Bis(2-sulfanylethyl)amino native peptide ligation

Article abstract of DOI:10.1021/ol102273u

The reaction of a peptide featuring a bis(2-sulfanylethyl)amino (SEA) group on its C-terminus with a cysteinyl peptide in water at pH 7 and 37 °C leads to the chemoselective and regioselective formation of a native peptide bond. This method called SEA lig

Full text of DOI:10.1021/ol102273u

ORGANIC
LETTERS
2010
Vol. 12, No. 22
5238-5241
Bis(2-sulfanylethyl)amino Native Peptide
Ligation
Nathalie Ollivier, Julien Dheur, Reda Mhidia, Annick Blanpain, and Oleg Melnyk*
CNRS UMR 8161, UniV Lille Nord de France, Institut Pasteur de Lille, IFR 142
Molecular and Cellular Medicine, 1 rue du Pr Calmette 59021 Lille Cedex, France
oleg.melnyk@ibl.fr
Received September 22, 2010
ABSTRACT
The reaction of a peptide featuring a bis(2-sulfanylethyl)amino (SEA) group on its C-terminus with a cysteinyl peptide in water at pH 7 and 37
°C leads to the chemoselective and regioselective formation of a native peptide bond. This method called SEA ligation enriches the native
peptide ligation repertoire available to the peptide chemist. Preparation of an innovative solid support which allows the straightforward synthesis
of peptide SEA fragments using standard Fmoc/tert-butyl solid phase peptide synthesis procedures is also described.
Peptide ligation methods such as native chemical ligation
(NCL),1 Staudinger ligation,2 or the decarboxylative con-
densation of N-alkylhydroxylamines and R-ketoacids3 lead
to the formation of a native peptide bond at the ligation site.4
In particular, NCL is a powerful method for synthesizing
native or modified proteins of moderate size (∼150 amino
acids). NCL is based on the reaction of a peptide thioester
with a cysteinyl peptide. A first transthioesterification step
is followed by an intramolecular S,N-acyl shift that results
in the formation of a native X-Cys peptide bond. Thiol
amino acid derivatives combined with a methylation, des-
ulfurization, or saponification step5 or the use of N-linked
thiol-containing removable auxiliaries6 were used to extend
the principle of NCL to sites other than Cys residues.
these approaches is to facilitate peptide thioester synthesis7
or find alternatives to these useful peptide derivatives.8 For
example, the elegant work of Aimoto et al. showed the
(5) Selenocysteine: (a) Gieselman, M. D.; Xie, L.; van Der Donk, W. A.
Org. Lett. 2001, 3, 1331. (b) Quaderer, R.; Hilvert, D. Chem. Commun.
(Cambridge, U. K.). 2002, 21, 2620. Methionine: Tam, J. P.; Yu, Q.
Biopolymer (Pept. Sci.) 1998, 46, 319. Selenomethionine: Roelfes, G.;
Hilvert, D. Angew. Chem., Int. Ed. 2003, 42, 2275. Recent review on
desulfurization-based ligation methods: Rohde, H.; Seitz, O. Biopolymers
2010, 94, 551. Alanine: Yan, L. Z.; Dawson, P. E. J. Am. Chem. Soc. 2001,
123, 526. (g) Wan, Q.; Danishefsky, S. J. Angew. Chem., Int. Ed. Engl.
2007, 46, 9248. (h) Phenylalanine: Crich, D.; Banerjee, A. J. Am. Chem.
Soc. 2007, 129, 10064. Valine: Haase, C.; Rohde, H.; Seitz, O. Angew.
Chem., Int. Ed. Engl. 2008, 47, 6807. Chen, J.; Wan, Q.; Yuan, Y.; Zhu,
J.; Danishefsky, S. J. Angew. Chem., Int. Ed. Engl. 2008, 47, 8521. Leucine:
Harpaz, Z.; Siman, P.; Kumar, K. S.; Brik, A. ChemBioChem 2010, 11,
1232. Glycopeptide synthesis: Brik, A.; Yang, Y. Y.; Ficht, S.; Wong, C. H.
J. Am. Chem. Soc. 2006, 128, 5626. Side-chain auxiliaries for Asp, Glu,
and Ser: Lutsky, M. Y.; Nepomniaschiy, N.; Brik, A. Chem. Commun.
(Cambridge, U. K.) 2008, 10, 1229. Kumar, K. S.; Harpaz, Z.; Haj-Yahya,
M.; Brik, A. Bioorg. Med. Chem. Lett. 2009, 19, 3870. Hojo, H.; Ozawa,
C.; Katayama, H.; Ueki, A.; Nakahara, Y.; Nakahara, Y. Angew. Chem.,
Int. Ed. Engl. 2010, 49, 5318.
Recently, the use of the reverse N,S-acyl shift has emerged
as a promising strategy for peptide thioester synthesis or for
designing novel native ligation methods relying on the in
situ generation of peptide thioesters. The key idea within
(6) (a) Canne, L. E.; Bark, S. J.; Kent, S. B. H. J. Am. Chem. Soc. 1996,
118, 5891. (b) Offer, J.; Dawson, P. E. Org. Lett. 2000, 2, 23. (c) Botti, P.;
Carrasco, M. R.; Kent, S. B. H. Tetrahedron Lett. 2001, 42, 1831.
(7) (a) Hojo, H.; Onuma, Y.; Akimoto, Y.; Nakahara, Y.; Nakahara, Y.
Tetrahedron Lett. 2007, 48, 25. (b) Nakamura, K.; Mori, H.; Kawakami,
T.; Hojo, H.; Nakahara, Y.; Aimoto, S. Int. J. Pept. Res. Ther. 2007, 13,
191. (c) Tsuda, S.; Shigenaga, A.; Bando, K.; Otaka, A. Org. Lett. 2009,
11, 823.
(1) (a) Dawson, P. E.; Muir, T. W.; Clark-Lewis, I.; Kent, S. B. H.
Science 1994, 266, 776. (b) Hackeng, T. L.; Griffin, J. H.; Dawson, P. E.
Proc. Natl. Acad. Sci. 1999, 96, 10068.
(2) (a) Saxon, E.; Bertozzi, C. R. Science 2000, 287, 2007. (b) Nilsson,
B. L.; Kiessling, L. L.; Raines, R. T. Org. Lett. 2000, 2, 1939. (c) Nilsson,
B. L.; Hondal, R. J.; Soellner, M. B.; Raines, R. T. J. Am. Chem. Soc.
2003, 125, 5268.
(3) Bode, J. W.; Fox, R. M.; Baucom, K. D. Angew. Chem., Int. Ed.
Engl. 2006, 45, 1248.
(8) (a) Kawakami, T.; Aimoto, S. Chem. Lett. 2007, 36, 76. (b)
Kawakami, T.; Aimoto, S. AdV. Exp. Med. Biol. 2009, 611, 117. (c)
Kawakami, T.; Aimoto, S. Tetrahedron 2009, 65, 3871. (d) Kawakami, T.;
Aimoto, S. Tetrahedron Lett. 2007, 48, 1903.
(4) For a recent review: Hackenberger, C. P.; Schwarzer, D. Angew.
Chem., Int. Ed. Engl. 2008, 47, 10030.
10.1021/ol102273u  2010 American Chemical Society
Published on Web 10/21/2010

capacity of cysteinyl prolyl ester peptides to form in situ
peptide thioesters through an intramolecular N,S-acyl shift.8
We describe hereinafter the chemoselective and regiose-
lective reaction of a peptide featuring a bis(2-sulfanylethy-
l)amino (SEA) group on its C-terminus with a cysteinyl or
homocysteinyl peptide in the presence of 4-mercaptopheny-
lacetic acid9 (MPAA) and tris(2-carboxyethyl)phosphine
(TCEP). This reaction occurs in water at pH 7 and leads to
the formation of a native peptide bond in good yield (Scheme
1).
Scheme 2. Synthesis of Solid Support 19
Scheme 1. SEA Ligation between a Bis(2-sulfanylethyl)amino
Peptide 1-4b and a Cysteinyl or Homocysteinyl Peptide
Leading to the Chemoselective and Regioselective Formation of
a Native Peptide Bonda
Chlorotrityl resin 17 (1.4 mmol/g) was used in excess to
amine 16 (molar ratio 17/16: 10/1) to lower the loading of
the solid support to a value compatible with standard Fmoc/
tert-butyl solid phase peptide synthesis protocols,11 as well
as to favor the bridging of polystyrene with amine 16. The
reaction was performed in the absence of base to allow amine
protection by protonation.12 A negative Ellman test for free
thiol groups showed the successful bridging of amine 16
through both thiol groups.13 Moreover, a positive chloranil
test ascertained the presence of a secondary amine on the
solid support.14 High resolution magic angle spinning H
1
NMR of solid support 18 was consistent with the proposed
structure (see Supporting Information). The loading was
determined by coupling Fmoc-protected amino acids to solid
support 18, followed by removal of the Fmoc group with
piperidine and UV quantification of the dibenzofulvene-
piperidine adduct. Amino acid activation using PyBOP or
HBTU was unsuccessful in accord with the low reactivity
of secondary amines in amide bond-forming reactions.
Alternately, Fmoc-amino acid fluorides15 or PyBrOP16
activation permitted the efficient acylation of solid support
18 to give resin 19. The loadings obtained for resin 19
(0.14-0.16 mmol/g) correspond to a nearly quantitative
immobilization of amine 16.
a Peptides 1-4b are formed in situ by TCEP reduction of peptides 1-4a.
We next examined the synthesis of peptides 1-4a. For
this, resin 19 was submitted to standard automated Fmoc/
tert-butyl SPPS (Scheme 3). Deprotection and cleavage of
the peptidyl resin furnished successfully peptides 1-4b in
excellent purity, which equilibrated with the thioester forms
1-4c in acidic solution. This equilibration occurred in about
1 h for the Gly analogue 1b but required up to 25 h for the
Val analogue 4b. The relationship between the aqueous
solution pH and the ratio between thioester 2c and amide
We first examined the synthesis of peptides 1-4a starting
from the innovative solid support 18, which features a bis(2-
sulfanylethyl)amino moiety linked to a trityl polystyrene resin
through both sulfur atoms (Scheme 2). For this, trityl-
protected bis(2-sulfanylethyl)amine 15 was prepared by
reacting bis(2-chloroethyl)amine hydrochloride with triph-
enylmethyl mercaptan in the presence of DBU.10 To
minimize the oxidation of 16 into the cyclic disulfide, 16
was immediately reacted with chlorotrityl polystyrene resin
17 under an inert atmosphere after deprotection in TFA.
(11) Fields, G. B.; Noble, R. L. J. Pept. Protein Res. 1990, 35, 161 Int.
(12) Conrad, R. A.; Cullinan, G. J.; Gerzon, K.; Poore, G. A. J. Med.
Chem. 1979, 22, 339.
(13) Ellman, G. L. Arch. Biochem. Biophys. 1959, 82, 70.
(14) Vojkovsky, T. Pept. Res. 1995, 8, 236.
(9) Johnson, E. C.; Kent, S. B. J. Am. Chem. Soc. 2006, 128, 664.
(10) Adapted from Banerjee, S. R.; Maresca, K. P.; Stephenson, K. A.;
Valliant, J. F.; Babich, J. W.; Graham, W. A.; Barzana, M.; Dong, Q.;
Fischman, A. J.; Zubieta, J. Bioconjugate Chem. 2005, 16, 885.
(15) Carpino, L. A.; Mansour, E.-S. M. E.; Sadat-Aalaee, D. J. Org.
Chem. 1991, 56, 2611.
(16) Coste, J.; Frerot, E.; Jouin, P. J. Org. Chem. 1994, 59, 2437.
Org. Lett., Vol. 12, No. 22, 2010
5239

Synthesis of Peptides 1-4aa b
Table 1. Ligation of Peptides 1-4a with Peptides 5-7
,
Scheme 3
.
peptide
peptide SEA
aminothiol fragment
yielda (%)
8
9
10
11
12
13
1a
2a
3a
4a
1a
1a
5
5
5
5
6
7
32b
77b
54b
58c
29b
42b
a Isolated yields. b 37 °C. c 45 °C.
a Isolated yields. b D-Aa content was determined by chiral GC-MS
analysis after acid hydrolysis.
2b is shown in Figure 1. This equilibrium complicated the
RP-HPLC purification of peptides 1-4b using an acidic
Figure 1. Influence of the aqueous solution pH on the equilibrium
between thioester 2c and amide 2b. C18 RP-HPLC (detection at
215 nm).
water/acetonitrile linear gradient containing 0.5‰ TFA (pH
1.8). To avoid this problem, peptides 1-4b were oxidized
by air into the cyclic disulfides 1-4a prior to the purification
step.
With peptides 1-4a in hand, we investigated their reaction
with cysteinyl peptide 5 in the presence of TCEP and MPAA.
TCEP was used to reduce in situ peptides 1-4a into peptides
1-4b. TCEP is still necessary when the reduced form 1-4b
is used directly in the ligation experiment, due to the ease
of oxidation of the bis(2-sulfanylethyl)amino moiety by
traces of molecular oxygen. Gratifyingly, ligation between
peptide 1-3a and cysteinyl peptide 5 proceeded cleanly
within 27 h at 37 °C and pH 7 to give the native peptides
8-10 (Table 1). Figure 2 shows the LC-MS analysis of the
ligation reaction for 3a. After 27 h, only cysteinyl peptide 5
used in slight excess and native peptide 10 could be detected.
Figure 2. RP-HPLC monitoring of the ligation reaction (100 mM,
pH 7.05, sodium phosphate buffer, TCEP 200 mM, MPAA 200
mM; peptide SEA: 7 mM; peptide 5: 10 mM). (A,B) 3a + 5 at 37
°C. (C, D) 4a + 5 at 45 °C. C18 Nucleosil column (detection at
215 nm). More data (LC-MS) can be found in the Supporting
Information.
Importantly, chiral GC-MS analysis of peptide 10 after acid
hydrolysis showed that racemization of the Tyr residue was
minimal (1.2% D-Tyr). Similar reactivities were observed
for Gly analogue 1a or Ala analogue 2a (1.7% D-Ala by
5240
Org. Lett., Vol. 12, No. 22, 2010

chiral GC-MS for peptide 9). NMR analysis of peptide 9
confirmed the presence of a native Ala-Cys peptide bond
(see Supporting Information). Ligation of Val analogue 4a
with Cys peptide 5 led also successfully to native peptide
11 (0.18 D-Val by chiral GC-MS, Figure 2C,D), but this
required 7 days at 45 °C. However, the purity of the crude
product must be highlighted and illustrates the high stability
of SEA fragments which do not hydrolyze even for prolonged
reaction times.
Reaction of peptide 1a with peptide 5 in the absence of
TCEP failed to give any ligation product 8. No reaction was
also observed when peptide 1a was incubated with the Ser
analogue of peptide 5 (data not shown). Moreover, reaction
of peptide 1a with peptide 7, which features two Cys residues
within its sequence, furnished successfully peptide 13. The
reaction is thus selective for the N-terminal Cys residue.
These experiments show the importance of both bis(2-
sulfanylethyl)amino and free ꢀ-aminothiol groups for the
ligation to proceed. Importantly, ligation of peptide 1a with
Hcy peptide 6 led successfully to native peptide 12. The
compatibility of SEA ligation with the Hcy residue extends
potentially the methodology to the assembly of native
peptides at X-Met junctions. Finally, MPAA is not abso-
lutely required for the ligation to proceed. The ligation rate
of peptide 2a with peptide 5 in the absence of MPAA was
reduced by a factor of 2.5 only.
The reactivity of peptide SEA fragments is unprecedented.
At pH 7, peptide thioesters 1-4c or transthioestherification
products with MPAA were not detected by RP-HPLC. Only
the reduced SEA fragments 1-4b are observed (Figure 2).
This is in contrast with 2-sulfenylethylamido peptide deriva-
tives reported to date, which show an amide-thioester
equilibrium at acidic pH too but do not ligate at pH 7 with
Cys peptides. Other systems such as the cysteinyl-prolyl
ester peptides (CPE) ligate with Cys peptides at pH 8.2.8 In
this case, the N,S-acyl shift equilibrium is displaced toward
the thioester form by an intramolecular amide bond forming
reaction involving the R-amino group of the Cys residue and
ester group. The corresponding cysteinyl-prolyl amide
peptides do not ligate showing the critical role played by
the ester functionality and thus the intramolecular locking
mechanism. Another strategy for favoring thioester formation
through an intramolecular acyl shift is to use more electro-
philic carbonyl derivatives.17 In particular, Botti et al.
reported that peptide-CRoxy-(2-mercapto-1-carboxyamide)
ethyl ester undergoes an O,S-acyl shift at neutral pH. The
resulting thioester reacted in situ with a cysteinyl peptide in
a NCL-like reaction, but hydrolysis of the starting ester was
found to be significant.
The structure of peptide SEA fragments does not permit
a priori an intramolecular locking reaction as in the CPE
strategy. Moreover, the peptide carbonyl group which
participates in the N,S-acyl shift is not activated compared
to N-alkyl cystein derivatives already reported.7a,b In this
context, a novel mechanism may operate that could involve
the participation of neighboring amino and sulfenyl groups
within thioesters 1-4c (Scheme 3). Few reports have
described a significant acceleration of the rate of ester
solvolysis by an intramolecular bifunctional general base-
general acid catalysis mechanism.18 In such a catalysis, one
functional group can activate the incoming nucleophile,
whereas another group stabilizes the transition state by
hydrogen bonding or ion pair formation. In this context, the
efficiency of the ligation reaction might be due to a
significant acceleration of the transthioesterification step due
to an intramolecular bifunctional catalysis, thereby overcom-
ing the low concentration of the thioester at pH 7.
In conclusion, SEA ligation is a chemoselective, regiose-
lective, and racemization free native ligation method that
enriches the existing native peptide ligation tool box. This
method can potentially be used in various chemical or
biochemical applications thanks to the ease of synthesis of
SEA peptide fragments and to the efficiency and simplicity
of this ligation method. The elucidation of the SEA ligation
mechanism as well as its application to the total synthesis
of proteins is in progress.
Acknowledgment. We acknowledge financial support
from Cance´ropoˆle Nord-Ouest, Re´gion Nord Pas de Calais,
and the European Community. We thank Emmanuelle Boll
(CNRS) for NMR experiments.
Supporting Information Available: Experimental pro-
cedures and characterization data for all compounds. This
material is available free of charge via the Internet at
http://pubs.acs.org.
OL102273U
(17) (a) Ollivier, N.; Behr, J. B.; El-Mahdi, O.; Blanpain, A.; Melnyk,
O. Org. Lett. 2005, 7, 2647. (b) Botti, P.; Villain, M.; Manganiello, S.;
Gaertner, H. Org. Lett. 2004, 6, 4861.
(18) (a) Kupchan, S. M.; Eriksen, S. P.; Shen, Y.-T. J. Am. Chem. Soc.
1963, 85, 350. (b) Maugh II, T.; Bruice, T. C. J. Am. Chem. Soc. 1971, 93,
3237.
Org. Lett., Vol. 12, No. 22, 2010
5241