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109
CChhaapptteerr 66
Synthesis of Amides of Amino Acids and Peptides from
their Thiol Esters using Magnesium and Ammonium
Formate
6.1 INTRODUCTION
Carboxylic acid amides represent a huge class of natural and
synthetic compounds including drugs and peptide hormones. Many
biologically active peptides possess the C-terminal amide structure, and in
most of the amidated peptides, such as oxytocin [1], gastrin [2], thyrotropin
releasing hormone [3] and calcitonin [4], the C-terminal amide structure is
shown to be essential for eliciting their full biological activity. Solid phase
synthesis of C-terminal peptide amides are normally achieved using
benzhydrylamino polystyrene resins and their substituted analogues [5-6].
Application of resins containing benzhydrylamine type linkers functionalized
with electron-donating alkoxy groups such as in the Rink-amide linker,
provides easy and efficient method for the solid phase synthesis of peptide
amides under very mild acidic cleavage conditions [7]. On the other hand,
there still remains a significant need to develop facile reagent systems for
Synthesis of Amides of Amino Acids and Peptides… Chapter 6
110
the mild conversion of carboxylic acids of amino acids, peptides and small
organic molecules to their corresponding amides.
6.2 RESULTS AND DISCUSSION
Ammonium formate has been extensively used as a source of
hydrogen for catalytic transfer hydrogenation reactions in presence of many
metal catalysts [8-10] and also as a source of ammonia for reductive
amination of carbonyl compounds [11,12]. Application of ammonium
formate in combination with magnesium provides an efficient system for the
removal of protecting groups in peptide synthesis [13]. The role of
ammonium formate as a hydrogen source for catalytic transfer
hydrogenation reactions has been explained by its dissociation producing
HCOO- and NH4+ (Scheme 6.1) [14]. Consequently, the hydrogen may be
transferred in the form of proton or hydride or both depending on the
reaction conditions employed.
HCOONH4 HCOO- + NH4+
HCOO- H- + CO2
NH4+ NH3 + H+
M
Scheme 6.1: Dissociation pattern of ammonium formate under catalytic transfer hydrogenation conditions.
Fukuyama et al. reported Pd-C catalyzed reduction of a wide variety
of thiol esters to their corresponding aldehydes using triethylsilane as
hydride source [15,16]. Initially, as a part of our programme to synthesize
amino acid and peptide aldehydes as potential HIV protease inhibitors, we
planned to mimic Fukuyama reaction to reduce amino acid and peptide thiol
esters to their corresponding aldehydes using magnesium/ammonium
formate system. We expected that ammonium formate would serve as a
hydride source in presence of inexpensive magnesium metal. To our
surprise, magnesium/ammonium formate system reduced amino acid and
peptide thiol esters into their corresponding amides in high yields at room
temperature (Scheme 6.2).
Synthesis of Amides of Amino Acids and Peptides… Chapter 6
111
Thiol esters can be easily prepared from their corresponding
carboxylic acids and thiols via the acid chlorides or mixed anhydrides, or by
utilizing various dehydrating agents such as DCC or EDCI [17]. For the
synthesis of N-protected amino acid thiol esters, the mixed anhydride
method using isobutyl chloroformate (IBCF) is found to be more adequate
and efficient. A wide range amino acid and peptide thiol esters were
synthesized and reduced conveniently to their corresponding amides using
magnesium/ammonium formate at room temperature (Table 6.1). The
course of the reaction was monitored by thin layer chromatography (TLC)
and IR spectra. The work-up and isolation of the products were easy. The
products were characterized by IR, NMR and elemental analysis. The
appearance of two strong stretching bands between 3350 and 3180 cm-1 due
to –NH2 group of primary amide clearly shows that the thiol esters were
reduced to their corresponding amides.
NH
OHR2
R1O
IBCF or EDCI/HOBt
R3SHNH
SR2
R1O
R3
Mg/HCOONH4
MeOH, r.t.
NH
NH2
R2
R1O
+ HS-R3
Where R1 = Fmoc or Boc protecting groupsR2 = corresponding side chain of amino acidsR3 = C2H5 or C6H5
Scheme 6.2: Mg/ammonium formate aided reduction of thiol esters to amides.
It was found that the, reduction of phenyl thiol esters is faster than
their corresponding ethyl thiol esters (Table 1; entries 8-11). The
commonly used N-α protecting groups of amino acids and peptides such as
Boc and Fmoc are found to be unaffected under the reaction condition
employed. However, many side chain protecting groups are not compatible
with magnesium/ammonium formate system viz. 2-ClZ, OBzl, Z [13].
Synthesis of Amides of Amino Acids and Peptides… Chapter 6
112
Table 6.1: Synthesis of Amides of Amino Acids and Peptides from Thiol Esters using Mg/HCO2NH4 System.
Thiol Ester Product Time (hrs)
Yield (%)a M.P (0C)
Boc-Ala-S-Et 1a Boc-Ala-NH2 2a 2.30 80 187.00
Boc-Phe-S-Et 1b Boc-Phe-NH2 2b 3.00 70 129.50
Boc-Leu-S-Et 1c Boc-Leu-NH2 2c 3.30 82 124-125
Boc-Lys(2-ClZ)-S-Et 1d Boc-Lys-NH2 2d 4.15 85 84-86
Boc-Glu(OcHx)-S-Et 1e Boc-Glu(OcHx)-NH2 2e 2.45 76 118-120
Boc-Gly-S-Et 1f Boc-Gly-NH2 2f 2.00 68 130-132
Boc-Pro-S-Et 1g Boc-Pro-NH2 2g 4.30 70 103-104
Boc-Val-S-Ph 1h Boc-Val-NH2 2h 1.45 65 157.40
Boc-Ala-S-Ph 1j Boc-Ala-NH2 2j 1.30 69 187.00
Boc-Phe-S-Ph 1k Boc-Phe-NH2 2k 1.40 72 129.50
Boc-Pro-S-Ph 1l Boc-Pro-NH2 2l 1.10 71 103-104
Boc-AVG-S-Et 1m Boc-AVG-NH2 2m 4.15 89 149.5
Boc-GGFP-S-Et 1n Boc-GGFP-NH2 2n 5.00 80 124.00
Boc-GVGVP-S-Et 1o Boc-GVGVP-NH2 2o 8.00 75 113.90
Fmoc-Ala-S-Et 1p Fmoc-Ala-NH2 2p 4.30 80 162.90
Fmoc-Phe-S-Et 1q Fmoc-Phe-NH2 2q 5.10 82 162-164
Fmoc-Gly-S-Et 1r Fmoc-Gly-NH2 2r 3.50 79 115-118
Fmoc-Leu-S-Et 1s Fmoc-Leu-NH2 2s 5.30 81 191-193
a. Isolated yields are based on single experiment and the yields were not optimized.
Reduction of thiol esters was also attempted in absence of
magnesium using only ammonium formate in methanol. Even after
prolonged reaction time (24 hours), the starting material was recovered
quantitatively. It was also observed that, there was no formation amino acid
or peptide aldehyde or even amino aicds. Further, we also observed that the
reduction of thiol esters do not occurs either with Zn/HCOONH4,
Synthesis of Amides of Amino Acids and Peptides… Chapter 6
113
Pb/HCOONH4 or Ra-Ni/HCOONH4 systems. A plausible mechanism of the
magnesium/ammonium formate promoted reduction of thiol esters to their
corresponding amides is shown in Scheme 6.3. The mechanism follows the
amine acylation methodology. The initial step involves attack of ammonia
(which is released by the dissociation of ammonium formate Scheme 6.1) on
the carbonyl group, so that the more powerful the electron-withdrawing
nature of the leaving group, the faster will the reaction occur. Hence the
reduction of phenyl thiol esters is rapid than ethyl thiol ester.
NH
S
OR3R1
R2NH3
MgNH
R1
R2
O
N
S
HHH
R3NH
R1O
R2
SNH2
R3-H+
-R3S-
NH
NH2
R2
OR1
Scheme 6.3: Plausible mechanism of magnesium/ammonium formate mediated reduction
of thiol esters to amides.
In summary, magnesium/ammonium formate system provides an
efficient general protocol for the synthesis of amides of amino acids and
peptides from their corresponding thiol esters. The ease of product
separation, safe reaction medium, high selectivity and high yield promote
this method as a promising alternative to the existing methods.
6.3 EXPERIMENTAL
GENERAL:
All the amino acids used were of L-configuration unless otherwise
specified. All tert-butyloxycarbonyl (Boc) amino acids, 9-
fluorenylmethyloxycarbonyl (Fmoc) amino acids, amino acid derivatives, 1-
hydroxybenzotrizole (HOBt) and trifluoroacetic acid (TFA) were purchased
from Advanced Chem. Tech., (Louisville, Kentucky, USA).
Isobutylchloroformate and N-methyl morpholine (NMM) were purchased from
Sigma Chemicals (St. Louis, USA). Thio phenol (PhSH) and Thio ethanol
Synthesis of Amides of Amino Acids and Peptides… Chapter 6
114
(EtOH) were purchased from Sigma Aldrich Chem. Pvt. Ltd. (Bangalore,
India).
The Thin layer chromatography (TLC) was carried out on silica gel plates
obtained from Whatman Inc., with the following solvent systems:
Rf1: CHCl3: CH3OH: CH3COOH (95:05:3)
Rf2: CHCl3: CH3OH: CH3COOH (90:10:3)
Rf3: CHCl3: CH3OH: CH3COOH (85:15:3)
The details of instruments used have been described in chapter 2.
6.3.1 PEPTIDE SYNTHESIS
The peptides required for the purpose of thiol ester preparation were
synthesized by a classical solution phase synthesis by stepwise approach.
The C-terminus carboxyl group was protected by the benzyl ester and its
removal was effected by hydrogenolysing using HCOONH4/Mg [13]. All
coupling reactions were achieved with isobutylchloroformate. The protected
peptides were purified by recrystalization and characterized by physical and
analytical techniques.
SYNTHESIS OF AVG (Scheme 6.4)
Boc-Val-Gly-OBzl (I): Boc-Val-OH (2.17 g, 0.01 mol) dissolved in acetonitrile
(20 mL) and cooled to 0 °C was added N-methylmorpholine (1.02 ml, 0.01
mol). The solution was cooled to -15 °C ± 1 °C and isobutylchloroformate
(1.5 mL, 0.01 mol) was added under stirring while maintaining the
temperature at -15°C. After stirring the reaction mixture for 10 minutes at
this temperature, a pre-cooled solution of HCl.H-Gly-OBzl (2.02 g, 0.01 mol)
and NMM (1.5 mL, 0.01 mol) in DMF (20 mL) was added slowly. After 20 min,
the pH of the solution was adjusted to eight by the addition of NMM and the
reaction mixture stirred over night at room temperature. Acetonitrile was
removed under reduced pressure and the residual DMF solution was poured
into about 100 mL ice-cold 90% saturated KHCO3 solution and stirred for
30 min. The precipitated peptide was filtered, washed with water, 1N HCl,
water and dried. The crude peptide was recrystalised from ether and
Synthesis of Amides of Amino Acids and Peptides… Chapter 6
115
petroleum ether to obtain 3 of I (yield 87%). Rf1 0.58, Rf
2 0.61 and Rf3 0.66,
m.p. 81-82 °C (Lit. 80-82 °C) [18].
Boc-Ala-Val-Gly-OBzl (II): The peptide I ( 2.9 g, 0.008 mol) was deblocked
with 4N HCl/dioxane (3 mL) for 1.5 hr. Excess HCl and dioxane were
removed under reduced pressure, triturated with ether, filtered, washed
with ether and dried (yield, 100%). The HCl.H-Val-Gly-OBzl was neutralized
with NMM (0.81 mL, 0.008 mol) and coupled to Boc-Ala (1.5 g, 0.008 mol) in
acetonitrile (15 mL) and NMM (0.81 mL, 0.008 mol) using
isobutylchloroformate (1.1 mL, 0.008 mol) and worked up the same as I to
obtain 3.12 g of II (yield 91.2%). Rf1 0.55 and Rf
2 0.64, m.p. 147-148 °C (Lit.
147.5 °C) [19].
GlyValAla
OBzlHBoc OH
Boc(i)
(ii)
(i)
(iii)
OBzl
OBzl
OBzl
OH
HBoc
Boc
OH
Boc
(i) IBCF / NMM(ii) 4N HCl / Dioxane(iii) HCOONH4 / Mg
Scheme 6.4: Schematic representation of synthesis of AVG
Boc-Ala-Val-Gly-OH (III): The peptide II (2.61 g, 0.006 mol) was
hydrogenolyzed using ammonium formate (2.0 equiv.) and Mg (1 equivalent)
in methanol (30 mL) for 2 hours at room temperature. The catalyst was
filtered and washed with methanol. The combined filtrate was evaporated
in vacuo and the residue taken into CHCl3, washed with water, and dried
over Na2SO4. The solvent was removed under reduced pressure and
triturated with ether filtered, washed with ether and dried to obtain 1.93 g
Synthesis of Amides of Amino Acids and Peptides… Chapter 6
116
of III (yield 93%). Rf2 0.27 and Rf
3 0.35, m.p. 190-191 °C (Lit. m.p. 190 °C)
[19].
SYNTHESIS OF GGFP (Scheme 6.5):
Boc-Phe-Pro-OBzl (IV). The Boc-Phe (2.65 g, 0.01 mol) dissolved in
acetonitrile (30 mL) and cooled to 0 °C was added N-methylmorpholine
(1.1 mL, 0.01 mol). The solution was cooled to -15 °C ± 1 °C and
isobutylchloroformate (1.37 mL, 0.01 mol) was added under stirring while
maintaining the temperature at -15 °C. After stirring the reaction mixture
for 10 minutes at this temperature, a pre-cooled solution of HOBt (1.53 g,
0.01 mol) was added. The reaction mixture was stirred for and additional 10
min and a pre-cooled solution of HCl.H-Pro-OBzl (2.42 g, 0.01 mol) and NMM
(1.1 mL. 0.001 mol) in DMF (25 mL) was added slowly. After 20 min, the pH
of the solution was adjusted to eight by the addition of NMM and reaction
mixture stirred over night at room temperature and worked up the same as I
to obtain IV (3.84 g, yield 86%). Rf1 0.75 and Rf
2 0.84, m.p. 101-102 °C, (Lit.
m.p. 100 °C) [20].
ProPheGlyGly
OBzlHBoc OH
Boc(i)
(ii)
(iii)
(iv)
(ii)
(iii)
OBzl
OBzl
OBzl
OBzl
OBzl
OH
HClBoc
Boc
Boc
Boc
OH
Boc
OH HCl
(i) IBCF / NMM/HOBt(ii) 4N HCl / Dioxane(iii) IBCF /NMM(iv) HCOONH4 /Mg
Scheme 6.5: Schematic representation of synthesis of GGFP
Synthesis of Amides of Amino Acids and Peptides… Chapter 6
117
Boc-Gly-Phe-Pro-OBzl (V). The peptide IV (3.64 g, 0.008 mol) was
deblocked with 4N HCl/dioxane (40 mL) for 1.5 hr. Excess HCl and dioxane
were removed under reduced pressure, triturated with ether, filtered,
washed with ether and dried (yield, 100%). The HCl.H-Phe-Pro-OBzl was
neutralized with NMM (0.81 mL, 0.008 mol) and coupled to Boc-Gly (1.4 g,
0.008 mol) in acetonitrile (15 mL) and NMM (0.81 mL, 0.008 mol) using
isobutylchloroformate (1.1 mL, 0.008 mol) and worked up the same as I to
obtain V (3.5 g, yield 88%). Rf1 0.64 and Rf
2 0.72, m.p. 86 °C (Lit. m.p. 86
°C) [20].
Boc-Gly-Gly-Phe-Pro-OBzl (VI). The peptides V (3.06 g, 0.006 mol) was
deblocked with 4 N HCl/dioxane (30 mL) for 1.5 hr. Excess HCl and dioxane
were removed under reduced pressure, triturated with ether, filtered,
washed with ether and dried (yield, 100%). The HCl.H-Gly-Phe-Pro-OBzl was
neutralized with NMM (0.66 mL, 0.006 mol) and coupled to Boc-Gly (1.05 g,
0.006 mol) in acetonitrile (15 mL) and NMM (0.66 mL, 0.006 mol) using
isobutylchloroformate (0.82 mL, 0.006 mol) and worked up the same as I to
obtain VI (3.1 g, yield 89%). Rf1 0.59 and Rf
2 0.68, m.p. 97 °C (Lit. m.p. 96
°C) [20].
Boc-Gly-Gly-Phe-Pro-OH (VII). The peptide VI (2.92 g, 0.005 mol) was
hydrogenolyzed using ammonium formate (2.0 equiv.) and Mg (1 equivalent)
in methanol (10 mL/g) for 2 hours at room temperature. The catalyst was
filtered and washed with methanol. The combined filtrate was evaporated
in vacuo and the residue taken into CHCl3, washed with water, and dried
over Na2SO4. The solvent was removed under reduced pressure and
triturated with ether filtered, washed with ether and dried to obtain VII
(2.32 g, yield 94%). Rf2 0.36 and Rf
3 0.48, m.p. 105-106 °C (Lit. m.p. 105 °C)
[20].
SYNTHESIS OF GVGVP (Scheme 6.6):
Boc-Val-Pro-OBzl (VIII): The HCl.H-Pro-OBzl (6.04, 0.04 mol) was
neutralized with NMM (4.4 mL mL, 0.04 mol) and coupled to Boc-Val (8.7 g,
0.04 mol) in acetonitile (60 mL) and NMM (4.4 mL, 0.04 mol) using
Synthesis of Amides of Amino Acids and Peptides… Chapter 6
118
isobutylchloroformate (5.2 mL, 0.04 mol), HOBt (6.12 g, 0.04 mol) and
worked up the same as IV to obtain VIII. The crude sample was recrystalised
from ethyl acetate and petroleum ether to obtain VIII (13.1 g, yield 81.2 %).
Rf1 0.84 and Rf
2 0.75 (m.p. 81-83°C), (Lit. 83°C) [18].
Boc-Gly-Val-Pro-OBzl. (IX). The peptide VIII (12.13 g, 0.03 mol) was
deblocked with 4N HCl/dioxane (120 mL) for 1.5 hr. Excess HCl and dioxane
were removed under reduced pressure, triturated with ether, filtered,
washed with ether and dried (yield, 100 %). The HCl.H-Val-Pro-OBzl was
neutralized with NMM (3.3 mL, 0.03 mol) and coupled to Boc-Gly (5.25 g,
0.03 mol) in acetonitrile (35 mL) and NMM (3.3 mL) using
isobutylchloroformate (3.9 mL, 0.03 mol) and worked up the same as
dipeptide to obtain IX (12.74 g, yield 92.3%). The same was recrystallized
from ether/petroleum ether. Rf1 = 0.42, Rf
2 = 0.58, m.p. 97 °C, (Lit. 99
°C) [21].
ProValGlyGly
OBzlHBoc OH
Boc(i)
(ii)
(iii)
(iv)
(ii)
(iii)
OBzl
OBzl
OBzl
OBzl
OBzl
OH
HBoc
Boc
Boc
Boc
OH
Boc
OH H
(i) IBCF / NMM/HOBt(ii) 4N HCl / Dioxane(iii) IBCF / NMM(iv) HCOONH4 /Mg
Val
Boc OH H
OBzl
Boc
OBzl(ii)
(iii)
Scheme 6.4: Schematic representation of synthesis of GVGVP
Synthesis of Amides of Amino Acids and Peptides… Chapter 6
119
Boc-Val-Gly-Val-Pro-OBzl. (X). The peptide IX (11.5 g, 0.025 mol) was
deblocked with 4N HCl/dioxane (110 mL) for 1.5 hr. Excess HCl and dioxane
were removed under reduced pressure, triturated with ether, filtered,
washed with ether and dried (yield, 100 %). The HCl.H-Gly-Val-Pro-OBzl was
neutralized with NMM (2.75 mL, 0.025 mol) and coupled to Boc- Val (5.43 g,
0.025 mol) in acetonitrile (35 mL) and NMM (2.75 mL) using
isobutylchloroformate (3.25 mL, 0.025 mol) and worked up the same as
dipeptide to obtain X (12.61 g, yield 90.3%). The same was recrystallized
from ether/petroleum ether. Rf1 = 0.61, Rf
2 = 0.72 m.p. 96-99 °C, (Lit. 96-98
°C) [22].
Boc-Gly-Val-Gly-Val-Pro-OBzl. (XI). The peptide X (11.21 g, 0.02 mol) was
deblocked with 4N HCl/dioxane (110 mL) for 1.5 hr. Excess HCl and dioxane
were removed under reduced pressure, triturated with ether, filtered,
washed with ether and dried (yield, 100%). The HCl.H-Val-Gly-Val-Pro-OBzl
was neutralized with NMM (2.2 mL, 0.02 mol) and coupled to Boc-Gly (3.5g,
0.02 mol) in acetonitrile (35 mL) and NMM (2.2 mL) using
isobutylchloroformate (2.6 mL, 0.02 mol) and worked up the same as
dipeptide to obtain XI (10.98 g, yield 89.2%). The same was recrystallized
from ether/petroleum ether. Rf1 = 0.69, Rf
2 = 0.64 m.p. 117-119°C, (Lit.
116-118°C) [18].
Boc-Gly-Val-Gly-Val-Pro-OH (XII). The peptide XI (6.17 g, 0.01 mol) was
hydrogenolysed in methanol (60 mL) using ammonium formate (2
equivalents) and Mg (1 equivalent) for 2 hours at room temperature. The
catalyst was filtered and washed with methanol. The combined filtrate was
evaporated in vacuo and the residue taken into CHCl3, washed with water,
and dried over Na2SO4. The solvent was removed under reduced pressure
and triturated with ether filtered, washed with ether and dried to obtain XII
(4.8 g, yield 90.9%). Rf2 = 0.68, Rf
3 = 0.78 m.p. 128 °C, (Lit. 127 °C) [23].
Synthesis of Amides of Amino Acids and Peptides… Chapter 6
120
6.3.2 GENERAL PROCEDURE FOR SYNTHESIS OF THIOL ESTERS OF AMINO
ACIDS AND PEPTIDES
To Boc-Xaa-OH or Fmoc-Xaa-OH (Xaa = amino acid or peptide) (10
mmol), dissolved in acetonitrile (30 mL, a little amount of DMF is added in
case of peptides) and cooled to 0°C was added N-methylmorpholine (1.1
mL, 10mmol). To this solution, isobutylchloroformate (1.3 mL, 10 mmol)
was added drop wise under stirring while maintaining the temperature at
0°C. After stirring the reaction mixture for 10 min at this temperature,
1-hydroxybenzotriazole (1.55 g, 10 mmol) was added. The reaction mixture
was stirred for an additional 10 min and thiol (ethyl thiol (EtSH) or phenyl
thiol (PhSH)) (10 mmol) was added slowly. After 20 min, the pH of the
solution was adjusted to 8 by the addition of N-methylmorpholine (NMM)
and the reaction mixture was stirred over night at room temperature.
Acetonitrile was removed under reduced pressure. The residue was taken
into chloroform, washed consecutively with 0.01 N cold HCL, aqueous
sodiumbicarbonate and water and dried over sodium sulphate. The crude
product was recrystallized from ethanol to obtain the corresponding amino
or peptide thiol ester; yield varied between 80-90% (Fig. 6.1 and Fig. 6.2;
crystal structures of Boc-Pro-S-Ph and Boc-Leu-S-Et respectively).
6.3.3 GENERAL PROCEDURE FOR THE REDUCTION OF THIOL ESTERS OF
AMINO ACIDS AND PEPTIDES TO AMIDES
A suspension of an appropriate thiol ester of a protected amino acid or
peptide (200 mg) and Mg (200 mg) in methanol (2.5 mL; little excess for
peptide thiol esters) was stirred with ammonium formate (2-4 equivalents)
at room temperature. After completion of the reaction (monitored by TLC),
the reaction mixture was filtered through celite and washed with solvent.
The combined filtrate and washings were evaporated under vacuum. The
residue was taken into ethyl acetate or chloroform and washed twice with
50% saturated brine and finally with water. The organic layer was dried over
anhydrous sodium sulphate and evaporation of organic layer followed by
purification through column chromatography (CHCl3: CH3OH; 96:4) to yield
desired product (amide).
Synthesis of Amides of Amino Acids and Peptides… Chapter 6
121
Fig 6.1: ORTEP diagram of Boc-Pro-S-Ph
Fig 6.2: ORTEP diagram of Boc-Leu-S-Et
Synthesis of Amides of Amino Acids and Peptides… Chapter 6
122
Table 6.2: Crystal data and structure refinement table for Boc-Pro-S-Ph
Empirical formula C16H21NO3S Formula weight 307.40 Temperature 293(2) K Wavelength 0.71073 Å Crystal system Triclinic Space group P1 Cell dimensions a = 6.0250(7) Å b = 8.2820(13) Å c = 8.7700(14) Å α = 102.352(4)° β = 102.993(11)° γ = 90.279(8)° Volume 415.89(10) Å3 Z 1 Density (calculated) 1.227 Mg/m3 Absorption coefficient 0.203 mm−1
F000 164 Crystal size 0.3 × 0.27 × 0.25 mm Theta range for data collection 2.44-32.61°
Index ranges -7 ≤ h ≤ 7 -12 ≤ k ≤ 12 -12 ≤ l ≤ 12
Reflections collected 3,284 Independent reflections 3,284 [R(int) = 0.0000]
Refinement method Full-matrix least-squares on F2
Data / restraints / parameters 3,284 / 3 / 194 Goodness-of-fit on F2 1.067 Final R indices [I > 2σ(I)] R1 = 0.0465, wR2 = 0.1343 R indices (all data) R1 = 0.0591, wR2 = 0.1586 Extinction coefficient 0.29(4) Largest diff. peak and hole 0.209 and -0.266 e. Å−3
Table 6.3: Crystal data and structure refinement table for Boc-Leu-S-Et
Empirical formula C13H25NO3S Formula weight 275.40 Temperature 293(2) K Wavelength 0.71073 Å Crystal system Orthorhombic Space group pbca
Table 6.3 continued...
Synthesis of Amides of Amino Acids and Peptides… Chapter 6
123
Table 6.3 continued...
Cell dimensions a = 9.6740(8) Å b = 10.6590(7) Å c = 16.2190(13) Å Volume 1672.4(2) Å3 Z 4 Density (calculated) 1.094 Mg/m3 Absorption coefficient 0.195 mm−1
F000 600 Crystal size 0.27 × 0.25 × 0.2 mm
Index ranges -11 ≤ h ≤ 11 -12 ≤ k ≤ 12 -19 ≤ l ≤ 19
Reflections collected 2838 Independent reflections 1682 [R(int) = 0.0167] Refinement method Full-matrix least-squares on F2
Data / restraints / parameters 1682 / 0 / 169 Goodness-of-fit on F2 1.215 Final R indices [I > 2s(I )] R1 = 0.0451, wR2 = 0.1256 R indices (all data) R1 = 0.0587, wR2 = 0.1544 Absolute structure parameter 10(10) Largest diff. peak and hole 0.225 and -0.349 e. Å−3
Boc-Ala-NH2 (2a & 2j): IR (KBr): ν =1248, 1619, 1650, 3202, 3346cm-1. 1H NMR (500 MHz, CDCl3, ppm) δ = 7.20 (s, 2H, -NH2), 7.40 (d, 1H, -NH-), 1.43
(s, 9H, Boc-CH3); 4.68 (m, 1H, α-CH); 1.42 (d, 3H, -CH3). 13C NMR (125 MHz,
CDCl3, ppm): 17.25, 28.54, 47.93, 79.52, 155.69(-CONH-) 175.45 (-CONH2).
Anal. Calcd for C8H16N2O3: C, 51.05; H, 8.57; N, 14.88. Found: C, 50.39; H, 8.02; N, 15.16.
Boc-Phe-NH2 (2b & 2k): IR (KBr): ν =1250, 1165, 1655, 3193, 3345 cm-1. 1H NMR (500 MHz, CDCl3, ppm): δ = 6.95 (s, 2H, -NH2), 7.30 (d, 1H, -NH-), 1.45
(d, 9H, Boc-CH3), 4.23 (d, 1H, α-CH), 3.20 (t, 2H, β-CH2); 7.21 (d, 2H, Ar-H);
7.41(d, 2H, Ar-H); 7.25 (d, 1H, Ar-H). 13C NMR (125 MHz, CDCl3, ppm): 28.6,
37.2 (β-CH2-), 47(α-CH-), 79.51, 155.65 (-CONH-) 176.23 (CONH2), 128,
128.52, 125.97, 136.65 (Ar).
Anal. Calcd for C14H20N2O3: C, 63.62; H, 7.63; N, 10.60. Found: C, 63.99; H,
7.08; N, 10.57.
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Boc-Leu-NH2 (2c): IR (KBr): ν =1250, 1615, 1655, 3193, 3345 cm-1. 1H NMR
(500 MHz, CDCl3, ppm): δ = 6.89 (s, 2H, -NH2); 7.39 (s, 1H, NH); 1.44 (s, 9H,
Boc-CH3); 4.20 (d, 1H, α-CH); 1.72 (t, 2H, β-CH2); 1.50 (t, 1H, γ-CH); 0.96 (d,
6H, δCH3). 13C NMR (125 MHz, CDCl3, ppm): 21.59 (γ-CH), 22.62, 24.42
(CH3), 27.99, 40.95 (β-CH2-), 52.26 (α-CH2-), 79.79, 155.51 (-CONH-), 175.30 (CONH2).
Anal. Calcd for C11H22N2O3: C, 57.37; H, 9.63; N, 12.16. Found: C, 56.98; H, 9.98; N, 12.26.
Boc-Lys-NH2 (2d): IR (KBr): ν =1252, 1619, 1654, 3320, 3337 cm-1. 1H NMR (500 MHz, CDCl3, ppm): δ = 6.20 (s, 2H, -NH2), 7.30 (d, 1H, Boc-NH), 1.43 (s, 9H, Boc-CH3), 4.53 (t, 1H, α-CH); 1.80 (m, 2H, β-CH2); 1.25 (m, 2H, γ-CH2); 1.52 (m, 2H, δ-CH2); 2.65 (m, 2H, ε-CH2), 2.2 (brs, 2H, -NH2). 13C NMR (125 MHz, CDCl3, ppm): 28.45, 79.55, 155.62 (Boc), 51.5, 31.10, 22.31, 28.59, 42.19, 174.92 (CONH2). Anal. Calcd for C11H23N3O3: C, 53.86; H, 9.45; N, 17.13. Found: C, 54.08; H, 9.78; N, 17.26.
Boc-Gly-NH2 (2e): IR (KBr): ν =1256, 1617, 1650, 3195, 3347 cm-1. 1H NMR (500 MHz, CDCl3, ppm): δ = 7.05 (s, 2H, -NH2); 7.38 (d, 1H, Boc-NH); 1.44 (s, 9H, Boc-CH3); 3.85 (d, 2H, CH2). 13C NMR (125 MHz, CDCl3, ppm): 28.41, 79.52, 43.24 (α CH-), 156.22 (-CONH-), 169.86 (CONH2). Anal. Calcd for C7H14N2O3: C, 48.26; H, 8.10; N, 16.08. Found: C, 48.37; H, 8.58; N, 16.22.
Boc-Glu(OcHx)-NH2 (2f): IR (KBr): ν =1255, 1100, 1615, 1652, 3194, 3345 cm-1. 1H NMR (500 MHz, CDCl3, ppm): δ = 6.97 (s, 2H, -NH2), 7.29 (d, 1H, NH), 1.43(d, 9H, Boc-CH3), 4.53 (d, 1H, αCH), 2.07 (t, 2H, βCH2), 2.45 (d, 2H, γCH2), 3.91 (m, 1H, -CH-), 1.43-1.52 (m, 4H, -CH2-), 1.55-1.68 (m, 4H, -CH2-), 1.47-1.49 (m, 2H, -CH2-). 13C NMR(125 MHz, CDCl3, ppm): 28.6,79.5,159.9(Boc); 57.5(αCH2-), 25.60(βCH2-), 34.20(γCH2), 173.5, 22.00, 28.00, 33.20, 75.5 (OcHx), 173.98 (CONH2). Anal. Calcd for C16H28N2O3: C, 58.52; H, 8.59; N, 8.53. Found: C, 58.87; H, 8.72; N, 8.44.
Boc-Pro-NH2 (2g & 2l): IR (KBr): ν =1260, 1615, 1652, 3198, 3346 cm-1. 1H NMR (500 MHz, CDCl3, ppm): δ = 6.97 (s, 2H, -NH2), 1.46 (s, 9H, Boc-CH3), 4.30 (d, 1H, CH), 1.73 (t, 2H, -CH2), 1.54 (t, 2H, CH2), 3.34 (d, 2H, CH2). 13C NMR (125 MHz, CDCl3, ppm): 24.20, 28.42 (Boc), 47.10, 60.25, 79.83, 154.9 2 (-CONH-), 178.19 (CONH2). Anal. Calcd for C10H18N2O3: C, 56.06; H, 8.47; N, 13.07. Found: C, 55.87; H, 8.68; N, 13.62.
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Boc-Val-NH2 (2h): IR (KBr): ν = 1258, 1617, 1653, 3199, 3348 cm-1. 1H NMR (500 MHz, CDCl3, ppm): δ = 7.10 (s, 2H, -NH2), 7.32(d, 1H, NH), 1.46 (d, 9H, Boc-CH3), 4.23 (d, 1H, α-CH), 1.79 (m, 1H, β-CH), 1.00 (d, 6H, γCH3). 13C NMR (125 MHz, CDCl3, ppm): 17.20 (CH3), 28.43, 30.10 (β-CH-), 62.5 (α-CH2-), 79.5, 155.75 (-CONH-), 176.68 (CONH2). Anal. Calcd for C10H20N2O3: C, 55.53; H, 9.32; N, 12.95. Found: C, 55.77; H, 9.68; N, 13.20.
Boc-AVG-NH2 (2m): IR (KBr): ν =1252, 1165, 1519, 1645, 3360 cm-1. 1H NMR (500 MHz, DMSO, ppm): δ = 7.15 (s, 2H, -NH2), 7.40 (d, 1H, Boc-NH), 8.31 (d, 1H, Val-NH), 8.98 (t, 1H, Gly-NH), 1.40 (s, 9H, Boc-CH3), 4.57 (m, 1H, α-H of Ala), 4.12 (m, 1H, α-H of Val), 3.83 (m, 2H, α-H’s of Gly), 1.89 (m, 1H, β-CH of Val), 1.48 (m, 3H, CH3 of Ala), 0.8-0.9 (m, 6H, CH3 of Val). 13C NMR (125 MHz, CDCl3, ppm): 28.6, 79.5, 155.9 (Boc), 17.20, 18.48, 30.10, 42.37, 47.87, 57.92, (alkyl), 171.6 (CONH), 169.8 (CONH2) Anal. Calcd for C15H28N4O5: C, 52.31; H, 8.19; N, 16.27. Found: C, 52.67; H, 8.48; N, 16.73.
Boc-GGFP-NH2 (2n): IR (KBr): ν =1255, 1167, 1520, 1650, 3355 cm-1. 1H NMR (500 MHz, DMSO, ppm): δ = 7.21 (s, 2H, -NH2); 7.39 (d, 1H, Boc-NH); 1.46 (s, 9H, Boc-CH3), 3.85-4.09 (m, 4H, α-H’s of Gly), 4.23 (m, 1H, α-H of Phe), 3.19-3.35 (m, 2H, β-H’s of Phe), 7.21-7.41 (m, 5H, Ar-H), 4.36 (t, 1H, CH), 1.92-2.02 (m, 2H, -CH2), 2.09-2.33 (m, 2H, -CH2), 3.41-3.51 (m, 2H, - CH2) 13C NMR (125 MHz, CDCl3, ppm): 28.6, 79.5, 155.83 (Boc), 24.13, 28.97, 47.00, 35.67, 42.72, 44.21, 50.12, 61.19 (alkyl & pyrolidne), 127.72-136.60 (Ar), 170.24, 170.72, 171.6 (CONH), 169.83 (CONH2). Anal. Calcd for C23H33N5O6: C, 58.09; H, 6.99; N, 14.73. Found: C, 58.31; H, 7.08; N, 15.13.
Boc-GVGVP-NH2 (2o): IR (KBr): ν =1257, 1159, 1635, 3349 cm-1. 1H NMR (500 MHz, DMSO, ppm): δ = 7.20 (s, 2H, -NH2), 7.40 (d, 1H, Boc-NH), 8.32 (d, 2H, NH of Val), 8.92 (t, 1H, NH of Gly), 1.38 (s, 9H, Boc-CH3), 3.85, 4.09 (d, 4H, α-H’s of Gly ), 4.23 (d, 2H, α-H’s of Val), 1.79 (m, 2H, β-H’s of Val), 1.00 (d, 12H, γ-CH3 of Val); 4.30 (d, 1H, CH); 1.91-2.02 (m, 2H, -CH2), 2.09-2.34 (m, 2H, CH2), 3.41-3.51 (m, 2H, CH2). 13C NMR (125 MHz, CDCl3, ppm): 28.6, 79.5, 155.89 (Boc), 170.69 (-CONH of Gly), 171.28, 171.56 (-CONH of Val), 43.09, 44.32 (α-C of Gly), 55.78, 56.68 (α-C’s of Val), 31.10, 31.35 (β-C’s of Val), 17.89 (γ-C’s of Val), 22.20, 47.10, 28.9, 65.00 (C’s pyrolidne), 169.89 (CONH2). Anal. Calcd for C24H42N6O7: C, 54.74; H, 8.04; N, 15.96. Found: C, 55.12; H, 7.88; N, 16.17.
Fmoc-Ala-NH2 (2p): IR (KBr): ν =1253, 1319, 1535, 1666, 3197, 3316 cm-1. 1H NMR (500 MHz, CDCl3, ppm): δ = 7.89 (d, 1H, NH), 6.96 (s, 2H, -NH2), 7.29 –
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7.75 (m, 8H, Fmoc), 4.27 (m, 2H, CH2), 4.20 (m, 1H, α-CH-), 4.01 (s, 1H, Fmoc-CH), 1.23 (d, 3H, -CH3). 13C NMR (125 MHz, DMSO, ppm): 18.43 (-CH3), 46.84 (Fmoc-CH), 50.045 (α-CH-), 65.74 (Fmoc-CH2), 120.23-144.06 (Fmoc), 155.81 (-CONH-), 174.66 (CONH2). Anal. Calcd for C18H18N2O3: C, 69.66; H, 5.85; N, 9.03. Found: C, 68.96; H, 6.43; N, 9.87.
Fmoc-Phe-NH2 (2q): IR (KBr): ν =1255, 1320, 1665, 3200, 3321 cm-1. 1H NMR (500 MHz, DMSO, ppm): δ = 7.91 (d, 1H, NH), 7.10 (s, 2H, -NH2), 7.28 – 7.84 (m, 8H, Fmoc), 4.46 (s, 1H, Fmoc-CH), 4.23 (d, 1H, α-CH); 3.20-3.44 (t, 2H, βCH2), 7.21 (d, 2H, Ar-H), 7.41(d, 2H, Ar-H), 7.25 (d, 1H, Ar-H). 13C NMR (125 MHz, DMSO, ppm): 46.84 (Fmoc-CH), 65.72 (Fmoc-CH2), 120.35-143.56 (Fmoc), 155.85 (CONH-), 47(α-CH-); 37.12(β-CH-); 175.61 (CONH2); 128, 128.5, 126, 139.6 (Ar). Anal. Calcd for C24H22N2O3: C, 74.59; H, 5.74; N, 7.25. Found: C, 75.06; H, 5.83; N, 7.37.
Fmoc-Gly-NH2 (2r): IR (KBr): ν =1253, 1318, 1662, 3195, 3329 cm-1. 1H NMR (500 MHz, DMSO, ppm): δ = 7.90 (d, 1H, NH), 6.98 (s, 2H, -NH2), 7.28 –7.84(m, 8H, Fmoc), 4.32 (s, 1H, Fmoc-CH), 3.85 (d, 2H, CH2). 13C NMR (125 MHz, DMSO, ppm): 47.14 (Fmoc-CH), 65.78 (Fmoc-CH2), 120.25-143.97 (Fmoc), 156.21 (CONH-), 47(α CH-), 174.85 (CONH2). Anal. Calcd for C17H16N2O3: C, 68.91; H, 5.44; N, 9.45. Found: C, 69.05; H, 5.53; N, 9.39.
Fmoc-Leu-NH2 (2s): IR (KBr): ν =1252, 1615, 1653, 3193, 3335 cm-1. 1H NMR (500 MHz, DMSO, ppm): δ = 7.88 (d, 1H, NH), 7.10 (s, 2H, -NH2), 7.28–7.87 (m, 8H, Fmoc), 4.40 (s, 1H, Fmoc-CH), 4.23 (d, 1H, αCH), 1.79 (t, 2H, βCH2), 1.50 (t, 1H, γCH), 1.00 (d, 6H, δCH3). 13C NMR (125 MHz, DMSO, ppm): 46.86 (Fmoc-CH), 65.75 (Fmoc-CH2), 120.25-143.97 (Fmoc), 155.85 (-CONH-), 55.0 (αCH2-), 41.00(βCH2-), 22.1(γCH), 22.4, 22.35 (CH3), 175.25 (-CONH2). Anal. Calcd for C21H24N2O3: C, 71.57; H, 6.86; N, 7.95. Found: C, 71.56; H, 6.97; N, 8.19.
Figure 6.3: IR spectra of Boc-Leu-NH2
Figure 6.4: 1H NMR spectra of Boc-Leu-NH2
Figure 6.5: 13C NMR spectra of Boc-Leu-NH2
Figure 6.6: 1H NMR spectra of Fmoc-Ala-NH2
Figure 6.7: 13C NMR spectra of Fmoc-Ala-NH2
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