Ca2+-mediated thiolysis of peptide-resin linkage as an option for preparing protected peptide thioesters

Article abstract of DOI:10.1016/j.tetlet.2008.04.081

A mild new procedure for preparing protected peptide thioesters, based on Ca²⁺-assisted thiolysis of peptide-Kaiser oxime resin (KOR) linkage, is described. Ac-Ile-Ser(Bzl)-Asp(OcHx)-SR (Ac: acetyl; Bzl: benzyl; cHx: cyclohexyl), model peptide, was readily released from the resin by incubating the peptide-KOR at 60 °C in mixtures of DMF with n-butanethiol [R = (CH₂)₃CH₃] or ethyl 3-mercaptopropionate [R = (CH₂)₂COOCH₂CH₃] containing Ca(CH₃COO)₂. After serine and aspartic acid side-chain deprotection under acid conditions, Ac-Ile-Ser-Asp-S(CH₂)₂COOCH₂CH₃ was successfully obtained with good quality and high yield. This type of C-terminal modified peptide may act as an excellent acyl donor in peptide segment condensation by the thioester method, native chemical ligation and enzymatic methods.

Full text of DOI:10.1016/j.tetlet.2008.04.081

Available online at www.sciencedirect.com
Tetrahedron Letters 49 (2008) 3853–3857
Ca2+-mediated thiolysis of peptide–resin linkage as an option
for preparing protected peptide thioesters
*
´
ˆ
Patrıcia B. Proti, M. Teresa M. Miranda
Department of Biochemistry, Institute of Chemistry, University of Sa˜o Paulo, PO Box 26077, 05513-970 Sa˜o Paulo, Brazil
Received 27 December 2007; revised 9 April 2008; accepted 14 April 2008
Available online 28 April 2008
Abstract
A mild new procedure for preparing protected peptide thioesters, based on Ca2+-assisted thiolysis of peptide–Kaiser oxime resin
(KOR) linkage, is described. Ac-Ile-Ser(Bzl)-Asp(OcHx)-SR (Ac: acetyl; Bzl: benzyl; cHx: cyclohexyl), model peptide, was readily
released from the resin by incubating the peptide–KOR at 60 °C in mixtures of DMF with n-butanethiol [R = (CH2)3CH3] or ethyl
3-mercaptopropionate [R = (CH2)2COOCH2CH3] containing Ca(CH3COO)2. After serine and aspartic acid side-chain deprotection
under acid conditions, Ac-Ile-Ser-Asp-S(CH2)2COOCH2CH3 was successfully obtained with good quality and high yield. This type of
C-terminal modified peptide may act as an excellent acyl donor in peptide segment condensation by the thioester method, native chemical
ligation and enzymatic methods.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: Solid-phase peptide synthesis; Kaiser oxime resin; Ethyl 3-mercaptopropionate; n-Butanethiol; Metal ion
Preliminary accounts of certain aspects of this work are
described as a communication in Ref. 35.
is an appropriate resin to prepare C-terminally modified
peptides,8,9 it has also been employed to furnish protected
peptide thioesters through aminolysis of oxime ester bond
with thioesterified amino acid.6
Peptide thioesters—the key building blocks in conver-
gent peptide synthesis through the thioester method,1,2
native chemical ligation3,4 or protease-mediated peptide
condensation5,6—can be prepared by a stepwise solid-
phase synthesis through t-butyloxycarbonyl (Boc) or fluo-
renylmethoxycarbonyl (Fmoc) strategy using highly effi-
cient procedures.
Regarding the Boc strategy, peptide thioesters can be
obtained by peptide elongation starting from Boc-amino
acid-S(CH2)2CO-4-methylbenzhydrylamine (MBHA) resin
or -hydroxymethylphenylacetamidomethyl resin (PAM
resin) followed by HF treatment of the resulting peptide–
resin.1,2 The use of 3-mercaptopropionic acid as a highly
versatile multidetachable linker, stable to the protocols
used in the Boc strategy and HF treatment, but cleavable
by thiol nucleophile, has also been explored.7 Since KOR
As to the Fmoc strategy, several attempts have over-
come the lability of the thioester functionality to bases.10
Such attempts include (i) using alternative Fmoc-deprotec-
tion cocktails, such as the one-to-one mixture of N-methyl-
pyrrolidone–DMSO containing 1-methylpyrrolidine (25%
v/v), hexamethyleneimine (2% v/v) and N-hydroxybenzo-
triazole (2% w/v);11,12 (ii) employing the backbone amide
linker (BAL) strategy in which the thioester moiety is intro-
duced at the end of the solid-phase peptide synthesis via
amino acid thioester coupled to the C-terminal residue;13,14
(iii) performing alkylaluminium-mediated thiolysis of pep-
tide–PAM or Wang resin linkage;15,16 (iv) carrying out the
solution-phase thioesterification of a fully protected pep-
tide obtained from 2-chlorotrityl resin (2ClTrt resin) in
the presence of coupling reagents.17–19 Employing a poten-
tially more general procedure, peptide thioesters have also
been prepared by Fmoc-based SPPS using ‘safety-catch’
linkers, which form with the growing peptide a linkage
*
Corresponding author. Tel.: +55 11 3091 3855; fax: +55 11 3815 5579.
E-mail address: mtmirand@iq.usp.br (M. T. M. Miranda).
0040-4039/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2008.04.081

3854
P. B. Proti, M. T. M. Miranda / Tetrahedron Letters 49 (2008) 3853–3857
stable to acidic and basic conditions. Among the examples
are (i) the sulfonamide linker that can be activated by
alkylation with trimethylsilyldiazomethane or iodoaceto-
nitrile and cleaved by a thiol nucleophile to release the
respective protected peptide thioester;20–23 (ii) the aryl-
hydrazine linker, which renders the resulting peptide
hydrazide resin capable of undergoing mild oxidation and
being converted into a reactive acyl diazene intermediate
that, in turn, can be cleaved by an a-amino acid S-alkyl
thioester providing the correspondent protected peptide
thioester.10,24
Evidently all the procedures cited above show advanta-
ges and disadvantages. Hence, new procedures for the syn-
thesis of protected peptide thioesters are of interest in
peptide and protein chemistry. While studying the prepara-
tion of protected peptide esters through Ca2+-mediated
alcoholysis of peptide–KOR linkage,25 we preliminarily
observed that the replacement of the alcohol by ethanethiol
led to the desired peptide thioester selectively under very
mild conditions. Nevertheless, the thiolysis was very slow
and the procedure was compatible only with the Boc chem-
istry. In fact, the Boc chemistry has been supplanted by the
Fmoc strategy. On the other hand, the Boc strategy is still
used due to its lower cost, the option of replacing HF by
other acids for peptide full deprotection/cleavage from
the resin26–28 and the fact that there are countries where
HF is still commercially available and the laboratories spe-
cialized in peptide synthesis use it only on a very small scale
mostly for scientific purposes.29–31 Therefore, in this work
we studied Ca2+-mediated thiolysis of peptide–KOR
linkage in an attempt to prepare an Ac-peptide-
S(CH2)2COOCH2CH3. As in our previous work,25 frag-
ment 22–24 of the gastrointestinal hormone cholecystoki-
nin-33 was chosen as a model peptide.
as solvent and 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetra-
methyluronium tetrafluoroborate (TBTU)/N,N-diisopro-
pylethylamine (DIEA) as coupling reagents.33 After
completion, Ac-Ile-Ser(Bzl)-Asp(OcHx)–KOR (1; substitu-
tion level of 0.32 mmol/g) was characterized by total
hydrolysis/amino acid analysis. Thiolysis reactions starting
from 1 were performed in 20% n-butanethiol/DMF in the
absence and presence of Ca(CH3COO)2 under the experi-
mental conditions described in Scheme 1. Their monitoring
by RP-HPLC and the analyses of 48 h-reaction aliquots
by liquid chromatography coupled to electrospray ioniza-
tion mass spectrometry (LC/ESI-MS) revealed that, as
expected, the reaction in the presence of Ca2+ furnished
more peptide thioester 2a {ESI-MS [MH]+: 620.6 (found);
621.2 (calcd)} than that conducted without Ca2+
.
Having confirmed our preliminary observation25 and,
hence, the ability of Ca2+ to mediate thiolysis of the oxime
ester bond of 1, attempts to prepare Ac-Ile-Ser(Bzl)-
Asp(OcHx)-S(CH2)2COOCH2CH3 were done. In fact, the
thioester corresponding to ethyl 3-mercaptopropionate is
one of the most commonly employed in convergent peptide
synthesis using either the thioester method17,19 or the
native chemical ligation.20,22 In addition, it may also be
used in enzyme-mediated peptide condensation due to its
high susceptibility to nucleophiles and, consequently, high
potential to form the acyl-enzyme complex.
Thiolyses of 1 in 20% ethyl 3-mercaptopropionate/DMF
were also performed at 60 °C for 6 h in the absence and
presence of Ca(CH3COO)2 (Scheme 1). Their monitoring
by RP-HPLC and the analyses of the 6 h-reaction aliquots
by LC/ESI-MS demonstrated that our procedure is appro-
priate for the thiolysis studied and indicated that the equi-
librium for the Ca2+-mediated reaction was attained before
6 h. The yields of peptide detachment from KOR were
determined through amino acid analyses of 1 before and
after thiolysis.33 Compound 2b {ESI-MS [MH]+: 664.6
(found); 664.8 (calcd)} was provided after 6 h in 49% (con-
trol reaction) and 94% (Ca2+-mediated reaction) yields.
Boc-Asp(OcHx)-OH (1.74 equiv to the reactive groups
in the resin) was first coupled to KOR (1 g; substitution
level of 0.87 mmol/g) in methylene chloride (DCM;
10 mL) for 24 h, using N,N0-dicyclohexylcarbodiimide
(DCC; 1.74 equiv),32 to produce Boc-Asp(OcHx)–KOR
(aminoacylation level of 0.53 mmol/g). Chain assembly
was carried out manually by Boc chemistry using DMF
To determine the minimum reaction time for the Ca2+
-
mediated thiolysis with ethyl 3-mercaptopropionate, two
new reactions were performed starting from 1 with
Scheme 1. Ca2+-mediated thiolysis of Ac-Ile-Ser(Bzl)-Asp(OcHx)–KOR oxime ester bond. Reagents and conditions: (i) 20% CH3(CH2)3SH/DMF, 60 °C
(2a); (ii) 20% CH3CH2OOC(CH2)2SH/DMF, 60 °C (2b); (iii) 50% CH3CH2OOC(CH2)2SH/DMF, 50 °C (2b). All the reactions were performed using
1 equiv of Ca(CH3COO)2/1 equiv of peptide under orbital shaking at 300 rpm.

P. B. Proti, M. T. M. Miranda / Tetrahedron Letters 49 (2008) 3853–3857
3855
Table 1
Ca(CH3COO)2 in either 20% thiol/DMF at 60 °C or in
higher thiol content and lower temperature (50% thiol/
DMF at 50 °C; Scheme 1). Control reactions (with no addi-
tive) were also promoted. Their monitoring by RP-HPLC
and the analyses of 2 h-reaction aliquots by LC/ESI-MS
demonstrated that (i) the reaction mediated by Ca2+
attained its equilibrium in 2 h; (ii) the desired product
2b {ESI-MS [MH]+: 664.6 (found); 664.8 (calcd)} was
obtained with good quality; (iii) the thiol dimer
CH3CH2OOC(CH2)2S–S(CH2)2COOCH2CH3 (3) {ESI-
MS [MH]+: 266.9 (found); 266.4 (calcd)} was the main
byproduct formed in either Ca2+-mediated thiolyses or
control reactions (3 was also produced when the solution
20% CH3CH2OOC(CH2)2–SH/DMF was incubated with
Ca(CH3COO)2 in the absence of the peptide–KOR). Figure
1 shows the RP-HPLC profiles for the reactions performed
in 50% thiol/DMF at 50 °C. The yields of peptide thioester
detachment from KOR described in Table 1 showed that
both conditions used were suitable for Ca2+-mediated
thiolysis.
Yields of peptide 2b detachment from KOR after oxime ester bond
thiolysis with ethyl 3-mercaptopropionate for 2 h
Mediator
Solvent system (V/V)
T (°C)
Peptide detachmenta (%)
—
20% thiol/DMF
20% thiol/DMF
50% thiol/DMF
50% thiol/DMF
60
60
50
50
28
93
53
92
Ca2+
—
Ca2+
a
The yields of peptide detachment from KOR were determined through
amino acid analysis of 1 before and after thiolysis.33
that only byproduct 3 was not eliminated. On the other
hand, its formation can be minimized using inert atmo-
sphere for the reactions, which would not be possible, for
example, in the aryl hydrazine linker procedure performed
under an oxidant condition.24
In order to be employed in convergent peptide synthesis
through the thioester method, native chemical ligation or
enzyme-mediated peptide condensation, our model peptide
should have its serine and aspartic acid side-chains depro-
tected. Thus, after Sep-Pak C18 pre-treatment, compound
2b contaminated with dimer 3 was submitted to HF treat-
ment for 1.5 h in the presence of anisol. The RP-HPLC
(Fig. 2) and LC/ESI-MS analyses of the resulting crude
peptide revealed that (i) the desired product Ac-Ile-Ser-
Asp-S(CH2)2COOCH2CH3 (4) {ESI-MS [MH]+: 492.3
(found); 492.6 (calcd)} was successfully obtained; (ii)
Ca2+-containing reaction media were pooled and then
placed in a 5 mL syringe connected to a Sep-Pak Plus
C18 (Waters) to eliminate calcium salt, DMF and thiol
from the desired protected peptide thioester. It was eluted
from the cartridge with aqueous solutions of acetonitrile
containing 0.1% trifluoroacetic acid (TFA). RP-HPLC
and LC/ESI-MS analyses of the eluted fractions indicated
Fig. 1. RP-HPLC monitoring of oxime ester bond thiolysis of 1 in 50% CH3CH2OOC(CH2)2SH/DMF at 50 °C. (A) control reaction; (B) Ca2+-mediated
reaction. (I) DMF; (II) CH3CH2OOC(CH2)2SH. Elution conditions: column, Vydac C18 (4.5 ꢀ 250 mm) at a flow rate of 1 mL/min; eluent, aqueous
acetonitrile containing 0.1% TFA.

3856
P. B. Proti, M. T. M. Miranda / Tetrahedron Letters 49 (2008) 3853–3857
In conclusion, we have demonstrated that Ca2+-medi-
ated thiolysis of the oxime ester bond of a peptide–KOR
using ethyl 3-mercaptopropionate may be an optional, sim-
ple and efficient one-step procedure for preparing precur-
sors of acyl donors suitable for chemical convergent
peptide syntheses. It employs mild conditions [Ca(CH3-
COO)2 as additive instead of acids or bases] avoiding sig-
nificant side-reactions and the use of an ozone-depleting
solvent (DMF as a solvent rather than methylene chloride).
Acknowledgements
This work was supported by grants from Fundaßcao de
˜
Amparo a Pesquisa do Estado de Sao Paulo, FAPESP
˜
(04/15376-7) and Conselho Nacional de Desenvolvimento
´
´
Cientıfico e Tecnologico, CNPq (481114/2004-1). P.B.P.
was a graduate fellow of FAPESP (02/13000-1). The
authors thank C. W. Liria for the amino acid analyses,
F. M. Prado for the help with the mass spectrometry anal-
yses and G. G. Bianco and G. C. Barazzone for the assis-
Fig. 2. RP-HPLC profile of the crude peptide thioester 4 remaining from
HF treatment. Elution conditions: column, Vydac C18 (4.5 ꢀ 250 mm) at
a flow rate of 1 mL/min; eluent, aqueous acetonitrile containing 0.1%
TFA.
1
tance with the H NMR spectra interpretation.
compound 3 did not hamper the side-chain deprotection
reaction, nor was detected in the final solution, having
probably been extracted in the ether phase during peptide
precipitation after the HF treatment; (iii) byproduct 5
{ESI-MS [MH]+: 492.3 (found); 492.6 (calcd)} seems to
be an isomer of 4, most likely a product from the N–O acyl
shift34 (although this secondary reaction can be reverted by
the treatment of O-acyl-peptide with basic aqueous solu-
tion, it was not done due to the known thioester moiety
lability to bases).11 Compounds 2b and 4 were purified by
RP-HPLC and further characterized by amino acid analy-
Supplementary data
This material contains the experimental procedures
employed and the analysis data for the purified peptide
thioesters. Supplementary data associated with this article
can be found, in the online version, at doi:10.1016/j.tetlet.
2008.04.081.
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