Convenient peptide synthesis using unprotected α-amino acids containing another hydrophilic moiety under basic conditions

Article abstract of DOI:10.1246/cl.2012.577

Carboxylic acids 1 and 7 reacted effectively with unprotected α-amino acids 2 containing another hydrophilic moiety under basic conditions via activation by ethyl chloroformate and triethylamine to afford the corresponding amides in 7499% yields.

Full text of DOI:10.1246/cl.2012.577

doi:10.1246/cl.2012.577
Published on the web May 12, 2012
577
Convenient Peptide Synthesis Using Unprotected ¡-Amino Acids
Containing Another Hydrophilic Moiety under Basic Conditions
Takuya Noguchi, Seunghee Jung, and Nobuyuki Imai*
Faculty of Pharmacy, Chiba Institute of Science, 15-8 Shiomi-cho, Choshi, Chiba 288-0025
(Received February 17, 2012; CL-120133; E-mail: nimai@cis.ac.jp)
R2
O
Carboxylic acids 1 and 7 reacted effectively with unpro-
ClCO2Et
Et3N
O
R2
O
O
R1
O
tected ¡-amino acids 2 containing another hydrophilic moiety
under basic conditions via activation by ethyl chloroformate and
triethylamine to afford the corresponding amides in 74-99%
yields.
R1
O
R1
H2N
OH
N
O
OEt
Cbz-HN
OH
H
Cbz-HN
OH
THF
Cbz-HN
THF-aq. base
Scheme 1.
Table 1. Amidation of 3-phenylpropanoic acid (1) with
unprotected ¡-amino acids 2a-2ea
Effective coupling reagents for N-acylation of unprotected
¡-amino acids via activation of carboxylic acids have been
reported.1 These types of active species are relatively stable in
water and easy to handle. For example, the coupling reactions
using unprotected ¡-amino acids and N-acyl derivatives ob-
tained by the combination of 1-hydroxysuccinimide (HOSu)
or 1-hydroxybenzotriazole (HOBt) and 1-ethyl-3-(3-dimethyl-
aminopropyl)carbodiimide hydrochloride (WSCI) proceed under
mild conditions.1b On the other hand, unprotected ¡-amino acids
react easily with p-nitrophenyl esters obtained by p-nitrophenyl
chloroformate (pNPCF) in the presence of triethylamine and
4-dimethylaminopyridine (DMAP), and no racemization was
observed.1d However, these types of activating reagents are
relatively expensive. We have just reported convenient prepa-
ration of dipeptides without protection of C-terminals via mixed
carbonic carboxylic anhydrides.1a
O
1) ClCO2Et, Et3N, THF, 0 °C, 30 min
Ph
OH
R
O
2)
, H2O, 0
H2N OH
°
C, 30 min
1
(1.0 mmol)
2
R
R
O
O
O
O
O
O
+
+
Ph
EtO
N
H
Ph
N
H
O
Ph
OH
OH
3
4
1'
Ratio of
3:4:1¤b
Yield of 3/%
Correctedc Crystal
Entry
R
2
Mother
solution
1
2
3
4
5
HOCH2
HSCH2
p-HOC6H4
H2NCOCH2CH2 2d
HO2CCH2CH2 2e
2a
2b
2c
89
86
73
73
50
87
86
71
73
45
19:55:26
16:71:13
10:6:83
0:0:100
13:24:64
Herein, we describe condensation of carboxylic acids with
unprotected ¡-amino acids containing hydrophilic groups under
basic conditions via activation by ethyl chloroformate and
triethylamine (Scheme 1).
aAll reactions were carried out with 1.0 mmol of 1, 1.4 mmol
of ethyl chloroformate, and 3.0 mmol of triethylamine in
20 mL of THF. After stirring for 30 min at 0 °C, 1.5 mmol of
unprotected ¡-amino acid 2 in 20 mL of H2O was added at
0 °C to the reaction mixture. bDetermined by 1H NMR analysis
of the crude concentrate of the mother solution. cCrystal + 3 in
the mother solution (3 + 4 + 1¤).
In a preliminary investigation, the reaction of 3-phenyl-
propanoic acid (1) with 1.5 equivalents of unprotected ¡-amino
acids 2a-2e in the presence of 1.4 equivalents of ethyl
chloroformate and 3.0 equivalents of triethylamine in tetra-
hydrofuran (THF)-H2O afforded the corresponding amide 3a-3e
as indicated in Table 1. The reaction of 1 with unprotected
¡-amino acids containing an alcohol (2a), a thiol (2b), a phenol
(2c), or an amide (2d) moiety gave the corresponding products
in 73-89% yields within a short reaction time. Small amounts of
in 67% yield with a 30% yield (based on 1) of the by-product 1¤
(Entry 7).
A possible pathway is shown in Scheme 2. A carboxylic
acid 2 reacted with a mixed carbonic carboxylic anhydride 5
to form the corresponding carboxylic anhydride intermediate 6
or 6-H+ predominantly. Under neutral or basic conditions, the
formation of 6 proceeds easily, followed by the intramolecular
reaction of 6 via five-membered transition state to afford the
corresponding amide, because both the carboxylate anion and
free amine are good nucleophiles (Entries 3-6 in Table 2). The
reaction does not work well under acidic conditions because the
carboxylic acid possesses low nucleophilicity and ammonium
ion is not nucleophilic (Entry 1 in Table 2).
Furthermore, condensation of 3-phenylpropanoic acid (1)
and N-benzyloxycarbonyl (Cbz)-L-phenylalanine (7) with sev-
eral kinds of unprotected ¡-amino acids 2a-2e in an aqueous
NaHCO3 solution via mixed carbonic carboxylic anhydride are
collected in Tables 3 and 4, respectively. The yields of the
1
by-products 4 and 1¤ were detected by H NMR analysis of the
concentrates of the mother solutions (Entries 1-4). However, the
reaction of 1 with L-Glu-OH (2e) slightly soluble in water
afforded 3e in only 50% yield with a 48% yield (based on 1) of
the by-product 1¤ (Entry 5).
Next, the amidation of 3-phenylpropanoic acid (1) with
L-Glu-OH (2e) under acidic or basic conditions was examined,
and the results are summarized in Table 2. The reaction of 1 with
2e under acidic conditions using 1.5 equivalents of HCl afforded
3e in 6% yield with a 76% yield of the corresponding carbonic
carboxylic anhydride 5 as shown in Entry 1. In contrast, the
reaction of 1 with 2e under basic conditions using 1.5
equivalents of NaHCO3, NaOH, Na2CO3, or K2CO3 gave 3e
in 94-99% yields (Entries 3-6). The amidation of 1 with 2e in
the presence of 20 equivalents of K2CO3 afforded the amide 3e
Chem. Lett. 2012, 41, 577-579
© 2012 The Chemical Society of Japan
www.csj.jp/journals/chem-lett/

578
Table 2. Effect of additives on the amidation of 3-phenyl-
propanoic acid (1) with L-glutamic acid (2e)a
Table 3. Amidation of 3-phenylpropanoic acid (1) with
unprotected ¡-amino acids 2a-2e under basic conditionsa
O
O
1) ClCO2Et, Et3N, THF, 0 °C, 30 min
1) ClCO2Et, Et3N, THF, 0 °C, 30 min
Ph
OH
Ph
OH
R
O
2) L-Glu-OH (2e), additive, H2O, 0 °C, 30 min
2)
, NaHCO3, H2O, 0 °C, 30 min
1
1
(1.0 mmol)
(1.0 mmol)
O
COOH
+
OH
H2N OH
O
COOH
O
O
2
O
O
+
EtO
N
H
4e
Ph
N
H
Ph
O
Ph
O
R
O
R
O
O
OH
O
O
+
+
Ph
EtO
N
H
4
Ph
N
H
O
Ph
3e
1'
OH
OH
3
1'
Ratio of
3e:4e:1¤b
Yield of 3e/%
Correctedd Crystal
Ratio of
3:4:1¤b
Entry Additive pH valuec
Yield of 3/%
Correctedc Crystal
Mother
solution
Entry
R
2
Mother
solution
1
2
3
4
5
6
7e
HCl
free
1.0
4.5
6.5
7.5
8.0
9.0
6
50
94
99
98
98
67
6
45
90
99
98
96
66
0:0:0
1
2
3
4d
5
HOCH2
HSCH2
p-HOC6H4
p-HOC6H4
H2NCOCH2CH2 2d
HO2CCH2CH2 2e
2a
2b
2c
2c
93
94
68
90
74
94
91
92
66
79
74
90
13:57:30
11:80:10
10:10:80
29:52:19
0:0:100
13:24:64
22:55:23
7:52:41
5:27:68
9:39:52
6:24:71
NaHCO3
NaOH
Na2CO3
K2CO3
K2CO3
6
22:55:23
12
aAll reactions were carried out with 1.0 mmol of 1, 1.4 mmol
of ethyl chloroformate, and 3.0 mmol of triethylamine in
20 mL of THF. After stirring for 30 min at 0 °C, a solution of
1.5 mmol of unprotected ¡-amino acid 2 and 1.5 mmol of
NaHCO3 in 20 mL of H2O was added at 0 °C to the reaction
mixture. bDetermined by 1H NMR analysis of the crude
aAll reactions were carried out with 1.0 mmol of 1, 1.4 mmol
of ethyl chloroformate, and 3.0 mmol of triethylamine in
20 mL of THF. After stirring for 30 min at 0 °C, a solution of
1.5 mmol of L-glutamic acid (2e) and 1.5 mmol of an additive
in 20 mL of H2O was added at 0 °C to the reaction mixture.
1
bDetermined by H NMR analysis of the crude concentrate of
c
the mother solution. cThe pH value of the solution of 2e and an
additive in 20 mL of H2O was measured with pH-test paper.
dCrystal + 3e in the mother solution (3e + 4e + 1¤). eThe
reaction was carried out with 20 mmol of K2CO3.
concentrate of the mother solution. Crystal + 3 in the mother
d
solution (3 + 4 + 1¤). NaOH was used instead of NaHCO3.
Table 4. Synthesis of dipeptides 8 with Cbz-L-Phe-OH (7) and
unprotected ¡-amino acids 2a-2e under basic conditionsa
1) ClCO2Et, Et3N, THF, 0 °C, 30 min
R
O
O
Ph
O
R
R
O
-
H+
R
O
O
HO
Ph
O
O
N
H
8
H3N
R
O
H3N
OH
H2N
2)
, NaHCO3, H2O, 0 °C, 30 min
2
Cbz-HN
OH
Cbz-HN
OH
7
H2N OH
2
O
O
fast
O
Et
5
Yieldb/%
fast
O
5
slow
O
Entry
R
2
Dipeptide 8
Ph
O
O
5
1
HOCH2
HSCH2
p-HOC6H4
H2NCOCH2CH2
HO2CCH2CH2
HO2CCH2CH2
2a
2b
2c
2d
2e
2e
8a
8b
8c
8d
8e
8f
95
91
93
82
93
83
O
O
Ph
R
O
Ph
H2N
2
Ph
Ph
O
H2N
O
NH3
6-H+
3c
4
O
R
6
R
6
fast
slow
fast
5d
6e
O
R
O
R
O
R
O
O
O
N
H
N
H
N
H
Ph
Ph
O
OH
OH
aAll reactions were carried out with 1.0 mmol of Cbz-L-
phenylalanine (7), 1.4 mmol of ethyl chloroformate, and
3.0 mmol of triethylamine in 20 mL of THF. After stirring
for 30 min at 0 °C, a solution of 1.5 mmol of ¡-amino acid 2
and 1.5 mmol NaHCO3 in 20 mL of H2O was added at 0 °C to
the reaction mixture. Isolated yield. NaOH was used instead
of NaHCO3. dDetails of experimental procedure in ref. 2. eThis
reaction was carried out with 0.5 mmol of Cbz-D-phenyl-
alanine (7¤), 0.7 mmol of ethyl chloroformate, and 1.5 mmol of
triethylamine in 10 mL of THF. After stirring for 30 min at
0 °C, 0.75 mmol of ¡-amino acid 2 in 10 mL of H2O was
added at 0 °C to the reaction mixture.
Scheme 2. Possible pathway of the amidation via mixed
carbonic carboxylic anhydride 5.
reactions with ¡-amino acids containing an alcohol (2a), a thiol
(2b), an amide (2d), or a carboxylic acid (2e) moiety were higher
in all cases under the basic conditions (Entries 1, 2, 5, and 6 in
Table 3) than those without base (Table 1). The reaction of 1
with L-Tyr-OH (2c) slightly soluble in an aqueous NaHCO3
solution afforded 3c in 68% yield (Entry 3 in Table 3). L-Tyr-
OH (2c) is dissolved in an aqueous NaOH solution and the
reaction of 1 with 2c gave the corresponding amide 3c in 90%
yield (Entry 4 in Table 3). Finally, the reaction of 7 with
unprotected ¡-amino acids 2a-2e under basic conditions
afforded the peptides 8a-8f in 82-95% yields as shown in
Table 4. L- and D-Cbz-Phe-OH with L-Glu-OH were converted
into the corresponding single diastereomers, which are checked
by 1H NMR analysis, in 93% and 83% yields, respectively
b
c
(Entries 5 and 6). These results indicate that racemization does
not proceed under the reaction conditions.
In conclusion, we have improved the peptide synthesis
using unprotected ¡-amino acids 2a-2e containing another
hydrophilic moiety under basic conditions to afford the
Chem. Lett. 2012, 41, 577-579
© 2012 The Chemical Society of Japan
www.csj.jp/journals/chem-lett/

579
corresponding dipeptide 8a-8f in 82-95% yields. Further
investigations about this type of condensation for preparation
of polypeptide are under way in our group.
After stirring for 30 min at 0 °C, a solution of 220 mg
(1.50 mmol, 1.5 equiv) of L-Glu-OH (2e) in 20 mL of
aqueous NaHCO3 (1.50 mmol, 1.5 equiv) solution was added
at 0 °C to the colorless suspension. The mixture was stirred
for 30 min at 0 °C, the colorless clear solution was
concentrated in vacuo. To the residue was added a 1.0 M
aqueous HCl solution to adjust to pH 2. The resulted
suspension was extracted with 100 mL of ethyl acetate, and
the organic layer was washed with 10 mL of brine, and dried
over MgSO4, and concentrated. The crude product was
recrystallized from 80 mL of a 1:7 mixture of EtOAc and
hexane to afford 399 mg (93% yield) of 8e (Cbz-L-Phe-L-
Glu-OH). 8e: colorless powder; 1H NMR (CD3OD): ¤ 1.89-
1.99 (1H, m, CH), 2.15-2.33 (1H, m, CH), 2.39 (2H, t,
J = 7.5 Hz, CH2), 2.85 (1H, dd, J = 9.7, 13.9 Hz, CHA),
3.15 (1H, dd, J = 5.1, 13.9 Hz, CHB), 4.40-4.47 (2H, m,
CH2), 5.01 (2H, s, CH2), 7.18-7.32 (10H, m, C6H5 © 2).
References and Notes
1
a) T. Noguchi, N. Tehara, Y. Uesugi, S. Jung, N. Imai, Chem.
Lett. 2012, 41, 42, and the references cited therein. b) F.
Fujisaki, M. Oishi, K. Sumoto, Chem. Pharm. Bull. 2007,
55, 124. c) A. R. Katritzky, K. Suzuki, S. K. Singh, Synthesis
2004, 2645. d) P. Gagnon, X. Huang, E. Therrien, J. W.
Keillor, Tetrahedron Lett. 2002, 43, 7717. e) J. Ottl, H. J.
Musiol, L. Moroder, J. Pept. Sci. 1999, 5, 103.
2
A typical procedure of the amidation of 7 with 2e by using
ethyl chloroformate is as follows. To a solution of 299 mg
(1.0 mmol) of Cbz-L-Phe-OH (7) in 20 mL of THF, 134 ¯L
(1.4 mmol, 1.4 equiv) of ethyl chloroformate and 415 ¯L
(3.0 mmol, 3.0 equiv) of triethylamine were added at 0 °C.
Chem. Lett. 2012, 41, 577-579
© 2012 The Chemical Society of Japan
www.csj.jp/journals/chem-lett/