C O M M U N I C A T I O N S
amino groups) in the same solvent system, both the R-amino group
and lysine residue were acylated (see Supporting Information).
Using a stoichiometric amount of N-hydroxysuccinimide ester, only
the lysine residue was acylated. To the best of our knowledge, the
present method is the first example of N-terminal R-amino group
acylation of peptides without lysine modification in aqueous
NaHCO3 buffer. Furthermore, 2f, aliphatic 2g, and internal 2h
alkynes were found to couple at the N-terminal R-amino group of
6a to give 7ab-ad (eq 2).
In summary, a new method for oxidative amide synthesis and
peptide ligation using the “1 + Oxone/H2O2” protocol has been
developed.
Acknowledgment. This work were supported by The University
of Hong Kong (University Development Fund), the Hong Kong
Research Grants Council (HKU7009/06P), and the Area of Excel-
lence Scheme (AoE/P-10-01) established under the University
Grants Committee (HKSAR). We thank Dr. W.-H. Cheung for
providing some of the peptide samples, Dr. K.-H. Sze and Dr. S.-
C. Yan for 2D NMR studies of peptides, and Mr. M.-K. Tse for
CD studies of peptides.
Supporting Information Available: Full experimental procedures,
1
characterization data, mass spectra, CD and 2D NMR studies, and H
and 13C NMR spectra. This material is available free of charge via the
References
N-Terminal acylations of five other peptides [GEQRKDVYVQ-
LYL, HDMNKVLDL, TYGPVFMSL, STSSSCNLSK, and SSC-
SSCPLSK] at 100 µM scale with phenylacetylene (2a) have also
been achieved (see Supporting Information). With these five
peptides, inter- and intramolecular disulfide bond formations were
observed at cysteine residues, and oxidation of methionine residues
to sulfoxides was observed. Nevertheless, the disulfide-bonded
cysteines and oxidized methionines could be reduced back to free
cysteines (this work) and methionines by treatment with dithio-
threitol and N-methylmercaptoacetamide,1a respectively. The present
method could be scaled up; for example, 5.3 mg of 2a-modified
SSCSSCPLSK [purified by preparative reversed-phase HPLC in
65% isolated yield based on 81% conversion and confirmed by
MS/MS] was obtained through a one-pot reaction; see Supporting
Information.
Ketenes are widely regarded as intermediates in alkyne oxida-
tion to carboxylic acids/esters.10,16 In this work, deutero-1-phenyl-
acetylene was oxidized under (a) aqueous NaHCO3 and (b) aqueous
NH4HCO3 conditions to afford deutero-2-phenyl acetic acid and
deutero-2-phenyl acetamide in 85 and 83% isolated yields, respec-
tively (eq 3). No crossover of deuterium in the amide products was
observed by GC-MS when a 1:1 ratio of deutero-1-phenylacetylene
and 2b was oxidized under aqueous NH4HCO3 conditions (see
Supporting Information). ESI-MS analysis of 1 in CH3CN solution
in the presence of Oxone/NaHCO3 showed ion cluster peaks
centered at m/z ) 958.9, which matched the [Mn(2,6-Cl2TPP)(O)]+
formulation.17 These findings are consistent with the mechanism
of inhibition of cytochrome P450 activities by alkynes, which was
proposed to occur through alkyne oxidation to generate oxirene
and ketene intermediates.10d,18
(1) (a) Lundblad, R. L. Chemical Reagents for Protein Modification, 3rd ed.;
CRC Press: Boca Raton, FL, 2000. (b) Dent, A. H.; Aslam, M.
Bioconjugation: Protein Coupling Techniques for the Biomedical Sci-
ences; Macmillan Reference Press: London, 1997; pp 50-100.
(2) (a) Gevaert, K.; Goethals, M.; Martens, L.; van Damme, J.; Staes, A.;
Thomas, G. R.; Vandekerckhove, J. Nat. Biotechnol. 2003, 21, 566. (b)
McDonald, L.; Robertson, D. H. L.; Hurst, J. L.; Beynon, R. J. Nat.
Methods 2005, 2, 955.
(3) (a) Tam, J. P. J. Immunol. Methods 1996, 196, 17. (b) Selo, I.; Negroni,
L.; Creminon, C.; Grassi, J.; Wai, J. M. J. Immunol. Methods 1996, 199,
127. (c) Gilmore, J. M.; Scheck, R. A.; Esser-Kahn, A. P.; Joshi, N. S.;
Francis, M. B. Angew. Chem., Int. Ed. 2006, 45, 5307.
(4) (a) Houseman, B. T.; Huh, J. H.; Kron, S. J.; Mrksich, M. Nat. Biotechnol.
2002, 20, 270. (b) Panicker, R. C.; Huang, X.; Yao, S. Q. Comb. Chem.
High Throughput Screening 2004, 7, 547.
(5) (a) Prescher, J. A.; Bertozzi, C. R. Nat. Chem. Biol. 2005, 1, 13. (b) van
Swieten, P. F.; Leeuwenburgh, M. A.; Kessler, B. M.; Overkleeft, H. S.
Org. Biomol. Chem. 2005, 3, 20. (c) van Maarseveen, J. H.; Reek. J. N.
H.; Back, J. W. Angew. Chem., Int. Ed. 2006, 45, 1841. For recent
examples on selective modifications/ligations of proteins and peptides,
see: (d) Antos, J. M.; Francis, M. B. J. Am. Chem. Soc. 2004, 126, 10256.
(e) McFarland, J. M.; Francis, M. B. J. Am. Chem. Soc. 2005, 127, 13490.
(f) Dantas de Araujo, A.; Palomo, J. M.; Cramer, J.; Kohn, M.; Schroder,
H.; Wacker, R.; Niemeyer, C.; Alexandrov, K.; Waldmann, H. Angew.
Chem., Int. Ed. 2006, 45, 296. (g) Bang, D.; Pentelute, B. L.; Kent, S. B.
H. Angew. Chem., Int. Ed. 2006, 45, 3985.
(6) (a) Humphrey, J. M.; Chamberlin, A. R. Chem. ReV. 1997, 97, 2243. (b)
Montalbetti, C. A. G. N.; Falque, V. Tetrahedron 2005, 61, 10827. (c)
Bode, J. W.; Fox, R. M.; Baucom, K. D. Angew. Chem., Int. Ed. 2006,
45, 1248.
(7) (a) Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B. Angew.
Chem., Int. Ed. 2002, 41, 2596. (b) Tornoe, C. W.; Christensen, C.; Meldal,
M. J. Org. Chem. 2002, 67, 3057. (c) Wang, Q.; Chan, T. R.; Hilgraf, R.;
Fokin, V. V.; Sharpless, K. B.; Finn, M. G. J. Am. Chem. Soc. 2003, 125,
3192. (d) Agard, N. J.; Prescher, J. A.; Bertozzi, C. R. J. Am. Chem. Soc.
2004, 126, 15046. (e) Zhang, L.; Chen, X.; Xue, P.; Sun, H. H. Y.;
Williams, I. D.; Sharpless, K. B.; Fokin, V. V.; Jia, G. J. Am. Chem. Soc.
2005, 127, 15998.
(8) (a) Knapton, D. J.; Meyer, T. Y. Org. Lett. 2004, 6, 687. (b) Uenoyama,
Y.; Fukuyama, T.; Nobuta, O.; Matsubara, H.; Ryu, I. Angew. Chem.,
Int. Ed. 2005, 44, 1075.
(9) (a) Cho, S. H.; Yoo, E. J.; Bae, I.; Chang, S. J. Am. Chem. Soc. 2005,
127, 16046. (b) Cassidy, M. P.; Raushel, J.; Fokin, V. V. Angew. Chem.,
Int. Ed. 2006, 45, 3154.
(10) (a) Curci, R.; Fiorentino, M.; Fusco, C.; Mello, R. Tetrahedron Lett. 1992,
33, 7929. (b) Zhu, Z.; Espenson, J. H. J. Org. Chem. 1995, 60, 7728. (c)
Che, C.-M.; Yu, W.-Y.; Chan, P.-M.; Cheng, W.-C.; Peng, S.-M.; Lau,
K.-C.; Li, W.-K. J. Am. Chem. Soc. 2000, 122, 11380. (d) Zeller, K.-P.;
Kowallik, M.; Haiss, P. Org. Biomol. Chem. 2005, 3, 2310.
(11) (a) Chan, W.-K.; Liu, P.; Yu, W.-Y.; Wong, M.-K.; Che, C.-M. Org. Lett.
2004, 6, 1597. (b) Chan, W.-K.; Wong, M.-K.; Che, C.-M. J. Org. Chem.
2005, 70, 4226.
(12) For optimization of reaction conditions, see Supporting Information.
(13) Lane, B. S.; Vogt, M.; DeRose, V. J.; Burgess, K. J. Am. Chem. Soc.
2002, 124, 11946.
(14) Chan, W.-K.; Yu, W.-Y.; Wong, M.-K.; Che, C.-M. J. Org. Chem. 2003,
68, 6576.
(15) Yamazaki, S. Bull. Chem. Soc. Jpn. 1997, 70, 877.
We have independently generated PhCHdCdO from photo-
Wolff rearrangement of PhC(O)CHN2. The same N-terminal
selectivity was observed in the coupling reactions of PhCHdCdO
with 6a, GEQRKDVYVQLYL, and HDMNKVLDL, and the lysine
residues remained intact (see Supporting Information). CD studies
(using the CONTINLL program for deconvolution) estimated the
R-helical, â-sheet, and turn content of 6a in solution to be 21.7,
6.6, and 12.6%, respectively, with 59.1% of random coil. Two-
dimensional NOESY analysis revealed an inter-residue NOE signal
between the K6 NH proton and the Y1 phenyl ring proton of 6a,
thus suggesting that the N-terminal selectivity in peptide ligation
may be in part due to the solution conformation of 6a.
(16) Tidwell, T. T. Ketenes 2nd; Wiley: New York, 2006.
(17) It is generally accepted that high-valent metal oxo species are generated
in manganese porphyrin-catalyzed oxidation with Oxone as an oxidant.
See: (a) Robert, A.; Meunier, B. New J. Chem. 1988, 12, 885. (b) Jin,
N.; Bourassa, J. L.; Tizio, S. C.; Groves, J. T. Angew. Chem., Int. Ed.
2000, 39, 3849.
(18) Beebe, L. E.; Roberts, E. S.; Fornwald, L. W.; Hollenberg, P. F.; Alworth,
W. L. Biochem. Pharmacol. 1996, 52, 1507.
JA064479S
9
J. AM. CHEM. SOC. VOL. 128, NO. 46, 2006 14797