Fmoc solid-phase synthesis of peptide thioesters using an intramolecular N,S-acyl shift

Article abstract of DOI:10.1021/ol050776a

(Chemical Equation Presented) We describe the Fmoc solid-phase synthesis of peptide thioesters based on the alkylation of the safety-catch sulfonamide linker with a protected 2-mercaptoethanol derivative. The thioester is generated on the solid phase afte

Full text of DOI:10.1021/ol050776a

ORGANIC
LETTERS
2005
Vol. 7, No. 13
2647-2650
Fmoc Solid-Phase Synthesis of Peptide
Thioesters Using an Intramolecular
N,S-Acyl Shift
Nathalie Ollivier,† Jean-Bernard Behr,‡ Ouafaˆa El-Mahdi,† Annick Blanpain,† and
Oleg Melnyk*,†
Institut Pasteur de Lille, UniVersite´ de Lille 2, UMR 8525 CNRS, 1 rue du Professeur
Calmette, 59021 Lille Cedex, France, and Laboratoire de Chimie Bioorganique,
UniVersite´ de Reims Champagne-Ardenne, BP 1039, 51687 Reims Cedex 2, France
oleg.melnyk@ibl.fr
Received April 11, 2005
ABSTRACT
We describe the Fmoc solid-phase synthesis of peptide thioesters based on the alkylation of the safety-catch sulfonamide linker with a
protected 2-mercaptoethanol derivative. The thioester is generated on the solid phase after the peptide chain assembly as a consequence of
an intramolecular N,S-acyl shift. Depending on the stability of the spacer separating the sulfonamide linker from the resin toward TFA, treatment
of the peptidyl resin with TFA led to a soluble or supported deprotected thioester.
Chemoselective ligation methods such as native chemical
ligation1 or Staudinger ligation2 allow the assembly of
proteins of moderate size (150 amino acids) and provide
ready access to natural as well as modified proteins. Native
chemical ligation is based on the reaction of a peptide bearing
a C-terminal thioester group with an N-terminal cysteinyl
peptide. Transthioesterification is followed by an intra-
molecular S,N-acyl shift that leads to the formation of a
peptide bond at an X-Cys junction. Application of this
chemistry to N-terminal selenocysteine peptides was also
described.3 Methods based on the use of N-linked, thiol-
containing, cleavable auxiliaries4 were used to extend the
principle of native chemical ligation to sites other than Cys
residues. Ligation of peptide thioesters with N-terminal
homocysteine5 or homoselenocysteine6 peptides followed by
methylation permitted the formation of X-Met or X-Seleno-
Met bonds.7
The key starting materials for native chemical ligation are
unprotected C-terminal peptide thioesters. Peptide thioesters
are often prepared using Boc/benzyl solid-phase peptide
chemistry.1c However, the widespread use of the Fmoc/tert-
butyl chemistry8 for peptide synthesis over the Boc/benzyl
method has stimulated the development of methods allowing
the preparation of peptide thioesters that are compatible with
the basic treatments used to remove the Fmoc R-amino
protecting group.
The thioester group is unstable in the presence of piper-
idine, which is usually employed for the removal of the Fmoc
† UMR 8525 CNRS.
‡ Universite´ de Reims Champagne-Ardenne.
(1) (a) Dawson, P. E.; Muir, T. W.; Clark-Lewis, I.; Kent, S. B. H.
Science 1994, 266, 776-779. (b) Dawson, P. E. Methods Enzymol. 1997,
287, 34-45. (c) Hackeng, T. L.; Griffin, J. H.; Dawson, P. E. Proc. Natl.
Acad. Sci. 1999, 96, 10068-10073.
(2) (a) Saxon, E.; Bertozzi, C. R. Science 2000, 287, 2007-2010. (b)
Nilsson, B. L.; Kiessling, L. L.; Raines, R. T. Org. Lett. 2000, 2, 1939-
1941. (c) Nilsson, B. L.; Hondal, R. J.; Soellner, M. B.; Raines, R. T. J.
Am. Chem. Soc. 2003, 125, 5268-5269.
(3) (a) Gieselman, M. D.; Xie, L.; van Der Donk, W. A. Org. Lett. 2001,
3, 1331-1334. (b) Quaderer, R.; Hilvert, D. Chem. Commun. (Cambridge,
UK) 2002, 21, 2620-1.
(4) (a) Canne, L. E.; Bark, S. J.; Kent, S. B. H. J. Am. Chem. Soc. 1996,
118, 5891-5896. (b) Offer, J.; Dawson, P. E. Org. Lett. 2000, 2, 23-26.
(c) Botti, P.; Carrasco, M. R.; Kent, S. B. H. Tetrahedron Lett. 2001, 42,
1831-1833.
(5) Tam, J. P.; Yu, Q. Biopolym. (Pept. Sci.) 1998, 46, 319-327.
(6) Roelfes, G.; Hilvert, D. Angew. Chem., Int. Ed. 2003, 42, 2275-
2277.
(7) For a review of the different ligation methods, see: Tam, J. P.; Xu,
J.; Eom, K. D. Biopolym. (Pept. Sci) 2001, 60, 194-205.
(8) Fields, G. B.; Noble, R. L. Int. J. Pept. Protein Res. 1990, 35, 161-
214.
10.1021/ol050776a CCC: $30.25
© 2005 American Chemical Society
Published on Web 06/02/2005

protection after the coupling of each amino acid. Li et al.9
and Clippingdale et al.10 have prepared peptide thioesters
by replacing piperidine with 1-methylpyrrolidine/hexamethyl-
eneimine/HOBt and DBU/HOBt mixtures, respectively.
Brask et al. have prepared peptide thioesters using the
backbone amide linker (BAL) and a thioester moiety masked
as a trithioortho ester throughout the Fmoc/tert-butyl peptide
synthesis.11 Other methods involve the formation of the
thioester group after chain assembly to avoid its exposure
to basic conditions. Barany et al. have used BAL or esters
resins and orthogonal allyl protection for the C-terminal
R-carboxylic group for generating thioesters after chain
assembly.12 Hilvert et al. have prepared peptide thioesters
using Lewis acid (Me2AlCl)-catalyzed thiolysis of ester
resins.13,14 Finally, two groups have developed an attractive
method exploiting the Kenner safety-catch sulfonamide
linker,15-17 which allowed the assembly of the peptide using
standard Fmoc/tert-butyl chemistry. Subsequent alkylation
of the acylsulfonamide linkage with diazomethane or iodo-
acetonitrile followed by displacement with an excess of a
thiol produced the protected thioester in solution that was
further deprotected with concentrated trifluoroacetic acid.18
The use of an excess of LiBr permitted improvement of the
thiolysis step.19
We report here a novel method for peptide thioester
synthesis that is based also on the use of the sulfonamide
safety-catch linker, but where the thioester function is
generated on the solid-phase as the consequence of an
intramolecular N,S-acyl shift as depicted in Scheme 1.
Our synthetic strategy begins with peptide assembly on a
3-carboxypropanesulfonamide linker using standard Fmoc/
tert-butyl chemistry. Next, alkylation of the acylsulfonamide
group is performed using Mitsunobu chemistry with protected
mercaptoethanol derivative 3.20 Removal of the thiol protect-
ing group gives supported intermediate 5, which features a
thiol nucleophile in close proximity to the activated carboxyl
group. Consequently, 5 was found to rearrange spontaneously
into 6 as a result of an intramolecular N,S-acyl shift which
Scheme 1. Synthesis of Thioesters Based on the Use of the
Kenner Safety-Catch Linker, Mitsunobu Alkylation with 3, and
Intramolecular N,S-Acyl Shift
involves a cyclic five-membered intermediate. Thus, a
protected peptide thioester is formed that is still attached to
the solid-support. Final deprotection of the peptide chain is
performed in TFA. The use of a Rink linker between the
3-carboxypropanesulfonamide arm and the solid support
leads to the liberation of the peptide thioester 7 in solution.
Alternately, the use of a TFA-resistant linker results in the
formation of a deprotected peptide thioester 7S still attached
to the resin that can be engaged in a native chemical ligation
from the solid phase.21
The triisopropyl group was chosen for the protection of
the thiol functionality of 2-mercaptoethanol because it is
compatible with Mitsunobu chemistry and can be removed
selectively in the presence of the standard side-chain protec-
tions. Key reagent 3 was prepared as described in Scheme 2
Scheme 2. Synthesis of Alcohol 3
(9) Li, X.; Kawakami , T.; Aimoto, S. Tetrahedron Lett. 1998, 39, 8669-
8672.
(10) Clippingdale, A. B.; Barrow, C. J.; Wade, J. D. J. Pept. Sci. 2000,
6, 225-234.
(11) Brask, J.; Albericio, F.; Jensen, K. J. Org. Lett. 2003, 5, 2951-
2953.
(12) (a) Tulla-Puche, J.; Barany, G. J. Org. Chem. 2004, 69, 4101-
4107. (b) Alsina, J.; Yokum, T. S.; Albericio, F.; Barany, G. J. Org. Chem.
1999, 64, 8761-8769. (c) Gross, C. M.; Lelie`vre, D.; Woodward, C. K.;
Barany, G. J. Pept. Res. 2005, 65, 395-410.
by reacting the potassium salt of triisopropylsilane thiol 822
with 2-bromoethanol 9 in anhydrous THF.
(13) Swinnen, D.; Hilvert, D. Org. Lett. 2000, 2, 2439-2442.
(14) Swinnen, A.; Hilvert, D. Angew. Chem., Int. Ed. 2001, 40, 3395-
3396.
In a first series of experiments, Fmoc-Phe-Ala dipeptide
was assembled on a 3-carboxypropanesulfonamide linker
attached to a Rink PEG-PS resin (NovaSyn). Peptidyl resin
2a (Scheme 1) was then treated with alcohol 3 in the presence
of diethylazodicarboxylate (DEAD) and triphenylphosphine
(8 equiv of each). Direct treatment of resin 4a with TFA led
to complex mixtures. Alternately, removal of the triisopro-
pylsilyl group of resin-bound peptide 4a in the presence of
(15) Kenner, G. W.; McDermott, J. R.; Sheppard, R. C. J. Chem. Soc.,
Chem. Commun. 1971, 118, 3055-3056.
(16) Bakes, B. J.; Ellman, J. A. J. Org. Chem. 1999, 64, 2322-2330.
(17) For a review on Kenner’s safety-catch linker, see: Heidler, P.; Link,
A. Bioorg. Med. Chem. 2005, 13, 585-599.
(18) (a) Ingenito, R.; Bianchi, E.; Fattori, D.; Pessi, A. J. Am. Chem.
Soc. 1999, 121, 11369-11374. (b) Shin, Y.; Winans, K.; Backes, B. J.;
Kent, S. B. H.; Ellman, J. A.; Bertozzi, C. R. J. Am. Chem. Soc. 1999, 121,
11684-11689.
(19) Quaderer R.; Hilvert D. Org. Lett. 2001, 3, 3181-3184.
(20) Willoughby, C. A.; Hutchins, S. M.; Rosauer, K. G.; Dhar, M. J.;
Chapman, K. T.; Chicchi, G. G.; Sadowski, S.; Weinberg, D. H.; Patel, S.;
Malkowitz, L.; Di Salvo, J.; Pacholok, S. G.; Cheng, K. Bioorg. Med. Chem.
Lett. 2002, 12, 93-96.
(21) Camarero, J. A.; Cotton, G. J.; Adeva, A.; Muir, T. W. J. Pept.
Res. 1998, 51, 303-316.
2648
Org. Lett., Vol. 7, No. 13, 2005

tetrabutylammonium fluoride buffered with acetic acid23
followed by treatment of the resin with TFA liberated 7a in
solution as the major product, together with peptide Fmoc-
Phe-Ala-NHSO2(CH2)3CONH2 10 due to incomplete Mit-
sunobu alkylation (7a/10 ratio determined to be 72/28 by
RP-HPLC). Peptide 7a was purified by RP-HPLC (50%
yield) and fully characterized.
with piperidine, and sequenced using Edman chemistry that
confirmed the formation of an alanine-cysteine peptide
bond. Taken together, these data strongly support the
structure proposed for peptide 7a. Additional results pre-
sented below (ligation experiments between peptides 7b,c
and peptides 12 and 14, solid-phase native chemical ligation
between peptidyl resin 7Sb and peptide 12) confirmed the
N,S-acyl shift following TBAF treatment and the formation
of a thioester group at the C-terminus of the peptide.
1
The H NMR COSY spectrum of 7a showed a SO2NH
proton at 5.27 ppm (triplet) linked to a CH2CH2 chain. This
signal was not observed in the spectrum of dipeptide 11
(Scheme 3), which was prepared by alkylation of dipeptide
Next, sequence Ac-ILKEPVHGA or Ac-ILKEPVHGV
was assembled on sulfonamide resin 1 (Rink PEG-PS,
NovaSyn) to give peptidyl resins 2b,c, which were alkylated
using 3 and Mitsunobu activation and treated with TBAF/
AcOH and then TFA as described before to give successfully
thioesters 7b,c. RP-HPLC profile of the crude thioesters 7b,c
showed that the conversions of resins 2b,c into alkylated
acylsulfonamide resins 4b,c were similar (ca. 67%) to the
conversion of peptidyl resin 2a into 4a. For comparison,
solution-phase alkylation of dipeptide 10 using similar
experimental conditions was complete after 3 h at room
temperature (Scheme 3). These results show that the alkyl-
ation of the sulfonamide did not depend on the nature of the
first amino acid Ala or Val, probably because the site of
alkylation is far enough from the CR. The whole process
worked equally well for both sequences (overall isolated
yields of purified products, including the SPPS, Mitsunobu
alkylation, TIPS cleavage and rearrangement, resin cleavage,
and deprotection steps: 23 and 21% for 7b and 7c, respec-
tively).
Scheme 3. Synthesis of Peptide 11a
a Alkylation was found to be complete after 3 h at room
temperature. Partial loss of the acid-sensitive TIPS protecting group
during silica gel chromatography accounted for the modest yield
obtained in this experiment.
Peptides 7b,c were then engaged in native chemical
ligation experiments with Cys-peptide 12 as described in
Scheme 4. Control experiments were performed in the
same way by treating peptides 7b,c with the peptide
H-SILKEPVHGV-NH2 14, where Ser has replaced the Cys
residue in peptide 12. Ligation proceeded well in the presence
of Cys-peptide 12, whereas no reaction was observed with
Ser-peptide 14. Peptides 13b and 13c were isolated with
73 and 49% yield, respectively, following RP-HPLC puri-
fication. They displayed the expected m/z values by MALDI-
TOF analysis and were found to be identical by RP-HPLC
and CZE to reference compounds prepared by standard
Fmoc/tert-butyl solid-phase peptide synthesis (see Supporting
Information).
10 in the presence of 3/DEAD/PPh3. Furthermore, an aliquot
of peptide 7a was reacted with peptide 12 (Scheme 4) in a
Scheme 4. Ligation of Peptides 7b,c with Peptide 12
The formation of supported C-terminal thioester following
removal of the TIPS group in the presence of TBAF opens
the possibility to perform a native chemical ligation from
the solid phase, which should result in the liberation of the
ligation product in solution. Camarero et al. have explored
this approach using a poly(ethylene glycol)-poly(N,N-dim-
ethylacrylamide) copolymer support and peptide thioesters
assembled using the Boc/benzyl strategy.21
pH 7.5 phosphate buffer in the presence of thiophenol/
benzylmercaptan. The resulting product Fmoc-FACILKEPVH-
GV-NH2 (MALDI-TOF [M + H]+ calcd monoisotopic
1533.8, found 1534.0) was isolated by RP-HPLC, treated
Fully deprotected peptidyl resin 7Sb was prepared as
described before starting from a NovaSyn TG amino resin.
Deprotection of the peptide chain was performed in con-
centrated TFA containing dimethyl sulfide, anisole, and water
as scavengers. Resin 7Sb was suspended under argon in pH
7.5 phosphate buffer and reacted with Cys-peptide 12 in
the presence of thiophenol and benzylmercaptan as for the
(22) Miranda, E. I.; Diaz, M. J.; Rosado, I.; Soderquist, J. A. Tetrahedron
Lett. 1994, 35, 3221-3224.
(23) Successful removal of the Si(iPr)3 group was confirmed by HR MAS
NMR. No cleavage of the peptide from the resin that could be due to TBAF-
mediated hydrolysis of supported thioester 6a was observed.
Org. Lett., Vol. 7, No. 13, 2005
2649

solution-phase ligation experiments. After 23 h of reaction
at room temperature, peptide 13b was isolated with a 23%
yield following RP-HPLC purification (calculated starting
from Fmoc-Ala-sulfonamide TG NovaSyn resin and includ-
ing the peptide elongation step, the Mitsunobu reaction with
3, TIPS removal, the N,S-acyl shift, the deprotection in TFA,
and the native chemical ligation reaction). Again, this product
was found to be identical to the reference peptide synthesized
using standard Fmoc/tert-butyl stepwise solid-phase chem-
istry.
In conclusion, we have presented a novel method for
synthesizing C-terminal peptide thioesters based on the use
of the safety-catch sulfonamide linker and an intramolecular
solid-phase N,S-acyl shift that results in the formation of the
thioester bond after the assembly of the peptide chain using
standard Fmoc/tert-butyl chemistry. The procedure worked
equally well for peptides presenting an Ala or Val residue
at the C-terminus and thus seems to be insensitive to the
bulkiness of the amino acid directly attached to the sulfon-
amide linker. The thioesters were found to be useful for
native chemical ligations. Interestingly, the methodology
could be applied to the preparation of a supported and
deprotected peptide thioester that permitted a native chemical
ligation to be performed from the solid-support.
Acknowledgment. We gratefully acknowledge financial
support from CNRS, Universite´ de Lille 2, and Institut
Pasteur de Lille. We thank Herve´ Drobecq for MALDI-TOF
mass spectra and Ge´rard Montagne for NMR experiments.
Supporting Information Available: Experimental sec-
1
tion; H or COSY, 13C, and HRMS data for compounds 3,
7a, 11; RP-HPLC for 10; and for 7b,c, 12, 13b,c (ligation
products and reference compounds), and 14, RP-HPLC using
two different columns and capillary zone electrophoresis
(CZE) and a copy of the coelutions between the ligation
products and reference compounds. This material is available
free of charge via the Internet at http://pubs.acs.org.
OL050776A
2650
Org. Lett., Vol. 7, No. 13, 2005