal hep-tapeptide

6.1.1 Design of more drug-like heptapeptide analogues As the next step of the investigation, we wanted to shorten the heptapeptide sequence 6.1 in order to produce analogues of the R2 C-terminus with more drug-like properties. Size restriction would make the derivatives more con-venient to evaluate by computational analysis methods.102 We hypothesized that the overall conformation of the truncated sequences should also be more affected by isomerization of the switch. To verify this we wanted to compare conformational preferences of shortened analogues with the flexible stilbene switch to those with the more rigid stiff-stilbene switch. However, in order to design these truncated sequences it was first necessary to determine which residues that were the most important for retained affinity. By studying the crystal structure of RNR (2BQ1) one can observe that the binding pocket is lined with positively charged residues whereas the inside of the pocket is hydrophobic (Figure 26).96

Figure 26. (Left) X-ray crystal structure of the R2 C-terminal heptapeptide 6.1 in-side the R1-R2 association site. Amino acid side chains are colored after type; hy-drophobic: white, polar: cyan, positive: blue, negative: red, aromatic: pink, proline: pale green glycine: green. (Right) Structure and sequence for heptapeptide 6.1.

The C-terminal Phe is located in the vicinity of several aromatic sidechains as well as an Arg residue which have the possibility of forming both π-π and π-cation interactions. Nurbo et al. have previously stated that

38

interactions between Trp and Phe residues and the aromatic region in the binding site are important for binding. Removal of negatively charged Asp-residues led to incremental decrease in affinity.102 Consequently, one can conclude that Trp and Phe are most important for binding and the addition of negatively charged residues can improve the affinity. For computational practicality we restricted ourselves to sequences of at most 4 amino acid residues including the conformational switch. With these restrictions in place one can in principle vary the position of the conformational switch in six different ways (Figure 27):

Figure 27. (1) N-terminal switch, (2) switch between the Asp (D) and Trp (W) resi-dues, (3) switch between W and Phe (F) residues, (4) switch in C-terminal position, (5) replacing the aromatic W with switch, or (6) have the switch replace the F resi-due.

Alternative 3 was deemed unfavorable since it would bring the important Trp and Phe residues too far away from each other. Alternative 4 was also considered less interesting since the resulting peptidomimetics would differ substantially from the heptapeptide mimetics 6.5 – 6.7. Conformational searches of the candidates from the remaining groups 1, 2, 5 and 6 were per-formed by Monte Carlo molecular mechanics (MCMM). For Z-isomers with a folded geometry, an intramolecular hydrogen bond was most often found be-tween the C and N-terminal chains. Such hydrogen bonding is substantially less likely to occur in the corresponding E-isomer and therefore it was chosen as an indicator of the overall geometry. The results indicate that flexibility of the stilbene switch 6.2 results in less distinct conformational differences be-tween the peptidomimetic photoisomers compared to corresponding mimetics containing 6.3 and 6.4. It is also indicated that the extra CH2-groups in 6.4 introduce a degree of flexibility which increases the H-bonding probability in the E-isomers, compared to 6.3. However, the overall effect of the more rigid switch is larger structural diversity in the photoisomers (Figure 28).

D W F

1 2 3 45 6

4 - 7 Å 2.5 Å

39

Figure 28. Probability of interstrand hydrogen bonding in peptidomimetics with E/Z isomers of stiff-stilbene amino acids 6.3 (left) and 6.4 (middle) and the stilbene derivative 6.2 (right).

To investigate the effect of shortening the peptide chain three stilbene containing candidates of varying length with large difference in hydrogen bond probability were chosen for synthesis together with the heptapeptide derivatives 6.8-6.10: Ac-Switch-F (6.8), Ac-Switch-D-F (6.9) and Ac-D-Switch-D-F 6.10 (Figure 29).

Figure 29. Shortened phototriggerable analogues 6.8-6.10 of the R2 C-terminal heptapeptide 6.1.

6.1.2 Synthesis of photomodulable peptidomimetics E-isomers of peptides 6.5-6.10 were prepared as carboxamides or carboxylic acids on 0.1-0.2 mmol scale, either manually, by automated peptide synthe-sizer or by microwave assisted SPPS, using standard Fmoc-chemistry. Standard coupling times for manual and automated syntheses were 90 minutes whereas microwave assisted couplings were performed for 30 min. All peptides were purified by reverse phase HPLC using gradients of ACN/H2O or MeOH/H2O. The Z-isomers of 6.5-6.10 were obtained photo-chemically by irradiation of DMSO-d6 solutions of the corresponding E with UV-light at 300 nm up to 120 min. The often stated issue of DHP forming as a side product during photoisomerization of stilbene (for details see Figure 6, p. 17) was not observed for the duration of our experiments. From these

40

results one can conclude that photochemical degradation of the stilbene chromophore is not an issue for this kind of application.

6.1.3 Conformational analysis by NMR spectroscopy In order to probe for the presence of inter-strand hydrogen bonding in 6.8-6.10 as indicated by the computational results, amide proton temperature coefficients were measured by variable temperature NMR.74 The results were in agreement with the calculations, showing hydrogen bonding in some of the Z-isomers but in none of the more linear E-isomers. This would sug-gest that the peptidomimetics are more flexible in solution in the E configu-ration as compared to the Z-form. Further efforts to characterize peptide conformational preference by nuclear Overhauser effects were unsuccessful. Owing to the flexibility of these short sequences, only sequential NOE:s were observed.

6.1.4 Binding studies of peptidomimetics The inhibitory potencies of compounds 6.5-6.7 were assessed in a competi-tive FP-assay as described in chapter 4. A fluorescent probe was synthesized by functionalizing the N-terminus of heptapeptide 6.1 with the fluorescent dansyl group. Concentrations of each peptidomimetic at 50% displacement of the fluorescent probe DC50 were calculated for 6.5-6.7 (Table 2).

Table 2. Inhibitory potency of phototriggerable peptidomimetic analogues 6.5-6.7 of the R2 C-terminal heptapeptide

Entry Inhibition DC50 (μM)

Relative inhibition

Increased inhibition upon E-Z photoisomerization

Ac-6.1 E-6.5 Z-6.5 E-6.6 Z-6.6 E-6.7 Z-6.7

8 0.35 0.36 0.11 0.29 0.09 0.12

1 23 22 73 28 94 67

3% 163% 33%

a Relative inhibition presented as DC50 (Ac-6.1) / DC50 (Entry x). b (DC50(Z) – DC50(E) / DC50(E) 100

All heptapeptides containing the stilbene conformational switch were more potent binders to RNR1 than the native heptapeptide Ac-6.1. As ex-pected, the linear E-isomers were the better binders for peptides 6.6 and 6.7. Compound 6.5 with its free N-terminal amine, showed no distinguishable difference in binding between the photoisomers. It would seem that the con-formational influence imposed by the fairly flexible stilbene phototrigger is insufficient to overcome the strong ionic interactions of the free amine

41

sidechain of 6.5. Moreover, the hydrophobic interactions of the stilbene chromophore itself appear to contribute positively with regards to the bind-ing affinity in both of its isomeric forms. This is in agreement with previous observations where an N-terminal Fmoc-residue increased binding affinity of 6.1.102 Inhibitory potencies of peptides 6.8-6.10 were evaluated in the same type of competitive FP assay as the heptapeptide analogues 6.5-6.7. Instead of the DC50 value, the dissociation constant KD2 was calculated. This was done in order to directly compare results to previously reported values from a small-molecule screening study.102

Table 3. Dissociation constants for phototriggerable drug like derivatives 6.8-6.10 a

Entry Inhibition KD2 (μM)

Relative inhibitionb

Increased inhibition upon E-Z photoisomerizationc

Ac-6.1 Fmoc-6.1 E-6.8 Z-6.8 E-6.9 Z-6.9 E-6.10 Z-6.10

8.3 0.7 23.9 15.2 9.9 3.9 8.2 5.3

1 12 0.35 0.55 0.83 2.12 1.01 1.57

57% 154% 55%

a Calculated with KD1 = 2.2 μM. b Relative inhibition presented as KD2 (Ac-6.1) /

KD2 (Entry x). c [(KD2(E) – KD2(Z)] / KD2(Z) 100.

Results reveal that truncation of the sequence decreases the overall inhibi-tory potency in both isomeric forms. However, the KD2 values were still lower than the previously reported natural peptides of similar length.102 The shorter derivatives also display differences between photoisomers in all three cases. In contrast to the longer analogues it was Z-isomers which displayed the strongest affinity. A possible explanation for this could be hydrophobic interactions which were observed exclusively with the Z-stilbene moieties in docking studies.

6.2 Conclusions We have demonstrated that the integration of a flexible small stilbene unit into a peptide inhibitor for Mtb RNR is a viable strategy for photochemical variation of binding affinity. In two out of three examples the E isomer proved to bind tighter to the target, and in one case a near 200 % increase in affinity was observed upon isomerization. Incorporation of the stilbene con-formational switch increases the binding affinity in both isomers as com-pared to the native heptapeptide Ac-6.1. This is likely due to favorable inter-action with aromatic residues in the binding site. A set of shorter phototrig-gerable peptidomimetics with the stilbene conformational switch have also

42

been designed computationally, synthesized and tested for affinity towards Mtb. Shortening of the peptide sequence resulted in decreased overall affini-ty and opposite binding preference i.e. KD2,Z > KD2,E. The stiff-stilbene switches 6.3 and 6.4 have yet to be incorporated into peptidomimetics but their impact on conformational divergence in peptidomimetics has been as-sessed computationally. Results indicate a correlation between the rigidity of the switch and increased difference in inter-strand hydrogen bond probability in photoisomers. The potential of the stiff-stilbene switch for peptidomimetic inhibitors deserves to be fully explored, especially now when recent results have shown its capability as a modulator of peptide secondary structure.103

43

7. Photomodulable units as linkers for bisporphyrin molecular tweezer hosts (Papers IV and V)

It has previously been demonstrated that a semi-rigid glycoluril based bisporphyrin tweezer 7.3 works as a supramolecular host system, capacity to accommodate a wide variety of diamine guest molecules.104,105 In continua-tion of these studies, we wanted to construct new molecular tweezers with photochromic linkers of different conformational flexibility, and investigate their behavior as host systems.

Figure 30. Bisporphyrin tweezers in this study (Ar= Ph).

The degree of preorganization in tweezers has been suggested as a ra-tionale for high binding constants. Consequently, the length and flexibility of the linker between the two porphyrin moieties are the two parameters, which affect the level of preorganization.106 With compounds 7.1-7.3 we wanted to

7.3

N

N

N

O

NNN

N Zn

NN

O

N N

N

N

N NZn

N

O

N

NN

ZnN

NH

O

NH

N N

NZnN

O

NNZn

NN

NH

O

NH

NN

N ZnN

NN Zn

NN

NH

O NNZn

NN

NH

O

NNZn

NN

NH

O

NNZn

NN

NH

O

Ar

Ar Ar

Ar

Ar Ar

ArAr

Ar

Ar

Ar

Ar

Ar

Ar

Ar

Ar

Ar Ar

ArAr

Ar Ar

ArAr

Ar Ar

ArAr

ArAr

Ar Ar

Ar

ArAr

ArOMe

OMe

PhPh

OMeArAr

Ar Ar OMe

-7.1-7.1

-7.2-7.2

44

explore the effect of a type of linkers which results in reduced preorganiza-tion, and/or restricted cavity size variation on binding affinity. For pho-toswitchable tweezers 7.1-7.2, the structural differences between photoiso-mers might enable functions such as photo induced stretching of long and flexible diamine guests. Another possibility could be different guest size preferences in the respective isomers.

7.1 Impact of linkers on tweezer characteristics (Paper IV) There are in principle four different degrees of freedom available for the porphyrin units in bis-porphyrin tweezers (Figure 31). The porphyrin dislo-cations are likely to occur interdependently, and are highly influenced by the structural characteristics of the interporphyrin linker.

distance variation and porphyrin rotation

porphyrin twisting and lateral dislocation

Figure 31. Available degrees of freedom for bisporphyrin tweezers. Top: porphyrin distance variation and rotation. Bottom: porphyrin twisting and lateral dislocation.

The enediyne linker of tweezer 7.1 (Figure 30) is relatively flexible and should therefore allow for all of the available distortion modes. The rigidity of the stiff-stilbene linker of tweezer 7.2 limits interporphyrin distance varia-tion when compared to the enediyne linker of 7.1. However the built in

45

skewedness of stiff-stilbene can compensate for this. Previous studies of tweezer 7.3 (Figure 30) have confirmed the conformational flexibility of its glycoluril linker with regard to distance variation. However, since the por-phyrin moieties of 7.3 are attached through 2 covalent bonds, porphyrin rota-tion is impossible and lateral dislocation might be impaired.

Table 4. Summary of degrees of freedom for porphyrin movement in tweezers 7.1-7.3. a

Degree of freedom Tweezer

Z-7.1 Z-7.2 7.3 Distance variation Porphyrin rotation Porphyrin twisting Lateral dislocation

✔ ✔ ✔ ✔

(✔) built-in built-in ✔

✔ ✖ ✔ ✖

a ✔: possible, ✖: not possible, (✔) not likely.

To probe the conformational flexibility of the tweezers and verify the as-sumptions in Table 4, in addition to conformational analysis we conducted a series of binding studies with flexible and rigid diamine guests and per-formed conformational analyses with MM of linkers, free tweezers and tweezer-guest complexes. Finally, conformational properties of the tweezers were probed via their capability of chirogenesis when binding chiral amino acid derivatives. Chirogenesis is dependent on such distortion modes in the tweezer that result in a helical bisporphyrin geometry, which can be detected by circular dichroism.

7.1.1 Conformational analysis of linker and tweezer geometries From the energy profiles of the linkers one can conclude that the stiff stil-bene linker of tweezer 7.2 has an intrinsically skewed conformation (9°) as opposed to the enediyne linker of 7.1 which has its local minimum at a dihe-dral angle of 0° (Figure 32 left). There is a dramatic difference between linkers in their capability of varying the bitesize. The stiff stilbene has a very narrow profile as opposed to the enediyne which displays a large flexibility (Figure 32, right). Tweezer 7.3 has been shown to have a relatively good capability of distance variation, although not as high as for enediyne.104

46

47

Figure 32. Energy profiles for the variation of dihedral angle (left) and of carbonyl distance in tweezers 7.1 (orange) and 7.2 (purple) and 7.3 (black).

With the properties of the linker backbones established, conformational searches were performed with the inclusion of the porphyrin moieties. For tweezer 7.1 the lowest energy conformer retained a 0° dihedral angle, and the distance between the Zn ions of the porphyrins was 4.9 Å. The backbone of tweezer 7.2 had a dihedral angle of 6.5° in the lowest energy conformer, and the Zn-Zn distance was slightly longer at 6.2 Å.

Based on the results from conformational analysis of the tweezers, we can summarize their properties as follows. Tweezer 7.1 possesses a high degree of conformational flexibility and allows all degrees of freedom described in Figure 31. This should in turn enable this tweezer to bind a wide variety of guest molecules with high binding constants. Because of the large flexibility in the enediyne backbone towards distance variation, skewing should not be necessary in order to accommodate larger sized guests. In contrast, the back-bone of tweezer 7.2 has an inherent skewing and a very restricted capability of distance variation. Thus it can only achieve the Zn-Zn distances required for ditopic binding of large guests by increased skewing and rotation of the porphyrin moieties. The X-ray crystal structure (Figure 33) obtained for 5.5d (for synthesis see Figure 19, p. 33) shows good agreement with the conformational analysis, displaying a double bond dihedral angle of 9.1° due to steric interactons between the alkene substituents.

0

1

2

3

4

5

6

7

8

9

10

4,5 6,5 8,5 10,5 12,5 14,50

1

2

3

4

5

6

7

8

9

10

-30 -24 -18 -12 -6 0 6 12 18 24 30

7.1

7.2

7.3

θ dihedral (o) C=O distance (Å)

kJ/m

ol

kJ/m

ol

Figure 33. Ortep representation of Z-5.5d viewed from above (left) and from the side (right). Thermal elipsoids are at 35% probability level.

Tweezer 7.3 is highly capable of distance variation due to the conforma-tional character of the glycoluril linker. However, the capability of skewing is smaller than for 7.1 and 7.2. Conformational analyses of host-guest com-plexes with tweezers 7.1-7.3 and a selection of dinitrogen guests were con-ducted in order to differentiate the structural impact of the linkers upon bind-ing.

Figure 34 Maximum N-N distances for the conformationally rigid and flexible guest molecules. Distances refer to minimum energy conformers (OPLS 2005 force field).

Guest molecules with both locked and variable N-N distances were stud-ied for better overview of conformational preferences in the tweezer com-plexes. The results (Table 5) indicate large Zn-Zn distance variations in tweezers 7.1 and 7.2 but somewhat less pronounced changes for tweezer 7.3. Table 5. Dihedral angles, intercarbonyl distances of linkers and Zn – Zn distances (Å) of tweezers Z-7.1, Z-7.3 and 7.3 with bound guests. Conformers were obtained from conformational analysis using the OPLS 2005 force field.

a Dihedral angle of linker C=C bond. bMinimizes into an in-out complex

Some interesting conformational dissimilarities are also apparent when the flexible guest 7.8 is bound to the tweezers (Figure 35).

Guest Host

Z-7.1 Z-7.2 7.3

dihedral angle a

CO – CO

distance

Zn-Zn distance

dihedral angle a

CO – CO

distance

Zn-Zn distance

Zn-Zn distance

(free tweezer) 7.6 7.7 7.8 7.9

0° 0.2° 0.1° 0.5°

b

8.5 7.9

10.6 7.1

b

4.9 7.0

11.3 8.4

b

6.5° 11.2° 11.5° 10.4° 10.5°

4.9 5.4 5.9 5.7 4.8

6.1 7.3

11.6 5.8 6.1

6.3 7.3

11.5 8.5 8.2

48

Figure 35. Global minima of porphyrin distortions in tweezers 7.1-7.3, obtained with molecular mechanics using the OPLS 2005 force field. Free tweezers are col-ored in purple and their host-guest complexes with 1,6-diaminohexane 7.8 are col-ored green and red for tweezer and guest, respectively.

As expected, tweezers 7.1 and 7.2 display some degree of porphyrin rota-tion in order to fit the guest 7.8 within the binding pocket. In addition to this, 7.1 also exhibit some lateral dislocation. In contrast, 7.3 achieves bitopic complex with 7.8 by twisting alone. The linker dihedral angles (Table 5) also suggest that tweezer 7.1 is not forced to skew over the central double bond like 7.2 in order to accommodate guests for ditopic binding. To evalu-ate the potential effect of isomerization on binding to diamine guests, con-formational analysis of the E-isomers of 7.1-7.2 were conducted. The results revealed that the cavity size increased considerably when compared to the Z-isomers (Table 6).

Table 6. Distances between porphyrin metal (Zn) centers (Å) obtained from con-formational analysis of host-guest complexes with 7.1-7.2.

Guest Host Zn-Zn distancea

dN-Nb E-7.1 ΔE-Z E-7.2 ΔE-Z

(free tweezer) 1,6 diaminohexane 7.8 1,12 diaminododecane 7.9 1,20 diaminoeicosane 7.10

- 8.8 Å

16.4 Å 26.6 Å

28.3 Å 29.6 Åc

18.9 Å

27.6 Å

23.4 Å 21.2 Å

- -

18.4 Å 18.3 Åc

17.9 Å

19.0 Å

12.3 Å 12.5 Å 11.8 Å

- a Global minimum; b fully stretched conformation; c monotopic 1:1 complex

Even though the tweezers have indicated some adaptation of sidewall dis-tances to the guest sizes in their Z-isomers (Table 5), the substantial differ-ence in cavity size of E and Z forms is still large enough to significantly

49

differentiate their binding properties. For example, diamine guests with an N-N distance considerably shorter than the Zn-Zn distance should be prohib-ited from ditopic binding with tweezers E-7.1 and E-7.2.

7.1.2 Synthesis of bisporphyrin tweezers The previously reported synthesis scheme for tweezer 7.3 involved 6 steps from meso-tetraphenylporphyrin (TPP) and 5 steps for the glycoluril linker with an overall yield of 4% from TPP.104 For tweezers 7.1 and 7.2 we sought to improve on this. Functionalized porphyrin units (TPP-NH2) for tweezers 7.1 and 7.2 were prepared in 4 steps starting from free base TPP (Paper IV).

Figure 36. Synthesis route to bisporphyrin tweezers 7.1 and 7.2; i) oxalyl chloride, DCM, r.t., 1-2 h, quant; ii) TPPNH2, CH2Cl2, r.t, 12 h (not isolated) ; iii) Zn(OAc)2

.H2O, CH2Cl2, MeOH, reflux, 30 min, 24%.

Dicarboxylic acid scaffolds Z-5.2 and Z-5.4 were quantitatively converted into their acid chlorides prior to coupling with TPP-NH2. Yields of tweezers 7.1 and 7.2 from TPP were 18 % and 19% respectively (24% for the cou-pling with TPP-NH2) (Paper IV).

7.1.3 Photostability of bisporphyrin linkers Photostability studies of the bisporphyrin linker units were performed on model compounds in order to verify their applicability as conformational switches for this application. For the dimethyl ester derivative of enediyne linker Z-5.2, a 55 % conversion to the E-isomer was obtained after selective irradiation at 340 nm for 7.5 hours. Importantly, while enediynes are known for undergoing Bergman cyclization primarily upon heating or UV-irradiation, Z-5.2-(COOMe) did not show any 1H NMR detectable byprod-ucts indicating this reaction. Such products would show characteristic higher order multiplets for symmetrically ortho-disubstituted benzene rings (Ap-pendix). The E-stiff stilbene diethylester 5.5d was exposed to selective UV-irradiation at 300 nm and reached 55 % conversion after 12.5 hours of irra-diation with no significant signs of breakdown products (Paper V).

Z-7.1

Z-7.2

5.2 7.1b

-5.4 -7.2b

TPP-NH2 =

NH2

NH

N

NH

N

COOH

COOH

COOH

COOH

COCl

COCl

COCl

COCl

i

i ii - iii

ii - iii

50

7.1.4 Isomerization of tweezers Initial irradiations of tweezers Z-7.1 and Z-7.2 at the absorption maxima of 340 (enediyne) and 360nm (stiff-stilbene) respectively did not result in

Figure 37. UV-Vis spectra of Z-7.2 before (solid) and after irradiation (dashed) at 254 nm for 150 min. Concentration of tweezer was 810-6 M in acetonitrile.

isomerization. This might be due to quenching of the C=C excited state of the linker, via energy transfer to the porphyrin units. However, when tweezer Z-7.2 was irradiated at 254 nm, a red shift of the Soret band could be ob-served together with a slight blue shift of the Q-bands (Figure 37). The shift of the Soret band to longer wavelengths is indicative of greater interporphy-rin distance as expected in the E-isomer of the tweezer.66

7.1.5 Binding studies with diamine guests (Paper IV and V) Binding studies were performed with diamine guests 7.6-7.9 (Figure 34, p. 47) in order to assess what impact the differing conformational flexibilities of 7.1-7.3 (Figure 30, p. 43) had on binding affinity. Titrations of tweezers 7.1-7.3 with the diamine guests resulted in typical red shifts of the Soret and Q-bands (Figure 38), together with clear isobestic points, which are indica-tive of well-defined complexes (Figure 38).108-110

51

Figure 38. Representative example of UV-titration curves with isobestic points for bisporphyrin tweezer 7.3.

The 1:1 stoichiometry i.e. ditopic guest binding in these complexes was supported by 1H NMR data which showed (n+1)/2 signals at the expected low chemical shifts. Increasing of UV/Vis red shifts observed with larger guest sizes was also indicative of ditopic binding.111 The observed shifts of the Soret and Q-bands towards longer wavelengths are the net of two coun-teracting processes: electronic effects upon binding of an amine guest which results in a red shift, and close interpoprhyrin distance which results in a blue shift.112,113 Thus, the observation of increasing red shifts with binding of larger guests is an indication that the porphyrin units are forced further apart in order to form a ditopic complex. Binding constants (UV/Vis titration) illustrate the result of cooperativity, displaying binding constants up to three orders of magnitude greater than the monotopic reference Zn-TPP (Table 7). Among the tweezers, 7.1 showed the highest binding constants followed by 7.3, and 7.2 which displayed the weakest binding.

Table 7. Binding constants (Ka, M-1) for ditopic binding of diamine guests to bisporphyrin tweezers Z-7.1, 7.2 and 7.3, and monotopic binding to a reference monoporphyrin host Zn-TPP.

Guest Host

Z-7.1 Z-7.2 7.3 Zn-TPP DABCO 7.6a

4,4’-bipyridyl 7.7 1,6-diaminohexane 7.8 1,12-diaminododecane 7.9

2104

4106

6106

8105

4105

2105

5105

2105

3106

2106 1106

2105

8103

- 5104

- a 1,4-Diazabicyclo[2.2.2]octane

The ability of 7.2 to adapt cavity bite-size by linker distortion alone is not sufficient to compensate for the reduced ability of distance variation. In con-trast, the larger binding constants observed for 7.1 might result from its

52

backbone flexibility, which can easily distort upon binding of guests. Dis-tance variation and porphyrin twisting in tweezer 7.3 are achieved through conformational changes of the glycoluril rings which require relatively low energies, resulting in high binding constants.

7.1.6 Chiral guest induced helicity To gain more information about the binding characteristics of tweezers Z-7.1, Z-2 and 7.3 (Figure 30, p. 43), their suceptibility for helicity induction was assessed by means of exciton coupled circular dichroism (ECCD). As-sessment of the degree of induced helicity from chiral guests to the achiral tweezers would provide more data on their conformational preferences. For this purpose, methyl esters of (L)-lysine and (L)-tryptophan (for synthesis see Appendix) were bound to the tweezers, followed by CD-spectroscopic analysis. The (L)-lysine methyl ester was chosen as a model chiral diamine with flexible N-N distance, allowing ditopic binding, whereas (L)-tryptophan methyl ester was selected as a monotopic ligand which previously has shown chirogenesis with bisporphyrin tweezers, depending on the structural proper-ties of the tweezer host.57 Tweezer Z-7.2 showed the strongest CD-response for both amino acid derivatives (Figure 39). This is likely a consequence of the already skewed stiff-stilbene linker which could favor unidirectional porphyrin twisting.

Figure 39. CD spectra of the complexes of L-Lysine methyl ester (left) and L-Tryptophan (right) with tweezers 7.1-7.3.

In addition to this, the restricted bite-size imposed by the linker should be beneficial for chiral recognition as this would enforce asymmetric porphyrin rotation and translocation to accommodate the chiral guest. The enediyne-linked tweezer Z-7.1 also produces a strong signal with the lysine derivative, but no significant response was obtained with the tryptophan analogue. This could be because the tweezer can adopt more than one equally contributing binding mode to the guest, which would cancel out the net CD-signal.114

53

Another possible explanation is that the flexibility of Z-7.1 prevents unidi-rectional rotation of the porphyrin units in a monotopic complex.57 Tweezer 7.3 displayed no significant chiral recognition of either guest, despite having the capability of porphyrin twisting. However, comparison of calculated structures of complexes between tweezers and the chiral guests revealed no porphyrin rotation for 7.3, whereas 7.1-7.2 showed significantly rotated por-phyrin sidewalls resulting from guest-induced helicity.

7.2 Conclusions Two new bisporphyrin tweezers with enediyne and stiff-stilbene conforma-tional switches have been designed, synthesized and compared to a previous-ly reported glycoluril based tweezer. The effect of tweezer rigidity on ditopic binding of various diamine compounds has been evaluated. Binding con-stants reveal that an unhindered ability of bitesize adaptation in the Z-isomers of the hosts is most important for strong binding. Circular dichroism studies of tweezers with chiral guests suggest that restricted inter-porphyrin distance is beneficial for chiral recognition since this promotes unidirectional helicity in the complexes. The switching ability of both linkers has been established and initial isomerization results have also been attained with tweezer 7.2. The potential of photo-induced bite-size alteration has also been evaluated computationally for enediyne and stiff-stilbene tweezers. Confor-mational searches indicate that substantial differences in interporphyrin dis-tance between isomers can be attained. Isomerization should therefore im-pact binding characteristics of the tweezers to a significant extent.

54

8. Outlook

This thesis covers the design, synthesis and subsequent incorporation of photochromic conformational switches into peptidomimetic guests and mo-lecular tweezer hosts. Photochemical modulation of the molecular structures of tweezers and peptidomimetics, and the impact on molecular recognition is then studied in protein-ligand and supramolecular host-guest systems.

We have demonstrated that a flexible stilbene amino acid moiety inserted into the backbone of known heptapeptide inhibitors for Mycobacterium tu-berculosis RNR allows for photochemical tuning of inhibitor affinities for the enzyme. In addition to this, we have designed and synthesized a series of shorter peptide derivatives, by qualitative analysis of the binding site and computational screening. (Paper I). For all peptidomimetics, the presence of the conformational switch has proven to greatly enhance the affinity of pep-tidomimetics of both isomeric forms. Ligand epitope mapping performed by STD-NMR has been probed as a method for obtaining more information on protein-ligand interactions (Paper III). This could be a promising comple-mentary method to screen for variations in binding behavior in upcoming investigations.

A synthesis route for a novel stiff-stilbene amino acid derivative with more prominent structural discrepancy between its photoisomers has been developed (Paper II). The enhanced potential for structure modulation of this new conformational switch compared to stilbene has also been demon-strated computationally (Paper III). The stiff-stilbene derivatives will be incorporated into peptidomimetics and their binding to RNR will be evaluat-ed. The structural rigidity combined with more red-shifted UV-Vis absorp-tion patterns makes stiff-stilbene a promising candidate for future therapeu-tic applications.

Two new bisporphyrin tweezers, linked by stiff-stilbene and enediyne moieties have been synthesized and their conformational characteristics as-sessed with regard to binding of diamine guests. A connection between link-er flexibility and guest affinity has been established. The helical nature and conformational restrictions of a stiff-stilbene linked tweezer proved to be favorable for chiral recognition with monotopic and bitopic guests (Paper IV). Initial efforts to photochemically vary the bitesize of our tweezers have been made (Paper V). These should be further explored and evaluated with regard to changes in binding behavior. The other isomeric form will have to be synthesized in order to provide more conclusive data.

55

9. Acknowledgements

I wish to express my sincere gratitude to the people who have helped me over the years, or just made my time as a PhD-student more enjoyable.

First of all, I would like to thank my excellent supervisor Prof. Adolf Gogoll. Thank you for all the enthusiasm and support you have showed me during these years. Your view of supervision and your philosophy of science have really allowed me to grow as a scientist. Most importantly, you have encouraged my inner curiosity, which is my greatest source of motivation.

I wish to thank my co-supervisor Prof. Helena Grennberg for all the great

advice you have given me, and for your patience listening to me during my darker spells.

I would also like to thank my second co-supervisor Prof. Anders Karlén for your input regarding the inhibitor project.

Thanks to past and present members of the AGHG group: Dr. Classe, my

fellow double dad for your laid back composure and for the excellent work during the tweezer project; Dr. Sara (Snorrehed) for the nice time, sharing the office space (excluding chocolate bars) p.s. I have finished your chloro-form now. Dr. Christoffer for the work done on the peptide paper. Thank you dear Hao for your passion and diligence both within and outside the lab, and for being a good friend “why drink coffee when you can drink beer?” Thanks Xiao, the other pole of the battery, for always being positive. Mi-chael, for all the fun times off work, and for all the sweat I’ve shed trying to keep up with you on the tennis court (and in the course lab). Anna for all our laughs and time spent drinking wine and talking about our kids and stuff. My current office-roomie Sandra destined for the one (porphyrin)-ring that binds them all! Thank you for being the voice of reason (and curses) in our office.

I would like to acknowledge the administrative and technical staff at the department: Johanna for the nice champagne evening and all the intriguing discussions about politics and Game of Thrones. Thank you Gunnar and Bosse for all your help. Johanna for fixing everything down at the course labs.

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I want to thank all the nice people at the department (there are so many new faces) for making my days nice and cheerful.

“I don't know half of you half as well as I should like; and I like less than half of you half as well as you deserve.” – J.R.R. Tolkien

Let’s start on the dark side! Thank you: Rabia, Kjell, Aleksandra, Rafael-lo, Jia-Fei, Maxim, Alexander, Lisa Å and Mia. The other side! Thanks to Carina, Karthik, Lina, Tobias, Emil, Huan, Åsa, Thilak, Jie and Alexandra B. Thanks to the nice people downstairs: Erika, Dirk, Eldar, Dr. Christian, and Dr. Sara S.

Jag vill också tacka några av mina kära vänner utanför BMC. Nisse, för

att du alltid lockar fram det bästa hos mig (även prickar). Peter, för alla mo-ments och ett stört trevligt bröllop. Micke, som förstår precis hur det känns just nu en knapp timme innan inlämning. Johanna för att du är så omtänk-sam och vet vad en avhandlings-zombie behöver. Christian, för alla stöd-kramar och för bra samtal om allt från kemi till brunsås. Tack allihop för att ni påmint mig om livets andra sidor.

Sedan vill jag tacka min familj. Tack Mamma och Pappa för att ni låtit

mig gå min egen väg och för att ni alltid uppmuntrat mig att ta reda på saker. Tack käre lillebror Marcus, som är en av få här på jorden som har samma sjuka humor som jag. Tack Frida och Astrid för att ni ställt upp och för era uppmuntrande ord. Anne och Håkan för all er hjälp, er värme och alla av-kopplande dagar ute på Gräsö.

Finally, I would like to thank the three most important people in my life. Tack Lisa. Du har haft ett minst lika tufft jobb som jag under den här senaste tiden. Jag finner inte ord för hur lycklig jag är tillsammans med dig och våra små gullungar Elsa och Viktor. Det är ni som lyser upp mitt liv. Jag älskar er.

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Acta Universitatis UpsaliensisDigital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Science and Technology 1329

Editor: The Dean of the Faculty of Science and Technology

A doctoral dissertation from the Faculty of Science andTechnology, Uppsala University, is usually a summary of anumber of papers. A few copies of the complete dissertationare kept at major Swedish research libraries, while thesummary alone is distributed internationally throughthe series Digital Comprehensive Summaries of UppsalaDissertations from the Faculty of Science and Technology.(Prior to January, 2005, the series was published under thetitle “Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Science and Technology”.)

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