N→S acyl-transfer-mediated synthesis of peptide thioesters using anilide derivatives

Article abstract of DOI:10.1021/ol8028093

N→S acyl-transfer-mediated synthesis of peptide thioesters utilizing an N-aminoacyl-N-sulfanylethylaminobenzoic acid derivative has been examined. The developed synthetic methodology for peptide thioesters is compatible with Fmoc solid-phase peptide synth

Full text of DOI:10.1021/ol8028093

ORGANIC
LETTERS
2009
Vol. 11, No. 4
823-826
NfS Acyl-Transfer-Mediated Synthesis
of Peptide Thioesters Using Anilide
Derivatives
Shugo Tsuda, Akira Shigenaga, Kiyomi Bando, and Akira Otaka*
Institute of Health Biosciences and Graduate School of Pharmaceutical Sciences, The
UniVersity of Tokushima, Tokushima 770-8505, Japan
aotaka@ph.tokushima-u.ac.jp
Received December 5, 2008
ABSTRACT
NfS acyl-transfer-mediated synthesis of peptide thioesters utilizing an N-aminoacyl-N-sulfanylethylaminobenzoic acid derivative has been
examined. The developed synthetic methodology for peptide thioesters is compatible with Fmoc solid-phase peptide synthesis (SPPS).
Peptide thioesters have played an indispensable role in native
chemical ligation (NCL) for the chemical synthesis of
peptides/proteins, whereby a thioester fragment chemose-
lectively reacts with the thiol group of an N-terminal cysteine
fragment followed by SfN acyl transfer to give a ligated
product.1 Success in NCL-mediated protein synthesis partly
depends on the accomplishment of preparing peptide thioesters.
Boc solid-phase peptide synthesis (Boc SPPS) on a thioester-
linked peptidyl resin has served as a standard protocol for
the preparation of peptide thioesters2 due to the instability
of the thioester linkage to base treatment such as 20%
piperidine/DMF used with Fmoc SPPS. However, the
increasing widespread use of Fmoc SPPS in the preparation
of various peptides including phosphorylated or glycosylated
materials has prompted the development of a synthetic
methodology for peptide thioesters via Fmoc protocols,3
including the use of Kenner’s safety catch linker3d,e,i or
orthothioester.3g Alternatively, NfS acyl transfer is seen in
naturally occurring thioester formations of the intein-extein
system,4,5 a feature which should have potential utility in
the chemical synthesis of thioesters.
Aimoto reported NfS acyl-transfer-mediated protocols
based on the side reaction observed in the acidolytic
deprotection of thiol-auxiliary-containing peptides.6-8 More-
over, we independently developed an acyl-transfer protocol
using a cysteine-derived N-peptidyloxazolidinone 18b in
(1) (a) Dawson, P. E.; Muir, T. W.; Clark-Lewis, I.; Kent, S. B. H.
Science 1994, 266, 776–779. (b) Dawson, P. E.; Kent, S. B. H. Annu. ReV.
Biochem. 2000, 69, 923–960.
(2) (a) Aimoto, S. Biopolymers 1999, 51, 247–265. (b) Hackeng, T. M.;
Giffin, J. H.; Dawson, P. E. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 10068–
10073.
(3) (a) Futaki, S.; Sogawa, K.; Maruyama, J.; Asahara, T.; Niwa, M.;
Hojo, H. Tetrahedron Lett. 1997, 38, 6237–6240. (b) Li, X.; Kawakami,
T.; Aimoto, S. Tetrahedron Lett. 1998, 39, 8669–8672. (c) Alsina, J.;
Yokumu, T. S.; Albericio, F.; Barany, G. J. Org. Chem. 1999, 64, 8761–
8769. (d) 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. (e)
Ingenito, R.; Bianchi, E.; Fattori, D.; Pessi, A. J. Am. Chem. Soc. 1999,
121, 11369–11374. (f) Swinnen, D.; Hilvert, D. Org. Lett. 2000, 2, 2439–
2442. (g) Brask, J.; Albericio, F.; Jensen, K. J. Org. Lett. 2003, 5, 2951–
2953. (h) Camarero, J. A.; Hackel, B. J.; de Yoreo, J. J.; Mitchell, A. R. J.
Org. Chem. 2004, 69, 4145–4151. (i) Mende, F.; Seitz, O. Angew. Chem.,
Int. Ed. 2007, 46, 4577–4580. (j) Blanco-Canosa, J. B.; Dawson, P. E.
Angew. Chem., Int. Ed. 2008, 47, 6851–6855.
(4) For reviews of intein-mediated protein splicing including mechanistic
consideration and synthetic applications, see: (a) Evans, T. C.; Xu, M. Q.
Chem. ReV. 2002, 102, 4869–4884. (b) Muir, T. W. Annu. ReV. Biochem.
2003, 72, 249–289. (c) Flavell, R.R.; Muir, T. W. Acc. Chem. Res. DOI:
10.1021/ar800129c
.
(5) For protein splicing bibliography, see: http://www.neb.com/neb/
inteins.html.
(6) Kawakami, T.; Sumida, M.; Nakamura, K.; Vorherr, T.; Aimoto, S.
Tetrahedron Lett. 2005, 46, 8805–8807
.
(7) For synthesis of peptide thioesters using OfS acyl transfer, see: (a)
Warren, J. D.; Miller, J. S.; Keding, S. J.; Danishefsky, S. J. J. Am. Chem.
Soc. 2004, 126, 6576–6578. (b) Botti, P.; Villain, M.; Manganiello, S.;
Gaertner, H. Org. Lett. 2004, 6, 4861–4864
.
10.1021/ol8028093 CCC: $40.75
Published on Web 01/22/2009
2009 American Chemical Society

which inhibition of delocalization of the amide nitrogen lone
pair to the exo-carbonyl π* orbital by the ring carbonyl
weakens the peptide-oxazolidine linkage and thereby leads
to the formation of peptide thioester 3 through nucleophilic
involvement of the thiol group neighboring to the activated
amide9 (Figure 1). We synthesized a peptide thioester using
Scheme 1
Figure 1. Formation of peptide thioester utilizing peptydyl oxazo-
lidinone and general concept for NfS acyl-transfer-mediated
synthesis of thioester.
resulting Ns-protected ally ester 9, a trityl (Trt)-protected
thiol unit 1011 was introduced under Mitsunobu conditions12
to give ethyl sulfide-modified compound 11. The Ns group
on 11 was removed by sulfanylacetic acid/LiOH in DMF to
give N-ethyl sulfide aniline 12. Reaction of 12 with Fmoc-
L- or -D-Ala-Cl in the presence of NaH in THF13 followed
by Fmoc-removal and subsequent coupling of Boc-Leu-OH
using 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium
hexafluorophosphate (HBTU)14/diisopropylethylamine (DIEA)
yielded dipeptide 14a (L-Ala form) or 14b (D-Ala form),
respectively, required for examination of the susceptibility
of the anilide derivatives to base treatment. Acylation of 12
with Fmoc-Gly-Cl (and 13C-carbonyl derivative) gave anil-
ides 13 (and 13′). The allyl group on 13 was removed by
treatment with Pd(PPh3)4/N-methylaniline (NMA)15 to give
the corresponding benzoic acid 15 which served as an
indispensable intermediate for the NfS acyl-transfer-medi-
ated preparation of peptide thioesters. For evaluation of the
the oxazolidinone system with Fmoc protocol; however,
partial loss of the peptide from the acylated oxazolidinone
and epimerization of the C-terminal amino acid during 20%
piperidine treatment were observed. Therefore, we have
sought a robust NfS acyl-transfer protocol for thioester
synthesis compatible with Fmoc-chemistry. On the basis of
the hypothesis that the presence of consecutive sp2-atoms
adjacent to an amide nitrogen (4 or 5 in Figure 1) should
induce nitrogen-pyramidalization and activate the amide
bond, we investigated the applicability of anilide derivatives
possessing thiol function at an appropriate position. Among
the potential structural units, we selected 4-(2-sulfanylethy-
lamino)benzene unit 6 as a core structure following prelimi-
nary examination.
Synthesis of requisite substrates 14-16 for evaluating the
NfS acyl-transfer-mediated preparation of thioesters is
shown in Scheme 1. Starting from 4-aminobenzoic acid 7,
the carboxy and amine units were protected by allyl and
o-nitrobenzenesulfonyl (Ns) groups,10 respectively. On the
(10) Fukuyama, T.; Jow, C. K.; Cheung, M. Tetrahedron Lett. 1995,
36, 6373–6374.
(11) Yeo, W-.O.; Min, D-.H.; Hsieh, R. W.; Greene, G. L.; Mrksich,
M. Angew. Chem., Int. Ed. 2005, 44, 5480–5483.
(8) For synthesis of peptide thioesters using NfS acyl transfer except
for ref 6, see: (a) Ollivier, N.; Behr, J.-B.; El-Mahdi, O.; Blanpain, A.;
Melnyk, O. Org. Lett. 2005, 7, 2647–2650. (b) Ohta, Y.; Itoh, S.; Shigenaga,
A.; Shintaku, S.; Fujii, N.; Otaka, A. Org. Lett. 2006, 8, 467–470. (c)
Nagaike, F.; Onuma, Y.; Kanazawa, C.; Hojo, H.; Ueki, A.; Nakahara, Y.;
Nakahara, Y. Org. Lett. 2006, 8, 4465–4468. (d) Hojo, H.; Onuma, Y.;
Akimoto, Y.; Nakahara, Y.; Nakahara, Y. Tetrahedron Lett. 2007, 48, 25–
28. (e) Kawakami, T.; Aimoto, S. Tetrahedron Lett. 2007, 48, 1903–1905.
(9) Romanelli, A.; Shekhtman, A.; Cowburn, D.; Muir, T. W. Proc. Natl.
Acad. Sci. U.S.A. 2004, 101, 6397–6402.
(12) Mistunobu, O. Synthesis 1984, 1–28.
(13) After coupling of Boc-Leu-OH to L- or D-Ala derivative, no
epimerization was detected, which indicates that coupling of Fmoc-Ala-Cl
with the sodium amide resulting from 12 and NaH does not induce an
observable level of racemization. This coupling protocol can be used for
other amino acids (Phe, Leu, Val, Ile), although application of the resulting
coupling products to the NfS acyl transfer is underway.
(14) Dourtoglou, V.; Gross, B. Synthesis 1984, 572–574.
(15) Payne, R. J.; Ficht, S.; Tang, S.; Brik, A.; Yang, Y. Y.; Case, D,
A.; Wong, C. H. J. Am. Chem. Soc. 2007, 129, 13527–13536.
824
Org. Lett., Vol. 11, No. 4, 2009

NfS acyl transfer, compound 16 was obtained from 13′ by
removal of Trt with TFA/Et3SiH at room temperature for
30 min.16
then conducted using standard Fmoc SPPS. Deprotection of
the completed resin 17 with TFA-Et3SiH-H2O (90:5:5, v/v)
at room temperature for 90 min gave crude anilide peptides
1816 which were then subjected to the treatment with 4 M
HCl/DMF20 in the presence of 1% (w/v) TCEP for 8 h at
37 °C to achieve the NfS acyl transfer. After HPLC
purification of the reaction mixture, peptide thioester 19 was
obtained in 33% isolated yield based on the protected resin.21
The resulting peptide 19 was proven to be thioester by the
conversion to another thioester 20 via sodium sulfanyle-
thanesulfonate treatment. Preliminary examination of the acyl
transfer revealed that reaction with 4 M HCl/DMF with a
powerful solubilizing performance allowed anilide peptide
18 to be efficiently converted to the corresponding thioester
19; however, the use of organic solvent on deprotected
peptides sometimes hampers the purification of peptides.
Therefore, we next examined an on-resin NfS acyl
transfer followed by the thiolytic release of peptide thioesters
using an aqueous solution (Scheme 3). Three model peptide
First, the stability of the anilide linkage and the risk of
epimerization of Ala in 14 under reaction conditions for
Fmoc-removal were confirmed. Compound 14 remained
unchanged after base treatment (20% piperidine/DMF) for
24 h at room temperature,17 which indicated that anilide 15
could be applicable to Fmoc SPPS. Next, trends of the NfS
acyl transfer were examined by exposure of 16 to acidic or
basic conditions followed by HPLC/MS analyses. Reaction
of 16 with 4 M HCl/dioxane in the presence of 1% (w/v)
tris(carboxyethyl)phosphine (TCEP) at room temperature for
2 h was completed to yield the corresponding S-acyl
compound. Furthermore, the observed trend in 4 M HCl/
dioxane was reconfirmed by 13C NMR measurements where
the 13C-signal at 167.8 ppm (amide carbonyl) was changed
to that at 197.3 and 199.6 ppm (thioester carbonyl).18 In
contrast to the cysteine-derived peptidyl oxazolidinone, no
NfS acyl transfer under basic conditions was observed.
Encouraged by these results, we next attempted to
synthesize pentapeptide thioester 19 (H-His-Arg-Phe-Ala-
Gly-SR) (Scheme 2). First, anilide 15 was introduced on Leu-
Scheme 3
Scheme 2a
a Amino acids are represented by boldface one-letter codes. HATU )
1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-ox-
ide hexafluorophosphate. Pbf ) 2,2,4,6,7-pentamethyldihydrobenzofuran-
5-sulfonyl.
incorporated NovaSyn TGR (Rink amide-type) resin with
the aid of HATU19/DIEA, and peptide-chain elongation was
resins 22 with an internal standard amino acid for amino
acid analysis (AAA) were prepared using aminomethyl
ChemMatrix resin.22 The internal standard (Ala for 22a and
(16) Little NfS acyl transfer occurred during short TFA treatment (at
room temperature for ca. 30-90 min) for removal of side-chain protecting
groups.
(17) Neither decomposition of the anilide linkage nor epimerization at
the Ala residue was observed by HPLC analyses of reactions of 14 with
20% piperidine/DMF; see the Supporting Information.
(18) Nakamura, K.; Sumida, M.; Kawakami, T.; Vorherr, T.; Aimoto,
S. Bull. Chem. Soc. Jpn. 2006, 79, 1773–1780.
(20) Compared to HCl/dioxane, HCl/DMF shows a powerful solubilizing
performance for deprotected peptides.
(21) Generally, a reversed reaction is encountered in the NfS acyl
transfer; however, little regeneration of the amides from the resulting
thioesters is observed in our system, which is probably due to low
nucleophilicity of the aniline nitrogen.
(19) Albericio, F.; Bofill, J. M.; El-Faham, A.; Kates, S. A. J. Org. Chem.
1998, 63, 9678–9683.
Org. Lett., Vol. 11, No. 4, 2009
825

Leu for 22b and 22c) and Fmoc-glycyl anilide 15 were
subsequently coupled on the resin with diisopropylcarbodi-
imide (DIPCDI)/1-hydroxybenzotriazole (HOBt) and HATU/
DIEA, respectively. Standard Fmoc SPPS on the resulting
resins afforded model peptide resins 22. Deprotection of each
resin with TFA-thioanisole-m-cresol-H2O-1,2-ethanedithi-
ol (EDT)-Et3SiH (80:5:5:5:2.5:2.5, v/v) at room temperature
for 1.5 h gave the side-chain-deprotected peptide resin 23.
Each resulting peptide resin was treated with 4 M HCl/
DMF-1% TCEP for 12 h at room temperature for the on-
resin NfS acyl transfer and was subsequently subjected to
a thiolytic release protocol using 3% (w/v) sodium sulfa-
nylethanesulfonate in the presence of 2% (v/v) thiophenol
in 6 M guanidine·HCl-100 mM phosphate buffer at pH 7.3.
In all cases, the sequence of treatments mentioned above
afforded peptide thioesters 25 with high purity in ap-
proximately 70% release yields (25a, 71%; 25b, 76%; 25c,
67%). Release yields of the thioesters were calculated based
on the internal standard whereby resin samples after thiol
treatment were hydrolyzed and the resulting hydrolysates
were analyzed by AAA.
preparation of peptide thioesters 25 to the protected resin
yielded phosphoamino acid-containing peptide thioester 26
in 67% release yield.
As mentioned in the experiment of treatment of 14 with
20% piperidine/DMF, no epimerization was observed during
the Fmoc procedure; however, partial epimerization was
detected on treatment of H-Phe-Ala-Ala-Ar peptide with 4
M HCl/DMF. Although the reason for the epimerization has
yet to be disclosed, reaction with TFA suppressed the
epimerization, although the NfS acyl transfer proceeded
slowly.24,25
In conclusion, we present an NfS acyl-transfer-mediated
synthesis of peptide thioesters using an N-substituted aniline
linker. The use of the linker allowed peptide thioesters to
be efficiently prepared by Fmoc-SPPS. Various applications
of the linker to peptide/protein synthesis via thioesters are
currently underway.
Acknowledgment. This research was supported in part
by a Grant-in-Aid for Scientific Research (KAKENHI). A.S.
is grateful for research grants from Takeda Science Founda-
tion.
The use of Fmoc-glycyl aniline derivative 15 in Fmoc
chemistry allowed peptide thioesters to be synthesized
efficiently. Therefore, we next attempted to synthesize
phosphoamino acid-containing peptide thioesters. On the
internal standard incorporated resins 21 was constructed a
protected peptide chain corresponding to RRVpSVAADG
26 using monobenzyl-protected phosphoamino acids.23 The
application of procedures identical to those used for the
Supporting Information Available: Experimental pro-
cedures, NMR charts for key compounds, and critical HPLC
charts including the analyses of NfS acyl-transfer reactions.
This material is available free of charge via the Internet at
http://pubs.acs.org.
OL8028093
(22) Aminomethyl ChemMatrix, totally poly(ethylene glycol) (PEG)-
based resin, was purchased from Aldrich. For the use of this resin in peptide
synthesis, see: Zhang, X.; Lu, X-.W.; Liu, C-.F. Tetrahedron Lett. 2008,
49, 6122–6125.
(24) NfS acyl transfer with HCl/DMF proceeds at about two times the
reaction rate compared with that with TFA; see the Supporting Informa-
tion.
(25) Treatment of HPLC-purified thioester sample (Phe-Ala-L-Ala-
thioester) with 4 M HCl/DMF at 37 °C for 4 h afforded a mixture of
diastereomers (parent peptide (80%) and epimerized Phe-Ala-D-Ala-thioester
(20%)).
(23) Wakamiya, T.; Togashi, R.; Nishida, T.; Saruta, K.; Yasuoka, J.;
Kusumoto, S.; Aimoto, S.; Kumagaye, K. Y.; Nakajima, K.; Nagata, K.
Bioorg. Med. Chem. 1997, 5, 135–145.
826
Org. Lett., Vol. 11, No. 4, 2009