Peptide coupling of unprotected amino acids through in situ p-nitrophenyl ester formation

Article abstract of DOI:10.1016/S0040-4039(02)01840-3

Several series of dipeptides and tripeptides were prepared via an activation-coupling method involving the in situ formation of a p-nitrophenyl ester of an (N-protected) amino acid, followed by coupling with an unprotected amino acid in partially aqueous solutions. The resulting peptide is easily isolated by precipitation. In general, the yields obtained are good to excellent and racemization is minimal. This method is particularly advantageous with respect to its simplicity and lack of obligatory side chain protection/deprotection steps.

Full text of DOI:10.1016/S0040-4039(02)01840-3

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 43 (2002) 7717–7719
Peptide coupling of unprotected amino acids through in situ
p-nitrophenyl ester formation
Paul Gagnon, Xicai Huang,† Eric Therrien and Jeffrey W. Keillor*
De´partement de chimie, Universite´ de Montre´al, CP 6128, Succursale centre-ville, Montre´al, Que´bec, Canada H3C 3J7
Received 16 May 2002; revised 24 July 2002; accepted 30 August 2002
Abstract—Several series of dipeptides and tripeptides were prepared via an activation–coupling method involving the in situ
formation of a p-nitrophenyl ester of an (N-protected) amino acid, followed by coupling with an unprotected amino acid in
partially aqueous solutions. The resulting peptide is easily isolated by precipitation. In general, the yields obtained are good to
excellent and racemization is minimal. This method is particularly advantageous with respect to its simplicity and lack of
obligatory side chain protection/deprotection steps. © 2002 Elsevier Science Ltd. All rights reserved.
Small peptides are important biomolecules and many
have therapeutic value. Unlike large peptides that are
commonly isolated from natural sources or produced
through recombinant techniques, small peptides are
usually prepared using organic synthetic methods.1
There exist many different methods for peptide coupling,
most of which require prior protection and subsequent
deprotection of various amino acid functional groups.2
One method that remains popular since its introduction
by Bodanszky in 1955 involves the activation of car-
boxylate groups through the formation of p-nitrophenyl
esters, which can typically be isolated as stable solids.3
The activation of the carboxylate afforded by the p-
nitrophenyl ester group is sufficiently strong to favor
efficient peptide coupling with most amino acids. Fur-
thermore, it is sufficiently mild to disfavor undesired side
reactions with amino acid side-chain functional groups
or adventitious water. Herein we report a coupling
method based on the in situ formation of p-nitrophenyl
esters of N-protected amino acids and the reaction of
these appropriately activated esters in aqueous/organic
solvent mixtures with unprotected amino acids. This
simple two-step process allows the rapid and efficient
peptide coupling and is broadly applicable.
In the first step, the esterification of N-protected amino
acids is easily effected through a protocol similar to one
previously reported by Sunggak and Kim.4 As shown in
Scheme 1, an N-protected amino acid is allowed to react
with p-nitrophenyl chloroformate (pNPCF) in the pres-
ence of triethylamine and 4-dimethylaminopyridine
(DMAP). The general procedure is as follows: To a
solution of 2.0 mmol of PG-Xaa in 100 mL of acetoni-
trile was added 0.31 mL (2.2 mmol) of Et3N. After
cooling in an ice bath, 0.44 g (2.2 mmol) of pNPCF was
added. After 5 min, 24 mg (0.2 mmol) of DMAP was
added. The reaction was then stirred for 50 min.
It has been suggested that the intermediate product of
this reaction is the mixed anhydride formed upon dis-
Scheme 1. In situ p-nitrophenyl ester formation and coupling.
* Corresponding author. Tel.: +1-514-343-6219; fax: +1-514-343-7586; e-mail: keillorj@chimie.umontreal.ca
† Present address: Medicinal Chemistry Department, Conjuchem, Montre´al, Canada.
0040-4039/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(02)01840-3

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P. Gagnon et al. / Tetrahedron Letters 43 (2002) 7717–7719
Table 1. Yields of some di- and tripeptides synthesized
placement of the chloride of pNPCF by the amino acid
carboxylate group. Presumably, this unstable mixed
anhydride then undergoes an intramolecular condensa-
tion reaction to give the more stable p-nitrophenyl ester
with the expulsion of an equivalent of CO2. However,
the mechanism has not been thoroughly explored.4 The
resulting p-nitrophenyl ester is formed cleanly and in
excellent yield. The reaction can be carried out in
various polar organic solvents, and in the presence of
different bases, of which DMAP appears to be the most
efficient. The ester can be isolated and purified, or used
directly in the following coupling step.
herein
Peptide
Yield (%)
Cbz-
Cbz-
Cbz-
L
L
L
-Phe-
-Phe-
-Phe-
L
L
L
-Cys
-His
-Ser
98a
88a
67a
Fmoc-
L
-Phe-
L
-Phe
82a
Cbz-
Cbz-
Cbz-
Cbz-
Cbz-
L
L
L
L
L
-Gln-
-Gln-
-Gln-
-Gln-
L
L
L
L
-Ala
-Leu
-Phe
-Val
-Phe-
66a
70a
79a
44a
-Phe-
L
L
-Val
66a,b
35c
Boc-
L
-Gln-Gly
-Gln-Gly
-Gln- -Ser
-Gln-Gly-Gly
-Gln-Gly
-Gln- -Leu
Cbz-Gly- -Leu-Gly
The coupling reaction shown in the second step of
Scheme 1 can be performed using many different
unprotected amino acids, dissolved in partially aqueous
solvent mixtures in the presence of Et3N as a base. The
general procedure for coupling is as follows: A clear
solution was prepared of 8.0 mmol of free amino acid
(Xaa%) in 100 mL of distilled water, and 1.12 mL (8.0
mmol) of Et3N. The ice bath was removed from the
activation solution and the solution of the second
amino acid was added to it dropwise with stirring over
5 min, and the solution was allowed to react for an
additional 100 min. The acetonitrile was then removed
under reduced pressure. To the remaining solution,
water was added to a final volume of 150 mL. This
solution was acidified with 6 M HCl to pH ꢀ2 and the
peptide product was allowed to precipitate at 4°C. The
solution was filtered and the remaining solid was rinsed
with 80 mL of 1 M HCl. The solid was dried under
vacuum for 24 h to give a white powder.
Cbz-
Cbz-
Cbz-
L
65c
L
L
L
31c
30b,c
15b,c
71b,c
43b,c
Cbz-Gly-
Cbz-
L
L
L
L
a Purified by precipitation.
b Overall yield for two activation/coupling and purification steps.
c Purified by chromatography.
accompanied by the formation of other by-products.
This is not surprising, since activated glutamine has
been shown previously to be susceptible to such side
reactions; it has been suggested previously that one of
the by-products formed could be the corresponding
glutarimidine.6
The tripeptides shown in Table 1 were obtained by first
making the Cbz-dipeptide by the above method, and
then coupling with the third free amino acid. Although
the coupling of Cbz-dipeptide with free amino acid
remains efficient, the reported yields for the purified
tripeptides are typically lower due to loss of material
during purification.
Although the dipeptide products can be purified by
chromatography, they can often be isolated more easily
by precipitation, after removal of the organic solvent by
rotary evaporation and acidification of the aqueous
solution. The use of Cbz and Fmoc as N-terminal
protecting groups particularly facilitates the isolation
and purification of the peptide products through pre-
cipitation. However, since the Fmoc group is known to
be slightly labile under basic conditions,5 the use of the
Cbz protecting group, given the presence of Et3N dur-
ing the coupling step, is preferable.
Given the mild conditions of this coupling method,
racemization was not anticipated to be a problem and
was in fact determined to be negligible by several
different methods. Firstly, the di- and tripeptides pre-
pared by this method were shown by NMR spec-
troscopy to be diastereomerically pure, as confirmed by
doping the samples with authentic diastereomer. Fur-
thermore, analysis of crude reaction product mixtures
by chiral HPLC also showed that negligible racemiza-
tion had taken place. Finally, analysis of reaction
product mixtures by capillary electrophoresis using a
chiral mobile phase7 verified the diastereomeric purity
of the dipeptide products. The authenticity and purity
of the peptide products were also established by com-
parison of some of their physical properties with values
reported in the literature.8–12 This comparison is sum-
marized in Table 2.
Many different aprotic organic solvents were found to
be suitable for the activation step and several different
organic solvents can be used with water for the cou-
pling step. Acetonitrile was found to be particularly
well-suited to this method since it is compatible with
the activation reaction, miscible with water for the
coupling step and easily removed under reduced
pressure.
Shown in Table 1 are some of the many peptides
prepared according to this method. In all cases the
desired peptide was obtained rapidly and easily, but the
yields were found to vary according to the different
amino acids used. The first four entries of Table 1
clearly show that amino acids with nucleophilic, basic
or bulky side chains may all be coupled using this
method. However, it should be noted that the activa-
tion of glutamine as a p-nitrophenyl ester may be
This rapid method of activation and coupling with free
amino acids is not without limitations. The side chain
hydroxyl group of serine was found to react with
pNPCF leading to a mixture of products.13 Further-
more, the activation of aspartic acid or glutamic acid as
p-nitrophenyl esters is not selective for the a-carboxyl-

P. Gagnon et al. / Tetrahedron Letters 43 (2002) 7717–7719
7719
Table 2. Comparison of physical properties of synthesized peptides to literature values
Peptide
aD
mp (°C)
Exp.
Lit.
Exp.
Lit.
Cbz-
Cbz-
Cbz-
Cbz-
Cbz-
Cbz-
L
L
L
L
L
L
-Phe-
-Phe-
-Gln-Gly
-Gln-
-Gln-
-Gln-
L
L
-His
-Ser
−8.0 (1.0, DMF)
−3 (2.0, DMF)
−3.9 (1.0, DMF)
−1.5 (1.5, DMF)
4.7 (1.0, DMF)
−5.1a (1.0, DMF)
−2.6b (1.0, DMF)
−3.0d (1.0, DMF)
−1.5a (1.5, DMF)
5.2a (1.0, DMF)
201–203
148–149
183–184
218–219
198–199
191–192
205–207a
147–148c
182–183d
219–220a
199–200a
193–194e
L
L
L
-Ala
-Phe
-Val
3 (1.1, 95% HOAc)
1.4e (1.1, 95% HOAc)
a Reference 8.
b Reference 9.
c Reference 10.
d Commercially available material (Sigma-Aldrich).
e Reference 12.
Acknowledgements
ate group. Finally, the coupling reaction with free
lysine is not selective for the a-amino group, and can
also take place with the o-amino group. Although these
features may be exploited for the synthesis of branched
peptides, they are undesirable for the typical synthesis
of linear, unbranched peptides, and indicate the neces-
sity of side-chain protecting groups for these amino
acids. However, overall this method offers the distinct
advantages of being simple, rapid and practical. Many
di-, tri- and tetrapeptides14 can be made quickly, with-
out the requirement of any additional protection and
deprotection steps. As a result, they can thus be pre-
pared in overall yields that compare favorably to other
peptide coupling methods. The key feature of this
method is that the in situ formation of p-nitrophenyl
esters apparently affords the appropriate level of activa-
tion of the C-terminal carboxylate group to allow
efficient subsequent aminolysis, without rendering the
activated amino acid susceptible either to adventitious
hydrolysis, racemization or reaction with side chain
functional groups. Finally, this method may prove to
be useful in solid-phase peptide synthesis. Preliminary
experiments14 have shown that crude reaction mixtures
of p-nitrophenyl esters of N-protected amino acids
prepared by this method can be used in standard Fmoc
(C“N) solid-phase peptide synthesis. Moreover, an
adaptation of this method may be highly amenable to
(N“C) solid-phase peptide synthesis. If the N-terminal
amino acid were anchored to a water compatible resin,
the peptide could be lengthened through successive
rounds of activation and coupling, without the neces-
sity of intermediate deprotection steps.15
We acknowledge the Natural Sciences and Engineering
Research Council (NSERC) and the Universite´ de
Montre´al for their financial support. PG also thanks
the Fonds pour les chercheurs et a` l’aide de la recherche
(FCAR) for a postgraduate scholarship.
References
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3. Bodanszky, M. Nature 1955, 175, 685.
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12. Klieger, E.; Gibian, H. Liebigs Ann. Chem. 1961, 183.
13. This mixture was observed in the synthesis of Fmoc-
Ser- -Phe, but not Cbz- -Ser-Gly.
14. Huang, X.; Keillor, J. W., unpublished observations.
L-
L
L
Supporting information available
15. Thieret, N.; Guibe´, F.; Albericio, F. Org. Lett. 2000, 2,
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Analytical data for all dipeptides and tripeptides.