Acid-catalyzed tandem thiol switch for preparing peptide thioesters from mercaptoethyl esters

Article abstract of DOI:10.1021/ol2007204

An efficient method compatible with Fmoc synthesis for preparing peptide thioesters via an acid-catalyzed tandem "thiol switch" of esters is described first by an intramolecular O-S acyl shift and then by an intermolecular S-S exchange, with concurrent deblocking of side chain protection groups.

Full text of DOI:10.1021/ol2007204

ORGANIC
LETTERS
2011
Vol. 13, No. 10
2610–2613
Acid-Catalyzed Tandem Thiol Switch for
Preparing Peptide Thioesters from
Mercaptoethyl Esters
Khee Dong Eom and James P. Tam*
School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive,
Singapore 637551
jptam@ntu.esu.sg
Received March 17, 2011
ABSTRACT
An efficient method compatible with Fmoc synthesis for preparing peptide thioesters via an acid-catalyzed tandem “thiol switch” of esters is
described first by an intramolecular OÀS acyl shift and then by an intermolecular SÀS exchange, with concurrent deblocking of side chain
protection groups.
Thioesters are versatile building blocks for various
synthetic schemes to prepare lactone and peptides.1 Thioe-
sters are susceptible to base and intolerant to piperidine in
Fmoc (fluorenylmethoxycarbonyl) chemistry and are cur-
rently the method of choice for many solid-phase syntheses
ofpeptides, particularly thosebearing glyco-and phospho-
modifications on the peptidyl side chains.2À4 These needs
have prompted the development of new methods for
preparing thioesters,5À9 and many employ a “thiol switch”
approach using a piperidine-resistant surrogate group
during the Fmoc peptide synthesis steps.2,4À9 Surrogate
groups such as sulfonamide, hydrazine, ester, and amide
with or without a thiol auxiliary2À7,9À15 are then activated
at the completion of peptide synthesis to undergo a thiol
switch reaction via an OÀS or NÀS acyl shift effected
either intra- or intermolecularly.
While each method has its merits, the use of a surrogate
is often complicated by synthetic complexity, by side
reactions at either the activation or the conversion step,
and in some cases, by the need of an additional step for its
removal. Consequently, a simple and practical method
would be valuable for preparing peptide thioesters under
an Fmoc-compatible scheme.
(1) (a) Dawson, P. E.; Muir, T. W.; Clark-Lewis, I.; Kent, S. B.
Science 1994, 266, 776–779. (b) Tam, J. P.; Lu, Y.-A.; Liu, C.-F. Proc.
Natl. Acad. Sci. U.S.A. 1995, 92, 12485–12489. (c) Tam, J. P.; Lu, Y.-A.;
Yang, J.-L.; Chiu, K.-W. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 8913–
8918. (d) Xu, J.; Eom, K. D.; Tam, J. P. Biopolymers 2001, 60, 206–211.
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Aimoto, S. Int. J. Pept. Res. Ther. 2007, 13, 191–202.
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r
10.1021/ol2007204
Published on Web 04/26/2011
2011 American Chemical Society

The NÀS and OÀS acyl shifts occur in the early stages of
protein splicing, and their biomimetic versions to afford a
thioester have been exploited.10À15 Recently, several
groups reported highly promising methods of thioester
preparations using the conformation-assisted NÀS acyl
shift of tertiary amides.12À15 In contrast, methods using
OÀS acyl shift of esters to prepare thioesters directly on
resin supports are less developed.
Scheme 2. Thioester Preparation under Acidic Conditions for
Tandem Thiol Switch via OÀS Acyl Shift and SÀS Acyl Shiftsa
Scheme 1. Preparation of Peptide-Thioesters on Hydro-
xyethylthiol (HET)-Resin
a Isolated yield (Supporting Information Figure 3-1).
deprotection step at the completion of peptide assemblage.
We reasoned that an ethoxythiol handle released during
the acid deprotection step as a C-terminal peptide ester 5
would permit an acid-catalyzed intramolecular OÀS acyl
shift 6f7 followed by an intermolecular SÀS exchange
7f8 with another thiol during the TFA (trifluoroacetic
acid) deprotection step under a timely activation by pro-
tonating the ester carbonyl 6 at the C-terminus (Scheme 2).
The proposed scheme would provide a solution to circum-
vent the issues for a separate step of activation and
auxiliary removal, and concurrently permit deprotection
of side chains under a one-pot reaction.
To determine the acidity requirement of the first OÀS
acyl step of our approach, we prepared a 9-residue peptide
ester KA9-OEt-SH 5awhich was readily obtainedfrom the
standard TFA cleavage of KA9-O-Et-S-Trt-resin 4a. TFA
treatment of the purified 5a without an external thiol for 2
h produced little rearranged product KA9-SEtÀOH
(<2%) and the startingmaterialKA-OEt-SH 5aremained
as the major product (>80%). Prolonged TFA treatment
for 18 h did not significantly increase the desired thioester.
These results were in contrast of acid-catalyzed thiol
switch reactions for preparing thioesters by NÀS acyl
shifts through tertiary amide surrogates which gave a
mixture of both amide and thioester products after TFA
treatment.11À15 However, our results are consistent with
the known basicity of the tertiary amide and ester moieties,
Esters (4 in Scheme 1) are generally stable during the
repetitive deprotection steps of Fmoc-chemistry and could
serve as a thioester surrogate during the Fmoc synthesis of
peptides. Although OÀS acyl shift has been exploited for
thioester preparation at pH 6.5 after the global deprotec-
tion of side chains,10 hydrolysis is a worrisome side reac-
tion. Hilvert’s group has also explored the use of Lewis
acid-thiolate on benzylic ester resin for preparing thioe-
sters under rather severe conditions.7 Here, we describe an
efficient method for preparing peptide thioester by a
tandem “thiol switch” of a peptide ester containing β-
mercaptoethanol handle (OEtSH) which enables an intra-
molecular OÀS acyl shift followed by an intermolecular
SÀS exchange reaction to afford a thioester.
Our approach makes use of the C-terminal alkoxythiol
linker that we have previously developed for cysteine-free
ligation.16 It has the advantage of being readily accessible
by reacting hydroxyethylthiol (HET) 1 or hydroxypropyl
thiol (HPT) with a trityl chloride resin 2 to form HET- or
HPT-resin, respectively (Scheme 1). The reactive thiol
linked to the resin as thiol ether 3 is protected during the
peptide synthesis stage and released as a latent thiolate
for the first “thiol switch” reaction during the acid
(16) Lu, Y.-A.; Tam, J. P. Org. Lett. 2005, 7, 5003–5006.
Org. Lett., Vol. 13, No. 10, 2011
2611

which have pKa of 0 and À3, respectively. Since the acidity
function of TFA is around À3, our results suggest that the
OÀS acyl shift will require an increase in acidity to proto-
nate the ester carbonyl for a smooth thioester conversion.
the thioester intermediate 5b, we used an increasing con-
centration of an external thiol, thiocresol (TC), in 0.1%
TfOH in TFA. With 5% TC in 2 h, the desired thioester 8a
was obtained in 70% yield (Figure 1).
For the acid-catalyzed SÀS exchange reactions, we
found that, under our proposed conditions, simple thiols
such as p-thiocresol and benzyl thiol were more suitable
than the bifunctional thiols, such as mecaptoethanol or
mecaptopropionic acid which gave a complicated product
profile because of side reactions of their O-moiety partici-
pating in the reverse OÀS acyl shift reactions.
Table 1. Effect of TfOH (v/v) in TFA on the Disappearance of
KA9-OEtSH 5a and the Formation of KA9-OH via 1,2-Elim-
ination to Form Ethylene Sulfide
KA9-OEt-SH (mol %)
KA9-OH (mol %)
TfOH (vol %)
1 h
8 h
1 h
8 h
In combining both steps, a direct one-step thioester
conversion from the peptide-HET resin of 4a, 4b, and 4c
was successfully achieved in 2 h affording 25À55% yield
with a deprotection mixture (TFA 9.3 mL, TC 0.5 mL, and
TfOH 18 μL within 0.18 mL TFA, 1:5%:0.18%, v/v/v)
from 60 mg of peptide resins. The conversion became
sluggish when a Lewis acid such as B(TFA)3 replaced
TfOH at the same concentration of 0.1 to 0.2% in TFA
(v/v).
The first of the two-thiol switch reactions is likely the
rate-determining step for the tandem thiol switch scheme
because it is an entropic-favored, intramolecular OÀS acyl
shift through a five-member ring intermediate. Control
experiments, requiring an intermolecular OÀS acyl shift
using methylbenzoate as a model compound, did not
afford thioester products under the proposed conditions
of 0.1À0.3% TfOH (v/v) in TFA and 5% TC for 2 h as
determined by HPLC. Similarly, the desired thiolactone
from the first tandem thiol switch reaction was not ob-
served with CG7-OEt-SH 5b containing an N-terminal
cysteine. Treatment of 5b with 0.15% TfOH in TFA
without TC for 8 h afforded CG7-OH as the main product
suggesting the intramolecular OÀS acyl shift by the
N-terminal thiol to form a thiolactone of 5a was unfavor-
able. Addition of 5% TC to the 0.15% TfOH/TFA solu-
tion for 4 h provided 42% CG7-TC 8b, which was
smoothly converted to the end-to-end cyclized lactam in
80% yield within 1 h at pH 7.5 or 8.5 (Supporting
Information Figure 1) with <6% the hydrolyzed product
CG7-OH.
0.01
0.06
0.1
0.2
1
92
74
57
53
48
72
21
2
1
52
62
62
44
19
30
31
34
1
0
To increase acidity of the TFA for the OÀS acyl shift of
5a, we added incrementally TfOH (trifluoromethane sul-
fonic acid) from 0.01 to 1% by volume to TFA (Table 1).
Since we were unable to observe a significant accumulation
of the desired thioester KA9-SEtÀOH 6a, the rates of SÀO
acyl shift in the conversion of KA9-SEtÀOH 6a from
KA9-OEt-SH 5a were deduced on the basis of the starting
material and the formation of the byproduct KA9-OH
which lost the OEtSH handle by a competitive acid-
catalyzed elimination reaction to form ethylene sulfide.
The use of 0.1À0.2% (v/v) of TfOH in TFA appeared to be
a suitable compromise so as to permit sufficient acid
strength to protonate the ester carbonyl to effect the
OÀS acyl shift but not too strong to increase competing
side reactions. We also concluded that the OÀS and SÀO
acyl shifts were likely reversible in stronger acidic solutions
than TFA alone. These results are consistent with our
previous findings that the OEtSH handle of peptide ester 5
is fairly stable in TFA but susceptible to degradation in
strong acids.16,17
Two side reactions associated with the current scheme
were also investigated. The first was the long-standing
problem of racemization during the esterification of the
first amino acid to a hydroxy resin such as the HET resin.
To determine the extent of racemization, we subjected the
esterification of Fmoc-Arg(Pbf)ÀOH or Fmoc-Ala-OH to
a high loading HET resin (1.4 mmol/g) to completion by
BOP-DIEA repetitively (three times) in the synthesis of
TIGGIR-TC 8c or KA9-TC 8a, which produced 85 and
11% racemization, respectively. Interestingly, the D and L
forms of both peptides 8c could be clearly identified in
HPLC in their thiocresol ester forms. For example, hex-
apeptide TIGGIR-TC 8c afforded two peaks (Supporting
Information Figure 3-1) in HPLC with the expected
molecular weight. Native ligation with a hexapeptide
Figure 1. Thioester conversion under 0.1% TfOH/TFA in (A)
1%, (B) 3%, (C) 5% of thiocresol from KA9-OEt-SH to KA9-
TC.
To investigate suitable conditions for the SÀS exchange
of transthioesterification in the second step of a tandem
thiol switch scheme to afford a stable thioester and to
terminate the equilibrations of OÀS and SÀO acyl shifts of
(17) Reynolds, D. D.; Fields, D. L.; Johnson, D. L. J. Org. Chem.
1961, 26, 5130–5133.
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Org. Lett., Vol. 13, No. 10, 2011

CALVIN-NH2 gave two ligated products TIGGIRCAL-
VIN 9c and TIGGIrCALVIN 9d with a different retention
time in HPLC. Direct synthesis of TIGGIRCALVIN 9c
and TIGGIrCALVIN 9d by solid phase synthesis con-
firmed D- and L-isomers of the C-terminal Arg position
(Supporting Information Figure 2). Several esterification
methods have been developed to address this side
reaction,7,18,19 and we recommended that prolonged ester-
ification under basic conditions should be avoided to
minimize racemization of the C-terminal amino acid dur-
ing the attachment step.
Arg by an electrophile such as NO to form Arg(NO),
Arg(EtSH) was reversible to Arg in a longer time reaction
>20 h (30%). It did not escape our attention that the facile
formation and the reversibility of Arg(EtSH) could pro-
vide a novel entry to peptide ligation of N-terminal Arg
peptides
In a preliminary study to support our results, we found
that the alkylation side reaction can be minimized in the
synthesis of TIGGIR-TC 8c by substituting 2-mercap-
toethanol with 3-mercaptopropanol to form HPT-trityl
resin. The acid-catalyzed conversion under our proposed
thiol-switch scheme to form the four-member ring propy-
lene sulfide was substantially slower than the three-mem-
ber ring ethylene sulfide to effect the alkylation reaction to
form Arg(PrSH).
Scheme 3. Reversible Alkylation Reaction of C-Terminal Arg to
Form C-Terminal Arg(EtSH)
In summary, the OÀS acyl shift of ester 5 was dependent
on acidity and the protonation state of the ester carbonyl.
Addition of a catalytic amount of TfOH accelerated the
OÀS acyl shift to afford a thioester 7. The addition of an
external thiol into the reaction mixture permits the second
“thiol switch” reaction and serves to favor the formation of
stable thioester 8 by terminating the equilibration of the acyl
migrations prone to the reverse reaction and degradation
between O and S moieties. Our proposed scheme bridges an
“acid gap” in preparing thioesters under acidic conditions. It
bears similarity to the “low TFMSA” and “low HF” condi-
tions which have an acidity function of À5.2 and could be
used for the synthesis of glycol- and phospho-peptides.2b,20
Our method based on convenient starting materials
provides a simple and practical solution to prepare thioe-
ster peptides compatible with Fmoc chemistry. It also
provides a choice of an ester 7 with a thiol handle or
thioesters for ligation, suitable for the cysteine-free,3,16
convergent synthesis,3 and the tandem ligation schemes.21
The second side reaction is a reversible alkylation of the
guanidino side chain of Arg of 8c by ethylene sulfide to
form TIGGI-Arg(EtSH) 10c, which was confirmed by
MS/MS sequencing (Supporting Information Figure 3-2
and 3-3). Ethylene sulfide is the elimination byproduct of
mercaptoethanol released from the SÀS exchange of the
HET handle or hydrolysis of peptide 6 (Scheme 3). It is an
electrophile and can act as an alkylating agent to form
adduct with nucleophilic side chains. Under our proposed
deprotection conditions containing 0.1À0.2% TfOH in
TFA with 5% TC, the alkylation appeared to be specific
for the guanidino side chain when tested against Fmoc-
Met, Fmoc-Tyr and Fmoc-Trp, and the large excess of TC
likely served as a scavenger for these nucleophilic side
chains. We found that the alkylation of TIGGIR-TC 8c
was 30% in 2 h, probably due to the proximal effect of the
C-terminal Arg to the OEtSH handle (Scheme 3), but
decreased to 1% in the model peptide GGRGG 5d with
an internally placed Arg. Similar to the acid-catalyzed
modification and removal of the guanidino side chain of
Acknowledgment. This research was supported in part
by Biomedical Research Council (BMRC 09/1/22/19/612)
of A*STAR and Academic Research Fund (ARC21/08) of
Ministry of Education in Singapore.
Supporting Information Available. General procedures,
additional HPLC and MS data. This material is available
free of charge via the Internet at http://pubs.acs.org.
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1983, 105, 6442–6455. (b) Tam, J. P.; Heath, W. F.; Merrifield, R. B. J.
Am. Chem. Soc. 1986, 108, 5242–5251.
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2003, 125, 73–82.
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H.; Carpino, L. A.; Bienert, M. J. Org. Chem. 1996, 61, 8831–8838.
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