Synthesis of thiazolidine thioester peptides and acceleration of native chemical ligation

Article abstract of DOI:10.1021/ol2002804

Thiazolidine thioester peptides were synthesized by reacting bis(2-sulfanylethyl)amido peptides with glyoxylic acid at pH 1. A significant increase in Native Chemical Ligation (NCL) rate was observed with thiazolidine thioesters compared to 3-mercaptoprop

Full text of DOI:10.1021/ol2002804

ORGANIC
LETTERS
2011
Vol. 13, No. 6
1560–1563
Synthesis of Thiazolidine Thioester
Peptides and Acceleration of Native
Chemical Ligation
Julien Dheur, Nathalie Ollivier, 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 January 28, 2011
ABSTRACT
Thiazolidine thioester peptides were synthesized by reacting bis(2-sulfanylethyl)amido peptides with glyoxylic acid at pH 1. A significant increase
in Native Chemical Ligation (NCL) rate was observed with thiazolidine thioesters compared to 3-mercaptopropionic acid-thioester analogues. The
method is of particular interest for accelerating valine-cysteine peptide bond formation.
Ligation chemistries are powerful synthetic tools for the
convergent and controlled assembly of complex molecular
scaffolds. In particular, native peptide ligation methods
allow the formation of native peptide bonds between
unprotected peptides and thus the total or hemi synthesis
of proteins.1a-d Native chemical ligation (NCL)1a,2 and
related extended methodologies3a-c are certainly the most
frequently used techniques for protein total synthesis.
NCL is based on the reaction of peptide thioesters with
cysteinyl peptides. The reaction proceeds through a series
of thiol-thioester exchanges first with an exogenous aro-
matic thiol used in excess and then with the side-chain
thiols of cysteine residues. N-terminal cysteine leads to a
key thioester-linked intermediate that rearranges sponta-
neously by acyl migration from sulfur to nitrogen, result-
ing in the formation of a native peptide bond.
avoiding long reaction times, which can be harmful to
sensitive biomolecules, and for compensating low reactant
concentrations. The discovery of chemical systems allow-
ing the acceleration ofligation chemistries is thusofutmost
importance.4a-f In particular, NCL was shown to be
accelerated by exogenous aromatic thiols which convert
the starting thioester, usually an alkylthioester, into a more
reactive aryl thioester.4e,f The other roles played by the
external thiol are to maintain cysteine thiols in a reduced
form and to permit the reversal of nonproductive thioester
intermediate formation with internal cysteine residues.
Acceleration of NCL by conformationally assisted posi-
tioning of reactive ends has also been described.5
In parallel, tremendous efforts have been focused on the
synthesis of peptide thioesters using Boc6 and later on
Fmoc-solid phase peptide synthesis (SPPS), which is now
One crucial aspect in ligation chemistry is the rate of
bond formation in water. Fast reactions are needed for
(4) (a) Dirksen, A.; Dirksen, S.; Hackeng, T. M.; Dawson, P. E. J.
Am. Chem. Soc. 2006, 128, 15602. (b) Dirksen, A.; Hackeng, T. M.;
Dawson, P. E. Angew. Chem., Int. Ed. 2006, 45, 7581. (c) Tornøe, C. W.;
Christensen, C.; Meldal, M. J. Org. Chem. 2002, 67, 3057. (d) Rostovtsev,
V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B. Angew. Chem., Int.
Ed. 2002, 41, 2596. (e) Johnson, E. C.; Kent, S. B. J. Am. Chem. Soc.
2006, 128, 6640. (f) Dawson, P. E.; Churchill, M. J.; Ghadiri, M. R.;
Kent, S. B. H. J. Am. Chem. Soc. 1997, 119, 4325.
(1) (a) Dawson, P. E.; Muir, T. W.; Clark-Lewis, I.; Kent, S. B.
Science 1994, 266, 776. (b) Saxon, E.; Armstrong, J. I.; Bertozzi, C. R.
Org. Lett. 2000, 2, 2141. (c) Saxon, E.; Bertozzi, C. R. Science 2000, 287,
2007. (d) Bode, J. W.; Fox, R. M.; Baucom, K. D. Angew. Chem., Int. Ed.
2006, 45, 1248.
(2) Hackeng, T. M.; Griffin, J. H.; Dawson, P. E. Proc. Natl. Acad.
Sci. U.S.A. 1999, 96, 10068.
(3) (a) For recent reviews see: Hackenberger, C. P.; Schwarzer, D.
Angew. Chem., Int. Ed. 2008, 47, 10030. (b) Rohde, H.; Seitz, O.
Biopolymers 2010, 94, 551. (c) Offer, J. Biopolymers 2010, 94, 530.
(5) (a) Severin, K.; Lee, D. H.; Kennan, A. J.; Ghadiri, M. R. Nature
1997, 389, 706. (b) Beligere, G. S.; Dawson, P. E. J. Am. Chem. Soc. 1999,
121, 6332.
(6) Hojo, H.; Aimoto, S. Bull. Soc. Chem. Jpn. 1991, 64, 111.
r
10.1021/ol2002804
Published on Web 02/24/2011
2011 American Chemical Society

the most popular technique for peptide synthesis. To
circumvent the instability of the thioester group in the
presence of bases, additives such as hydroxybenzotriazole
(HOBt) were used in combination with piperidine or other
bases to remove the Fmoc group on thioester-linked
peptidyl resin.7a,b Alternately, various methods introduce
the thioester functionality after the peptide elongation
step. This can be done either on the solid phase,8a-e during
the cleavage from the solid support,9 in solution or in situ
during NCL.9c,10 In particular, N,S-acyl shift-based meth-
ods have gained increasing importance recently for peptide
thioester synthesis,8c,9d,10b,10e,10j,10k or for designing novel
native ligation methods relying on the in situ generation of
peptide thioesters.10a,d,g-i,l,11
Scheme 1. Conversion of Bis(2-sulfanylethyl)amido Peptides
1a-d into Thiazolidine Thioesters 3a-d in the Presence of
Glyoxylic Acid
Previous works have often targeted the synthesis of
peptide thioesters derived from 3-mercaptopropionic acid
or other simple alkylthiols.2,6,11 A significant advance in
the field would be to create a novel peptide thioester
scaffold with enhanced reactivity in NCL compared to
3-mercaptopropionic acid-thioesters, to facilitate ligation
at encumbered residues. The use of Fmoc-SPPS to access
these peptide thioesters would be a plus.
thiazolidine thioesters compared to 3-mercaptopropionic
acid-thioester analogues.
We describe hereinafter a potential solution to these
highly challenging goals. First we describe a simplemethod
for the generation of thioesters 3 featuring a thiazolidine
moietyon the thiol handle (Scheme 1). Bis(2-sulfanylethyl)-
amido (SEA) peptides 1 at the basis of the method
described here are easily synthesized by using Fmoc-
SPPS.10a,l Second, we report that a significant increase in
Native Chemical Ligation (NCL) rate was observed with
Peptides 1 used in this study (Scheme 1) were synthesized
on the solid phase by using a supported bis(2-sulfanylethyl)-
amino reagent as described elsewhere.10a In brief, this
solid support, which features a bis(2-sulfanylethyl)amino
moiety linked to a trityl polystyrene resin through both
sulfur atoms, is fully compatible with standard automated
Fmoc-SPPS. Deprotection and cleavage of the peptide
from the resin furnished peptide amides 1. To avoid any
interference of peptide amide 1-peptide thioester 2 equili-
brium during purification, 1 was converted into the cyclic
disulfide 4 by air oxidation in basic medium.
The method described in Scheme 1 is based on the
interconversion between amide form 1 and thioester form
2. Formation of thioester 2, which is detected only below
pH 4 by RP-HPLC, is driven by the protonation of the
amino group within the thiol handle. Thioester 2 is the
major speciebelow pH∼3 for Ala, Tyr, and Val analogues,
and below pH ∼2 for Gly derivative.10a
(7) (a) Clippingdale, A. B.; Barrow, C. J.; Wade, J. D. J. Pept. Sci.
2000, 6, 225. (b) Li, X.; Kawakami, T.; Aimoto, S. Tetrahedron Lett.
1998, 39, 8669.
(8) (a) Alsina, J.; Yokum, T. S.; Albericio, F.; Barany, G. J. Org.
Chem. 1999, 64, 8761. (b) Brask, J.; Albericio, F.; Jensen, K. J. Org. Lett.
2003, 5, 2951. (c) Ollivier, N.; Behr, J. B.; El-Mahdi, O.; Blanpain, A.;
Melnyk, O. Org. Lett. 2005, 7, 2647. (d) Gross, C. M.; Lelievre, D.;
Woodward, C. K.; Barany, G. J. Pept. Res. 2005, 65, 395. (e) Tulla-
Puche, J.; Barany, G. J. Org. Chem. 2004, 69, 4101.
(9) (a) Ingenito, R.; Bianchi, E.; Fattori, D.; Pessi, A. J. Am. Chem.
Soc. 1999, 121, 11369. (b) Ohta, Y.; Itoh, S.; Shigenaga, A.; Shintaku, S.;
Fujii, N.; Otaka, A. Org. Lett. 2006, 8, 467. (c) Blanco-Canosa, J. B.;
Dawson, P. E. Angew. Chem., Int. Ed. 2008, 47, 6851. (d) Nagaike, F.;
Onuma, Y.; Kanazawa, C.; Hojo, H.; Ueki, A.; Nakahara, Y.; Nakahara,
Y. Org. Lett. 2006, 8, 4465. (e) Swinnen, D.; Hilvert, D. Org. Lett. 2000,
2, 2439. (f) Sewing, A.; Hilvert, D. Angew. Chem., Int. Ed. 2001, 40, 3395.
(g) Tofteng, A. P.; Sorensen, K. K.; Conde-Frieboes, K. W.; Hoeg-
Jensen, T.; Jensen, K. J. Angew. Chem., Int. Ed. 2009, 48, 7411. (h)
Mende, F.; Beisswenger, M.; Seitz, O. J. Am. Chem. Soc. 2010, 132,
11110. (i) Quaderer, R.; Hilvert, D. Org. Lett. 2001, 3, 3181.
(10) (a) Ollivier, N.; Dheur, J.; Mhidia, R.; Blanpain, A.; Melnyk, O.
Org. Lett. 2010, 12, 5238. (b) Tsuda, S.; Shigenaga, A.; Bando, K.;
Otaka, A. Org. Lett. 2009, 11, 823. (c) Botti, P.; Villain, M.; Manganiello,
S.; Gaertner, H. Org. Lett. 2004, 6, 4861. (d) Kawakami, T.; Aimoto, S.
Tetrahedron 2009, 65, 3871. (e) Hojo, H.; Onuma, Y.; Akimoto, Y.;
Nakahara, Y.; Nakahara, Y. Tetrahedron Lett. 2007, 48, 25. (f) Warren,
J. D.; Miller, J. S.; Keding, S. J.; Danishefsky, S. J. J. Am. Chem. Soc.
2004, 126, 6576. (g) Kawakami, T.; Aimoto, S. Chem. Lett. 2007, 36, 76.
(h) Kawakami, T.; Aimoto, S. Tetrahedron Lett. 2007, 48, 1903. (i)
Kawakami, T.; Aimoto, S. Adv. Exp. Med. Biol. 2009, 611, 117. (j)
Nakamura, K.; Mori, H.; Kawakami, T.; Hojo, H.; Nakahara, Y.;
Aimoto, S. Int. J. Pept. Res. Ther. 2007, 13, 191. (k) Nakamura, K.;
Kanao, T.; Uesugi, T.; Hara, T.; Sato, T.; Kawakami, T.; Aimoto, S. J.
Pept. Sci. 2009, 15, 731. (l) Hou, W.; Zhang, X.; Li, F.; Liu, C. F. Org.
Lett. 2011, 13, 386.
To displace efficiently the equilibrium between 1 and 2
toward a stable thioester form, we envisaged blocking the
β-aminothiol moiety within 2 by forming a thiazolidine
ring in the presence of an aldehyde. Indeed, thiazolidine
ligation between cysteinyl peptides and aldehyde deriva-
tives is a robust ligation method that proceeds efficiently
below pH 4,12 i.e., at a pH where the fraction of thioester
form 2 is significant. This chemistry has met a lot of success
with glyoxylic acid derivatives as the aldehyde partners,
due to high reactivity and compatibility with peptides and
other biomolecules.13
Toward this goal, peptides 4a-d were reduced back to
peptide amides 1 with zinc at pH 1 and simply filtered to
remove excess zinc.14 At pH 1, thioester form 2 is the major
species.10a Thus, thiazolidine formation was carried out at
(12) Liu, C. F.; Tam, J. P. Proc. Natl. Acad. Sci. U.S.A. 1994, 91,
6584.
(13) Melnyk, O.; Fehrentz, J. A.; Martinez, J.; Gras-Masse, H.
Biopolymers 2000, 55, 165.
(11) For a recent application of 3-mercaptopropionic acid-thioesters
see: Erlich, L. A.; Kumar, K. S.; Haj-Yahya, M.; Dawson, P. E.; Brik, A.
Org. Biomol. Chem. 2010, 8, 2392.
Org. Lett., Vol. 13, No. 6, 2011
1561

Table 1. Synthesis of Thiazolidine Peptides 3a-d
X
peptide reaction time (h) yielda (%) % D-enantiomerb
Gly
Ala
Tyr
Val
3a
3b
3c
3d
16
16
24
72
52
62
56
45
0.14
0.32
<0.1
a Isolated yields. b Determined by chiral GC-MS after acid
hydrolysis.15
this pH by just adding an excess of glyoxylic acid to the
resulting solution. In these conditions, reoxidation of
amide form 1 into cyclic form 4 by molecular oxygen was
not observed probably due to the low aqueous solution pH
and the use of an inert atmosphere. Gratifyingly, the
capping of β-aminothiol moiety within peptide thioester
2 by glyoxylic acid appeared to be an efficient process.
Thiazolidine thioester peptides 3a-d were purified by
using standard RP-HPLC techniques and isolated in good
yield (Table 1). Moreover, chiral GC-MS analysis of
peptides 3b-d after acid hydrolysis showed that thiazo-
lidine formation proceeded without significant racemiza-
tion of C-terminal residue.15
Scheme 2. NCL between Peptide Thioesters 3c,d and Cysteinyl
Peptide 5
Figure 1. Time-course of the ligation reaction for peptide
thioesters 3c, 6c (a) or 3d, 6d (c). The dotted line corresponds to a
first-order rate fit. Peptide thioesters (1 mM) were reacted at
room temperature with cysteinyl peptide 5 (1.5 mM) in the
presence of MPAA (10 mM) and TCEP (80 mM) at pH 7.4. (b)
3c or 6c after 1 h. (d) 3d or 6d after 20 h.
reduced form.4e The reactions were followed by RP-HPLC
(215 nm). The resulting data for peptide thioesters 3d, 6c,
and 6d could be fitted to the first-order rate equation
(dotted curves in Figure 1a,c), by considering peptide
thioesters 3/6 as limiting reactants. The data for thiazoli-
dine peptide thioester 3c could not be fitted to a simple
first- or second-order rate equation.
For thioester 6c, the half-time (t1/2) of the reaction, that
is, the time required to reach 50% conversion, was 120 min
(kobs = 6.5 ( 0.16 ꢀ 10-5 s-1). The t1/2 for thiazolidine
thioester 3c was, however, dramatically reduced to 2 min,
showing a ∼60-fold increase in the reaction rate compared
to thioester 6c. The fraction ligated after 1 h for thiazoli-
dine thioester peptide 3c was 84%, which must be com-
pared to 35% for thioester 6c (Figure 1b). Chiral GC-MS
analysis of peptide 7c after acid hydrolysis showed, how-
ever, 7% of D-Tyr, and thus a partial racemization of
C-terminal Tyr residue during the ligation step.
Similarly, the t1/2 for thioester 6d was 44 h (kobs = 4.4 (
0.5 ꢀ 10-6 s-1), whereas the t1/2 for thiazolidine thioester
3d was 10 h only (kobs = 1.9 ( 0.01 ꢀ 10-5 s-1), showing a
∼4.4-fold acceleration of the ligation reaction for Val
C-terminal residue. The fraction ligated after 20 h for
thiazolidine thioester peptide 3d was as high as 78%, but
only 26% for thioester 6d (Figure 1d). Gratifyingly, chiral
We next examined NCL of thiazolidine peptide thio-
esters3 with model cysteinyl peptide 5 (Scheme 2). Wewere
particularly interested in comparing the ligation rates
between thioesters 3 and peptide thioesters frequently used
in NCL, i.e., 3-mercaptopropionic acid-derived thioesters
6 (Figure 1).11 The comparison was done with thioester
peptides presenting a Tyr (3c/6c) or Val (3d/6d) residue on
the C-terminus. The reactions were performed by using
typical experimental conditions for NCL, i.e., at pH 7 with
each peptide thioester at 1 mM, in the presence of an excess
of (4-carboxymethyl)thiophenol (MPAA) as the exogen-
ous aromatic thiol and of TCEP to maintain all thiols in
(14) The use of TCEP for disulfide bond reduction was unsatisfac-
tory. Phosphines are known to react with aldehydes, see: Darensbourg,
D. J.; Joo, F.; Katho, A.; Stafford, J. N. W.; Benyei, A.; Reibenspies,
J. H. Inorg. Chem. 1994, 33, 175.
(15) Amelung, W.; Brodowski, S. Anal. Chem. 2002, 74, 3239.
1562
Org. Lett., Vol. 13, No. 6, 2011

GC-MS analysis of peptide7dafter acid hydrolysis showed
0.2% of D-Val only, and thus the absence of racemization
of C-terminal Val residue during the ligation step. NCL
with thiazolidine thioester 3d furnished peptide 7d with an
isolated yield of 47% after 48 h (Scheme 2). For compar-
ison, NCL of thioester 6d with peptide 5 required up to
seven days to go to completion (55% conversion after 72 h)
and furnished 7d with only 33% isolated yield. NCLs with
peptide thioesters featuring a C-terminal valine residue
require unusually long reaction times (>48 h) and are
often challenging. Indeed, the bulkinessofvaline sidechain
induces a shielding of valine carbonyl group toward
nucleophilic attack by exogenous thiol or cysteine peptide.
The work presented here shows that the use of thiazolidine
thioester peptides is a potential solution to sluggish NCLs
involving sterically demanding amino acids.
It is widely accepted that the rate limiting step in Cys
NCL is the thiol-thioester exchange between peptide al-
kylthioesters and the aromatic thiol such as MPAA.4e In
accord with this hypothesis, MPAA-thioesters were never
shown to accumulate during NCL involving 3 or 6. Once
formed, they are rapidly consumed in the reaction mixture.
Thus, the acceleration of NCL with thiazolidine thioester
peptide 3 relative to thioester 6 might be due to an
acceleration of the MPAA-thioester forming step.
The reaction of a thiol anion RS- with a thioester
R00COSR0 proceeds through formation of a tetrahedral
intermediate. When the attacking thiol RSH is less basic,
i.e., a poorer nucleophile and a better leaving group than
the thiol R0SH of the thiol ester, expulsion of the thioester
thiolate R0S- is rate determining.16 This is probably the
case in this study since pKa of thiol MPAA is 6.6,4e whereas
pKa of 3-mercaptopropionic acid thiol’s group is 10.217
and pKa of thiol 8 (Scheme 2) was estimated to be 9.4 using
SPARC18 algorithm. On this basis, thiol 8 is expected to be
a slightly better leaving group than 3-mercaptopropionic
acid. However, the question whether a ΔpKa value of 0.8
between the two thiols can account for the 60-fold increase
in ligation rate observed for Tyr derivatives 3c/6c remains
to be explored. Other factors may contribute to the accel-
eration of MPAA-thioester formation. Indeed, previous
work from Dawson’s group has shown that peptide thio-
esters featuring a histidine or cysteine residue on the
C-terminus react faster in Cys NCL than alanine
analogue.2 Internal participation of histidine or aspartic
acid residues in sugar-assisted ligation was also noted.19
These residues were shown to accelerate the rate limiting S,
N-acyl transfer step through probably an intramolecular
general base catalysis mechanism. Internal participation of
thiazolidine and/or carboxy groups present within thiol
handle 8 might increase the rate of MPAA-thioester for-
mation and thus of Cys NCL observed with thiazolidine
thioester peptides 3c/d. Studies aimed at deciphering the
factors involved in this acceleration effect are underway.
In conclusion, we show that thiazolidine thioester pep-
tides are easily prepared by combining Fmoc-SPPS of SEA
peptides and a simple chemical step in solution with
glyoxylic acid. The rate of NCL with thiazolidine thioester
peptides is significantly higher than that with 3-mercapto-
propionic acid-thioester analogues. The acceleration is
significant even for a sterically demanding amino acid such
asvaline. Inthiscase, noracemizationofvaline residue was
observed. We believe that the ease of preparation of
thiazolidine thioester peptides as well as their high reactiv-
ity will extend the frontier attainable to NCL, and in
particular constitute a potential solution to “difficult”
NCLs.
Acknowledgment. We acknowledge financial support
ꢀ
^
ꢀ
from Canceropole Nord-Ouest, Region 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.
(16) Hupe, D. J.; Jencks, W. P. J. Am. Chem. Soc. 1977, 99, 451.
(17) Danehy, J. P.; Noel, C. J. J. Am. Chem. Soc. 1960, 82, 2511.
(18) Lee, P. H.; Ayyampalayam, S. N.; Carreira, L. A.; Shalaeva, M.;
Bhattachar, S.; Coselmon, R.; Poole, S.; Gifford, E.; Lombardo, F. Mol.
Pharmaceutics 2007, 4, 498.
(19) (a) Brik, A.; Ficht, S.; Yang, Y.-Y.; Wong, C.-H. J. Am. Chem.
Soc. 2006, 128, 15026. (b) Lutsky, M.-Y.; Nepomniaschiy, N.; Brik, A.
Chem. Commun. 2008, 1229.
Org. Lett., Vol. 13, No. 6, 2011
1563