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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).


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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].


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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,


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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


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(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)



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


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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


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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


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


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


washed with ether and dried (yield, 100%). The HCl.H-Gly-Phe-Pro-OBzl was


0.006 mol) in acetonitrile (15 mL) and NMM (0.66 mL, 0.006 mol) using


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


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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



washed with ether and dried (yield, 100 %). The HCl.H-Val-Pro-OBzl was


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


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Boc-Val-Gly-Val-Pro-OBzl. (X). The peptide IX (11.5 g, 0.025 mol) was



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,



dipeptide to obtain X (12.61 g, yield 90.3%). The same was recrystallized


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



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,



dipeptide to obtain XI (10.98 g, yield 89.2%). The same was recrystallized


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].


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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).


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Fig 6.1: ORTEP diagram of Boc-Pro-S-Ph

Fig 6.2: ORTEP diagram of Boc-Leu-S-Et


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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...


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.


125

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 –


126

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


132

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