Cysteine-derived S-protected oxazolidinones: Potential chemical devices for the preparation of peptide thioesters

Article abstract of DOI:10.1021/ol052755m

An N-S acyl-transfer-mediated preparation of peptide thioesters using the S-protected oxazolidinone derived from cysteine has been developed and applied to the synthesis of a 32-mer biologically active peptide by native chemical ligation protocols.

Full text of DOI:10.1021/ol052755m

ORGANIC
LETTERS
2006
Vol. 8, No. 3
467-470
Cysteine-Derived S-Protected
Oxazolidinones: Potential Chemical
Devices for the Preparation of Peptide
Thioesters
Yusuke Ohta,† Saori Itoh,† Akira Shigenaga,‡ Saori Shintaku,‡
Nobutaka Fujii,† and Akira Otaka*,†,‡
Graduate School of Pharmaceutical Sciences, Kyoto UniVersity,
Sakyo-ku, Kyoto 606-8501, Japan, and Graduate School of Pharmaceutical Sciences,
The UniVersity of Tokushima, Shomachi, Tokushima 770-8505, Japan
aotaka@ph.tokushima-u.ac.jp
Received November 14, 2005
ABSTRACT
An N−S acyl-transfer-mediated preparation of peptide thioesters using the S-protected oxazolidinone derived from cysteine has been developed
and applied to the synthesis of a 32-mer biologically active peptide by native chemical ligation protocols.
Intein-mediated protein splicing is a self-catalytic editing
system of proteins in which an intervening sequence (termed
“intein”) is cleaved off from a protein precursor with
simultaneous ligation between the flanking fragments (N-
and C-exteins) to form a mature extein protein and the free
intein.1 These intein-mediated splicing systems have received
increasing attention in the field of protein chemistry including
purification,2 labeling,3 and semisynthesis of proteins.4 In this
splicing process, three consecutive acyl-transfer steps are
involved.5 The first N-S acyl transfer on the cysteine residue
at intein N-terminus has been extensively applied to the
biochemical preparation of protein (or peptide) thioesters.
These have shown great utility in the preparation of a wide
variety of proteins by native chemical ligation (NCL)
procedures.4,6 Therefore, we anticipated that mimicking the
intein-mediated N-S acyl-transfer step by chemical means
could provide an unprecedented alternative to reported
chemical methods for the synthesis of peptide thioesters.7 A
recent elegant NMR study8 on this acyl transfer revealed that
“ground-state destabilization” by inducing nonplanarity in
the scissile amide bond was responsible for activation of the
† Kyoto University.
‡ The University of Tokushima.
(1) For protein splicing bibliography, see: (a) Perler, F. B. Nucleic Acids
Res. 2002, 30, 383-384. (b) http://www.neb.com/neb/inteins.html.
(2) (a) Chong, S.; Mersha, F. B.; Comb, D. G.; Scott, M. E.; Landry,
D.; Vence, L. M.; Perler, F. B.; Benner, J.; Kucera, R. B.; Hirvonen, C. A.;
Pelletier, J. J.; Paulus, H.; Xu, M. Gene 1997, 192, 271-281. (b) Chong,
S.; Montello, G. E.; Zhang, A.; Cantor, E. J.; Liao, W.; Xu, M. Q.; Benner,
J. Nucleic Acids Res. 1998, 26, 5109-5115. (c) Evans, T. C., Jr.; Benner,
J.; Xu, M. Q. J. Biol. Chem. 1999, 274, 3923-3926.
(3) (a) Miller, L. W.; Cornish, V. W. Curr. Opin. Chem. Biol. 2005, 9,
56-61. (b) Zuger, S.; Iwai, H. Nat. Biotechnol. 2005, 23, 736-740. (c)
Girish, A.; Sun, H.; Yeo, D. S.; Chen, G. Y.; Chua, T. K.; Yao, S. Q.
Bioorg. Med. Chem. Lett. 2005, 15, 2447-2451.
(4) (a) Alexandrov, K.; Heinemann, I.; Durek, T.; Sidorovitch, V.; Goody,
R. S.; Waldmann, H.J. Am. Chem. Soc. 2002, 124, 5648-5649. (b) Muir,
T. W. Annu. ReV. Biochem. 2003, 72, 249-289. (c) Clayton, D.; Shapovalov,
G.; Maurer, J. A.; Dougherty, D. A.; Lester, H. A.; Kochendoerfer, G. G.
Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 4764-4769. (d) Machova Z.; Beck-
Sickinger, A. G. Methods Mol. Biol. 2005, 298, 105-135.
(5) (a) Poland, B. W.; Xu, M. Q.; Quiocho, F. A. J. Biol. Chem. 2000,
275, 16408-16413. (b) Evans, T. C.; Xu, M. Q. Chem. ReV. 2002, 102,
4869-4884.
(6) (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.
10.1021/ol052755m CCC: $33.50
© 2006 American Chemical Society
Published on Web 01/10/2006

amide leading to the N-S acyl transfer.9 On the basis of
this finding, we attempted to use an N-acyloxazolidinone
derivative to prepare thioesters by a “ground-state destabi-
lization” mechanism. In an acyloxazolidinone, the carbonyl
in the oxazolidinone ring is likely to influence the nN-π*CO
delocalization and to result in the activation (ground-state
destabilization) of the exo-amide linkage. Actually, N-
acyloxazolidinones have been documented to show active
ester character which is regulated by distortion of the amide
planarity.10 Because neighboring thiol group participation in
the cleavage of the acyloxazolidinone bond could facilitate
the formation of thioesters,11 an S-protected oxazolidinone
derived from cysteine was selected as a chemical device for
the preparation of peptide thioesters by an intein mimicking
mechanism. In this study, we examined the applicability of
cysteine-derived acyloxazolidinones to the N-S acyl shift-
mediated synthesis of peptide thioesters with application to
peptide synthesis using NCL.12 Very recently, two groups
have reported N-S acyl-transfer-mediated preparation of
peptide thioesters using chemical devices other than the
oxazolidinones.13,14
the S-protection on the oxazolidinone. Concomitant release
of the peptidyloxazolidinone from the resin yields the
S-protected peptide oxazolidinone as a precursor to the
thioester. Removal of the S-protection and subsequent N-S
acyl transfer affords the peptide thioester.
The p-methoxybenzyl (MBzl) group was selected as an
oxazolidinone S-protecting group because it remains intact
during standard Fmoc-based SPPS protocols (20% piperidine
treatment for Fmoc-removal and trifluoroacetic acid (TFA)
treatment for the final deprotection). Preparation of a
protected oxazolidinone derivative suitable for attaching to
an amino-functionalized resin and its use in the synthesis of
the peptide thioester precursor are shown in Scheme 1.
Scheme 1
The use of S-protected oxazolidinones in the synthesis of
peptide thioesters is conceptually outlined in Figure 1. A
Figure 1. Concept of N-S acyl transfer-mediated synthesis of
peptide thioesters using an S-protected oxazolidinone.
protected peptide chain is assembled on the S-protected
oxazolidinone-type linker by solid-phase peptide synthesis
(SPPS) followed by removal of protecting groups except for
Starting from the protected cysteine derivative 1, syn-1,2-
amino alcohol derivative 3 was obtained exclusively by a
sequence of reactions consisting of Weinreb-amidation using
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC),15 di-
isobutylaluminum hydride (DIBAL) reduction, and Refor-
(7) For synthesis of peptide thioesters using Boc strategy, see: (a)
Aimoto, S. Biopolymer 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.
For synthesis of peptide thioesters using the Fmoc strategy, see: (c) Ingenito,
R.; Bianchi, E.; Fattori, D.; Pessi, A. J. Am. Chem. Soc. 1999, 121, 11369-
11374. (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)
Sewing, A.; Hilvert, D. Angew. Chem., Int. Ed. 2001, 40, 3395-3396. (f)
Brask, J.; Albericio, F.; Jensen, K. J. Org. Lett. 2003, 5, 2951-2953.
(8) Romanelli, A.; Shekhtman, A.; Cowburn, D.; Muir, T. W. Proc. Natl.
Acad. Sci. U.S.A. 2004, 101, 6397-6402.
(9) Mujika, J. I.; Mercero, J. M.; Lopez, X. J. Am. Chem. Soc. 2005,
127, 4445-4453.
(10) Evans, D. A.; Anderson, J. C.; Taylor, M. K. Tetrahedron Lett. 1993,
34, 5563-5566.
468
Org. Lett., Vol. 8, No. 3, 2006

matsky reaction of allyl bromoacetate. Deprotection of
the Boc group of 3 with 4 N HCl-dioxane followed
by carbonyldiimidazole (CDI)/4-(dimethylamino)pyridine
(DMAP)-mediated cyclization of the resulting deprotected
compound afforded the S-MBzl oxazolidinone 4. Acylation
of lithiated 4 with Fmoc-Gly-OH in the presence of pivaloyl
chloride (Piv-Cl) and Et3N gave the Fmoc-glycyloxazolidi-
none 5a. For attachment of the acyloxazolidinone to the resin,
allyl ester on 5a was quantitatively removed by treatment
with Pd(PPh3)4 in CHCl3/AcOH/N-methylmorpholine (NMM)
(37:2:1, v/v).16
Fmoc-glycyloxazolidinone such as 6a can be applicable to
the N-S acyl-transfer-mediated methodology, extensive
efforts are in progress in our laboratory to develop depro-
tection conditions suitable for chiral C-terminal aminoacy-
lated oxazolidinones.
Next, to evaluate the practical usefulness of 6a in
application to the NCL-mediated preparation of peptides, we
undertook the synthesis of hBNP32-NH2 14. The peptide
chain assembly for the peptide thioester precursor fragment
9 (corresponding to hBNP32-NH2 (1-9)) was conducted
using the Fmoc-Rink linker-Leu resin, on which Fmoc-
glycyloxazolidinone 6a and Fmoc-amino acids were suc-
cessively coupled with the aid of diisopropylcarbodiimide
(DIPCDI)-HOBt (or 1-hydroxy-7-azabenzotriazole (HOAt)).
Treatment with Aimoto’s reagent cocktail (20 min reaction/
each step) was utilized for the removal of Fmoc groups.
Amino acid analysis of the hydrolysate resulting from the
completed resin 8 revealed that the peptide chain assembly
proceeded efficiently without significant decomposition of
the acyloxazolidinone linkage.22 Treatment of the completed
resin 8 with TFA-thioanisole-m-cresol-Et3SiH-H2O (80:
5:5:5:5, v/v) at room temperature for 2 h, followed by HPLC
purification, gave the S-protected peptidyloxazolidinone 9
as a thioester precursor in 22% yield.
The resulting carboxylic acid 6a can be coupled to an
amino-functionalized resin using standard peptide coupling
conditions. Before elongation of the peptide chain, the
stability of the acyloxazolidinone linkage toward basic
treatment needed for Fmoc-removal was examined. For this
purpose, Rink amide linker-functionalized resin possessing
an internal standard amino acid (Leu) for amino acid analysis
following acid hydrolysis was prepared by successive
coupling of Fmoc-Leu-OH and Fmoc-Rink linker17 on the
amino methyl resin. On this resin was coupled the Boc-
glycyloxazolidinone 6b, which was prepared by reactions
identical to those used for 6a. Treatment of the resulting resin
7b with basic reagent systems followed by amino acid
analysis after acid hydrolysis indicated that treatment with
20% piperidine in N,N-dimethylformamide (DMF) (standard
Fmoc deprotection) partially induced the decomposition of
the amide linkage (ca. 20% of the linkage was broken after
5 h treatment). On the other hand, the use of Aimoto’s
reagent mixture18 consisting of 1-methylpyrrolidine-
hexamethyleneimine-1-hydroxybenzotriazole (HOBt) (25%
(v/v)-2% (v/v)-3% (w/v) in 1-methylpyrrolidin-2-one
(NMP)-dimethyl sulfoxide (DMSO) (1:1)) proved to be
compatible with the resin in the presence of the acyloxazo-
lidinone linkage.19 This basic reagent system has been
successfully applied to the Fmoc-based synthesis of peptide
thioesters without affecting thioester linkages. However, one
potential limitation with the use of this procedure in the
synthesis of thioesters is racemization of chiral thioester-
linked C-terminal amino acids.20 This is the case with
aminoacylated oxazolidinones.21 At this stage, since only
The resulting peptidyloxazolidinone was then subjected
to the NCL-mediated synthesis of hBNP32-NH2 (Scheme
2). HPLC-purified S-protected derivative 9 was treated with
Scheme 2
(11) For nucleophilic involvement of hydroxy group leading to N-O
acyl transfer, see: Bew, S. P.; Bull, S. D.; Davies, S. G.; Savory, E. D.;
Watkin, D. J. Tetrahedron 2002, 58, 9387-9401 and references cited herein.
(12) A 32-mer disulfide-containing peptide, human brain natriuretic
peptide derivative (amide form: hBNP32-NH2 14), was selected as model
synthetic peptide. For hBNP32, see: Kambayashi, Y.; Nakao, K.; Mukoya-
ma, M.; Saito, Y.; Ogawa, Y.; Shiono, S.; Inoue, K.; Yoshida, M.; Imura,
H. FEBS Lett. 1990, 259, 341-345.
(13) Olliver, N.; Behr, H.-B.; Ei-Mahdi, O.; Blanpain, A.; Melnyk, O.
Org. Lett. 2005, 7, 2647-2650.
0.5 M trimethylsilyl bromide (TMSBr)-thioanisole (1:1) in
TFA and m-cresol23 at -10 °C for 1 h to afford the
S-deprotected peptidyloxazolidinone 10, which was then
purified by HPLC. NCL of the purified 10 with the
(14) Kawakami, T.; Sumida, M.; Nakamura, K.; Vorherr, T.; Aimoto,
S. Tetrahedron Lett. 2005, 46, 8805-8807.
(15) Wang, G.; Mahesh, U.; Chen, G. Y. J.; Yao, S. Q. Org. Lett. 2003,
5, 737-740 and references cited herein.
(16) Kates, S. A.; Daniels, S. B.; Sole, N. A.; Barany, G.; Albericio, F.
In Peptides: Chemistry, Structure and Biology; Hodges, R. S., Smith, J.
A., Eds.; ESCOM: Leiden, 1994; pp 113-115.
(20) Hasegawa, K.; Sha, Y. L.; Bang, J. K.; Kawakami, T.; Akaji, K.;
Aimoto, S. Lett. Peptide Sci. 2002, 8, 277-284.
(21) Oxazolidinone derivative 4 was derivatized with Fmoc-L-Ala-OH.
Then, treatment of the resulting Fmoc-L-Ala-linked derivative with the
Aimoto’s reagent cocktail afforded the mixture of H-L-Ala- or H-D-Ala-
linked oxazolidinone derivative. Results of quantitative HPLC analysis of
the reaction mixture are shown as a graph in the Supporting Information.
(17) Bernatowicz, M. S.; Daniels, S. D.; Kster, H. Tetrahedron Lett. 1989,
30, 4645-4648.
(18) Li, X. Q.; Kawakami, T.; Aimoto, S. Tetrahedron Lett. 1998, 39,
8669-8672.
(19) Treatment of 7b with Aimoto’s reagent cocktail for 5 h induced ca.
3% cleavage of the linkage. See the Supporting Information.
Org. Lett., Vol. 8, No. 3, 2006
469

N-terminal cysteine peptide (hBNP32-NH2 (10-32)) 12,
which was prepared by standard Fmoc-based peptide syn-
thesis, proceeded efficiently in phosphate buffer (pH 7.6)
containing 6 M guanidine‚HCl in the presence of 1% (v/v)
thiophenol to yield the ligated 2Cys-SH hBNP32-NH2 13.
At this step in the NCL protocol, the N-acylated compound
10 (retention time on HPLC analysis ) 10.8 min) disap-
peared completely within 1 min and a new compound
(putatively 11) with the same m/z value as that of 10 eluted
at 7.8 min (see the Supporting Information).24 After dilution
with phosphate buffer to three times volume, DMSO (10%,
v/v) was added to effect disulfide bond formation.25 HPLC
purification of the crude product gave purified hBNP32-NH2
14 in 78% yield calculated from the NCL step.
for N-S acyl-transfer-mediated synthesis of thioesters using
acylated oxazolidinones derived from S-protected cysteine.
Removal of peptidyloxazolidinone S-protection provides the
corresponding peptide thioesters through N-S acyl transfer
involving the thiol group in the adjacent activated acyl-
oxazolidinone linkage. This synthetic protocol was success-
fully applied to the NCL-mediated synthesis of hBNP32-
NH2. The use of cysteine-derived oxazolidinones as chiral
auxiliaries with application to the preparation of thioesters
could provide a new access to chiral thioester derivatives,
which could have potential utility in the synthesis of a wide
variety of compounds.
Acknowledgment. We thank Dr. Terrence R. Burke, Jr.,
NCI, NIH, for proofreading this manuscript. This research
was supported in part by the 21st Century COE Program
“Knowledge Information Infrastructure for Genome Sci-
ence”, a Grand-in-Aid for Scientific Research (KAKENHI).
A.O. is grateful for research grants from Sekisui Integrated
Research and Suzuken Memorial Foundation.
Based on the reaction mechanism of intein-mediated
formation of peptide thioesters, we have developed a method
(22) The decomposition of the acyloxazolidinone linkage is attributed
to both base-induced cleavage of the activated bond (ca. 3% decomposition
after 5 h treatment)19 and diketopiperazine (DKP) formation. These two
factors were probably responsible for the partial loss of peptide chain (Glu
(in the peptide)/Leu (in the solid support) ) 0.9:1.0). We have yet to prove
to attribute the loss to the base-induced cleavage, DKP formation, or
precision in amino acid analysis.
(23) Yajima, H.; Fujii, N.; Funakoshi, S.; Watanabe, T.; Murayama, E.;
Otaka, A. Tetrahedron 1988, 44, 805-819.
Supporting Information Available: Experimental pro-
cedures, NMR charts for key compounds, and HPLC charts
of the analyses of N-S acyl-transfer-mediated synthesis of
hBNP32-NH2. This material is available free of charge via
the Internet at http://pubs.acs.org.
(24) Removal of the thiol protection is needed for the coupling with
N-terminal cysteine peptide. Subjection of S-protected peptidyloxazolidinone
such as 9 to the NCL condition does not afford the ligated product.
(25) (a) Otaka, A.; Koide, T.; Shide, A.; Fujii, N.; Tetrahedron Lett,
1990, 32, 1223-1226; Tam, J. P.; Wu, C.; Liu, W.; Zhang, J. J. Am. Chem.
Soc. 1991, 113, 6657-6662. (c) Ueda, S.; Fujita, M.; Tamamura H.; Fujii,
N.; Otaka, A. ChemBioChem 2005, 6, 1983-1986.
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