Polymer-bound N-hydroxysuccinimide as a solid-supported additive for DCC-mediated peptide synthesis

Article abstract of DOI:10.1016/S0040-4039(01)00763-8

Polymer-supported N-hydroxysuccinimide (P-HOSu) has been prepared from a styrene/maleic anhydride co-polymer and used as an easily removable activating and racemization-reducing additive for the peptide coupling of amino acid derivatives using DCC.

Full text of DOI:10.1016/S0040-4039(01)00763-8

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 42 (2001) 4487–4489
Polymer-bound N-hydroxysuccinimide as a solid-supported
additive for DCC-mediated peptide synthesis
Rafael Chinchilla, David J. Dodsworth, Carmen Na´jera* and Jose´ M. Soriano
Departamento de Qu´ımica Orga´nica, Universidad de Alicante, Apartado 99, 03080 Alicante, Spain
Received 12 March 2001; revised 7 May 2001; accepted 9 May 2001
Abstract—Polymer-supported N-hydroxysuccinimide (P-HOSu) has been prepared from a styrene/maleic anhydride co-polymer
and used as an easily removable activating and racemization-reducing additive for the peptide coupling of amino acid derivatives
using DCC. © 2001 Elsevier Science Ltd. All rights reserved.
The use of 1,3-dicyclohexylcarbodiimide (DCC) in the
presence of N-hydroxysuccinimide (HOSu) or other
HOX compounds as racemization-reducing additives is
a classical method for peptide bond formation, which
involves the formation of active esters,1 derivative 1
being an example of one of them prepared from HOSu.
These activated species usually subsequently undergo
smooth coupling reactions with amines for the forma-
tion of amide bonds, and have been widely used in both
solution and solid-phase peptide synthesis.2 Isolated
HOSu-derived active esters have been employed, for
instance, for the preparation of leucyldemethylblasti-
cidin S as part of a study of self-resistance mechanisms
of Streptomyces bacteria,3 or for the synthesis of a
novel family of hairpin cyclic peptides.4 In addition,
they have been used in the final step of the synthesis of
the immunomodulator galactosylceramide AGL-537,5
and for the preparation of biotinylated ligands for
asymmetric hydrogenations,6 tetramine calixarenes as
selective extractants of actinide ions,7 or molecular
umbrellas.8 Typically, the formation of isolated or in
situ formed active HOSu-derived esters (1) is achieved
by the use of DCC as the initial coupling agent,1,2
although a uronium salt such as the commercially
available 2-succinimido-1,1,3,3-tetramethyluronium tet-
rafluoroborate (TSTU)9 or HOSu-derived carbonates
or phosphates have also been used.1 In addition, HOSu
esters have been isolated via transesterification reac-
tions mediated by polystyrene-supported 1-hydroxyben-
zotriazole-derived esters.10
Few examples of solid-supported HOSu esters have
been reported in the literature. Thus, polymer-bound
HOSu from a poly(ethylene-co-N-hydroxymaleimide)
polymer 2 cross-linked with aliphatic11 or aromatic
diamines12 have been prepared using DCC, and
employed for the synthesis of amides and peptides.
More recently, supported HOSu (3) has been prepared
from rather expensive Merrifield and ArgoPore™ resins
and used for the formation of resin-bound active HOSu
esters from labeling reagents such as fluorescein, cou-
marin, acridinium and biotin, followed by coupling
with amines such as striol, thyroxine, phenytoin and
desipramine haptens for clinical immunoassays.13 In
most cases, these HOSu-supported reagents were used
for the formation of the peptide bond through an
isolated polymeric succinimidyl ester.
In this context, we thought of using a supported HOSu
of type (2) as an additive for the DCC-mediated direct
synthesis of peptides. This polymeric reagent would
promote the acylation step and would allow a facile
O
Ph
n
OH
N
n
O
O
O
N
O
S
O
O
N
O
O
O
N
OH
OH
R
4
3
2
1
Keywords: amino acids; coupling reagents; polymers; peptides.
* Corresponding author. Fax: +34 96 5903548; e-mail: cnajera@ua.es
0040-4039/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(01)00763-8

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R. Chinchilla et al. / Tetrahedron Letters 42 (2001) 4487–4489
case of sterically hindered amino acids, which required
warming of the reaction mixture to 40°C in order to
achieve acceptable yields. Filtration and washing of the
reaction mixture allowed the separation of the P-HOSu,
which was then reused without appreciable loss of
activity.
purification and recovery by simple filtration. Thus,
polymer-supported HOSu [P-HOSu (4)] was obtained
by the reaction of commercially available poly(styrene-
alt-maleic anhydride)14 with an 50% aqueous solution
of hydroxylamine at 90°C for 1 day. After addition of
hexane and acidification with HCl, the resulting P-
HOSu (4) was filtered and dried in vacuo. This final
polystyrene copolymer was easy to handle in different
solvents and presented good mechanical stability, the
cross-linking with diamines, as in the case of
poly(ethylene-co-N-hydroxymaleimide) (2)11,12 was not
found to be necessary.
The presence of P-HOSu generally increased the iso-
lated yield of the obtained peptide, as shown for
instance in the synthesis of BocGly-PheOEt and
BocAla-ValOMe in 98 and 95% isolated yields, respec-
tively (Table 1, entries 1 and 8). When only DCC was
employed, however, the yields were 70 and 68%, respec-
tively (Table 1, entries 2 and 5). The effect of P-HOSu
was, in general, similar to that of adding free HOSu,
although in some cases P-HOSu afforded better yields
(Table 1, compare entries 1 and 2 and entries 8 and 9).
When difficult couplings using sterically hindered a,a-
dialkylamino acids such as a-aminoisobutyric acid
(Aib) were performed, the process was clearly favored
when adding P-HOSu to the DCC-mediated reaction.
Thus, BocAib-ValOMe was isolated in 63% yield when
using P-HOSu/DCC as compared with only 40% yield
when using DCC (Table 1, entries 13 and 14). In
addition, hindered N-methylated amino acids such as
CbzN-MeValOH could also be coupled using this
methodology (Table 1, entry 16). The use of diiso-
propylcarbodiimide (DIC) instead of DCC as coupling
agent afforded similar or even slightly lower results.
The loading of P-HOSu (4) was determined by the
assay of the OH group content.15 Thus, treatment of
450 mg of P-HOSu with acetic anhydride, subsequent
filtration and washing of the acetylated polymer and
further reaction with N-isopropylamine afforded 67 mg
of the corresponding N-isopropylacetamide, which
indicated an activity for the P-HOSu of 1.5 mmol g−1.
The obtained P-HOSu (4) was employed as an additive
in the DCC-mediated peptide coupling reaction
between Boc, Cbz and Fmoc-protected amino acids and
amino acid esters. The results obtained for different
peptide couplings using P-HOSu, and the comparable
reaction using HOSu and no additive, are presented in
Table 1. The base added to the reaction mixture exerted
a slight influence on the final yield. Thus, when pyridine
was used in the reaction with P-HOSu, the dipeptide
BocGly-PheOEt was obtained in 98% yield (Table 1,
entry 1), however, when triethylamine was used a 90%
yield was isolated (Table 1, entry 3). The reaction was
normally performed at room temperature, except in the
The extent of racemization employing this additive was
examined by using Anteunis’ test16 (the coupling of
CbzGlyPheOH and ValOMe, Table 1, entries 17 and
18). When Anteunis’ tripeptide was prepared using
Table 1. Peptides prepared by DCC-mediated coupling using P-HOSu (4) or HOSu as additives
Entry
Peptidea
Additive
T (°C)
t (h)
Base
Yield (%)b,c
1
2
3
4
5
6
7
8
BocGly-PheOEt
BocGly-PheOEt
BocGly-PheOEt
P-HOSu
HOSu
P-HOSu
P-HOSu
HOSu
P-HOSu
HOSu
P-HOSu
HOSu
P-HOSu
HOSu
P-HOSu
P-HOSu
HOSu
P-HOSu
P-HOSu
P-HOSu
HOSu
25
25
25
25
25
25
25
25
25
40
40
40
40
40
40
40
25
25
6
6
6
7
7
7
7
7
7
24
24
24
24
24
24
24
7
Pyridine
Pyridine
Et3N
98
85 (70)
90
94
96 (93)
83
94 (85)
95
83 (68)
47
58 (42)
65
63
CbzGly-GlyOMe
CbzGly-GlyOMe
CbzGly-AlaOMe
CbzGly-AlaOMe
BocAla-ValOMe
BocAla-ValOMe
FmocAla-GlyOEt
FmocAla-GlyOEt
BocVal-AibOMe
BocAib-ValOMe
BocAib-ValOMe
CbzAib-ValOMe
CbzNMeVal-ValOMe
CbzGlyPhe-ValOMed
CbzGlyPhe-ValOMed
Pyridine
Pyridine
Pyridine
Pyridine
Pyridine
Pyridine
Pyridine
Pyridine
Pyridine
Pyridine
Pyridine
Pyridine
Pyridine
Pyridine
Pyridine
9
10
11
12
13
14
15
16
17
18
70 (40)
59
56
90e
7
88f (84g)
a The formed bond is indicated.
b Isolated pure peptides (1H NMR).
c In parenthesis yields obtained without any additive under the same reaction conditions.
d Anteunis’s test.16
e A 18:1 mixture of epimers was detected (1H NMR).
f A 25:1 mixture of epimers was detected (1H NMR).
g A 9:1 mixture of epimers was detected (1H NMR).

R. Chinchilla et al. / Tetrahedron Letters 42 (2001) 4487–4489
4489
DCC without any additive, the observed degree of
epimerization was 9:1, as measured by H NMR (Table
2780; (b) Williams, A.; Ibrahim, I. T. Chem. Rev. 1981,
81, 589–636.
1
1, entry 18, footnote g), whereas when the reaction was
performed in the presence of P-HOSu the level of
epimerization was reduced to 18:1 (1H NMR) (Table 1,
entry 17, footnote e). By way of comparison, a 25:1
mixture was detected adding HOSu (Table 1, entry 18,
footnote f).
2. (a) Bodanszky, M. In The Peptides: Analysis, Synthesis,
Biology; Grass, E.; Meinhofer, J., Eds.; Academic Press:
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Z.; Barany, G. In Synthetic Peptides: A User’s Guide;
Grant, G. A., Ed.; Freeman: New York, 1992; p. 77; (c)
Bodanszky, M. Principles of Peptide Synthesis; Springer-
Verlag: Berlin, 1993; (d) Albericio, F.; Carpino, L. A.
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Lloyd-Williams, P.; Giralt, E. Methods Enzymol. 1997,
289, 313–336; (f) Lloyd-Williams, P.; Albericio, F.;
Giralt, E. Chemical Approaches to the Synthesis of Pep-
tides and Proteins; CRC Press: Boca Raton, 1997; (g)
Albericio, F.; Kates, S. A. In Solid-Phase Synthesis. A
Practical Guide; Kates, S. A.; Albericio, F., Eds.; Marcel
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We conclude that the economical, simple and easily
recoverable polymer-supported HOSu represents a con-
venient additive which improves the applicability of the
DCC-peptide coupling methodology. Further studies
on the use of this polymeric HOSu for the preparation
of other polymeric HOSu-derived reagents are now
underway.
In a typical peptide coupling reaction using P-HOSu
(4), a suspension of the protected amino acid (0.5
mmol), aminoester hydrochloride (0.5 mmol), P-HOSu
(367 mg, 0.55 mmol of active HOSu), DCC (103 mg,
0.5 mmol) and pyridine (122 mL, 1.5 mmol) in MeCN
(15 mL) was stirred at rt or 40°C for the time indicated
in Table 1. Hexane (10 mL) was added and the result-
ing precipitate was washed with hexane/AcOEt, 1/1 (10
mL) and filtered. The filtered solid consisted of P-
HOSu, which was recovered almost quantitatively. To
the filtrate was added saturated NaCl (20 mL) and the
mixture was extracted with AcOEt (3×10 mL). The
organic layer was washed with 2 M HCl (3×10 mL),
saturated NaHCO3 (3×10 mL) and water (3×10 mL).
The solvent was dried (Na2SO4) and evaporated (15
Torr) affording the corresponding crude peptides.
8. (a) Janout, V.; Lanier, M.; Regen, S. L. J. Am. Chem.
Soc. 1996, 118, 1573–1574; (b) Janout, V.; Lanier, M.;
Regen, S. L. J. Am. Chem. Soc. 1997, 119, 640–647.
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Tetrahedron Lett. 1989, 30, 1927–1930.
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Acknowledgements
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12. Andreev, S. M.; Tsiryapkin, V. A.; Samoilova, N. A.;
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Synthesis 1977, 303–304.
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Bioorg. Med. Chem. Lett. 1999, 9, 217–220; (b) Adam-
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We thank the Direccio´n General de Ensen˜anza Supe-
rior e Investigacio´n Cient´ıfica (project no. 1FD97-0721)
of the Ministerio de Educacio´n y Cultura (MEC) and
the Conselleria de Cultura Educacio´ i Cie`ncia of the
Generalitat Valenciana (project no. GV99-33-1-02) for
financial support.
14. Average MW ca. 550,000, containing 10–15%
monomethyl ester. Available from Aldrich.
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