Facile, fmoc-compatible solid-phase synthesis of peptide C-terminal thioesters.

Article abstract of DOI:10.1021/ol0060836

A short route to peptide C-terminal thioesters was developed that does not require the use of special linkers or resins and is compatible with standard Fmoc chemistry. Following conventional solid-phase peptide synthesis, an excess of Me(2)AlCl and EtSH i

Full text of DOI:10.1021/ol0060836

ORGANIC
LETTERS
2000
Vol. 2, No. 16
2439-2442
Facile, Fmoc-Compatible Solid-Phase
Synthesis of Peptide C-Terminal
Thioesters
Dominique Swinnen† and Donald Hilvert*
Laboratory of Organic Chemistry, Swiss Federal Institute of Technology (ETH),
UniVersita¨tstrasse 16, CH-8092 Zu¨rich, Switzerland
hilVert@org.chem.ethz.ch
Received May 19, 2000
ABSTRACT
A short route to peptide C-terminal thioesters was developed that does not require the use of special linkers or resins and is compatible with
standard Fmoc chemistry. Following conventional solid-phase peptide synthesis, an excess of Me2AlCl and EtSH in dichloromethane cleaves
peptides from Wang or Pam resins to give the corresponding thioesters directly in good yield and purity.
C-Terminal peptide thioesters are key intermediates in
enzymatic and nonenzymatic peptide bond forming reactions.
Their utility in peptide fragment condensations, in particular,
has made small and medium-sized proteins readily accessible
to total chemical synthesis.1 These activated species are also
valuable for the construction of proteins containing non-
natural backbones2 and for the synthesis of cyclic peptides3
and peptide dendrimers.4
and protein domains, molecular biologically with intein
technology.6 Synthetic methods compatible with widely used
9-fluorenylmethoxycarbonyl (Fmoc)-based chemistry7 would
complement these approaches, especially for peptides con-
taining functionalities incompatible with Boc chemistry.
However, resin-bound thioesters are unstable to repeated
exposure to piperidine, which is used to remove Fmoc
groups. The susceptibility of thiol esters to epimerization
under the basic conditions of Fmoc-SPPS is an additional
concern.
Peptide C-terminal thioesters can be prepared by standard
solid-phase peptide synthesis (SPPS) using tert-butoxycar-
bonyl (Boc) methodology5 or, for larger peptide fragments
Several strategies for solving these problems have been
reported. In one approach,8 a thioester-compatible Fmoc-
cleavage cocktail [i.e., 1-methylpyrrolidine (25%), hexa-
† Current address: Serono Pharmaceutical Research Institute, 14 chemin
des Aulx, CH-1228 Plan-Les-Ouates, Geneva, Switzerland.
(1) (a) Dawson, P. E.; Muir, T. W.; Clark-Lewis, I.; Kent, S. B. H.
Science 1994, 266, 776-779. (b) Wilken, J.; Kent, S. B. H. Curr. Opin.
Biotechnol. 1998, 9, 412-426.
(2) (a) Schno¨lzer, M.; Kent, S. B. H. Science 1992, 256, 221-225. (b)
Baca, M.; Muir, T. W.; Schno¨lzer, M.; Kent, S. B. H. J. Am. Chem. Soc.
1995, 117, 1881-1887. (c) Muir, T. W.; Williams, M. J.; Ginsberg, M. H.;
Kent, S. B. H. Biochemistry 1994, 33, 7701-7708. (d) Liu, C.-F.; Rao, C.;
Tam, J. P. Tetrahedron Lett. 1996, 37, 933-936. (e) Liu, C.-F.; Rao, C.;
Tam, J. P. J. Am. Chem. Soc. 1996, 118, 307-312.
(3) (a) Shao, Y.; Lu, W.; Kent, S. B. H. Tetrahedron Lett. 1998, 39,
3911-3914. (b) Zhang, L.; Tam, J. P. J. Am. Chem. Soc. 1999, 121, 3311-
3320.
(5) (a) Blake, J.; Li, C. H. Proc. Natl. Acad. Sci. U.S.A. 1981, 78, 4055-
4058. (b) Canne, L. E.; Walker, S. M.; Kent, S. B. H. Tetrahedron Lett.
1995, 36, 1217-1220. (c) Hojo, H.; Kwon, Y.; Kakuta, Y.; Tsuda, S.;
Tanaka, I.; Hikichi, K.; Aimoto, S. Bull. Chem. Soc. Jpn. 1993, 66, 2700-
2706. (d) Hackend, T. M.; Griffin, J. H.; Dawson, P. E. Proc. Natl. Acad.
Sci. U.S.A. 1999, 96, 10068-10073.
(6) Noren, C. J.; Wang J.; Perler, F. B. Angew. Chem., Int. Ed. 2000,
39, 450-466.
(7) (a) Atherton, E.; Sheppard, R. C. Solid-phase peptide synthesis, a
practical approach.; IRL Press: Oxford, 1989. (b) Fields, G. B.; Noble, R.
L. Int. J. Pept. Res. 1990, 35, 161-214.
(4) (a) Shao, J.; Tam, J. P. J. Am. Chem. Soc. 1995, 117, 3893-3899.
(b) Zhang, L.; Tam, J. P. J. Am. Chem. Soc. 1997, 119, 2363-2370.
(8) Li, X.; Kawakami, T.; Aimoto, S. Tetrahedron Lett. 1998, 39, 8669-
8672.
10.1021/ol0060836 CCC: $19.00 © 2000 American Chemical Society
Published on Web 07/08/2000

methyleneimine (2%), and 1-hydroxybenzotriazole (HOBt,
2%) in NMP-DMSO (1:1)] has been successfully applied
to the synthesis of an unprotected 25-residue peptide thioester
in 24% yield. Alternatively, the labile thioester can be
introduced at the end of the synthesis, for example, using
the backbone amide linker (BAL) strategy.9 The C-terminal
residue of a peptide, anchored to a solid support through its
backbone nitrogen, is activated and coupled to an amino acid
thioester immediately prior to final cleavage and deprotec-
tion. A 7-aa peptide was prepared in this way in excellent
yield and acceptable levels of epimerization.9 A potentially
more general method relies on a recent modification of
Kenner’s sulfonamide “safety-catch” linker.10 Normal Fmoc-
SPPS is followed by activation of the sulfonamide linker by
alkylation and cleavage of the peptide from the resin with a
thiol nucleophile. This strategy has been used to prepare
thioesters of several peptides,11 including a 24-residue
glycopeptide in 21% yield.12
As an alternative to these published methods, which may
require extensive optimization or exploit expensive or
commercially unavailable linkers, we report preliminary
results on a short and simple route to unprotected peptide
C-terminal thioesters using Fmoc-SPPS on the Wang13 and
Pam14 resins. Corey has shown that alkylaluminum thiolate,
prepared from trimethylaluminum and the corresponding
mercaptan, reacts with simple esters in CH2Cl2 to produce
thioesters in high yield.15 We reasoned that a peptide
assembled by Fmoc procedures on conventional hydroxy-
methyl resins would similarly yield peptide thioesters upon
treatment with an alkylaluminum thiolate. To our knowledge,
this has never been tried on peptide esters, in solution or on
solid support.16
1a with 2 equiv of Me3Al and 2 equiv of EtSH in CH2Cl2
for 5 h at room temperature gave the desired thioester in
good yield but with a poor enantiomeric excess (ee) of 67%
(Table 1, entry 1). Note that thio-orthoester 3a is also formed
Table 1. Solution-Phase Synthesis of Amino Thioesters
X
EtSH
time
(h)
ee
(%)a
entry
R
(equiv) (equiv)
% 2
78
% 3b
1
2
3
4
5
6
H
Me (2)
Me (6)
Me (2)
Me (2)
Cl (2)
2
18
6
30
6
5
6
67
8
77
5
traces
traces
traces
H
H
H
H
traces nd
3.5
3
1.75 86
1.75 87
83
84
77
86
99.7c
98
Ph Cl (2)
6
a Determined by optical rotation unless otherwise specified. b ee of 3a,b
not determined. c Determined by chiral gas chromatography.
as a byproduct in the reaction. The latter becomes the major
product if longer reaction times and an excess of Me3Al and
EtSH are used (Table 1, entry 2).
To circumvent the problem of racemization, less basic
conditions were sought. To “buffer” the thiolate solution,
an excess of thiol was added (Table 1, entries 3 and 4). When
the EtSH/Me3Al ratio was raised from 2:2 to 30:2, the ee
increased to 86%. The results were further improved by
replacing Me3Al with Me2AlCl. Under these new conditions
(2 equiv of Me2AlCl, 6 equiv of EtSH, 1.75 h., rt), 1a and
Boc-phenylalanine benzyl ester (1b) were easily converted
to thioesters 2a and 2b in good yields (86-87%) and
excellent optical purities (99.7% and 98% ee, respectively)
(Table 1, entries 5 and 6).
The optimized conditions were subsequently applied to
resin-bound peptides. However, only low yields of peptide
thioester were obtained. Larger excesses of Me2AlCl and
EtSH were needed to effect the reaction. For example,
peptide-resin 4a′ (Leu-Tyr(OtBu)-Arg(Pbf)-Ala-Gly-O-
Wang resin), prepared by standard Fmoc solid-phase pro-
tocols, was treated with 20 equiv of Me2AlCl and 60 equiv
of EtSH for 5 h. After evaporation of the solvent under
vacuum, cleavage of side chain protecting groups [TFA-
PhOH-H2O (95:2.5:2.5), 2.5 h]17 and purification by reverse-
phase HPLC, the desired peptide C-terminal thioester Leu-
Tyr-Arg-Ala-Gly-SEt 5a (34%) and the corresponding acid
Leu-Tyr-Arg-Ala-Gly-OH 6a (20%) were isolated (Table 2,
entry 1). Use of the more acid-stable Pam resin instead of
the Wang resin decreased the amount of free acid formed
(3-5%) and thus led to significantly higher yields of peptide
thioesters such as 5a and 5b (60-63%, Table 2, entries 2
and 3).
To investigate the efficiency of the alkylaluminum thiolate-
mediated cleavage, several concerns need to be addressed.
First, racemization of the C-terminal thioester residue is
conceivable under cleavage conditions. Second, poor solva-
tion of the peptide linked to the resin under the reaction
conditions (e.g., in CH2Cl2) might limit access of the reagent
to the cleavage site.7 Finally, the alkylaluminum reagent
might promote undesired reactions on the backbone and/or
the side chains of the peptide.
To study the first issue, we applied Corey’s conditions15
to Boc-alanine benzyl ester (1a) as a model. Treatment of
(9) Alsina, J.; Yokum, T. S.; Albericio, F.; Barany, G. J. Org. Chem.
1999, 64, 8761-8769.
(10) Backes, B. J.; Ellman, J. A. J. Org. Chem. 1999, 64, 2322-2330.
(11) Ingenito, R.; Bianchi, E.; Fattori, D.; Pessi, A. J. Am. Chem. Soc.
1999, 121, 11369-11374.
(12) Shin, Y.; Winans, K. A.; Backes, B. J.; Kent, S. B. H.; Ellman, J.
A.; Bertozzi, C. R. J. Am. Chem. Soc. 1999, 121, 11684-11689.
(13) Wang, S. J. Am. Chem. Soc. 1973, 95, 1328-1333.
(14) Mitchell, A. R.; Erickson, B. W.; Ryabtsev, M. N.; Hodges, R. S.;
Merrifield, R. B. J. Am. Chem. Soc. 1976, 98, 7357-7362.
(15) (a) Corey, E. J.; Beames, D. J. J. Am. Chem. Soc. 1973, 95, 5829-
5831. (b) Corey, E. J.; Kozikowski, A. P. Tetrahedron Lett. 1975, 925-
928. (c) Hatch, R. P.; Weinreb, S. M. J. Org. Chem. 1977, 42, 3960-
3961. (d) Cohen, T.; Gapinski, R. E. Tetrahedron Lett. 1978, 4319-4322.
(16) The reaction of alkylaluminum thiolate with aziridine-2-carboxylic
acid esters has been reported (Haener, R.; Olano, B.; Seebach, D. HelV.
Chim. Acta 1987, 70, 1676-1693) and for proline esters (Moss, W. O.;
Jones, A. C.; Wisedale, R.; Mahon, M. F.; Molloy, K. C.; Bradbury, R. H.;
Hales, N. J.; Gallagher, T. J. Chem. Soc., Perkin Trans. 1 1992, 2615-
2624).
(17) The use of (i-Pr)3SiH or EDT as scavenger led to formation of side
products.
2440
Org. Lett., Vol. 2, No. 16, 2000

Table 2. Solid-Phase Synthesis of Peptide Thioesters
entry
substrate
peptide sequence
LYRAG
LYRAG
HWYQQKSG
IFKDG
resin
time (h) a
% 5b
% 6b
% bis-thioester 7b,c
1
2
3
4
5
4a ′
4a
4b
4c
4d
Wang
Pam
Pam
Pam
Pam
5
5
5
5
34
60
63
26
24
20
3
5
5
nd
8
d
YTKYNDDDTFTVKVG
3.5
a Peptides were cleaved from the resin and the protecting groups subsequently removed. Deprotection conditions: 2.5 h treatment at rt with TFA-
PhOH-H2O (95:2.5:2.5) for 4a; TFA-thioanisole-EtSH-H2O (92.5:2.5:2.5:2.5) for 4b TFA-H2O (95:5) for 4c and TFA-EtSH-H2O (95:2.5:2.5) for
4d. b Isolated yield calculated from the loading capacity of the commercial resin. c Ile-Phe-Lys-Asp(SEt)-Gly-SEt. d Peptide thioester 5d was separated from
a mixture of other noncharacterized derivatives, probably poly-thioesters, by reverse-phase HPLC.
Aspartic acid-containing 4c (Ile-Phe-Lys(Boc)-Asp(OtBu)-
Gly-O-Pam resin) was used to test the utility of the method
with peptides bearing additional ester functionality. The
cleavage conditions not only gave the desired thioester 5c
(26%) but also some acid 6c (5%) and bis-thioester (7c, Ile-
Phe-Lys-Asp(SEt)-Gly-SEt) (8%) generated by reaction with
the t-Bu ester of the aspartic acid side chain (Table 2, entry
4), as well as significant amounts of aspartimide rearrange-
ment product.18,19 Despite these complications, we were able
to isolate the 15-residue peptide thioester 5d containing three
aspartic acid residues in 24% yield (Table 2, entry 5). The
yield from this procedure is comparable to that reported by
Li et al. for the same peptide using a customized linker and
special reagents for the removal of Fmoc protecting groups.8
To investigate the suitability of our new method for
cleaving peptides with an epimerizable C-terminal residue,
Z-Gly-Ala-Phe-O-Pam (4e) was synthesized. Cleavage of
4e under the above conditions (Scheme 1), followed by
treatment with TFA to quench the reaction, gave Z-Gly-Ala-
Phe-SEt (5e, 32% yield) together with deprotected Gly-Ala-
Phe-SEt (40%). Normal-phase HPLC analysis showed that
5e was produced as a 9:1 mixture of LL/LD diastereoisomers
(80% diastereomeric excess or de). This result contrasts with
the low level of epimerization observed for the reaction in
solution and reflects the different conditions employed. The
large excess of Me2AlCl and EtSH (20 equiv and 60 equiv,
respectively) used to cleave esters from the resin enhances
the overall efficiency of cleavage but also favors formation
of thio-orthoesters and ketene-thioacetals (see Table 1).
Conversion of these adducts to the thioester during acid
workup provides an opportunity for epimerization (Scheme
1). Indeed, when the cleavage of Z-Gly-Ala-Phe-O-Pam (4e)
was quenched with water instead of TFA, we observed three
Scheme 1
(18) Asp-Gly and Asp-Asp are known to be problematic motifs prone
to aspartimide formation (Quibell, M.; Owen, D.; Packman, L. C.; Johnson,
T. J. Chem. Soc., Chem. Commun. 1994, 2343-2344). In this particular
reaction, the aspartimide peptide thioester generated by cyclization of the
aspartyl residue during the cleavage reaction is the major product [50% of
the total peptide material as judged by integration of HPLC chromatograms
and electrospray mass spectrometry (ESMS)].
(19) To suppress the formation of side products (aspartimide and bis-
thioester), THF (20 equiv) was added as a weak Lewis base to coordinate
the aluminum reagent. This modification reduced the amount of aspartamide
side product formed (only 11% of the total peptide material, as determined
by HPLC) and increased the overall yield of thioester 5c up to 37%, but
the formation of bis-thioester 7c was also enhanced somewhat (20%).
Org. Lett., Vol. 2, No. 16, 2000
2441

products by HPLC and ESMS: thio-orthoester 8e, thioester
5e, and ketene thioacetal 9e in an 8:1:1 ratio (Scheme 1).
After reverse-phase HPLC purification of these com-
pounds, thio-orthoester 8e and ketene-thioacetal 9e were
separately hydrolyzed by treatment with TFA-H2O (95:5)
for 2.5 h at room temperature. Interestingly, orthoester 8e
was converted to peptide thioester 5e in 57% yield with little
epimerization (LL/LD 97:3 or 94% de) (Scheme 1). This result
is remarkable since acid hydrolysis of thio-orthoesters is
known to give a stabilized thioacetal cation that readily
undergoes â-hydrogen elimination to generate a ketene
thioacetal.20 Analogous treatment of ketene thioacetal 9e gave
rise to the thioester with very low de (30%) (Scheme 1).
In conclusion, Me2AlCl-mediated thiolate cleavage of
peptides from solid-phase supports provides direct access to
peptide C-terminal thioesters. This one-pot procedure is
compatible with Fmoc-SPPS and does not require special
linkers, resins, or complicated protocols. Side reactions with
aspartic acid side chains may reduce yields, although the
byproducts are easily separated by HPLC. Some epimeriza-
tion at the site of cleavage also currently limits the method
to the preparation of peptides having a C-terminal glycine.
Work is in progress to overcome these limitations and further
define the scope and the generality of the method. However,
given its simplicity, this approach may already prove useful
for those ligations involving peptide segments with an
activated C-terminal glycine.
Acknowledgment. We thank A. Sewing for chiral gas
chromatography measurements. The authors are grateful to
the ETH-Zu¨rich and Novartis Pharma AG for support of this
work.20
Supporting Information Available: Representative pro-
cedures, experimental details, and characterization of the
products from the cleavage of Lys(Boc)-Tyr(OtBu)-Arg(Pbf)-
Ala-Gly-O-Pam resin (4a) and of Z-Gly-Ala-Phe-O-Pam
(4e) with Me2AlCl. This material is available free of charge
via the Internet at http://www.pubs.acs.org.
(20) (a) Okuyama, T.; Kawao, S.; Fueno, T. J. Org. Chem. 1984, 49,
85-88. (b) Okuyama, T.; Fueno, T. J. Am. Chem. Soc. 1985, 107, 4224-
4229.
OL0060836
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Org. Lett., Vol. 2, No. 16, 2000