Efficient synthesis of peptides by extension at the N- and C-terminii of arginine

Article abstract of DOI:10.1021/jo800805w

(Chemical Equation Presented) L-N^ω-Nitroarginine and L-arginine were coupled with N-(Cbz-α-aminoacyl)benzotriazoles and N-Cbz-dipeptidoylbenzotriazoles to provide arginine LL-dipeptides 9a-e, 11a-d; LLL-tripeptides 18a-c, 20; and diastereomeric mixtures (9b+9b′), (9c+9c′), (11b+11b′) and (18c+18c′) [compound numbers written within parentheses represent a diastereomeric mixture or racemate; compound numbers without parentheses represent an achiral compound or a single enantiomer] by extension at the N-terminus of arginine, in isolated yields of 66-95% with complete retention of chirality as evidenced by NMR and HPLC analysis. Arginine LL-dipeptides 15a-d were synthesized by extension at the C-terminus of arginine in isolated yield of 66-80%, using benzotriazole activated arginine L-^ωNO₂-Arg-Bt, 13. Our methodology has also been used to synthesize the protected RGD peptide (Cbz ^α-L-^ωNO₂-Arg-Gly-L-Asp-(OH) ₂) 21.

Full text of DOI:10.1021/jo800805w

Efficient Synthesis of Peptides by Extension at the N- and
C-Terminii of Arginine
Alan R. Katritzky,* Geeta Meher, and Tamari Narindoshvili
Center for Heterocyclic Compounds, Department of Chemistry, UniVersity of Florida,
GainesVille, Florida 32611-7200
katritzky@chem.ufl.edu
ReceiVed April 10, 2008
L-Nω-Nitroarginine and L-arginine were coupled with N-(Cbz-R-aminoacyl)benzotriazoles and N-Cbz-
dipeptidoylbenzotriazoles to provide arginine LL-dipeptides 9a-e, 11a-d; LLL-tripeptides 18a-c, 20;
and diastereomeric mixtures (9b+9b′), (9c+9c′), (11b+11b′) and (18c+18c′) [compound numbers written
within parentheses represent a diastereomeric mixture or racemate; compound numbers without parentheses
represent an achiral compound or a single enantiomer] by extension at the N-terminus of arginine, in
isolated yields of 66-95% with complete retention of chirality as evidenced by NMR and HPLC analysis.
Arginine LL-dipeptides 15a-d were synthesized by extension at the C-terminus of arginine in isolated
yield of 66-80%, using benzotriazole activated arginine L-ωNO2-Arg-Bt, 13. Our methodology has also
been used to synthesize the protected RGD peptide (CbzR-L-ωNO2-Arg-Gly-L-Asp-(OH)2) 21.
Introduction
inflammatory diseases such as arthritis5 and colitis.6 On the other
hand, impaired NO production can cause hypertension7 and
atherosclerosis.8 Therefore, many studies have been conducted
on novel substrates and isoform-selective NOS inhibitors in
attempts to find treatments for pathological NO production in
biological systems. Nω-Methyl-L-arginine and Nω-ethyl-L-argi-
nine show limited selective inhibition of the NOS isoforms;9
however, high selectivity was estimated for dipeptides and
dipeptide esters containing Nω-nitroarginine and phenylalanine.10
Important selective inhibitors of neuronal NO synthase (nNOS)
over endothelial cell NO synthase (eNOS) include nonbiological
dipeptide amides and peptidomimetics, built on an L-Nω-
nitroarginine scaffold (1a-c, Figure 1).4e
The essential amino acid L-arginine with its guanidine group
is involved in numerous diverse biological processes connected
inter alia with cell division, healing wounds, removal of
ammonia, immune functions, and hormone release.1
L-Arginine is an immediate precursor of nitric oxide (NO) in
a reaction catalyzed by nitric oxide synthase (NOS) isoforms.2
Nitric oxide is a potent biological signal for diverse physiologi-
cal processes within the cardiovascular, immune, and nervous
systems.3 Overproduction of NO can lead to chronic neurode-
generative diseases including Alzheimer’s, Parkinson,4a–e and
Arginine is the preferred residue at the P1 position of serine
protease substrates11 such as trypsin,12 factor Xa,13 and others
of the coagulation cascade; it is an essential residue in the
integrin recognition sequence Arg-Gly-Asp.14a,b
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10.1021/jo800805w CCC: $40.75
Published on Web 08/12/2008
2008 American Chemical Society
J. Org. Chem. 2008, 73, 7153–7158 7153

Katritzky et al.
FIGURE 1. Selective inhibitors based on L-Nω-nitroarginine scaffold 1a-c.
Its geometry, charge distribution, and ability to form multiple
hydrogen bonds make arginine ideal for binding negatively
charged groups; when located on the outside of a protein it can
interact effectively with a polar environment. Certain peptides
containing high percentages of cationic amino acids efficiently
translocate through a cell membrane, as can short oligomers of
arginine.15a,b Synthetic arginine-rich peptides are efficient
transporters of diverse biomolecules including nucleic acids,17a,b
peptides, and proteins18 into the cytoplasmic and nuclear regions
of living cells.16 As a result of their extensive physiological
functions, arginine-containing peptides and conjugates show
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anticancer therapy.20a–d
Considerable effort has been devoted to the synthesis of
arginine peptides and peptidomimetics21a,b utilizing solution and
solid phase methodologies.22a–e The highly basic nature and
nucleophilic character of the guanidine moiety in arginine often
requires appropriate protection before chemical manipulations.
The mixed anhydride method was used to prepare various
C-terminal arginine peptides from Cbz-L-Nω-nitroarginine,23a–d
N-acetylated peptides from L-Nω-nitroarginine ester,24 the selec-
tive anticoagulant tripeptide D-Phe-Pro-Arg25 and peptides from
tribenzoxycarbonyl-L-arginine.26a,b Other approaches utilized
Arg(Cbz)2-OH with TFFH/collidine in CH2Cl2;27 coupling
agents were DCC,28 DCC/DNP, DCC/HOBt, DPPA,29 and
N-carboxyanhydride30 or pyrophosphate.31 Protected Leu-Arg-
Pro tripeptides are prepared with NMM/pivaloyl chloride/HOBt
32
in DMF or CH2Cl2. Unprotected arginine couples with
activated amino acid pentafluorophenyl ester (Pfp) in DMF and
utilization of orthogonal protection affords free tripeptide 4
(Scheme 1).33
With carboxyl-activated protected arginines, intramolecular
δ-lactam formation competes with coupling29,34 to an extent
depending on both the nature of the carboxyl activation and
the amino acid component. Mixed anhydride coupling favors
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MeCN (Scheme 2).34b Lactam formation was favored by
deprotection of the guanidino function27 and minimized by the
DPPA method.29
We have used N-acylbenzotriazoles extensively for N-
acylation of amines35a–e and amides,36 for C-acylation,37a–c and
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Synthesis of Peptides by Extension of Arginine
SCHEME 1
SCHEME 2
maintained during coupling reactions by means of HPLC and
NMR analysis.
1H NMR analysis of dipeptides 9a-e revealed that each LL-
dipeptide displayed two sets of doublets for two amide NH
protons ranging from 7.30 to 8.40 ppm, supporting their
enantiopurity. However, for each of the diastereomeric mixtures
(9b+9b′) and (9c+9c′), one of the amide NH protons showed
as a multiplet. The 13C NMR of (9b+9b′) and (9c+9c′) each
showed doubled signals for each aliphatic and carbonyl carbon.
The guanidine NH2 protons all appeared as broad signals ranging
from 7.30 to 8.30 ppm. The NH proton of the guanidine group
is a broad singlet at ∼8.50 ppm.
SCHEME 3
HPLC Analysis. The enantiopurity of each of the LL-dipetides
9a-e was further supported by HPLC analyses by using a
Chirobiotic T column [detection at 220 nm, flow rate 0.4-1.0
mL/min; eluting with MeOH/H2O (1:1) for 9a,c,d and (9c+9c′),
MeOH/H2O (9:1) for 9b and (9b+9b′); MeOH for 9e].
Enantiopure compound 9b showed a single retention time at
10.13 min, whereas the corresponding diastereomeric mixture
(9b+9b′) showed two retention times with equal intensity at
9.42 and 10.03 min. Similarly in the case of compound 9c one
single retention time at 10.79 min was observed, whereas the
diastereomeric mixture (9c+9c′) showed two retention times
at 8.64 and 10.45 min. Enantiopure compounds 9a, 9d, and 9e
showed single retention times at 11.16, 11.12, and 3.04 min,
respectively.
Preparation of LL-Dipeptides 11a-d and Diastereomeric
Mixture (11b+11b′) by Chain Elongation at the r-N-Terminus
of L-Arg-OH. L-Arginine-containing dipeptides 11a-d and
diastereomeric mixture (11b+11b′) were prepared by extension
at the N-terminus of L-arginine 10 by coupling with N-(Cbz-
R-aminoacyl)benzotriazoles 7a-d, (7b+7b′) in aqueous aceto-
nitrile without Et3N at 20 °C for 6 h (the strongly basic
guanidine group remains protonated at all pH values up to pH
9). After evaporation of solvent, the residue was purified by
reprecipitation from MeOH/Et2O. Repetition of this procedure
three times afforded complete removal of byproduct BtH and
gave pure dipeptides 11a-d and the diastereomeric mixture
(11b+11b′) in yields of 75-83% (Scheme 4, Table 2). NMR
analysis of the compounds revealed no detectable racemization
(<5%). For each of the enantiopure compounds 11a-d, two
sets of doublets were observed for the amide NH protons. For
the diastereomeric mixture (11b+11b′), one of the amide NH
protons appeared as a multiplet and the 13C NMR spectrum also
showed doubling of the signals for the aliphatic and carbonyl
carbons.
for O-acylation.38a,b Chiral di-, tri-, and tetrapeptides were
prepared using (R-Boc-, Cbz-, and Fmoc-aminoacyl)benzotria-
zoles in aqueous acetonitrile in good to high yields from
carboxyl-unprotected amino acids, both without side chain
functionality (Ala, Phe, Val, Leu)39a and with unprotected (Trp,
Tyr, Gln, Ser, Cys, Asn)39b–d and protected (Nω-Cbz-L-Lys-OH)
side chain functionality.40 The original chirality was preserved
in all cases (>95% as evidenced by NMR and >99% by HPLC).
Herein, we present convenient procedures for the coupling
of arginine and Nω-nitroarginine to the C- and N-termini of
amino acids and peptides using benzotriazole methodology.
Results and Discussion
Preparation of LL-Dipeptides 9a-e and Diastereomeric
Mixtures (9b+9b′) and (9c+9c′) by Extension at the
r-N-Terminus of L-Nω-NO2-Arg-OH. L-Nω-NO2-Arg-OH 8
couples with N-(R-Cbz-aminoacyl)benzotriazoles 7a-e derived
from chiral L-Phe, L-Met, L-Ala, L-Trp, and di-Cbz-L-cystine-
di-Bt and corresponding racemic mixtures (7b+7b′) and
(7c+7c′) in aqueous acetonitrile (CH3CN/H2O, 1:2) containing
Et3N in 30 min at 20 °C to give dipeptides 9a-e and
diastereomeric mixtures (9b+9b′) and (9c+9c′) (75-95%), all
isolated without column chromatography (Scheme 3, Table 1).
Diastereomeric mixtures were prepared to confirm that the
original chirality of the amino acids and peptides used is
(37) (a) Katritzky, A. R.; Meher, N. K.; Singh, S. K. J. Org. Chem. 2005,
70, 7792–7794. (b) Katritzky, A. R.; Abdel-Fattah, A. A. A.; Wang, M. Y. J.
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He, H. Y. J. Org. Chem. 2003, 68, 5720–5723.
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2652. (b) Katritzky, A. R.; Angrish, P.; Hur, D.; Suzuki, K. Synthesis 2005,
397–402. (c) Katritzky, A. R.; Angrish, P.; Suzuki, K. Synthesis 2006, 411–
424. (d) Katritzky, A. R.; Todadze, E.; Cusido, J.; Angrish, P.; Shestopalov,
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HPLC Analysis. HPLC analyses (flow rate 1.0 mL/min with
MeOH as eluent) of LL-dipeptides 11a, 11c, and 11d each
showed single retention times at 1.91, 1.92, and 2.88 min,
respectively.
Preparation from Cbzr-L-ωNO2-Arg-Bt 13 of Arginine
LL-Dipeptides 15a-c and Diastereomeric Mixture (15a+15a′)
by Extension at the C-Terminus of L-Arginine. To extend at the
arginine C-terminus, CbzR-L-ωNO2-Arg-OH 12 was treated with
J. Org. Chem. Vol. 73, No. 18, 2008 7155

Katritzky et al.
TABLE 1. Preparation of Arginine Dipeptides 9a-e, (9b+9b′), and (9c+9c′) from L-Nω-NO2-Arg-OH, 8
f
reactant
product
yielda (%)
mp (°C)
[R]23
tR (min)
D
Cbz-L-Phe-Bt 7a
Cbz -L-Met-Bt 7b
Cbz-DL-Met-Bt (7b+7b′)
Cbz-L-Ala-Bt 7c
Cbz-DL-Ala-Bt (7c+7c′)
Cbz-L-Trp-Bt 7d
Di-Cbz-L-cystine-di-Bt 7e
Cbz-L-Phe-L-ωNO2-Arg-OH 9ab
Cbz-L-Met-L-ωNO2-Arg-OH 9bc
Cbz-DL-Met-L-ωNO2-Arg-OH (9b+9b′)
Cbz-L-Ala-L-ωNO2-Arg-OH 9cd
90
80
95
93
92
80
75
173-175
141-143
61-64
-6.2
-4.2
11.16
10.13
9.42, 10.03
10.79
8.64, 10.45
11.12
-2.7
168-169
133-134
65-68
+3.8
-2.3
-19.6
-84.3
Cbz-DL-Ala-L-ωNO2-Arg-OH (9c+9c′)
Cbz-L-Trp-L-ωNO2-Arg-OH 9de
Di-Cbz-L-cystine-di-L-ωNO2-Arg-OH 9e
105-108
3.04
a Isolated yields. b Lit.23d mp 174-176 °C. c Lit.23d mp 140-143 °C. d Lit.23b mp 171-172 °C. e Lit.23d mp 68-75 °C. f Retention time for HPLC
TABLE 2. Preparation of Arginine Dipeptides 11a-d and (11b+11b′) from L-Arg-OH, 10
reactant
product
yielda (%)
mp (°C)
[R]23
D
Cbz-L-Phe-Bt 7a
Cbz-L-Phe-L-Arg-OH 11ab
Cbz-L-Met-L-Arg-OH 11b
Cbz-DL-Met-L-Arg-OH (11b+11b′)
Cbz-L-Trp-L-Arg-OH 11cc
Cbz-L-Val-L-Arg-OH 11dd
83
81
76
83
75
130-132
143-144
121-124
135-137
121-123
-10.4
-7.2
+5.9
-18.3
-1.0
Cbz-L-Met-Bt 7b
Cbz-DL-Met-Bt (7b+7b′)
Cbz-L-Trp-Bt 7c
Cbz-L-Val-Bt 7d
a Isolated yields, b Lit.33b mp 131-133 °C. c Reference 33c. d Reference 33d.
SCHEME 4
17a-c, (17c+17c′) in aqueous acetonitrile in the presence of
Et3N for 2 h to give tripeptides 18a-c and diastereomeric
mixture (18c+18c′) in 66-85% yields (Scheme 6, Table 4).
1
The H and 13C NMR spectra of the optically pure LLL-
tripeptides 18a-c showed the absence of racemization (<5%).
1H NMR showed three sets of doublets for the amide NH
protons for each of the enantiopure compounds. In the case of
the diastereomeric mixture (18c+18c′), each set of amide NH
protons appeared as split doublets and 13C NMR showed
doubling of the signals for the aliphatic and carbonyl carbons.
1H-benzotriazole and thionyl chloride in THF at 20 °C to give
benzotriazole derivative CbzR-L-ωNO2-Arg-Bt 13 (97%); this
reaction completed in 45 min without formation of side products.
Coupling CbzR-L-ωNO2-Arg-Bt 13 with free amino acids 14a-
d, (14a+14a′) gave chiral N-terminal arginine dipeptides 15a-d
and the diastereomeric mixture (15a+15a′) in yields of 65-80%
(Scheme 5, Table 3). The procedure was similar to that utilized
above for the preparation of 9a-c (Scheme 3). Purification of
crude product by reprecipitation in MeOH/Et2O gave pure
dipeptides 15a-c, (15a+15a′), while 15d was isolated by
acidifying at -15 °C. NMR analysis showed no detectable
racemization (<5%) for the LL-dipeptides 15a-d and diaster-
eomeric mixture (15a+15a′). However monitoring by TLC
disclosed that a side product 16 (10-30%) was formed in all
of these reactions and could be isolated from the filtrate. The
1
The room temperature H NMR for 18b showed the existence
of two rotameric forms, which underwent coalescence in a high
temperature 1H NMR experiment. HPLC analysis of tripeptide
18a shows one retention time at 3.17 min supporting its
enantiopurity.
Tripeptide 18c is a protected analogue of H-Asp-Phe-Arg-
OH 4, recently adopted as a catalyst for asymmetric Michael
addition reactions.33 The classical method33 for the preparation
of 4 (Scheme 1), utilizing the pentafluorophenyl ester of amino
acid in DMF, requires prolonged reaction times and complicated
isolation procedures; our methodology advantageously includes
coupling of benzotriazole activated amino acid in aqueous
media, short reaction times, and simple workup procedures
affording final chiral tripeptide 18c and diastereomeric mixture
(18c+18c′) in 82-84% yields.
Application of Benzotriazole Methodology in the
Synthesis of Protected RGD Peptide. We used benzotriazole
methodology to synthesize protected arginyl-glycyl-R-aspartyl
“RGD” tripeptide Cbz-Arg(NO2)-Gly-R-Asp(OH)2 21 (Scheme
7). The recent literature41 preparation of dibenzyl ester derivative
Cbz-Arg(NO2)-Gly-R-Asp(ꢀ-OBz)OBz utilizes amino acid es-
ters and requires low temperatures of -5 to +8 °C, prolonged
reaction times (14-16 h), and coupling reagents (HOBt, DCCI).
Dibenzyl ester protected tripeptide was finally deprotected by
a conventional method to give Arg-Gly-Asp-(OH)2.41 We now
show that our methodology utilizing benzotriazole activated
amino acid 13 enables synthesis of protected RGD peptide 21
from CbzR-L-ωNO2-Arg-Gly-Bt 19 and free aspartic acid 20 by
modification of the coupling procedure (THF was added to the
MeCN/H2O solvent system) adopted for the preparation of other
tripeptides.
1
structure of 16 was revealed (by H and 13C NMR) to be the
intramolecular cyclization product (δ-lactam); 16 formed com-
petitively with the expected dipeptide during coupling of
benzotriazole activated nitroarginine 13 with free amino acids,
other than Gly-OH.
HPLC Analysis. HPLC analyses for the enantiopure LL-
peptides 15a-d showed single retention times, while diaster-
eomeric mixture (15a+15a′) showed two retention times at 3.16
and 3.94 min (Table 3).
Preparation of C-Terminal Arginine Tripeptides 18a-c
and (18c+18c′) by Extension at the r-N-Terminus of
L-Nω-NO2-Arg-OH 8. N-Cbz-dipeptidoylbenzotriazoles 17a,b
were obtained as reported previously.40 Analogs 17c and
(17c+17c′) were prepared similarly to a described procedure39d
from Cbz-L-Asp(OBz)Bt 7f (synthesis of 7f is described in
Supporting Information). L-Nω-NO2-Arg-OH 8 was coupled with
(41) Abo-Ghalia, M.; Abd El-Rahman, S.; El-Kafrawy, A.; Kalomuch, A.
Amino Acids 2003, 24, 405–411.
7156 J. Org. Chem. Vol. 73, No. 18, 2008

Synthesis of Peptides by Extension of Arginine
SCHEME 5
TABLE 3. Preparation from Cbzr-L-ωNO2-Arg-Bt 13 of Arginine Dipeptides 15a-d, (15a+15a′)
c
reactant
product
yielda (%)
mp (°C)
[R]23
tR (min)
D
L-Phe-OH 14a
DL-Phe-OH (14a+14a′)
L-Met-OH 14b
L-Ser-OH 14c
CbzR-L-ωNO2-Arg-L-Phe-OH 15a
CbzR-L-ωNO2-Arg-DL-Phe-OH (15a+15a′)
CbzR-L-ωNO2-Arg-L-Met-OH 15b
CbzR-L-ωNO2-Arg-L-Ser-OH 15c
CbzR-L-ωNO2-Arg-Gly-OH 15db
68
65
66
65
80
217-219
205-207
144-146
83-85
+5.3
+8.1
-4.6
-5.6
-1.5
3.01d
3.16, 3.94d
3.34d
3.57d
Gly-OH 14d
115-117
4.15d
a Isolated yields. b Lit.41 mp 114-117 °C. c Retention time. d Flow rate 1.0 mL/min, eluent MeOH.
SCHEME 6
TABLE 4. Preparation of L-Arginine Tripeptides 18a-c and (18c+18c′)
reactant
product
yielda (%)
mp (°C)
[R]23
D
Cbz-L-Ala-L-Trp-Bt 17a
Cbz-L-Ala-L-Trp-L-ωNO2-Arg-OH 18a
66
75
84
82
150-151
77-79
128-129
60.4-65.0
-10.2
-8.0
-11.7
-6.3
Cbz-L-Phe-L-Met-Bt 17b
Cbz-L-Phe-L-Met-L-ωNO2-Arg-OH 18b
Cbz-L-Asp(OBz)-L-Phe-Bt 17cb
Cbz-L-Asp(OBz)-DL-Phe-Bt (17c+17c′)b
Cbz-L-Asp(OBz)-L-Phe-L- ωNO2-Arg-OH 18c
Cbz-L-Asp(OBz)-DL-Phe-L-ωNO2-Arg-OH (18c+18c′)
a Isolated yields; b Syntheses of 17c and (17c+17c′) are described in Supporting Information.
SCHEME 7. Preparation of Protected RGD Peptide 21
In comparison, our methodology offers simple preparative
and workup procedures, takes less time to complete, uses
inexpensive reagents, gives high yields, and allows the use of
free amino acids as coupling components, affording 21 in good
yield.
tripeptides in short reaction times utilizing simple preparative
and workup procedures, inexpensive reagents, and free amino
acids as coupling components. The peptides can be prepared
by chain elongation at either the N- or C-terminus of L-arginine.
We have successfully synthesized protected RGD peptide
sequence with our benzotriazole methodology.
Conclusions
Experimental Section
The methodology described in the present paper provides for
the convenient and efficient preparation of arginine di- and
General Procedure for the Preparation of LL-Dipeptides
9a-e and Diastereomeric Mixtures (9b+9b′), (9c+9c′). N-(Cbz-
J. Org. Chem. Vol. 73, No. 18, 2008 7157

Katritzky et al.
R-aminoacyl)benzotriazoles 7a-d and (7b+7b′) (0.5 mmol) were
added at 20 °C to a solution of L-ωNO2-Arg-OH 2 (0.5 mmol) in
MeCN (5 mL)/H2O (10 mL) in the presence of Et3N (0.6 mmol).
The reaction mixture was then stirred at 20 °C until the starting
material was completely consumed as observed by TLC using
EtOAc/hexanes (1:2) as the eluent. After addition of 4 N HCl (1
mL), the solution was concentrated under reduced pressure to
remove acetonitrile. Residue was extracted with EtOAc (20 mL),
and the organic extract was washed with 4 N HCl (5 mL) and
saturated NaCl (10 mL) and then dried over anhydrous MgSO4.
Evaporation of the solvent gave the desired product in pure form,
which was further recrystallized from MeOH/Et2O unless specified
otherwise.
δ 1.30-1.80 (m, 4H), 2.80-2.98 (m, 1H), 2.98-3.22 (m, 3H),
3.90-4.15 (m, 1H), 4.37-4.54 (m, 1H), 5.02 (s, 2H), 7.07-7.50
(m, 11H), 7.60-8.25 (m, 2H), 8.12 (d, J ) 7.4 Hz, 1H), 8.48 (br
s, 1H), 12.78 (br s, 1H). 13C NMR (DMSO-d6) δ 24.7, 29.2, 36.7,
53.3, 54.2, 65.5, 126.5, 127.7, 128.2, 128.4, 129.2, 137.0, 137.4,
155.9, 159.3, 171.7, 172.8. Anal. Calcd for C23H28N6O7: C, 55.19;
H, 5.64; N, 16.79. Found: C, 54.84; H, 5.76; N, 16.63.
(S)-2-[(S)-2-((S)-2-Benzyloxycarbonylaminopropanoylamino)-3-
(1H-indol-3-yl)propanoyl-amino]-5-nitroguanidinopentanoic Acid
(Cbz-L-Ala-L-Trp-L-ωNO2-Arg-OH, 18a). White microcrystals (66%),
mp 150-151 °C, [R]23D ) -10.2 (c 1.0, DMF); 1H NMR (DMSO-
d6) δ 1.13 (d, J ) 7.1 Hz, 3H), 1.43-1.85 (m, 4H), 2.90-3.15 (m,
1H), 3.15-3.25 (m, 3H), 3.95-4.10 (m, 1H), 4.15-4.20 (m, 1H),
4.50-4.65 (m, 1H), 4.98 (d, J ) 12.4 Hz, 1H, A part of AB
system,), 5.03 (d, J ) 12.6 Hz, 1H, B part of AB system), 6.96 (t,
J ) 7.4 Hz, 1H), 7.05 (t, J ) 7.4 Hz, 1H), 7.15 (s, 1H), 7.20-7.40
(m, 6H), 7.41 (d, J ) 7.4 Hz, 1H), 7.58 (d, J ) 7.7 Hz, 1H),
7.65-8.40 (m, 2H), 7.96 (d, J ) 8.0 Hz, 1H), 8.23 (d, J ) 8.0 Hz,
1H), 8.54 (br s, 1H), 10.82 (s, 1H), 12.71 (br s, 1H). 13C NMR
(DMSO-d6) δ 18.1, 24.8, 27.5, 28.4, 50.1, 51.6, 53.1, 65.4, 109.8,
111.2, 118.2, 118.4, 120.8, 123.6, 127.4, 127.8, 128.4, 136.0, 137.0,
155.7, 159.3, 171.5, 172.3, 173.3. Anal. Calcd for C28H34N8O8 ·H2O:
C, 53.50; H, 5.77; N; 17.82. Found: C, 53.18; H, 5.70; N, 17.37.
HRMS calcd for [C28H34N8O8 + Na]+: 633.2391. Found: 633.2371.
Procedure for Preparation of Cbzr-L-ωNO2-Arg-Gly-L-Asp-
OH, 21. CbzR-L-ωNO2-Arg-Gly-Bt (19, 0.5 mmol) was dissolved
in a minimum amount of THF and added dropwise at -15 °C to a
solution of L-Asp-OH (0.55 mmol) in MeCN (5 mL/3 mL) in the
presence of Et3N (2.1 mmol). The reaction mixture was stirred at
-15 °C, and progress was monitored using TLC by disappearance
of 19. After 3.5 h reaction mixture was concentrated under reduced
pressure to remove MeCN. The reaction mixture was acidified with
4 N HCl (2 mL) under cold condition, and the solution was extracted
with EtOAc (100 mL) after adding solid NaCl to the acidified
solution. The organic extract was washed with 4 N HCl and
saturated NaCl (10 mL) and then dried over anhydrous MgSO4.
Evaporation of solvent under reduced pressure gave the product,
which was purified by reprecipitation from MeOH/Et2O.
(S)-2-((S)-2-Benzyloxycarbonylamino-3-phenylpropanoylamino)-
5-nitroguanidinopentanoic Acid (Cbz-L-Phe-L-ωNO2-Arg-OH, 9a).
White microcrystals (90%), mp 173-175 °C (lit.23d 174-176 °C),
1
[R]23 ) -6.2 (c 1.0, DMF); H NMR (DMSO-d6) δ 1.40-1.70
D
(m, 3H), 1.70-1.87 (m, 1H), 2.60-2.80 (m, 1H), 2.90-3.05 (m,
1H), 3.05-3.25 (m, 2H), 4.10-4.35 (m, 2H), 4.92 (s, 2H),
7.00-7.40 (m, 10H), 7.51 (d, J ) 8.4 Hz, 1H), 7.60-8.25 (m,
2H), 8.32 (d, J ) 7.4 Hz, 1H), 8.56 (br s, 1H), 12.65 (br s, 1H).
13C NMR (DMSO-d6) δ 24.9, 28.3, 37.4, 40.2, 51.7, 56.0, 65.2,
126.3, 127.5, 127.7, 128.1, 128.3, 129.2, 137.0, 138.1, 155.9, 159.3,
171.8, 173.4. Anal. Calcd for C23H28N6O7: C, 55.19; H, 5.64; N,
16.79. Found: C, 54.88; H, 5.76; N, 16.63.
General Procedure for the Preparation of LL-Dipeptides 11a-d
and Diastereomeric Mixture (11b+11b′). N-(Cbz-R-aminoacyl-
)benzotriazoles 7a-d and (7b+7b′) (0.5 mmol) were added at 20
°C to a solution of L-Arg-OH 10 (0.5 mmol) in MeCN (5 mL)/
H2O (3 mL) The reaction mixture was then stirred at 20 °C until
the starting material was completely consumed as observed by TLC
using EtOAc/hexanes (1:2) as the eluent. The reaction mixture was
evaporated under reduced pressure, the residue was dissolved in a
minimum amount of MeOH, and then product was reprecipitated
with Et2O. This was repeated three times to remove all BtH from
reaction mixture.
(S)-2-((S)-2-Benzyloxycarbonylamino-3-phenyl-propionylamino)-
5-guanidinopentanoic Acid (Cbz-L-Phe-L-Arg-OH, 11a). White
microcrystals (83%), mp 131-132 °C (lit.33b 131-133 °C), [R]23
D
(S)-2-(2-((S)-2-(Benzyloxycarbonylamino)-5-(2-nitroguanidino)-
1
) -10.4 (c 1.0, DMF); H NMR (DMSO-d6) δ 1.35-1.75 (m,
pentanamido)acetamido)succinic Acid (Cbzr-L-ωNO2-Arg-Gly-L-
4H), 1.65-1.80 (m, 1H), 2.95-3.18 (m, 3H), 3.85-4.00 (m, 1H),
4.12-4.25 (m, 1H), 4.93 (s, 2H), 7.05-7.35 (m, 10H), 7.35-7.55
(m, 3H), 7.55-7.70 (m, 2H), 9.41 (br s, 1H). 13C NMR (DMSO-
d6) δ 25.3, 29.8, 37.4, 40.4, 53.7, 56.7, 65.2, 126.2, 127.3, 127.7,
128.1, 128.3, 129.2 137.1, 138.4, 155.9, 157.4, 170.4, 175.5.
Synthetic Procedure for Preparation of Cbzr-L-ωNO2-Arg-Bt,
13. CbzR-L-ωNO2-Arg-Bt was prepared from CbzR-L-ωNO2-Arg-OH
using a reported procedure.40 Reaction was completed in 45 min.
Benzyl N-[(1S)-4-{[Amino(nitroimino)methyl]amino}-1-(1H-
1,2,3-benzotriazol-1-ylcarbonyl)-butyl]carbamate (Cbzr-L-ωNO2-
Asp-(OH)2, 21). White microcrystals (60%), mp 75-79 °C, [R]23
D
1
) -13.7 (c 1.0, DMF); H NMR (DMSO-d6) δ 1.40-1.80 (m,
4H), 2.59 (dd, J ) 16.8, 6.6 Hz, 1H), 2.70 (dd, J ) 16.5, 5.5 Hz,
1H), 3.03-3.22 (m, 2H), 3.65-3.83 (m, 2H), 3.95-4.08 (m, 1H),
4.50-4.62 (m, 1H), 5.00 (d, J ) 12.6 Hz, A part of AB system,
1H), 5.05 (d, J ) 12.6 Hz, B part of AB system, 1H), 7.25-7.42
(m, 5H), 7.52 (d, J ) 7.4 Hz, 1H), 7.55-8.10 (m, 2H), 8.07-8.30
(m, 2H), 8.48 (br s, 1H) 12.60 (br s, 2H). 13C NMR (DMSO-d6):
δ 24.7, 29.0, 36.1, 41.6, 45.6, 48.5, 54.4, 65.0, 127.8, 128.4, 136.9,
156.1, 159.3, 168.6, 171.6, 172.0, 172.3. HRMS calcd for
[C21H31N7O10 + 2Na]+: 570.1531. Found: 570.1531.
Arg-Bt, 13). White microcrystals (97%), mp 146-148 °C, [R]23
D
1
) -16.2 (c 1.0, DMF); H NMR (DMSO-d6) δ 1.58-2.10 (m,
4H), 3.08-4.23 (m, 2H), 5.05 (s, 2H), 5.43-5.55 (m, 1H),
7.28-7.44 (m, 5H), 7.66 (t, J ) 7.7 Hz, 1H), 7.83 (t, J ) 7.7 Hz,
Acknowledgment. We thank Dr. Parul Angrish for her initial
help and Dr. C. Dennis Hall for helpful suggestions.
1H), 7.74-8.16 (m, 2H), 8.20-8.36 (m, 3H), 8.45 (br s, 1H). 13
C
NMR (DMSO-d6) δ 24.8, 28.1, 40.2, 54.1, 65.9, 114.0, 120.3, 126.8,
127.9, 128.0, 128.4, 130.6, 131.2, 136.7, 145.4, 156.4, 159.3, 171.8.
Anal. Calcd for C20H22N8O5: C, 52.86; H, 4.88; N, 24.66. Found:
C, 52.96; H, 4.89; N, 24.50.
(S)-2-((S)-2-Benzyloxycarbonylamino-5-(nitroguanidino)pen-
tanoylamino)-3-phenylpropanoic Acid (Cbzr-L-ωNO2-Arg-L-Phe-
OH, 15a). White microcrystals (68%), mp 220-222 °C (lit.23a
225-226 °C), [R]23D ) +5.3 (c 1.0, DMF); 1H NMR (DMSO-d6)
Supporting Information Available: Compound character-
ization data for 7f, 9b-e, (9b+9b′), (9c+9c′), 11b-d,
(11b+11b′), 15b-d, (15a+15a′), 17c, (17c+17c′), 18b,c,
(18c+18c′), 19, 22a, (22a+22a′). This material is available free
of charge via the Internet at http://pubs.acs.org.
JO800805W
7158 J. Org. Chem. Vol. 73, No. 18, 2008