oxyethylene units in acetylenic cross-linking agents and the Lys
positions in short peptides. The alkyl type 3 could bridge both the
peptides well (488%) within 60 min regardless of the alkyl spacer
lengths and the positions of the two Lys residues. On the other hand,
the oxyethylene type 4 showed much lower reactivities. The shorter
4a reacted with B (i2i+7) in only poor yield (38%), while both the
peptides remained unchanged over 60 min in the case of 4b from
the HPLC profiles.†
negligible extent as the temperature was raised. In the case of the
peptide B, the order of the a-helical contents changed to that of
2 > 1a > 1b over the entire temperature range. The peptide cross-
linked by 2 kept ca. ~ 30% of a-helical structures at 35 °C.
Although the peptides cross-linked by 3 and 4 almost became the
unfolded states at 60 °C, the one cross-linked by the diacetylenic 2
still included ca. 15% of the folded structure even at 70 °C. Thus,
the acetylenic cross-linking agents are superior to 3 and 4 for
stabilising the a-helix structures of both the peptides within a
temperature range covering most natural events and artificial
experiments.
We have demonstrated a new class of cross-linking agents
composed of actylenic cores for short peptides. The cross-linking
agents can be prepared by standard organic reactions in good yields.
The peptides cross-linked by the acetylenic agents showed higher
a-helical contents and thermal-stabilities than conventionally
cross-linked and native ones. In future investigations, these new
cross-linking agents might be applied to many biological recogni-
tion events in which a-helical peptides participate.
Fig. 3 shows CD spectra of the cross-linked peptides thus formed
at 5 °C. The a-helical contents of the peptides were evaluated on the
basis of the mean residue ellipticity at 222 nm.5,14 The cross-linked
peptides by the acetylenic 1 and 2 revealed higher a-helical
contents for both the peptides than those by 3 and 4. For peptide A
(i2i+4), the monoacetylenic 1 stabilised the a-helical structure
most effectively (435%), whereas ~ 15% of the native A folded up
under the same conditions. The distance of the rigid spacer of the
diacetylenic 2 might be longer than that of the short Lys–Lys
interval in A, which could cause the peptide cross-linked by 2 to be
relatively unstable. On the other hand, the peptide B revealed a
different but clear tendency. Especially, in the combination of the
diacetylenic 2 and the peptide B (i2i+7), characteristic Cotton
effects were remarkably strong at 191–193, 208, and 222 nm, and
a higher a-helical content ( ~ 65%) was observed than any other
combination. Since this value was ca. 1.3 times that by the
monoacethylenic 1, the number of acetylene units is considered to
contribute to the stabilisation of the a-helices. Although 3 and 4
stabilised the a-helical structures of both the peptides to a certain
extent, the acetylenic 1 and 2 were found to be more effective
stabilisers for these short peptides.
Noteworthy is that the a-helices thus formed survived up to
substantially elevated temperature. Fig. 4 depicts the thermal-
profiles of a-helical contents of the cross-linked peptides. As the
temperature of a solution containing the native A was slowly raised,
the secondary structure entirely turned into the unfolded (random-
coiled) state at 35 °C, near human body temperature, while the
corresponding ones cross-linked by 1 and 2 maintained ca. ~ 15%
of the folded structures at the same temperature. At 5 °C, the
stabilising abilities of the cross-linking agents decreased in the
following order: 1b > 1a > 2, but the difference became small to a
We are grateful to Professor Hiroaki Shinohara and Dr. Makoto
Genmei (Toyama University) for measurements of IR spectra. This
work was partly supported by the Sasakawa Scientific Research
Grant from The Japan Science Society.
Notes and references
1 Peptides: Chemistry and Biology, ed. N. Sewald and H.-D. Jakubke,
Wiley-VCH, Weinheim, 2002, ch. 3 and 9.
2 (a) S. Marqusee, V. M. Robbins and R. L. Baldwin, Proc. Natl. Acad.
Sci. USA, 1989, 86, 5286; (b) A. V. Finkelstein, A. Y. Badretdinov and
O. B. Btitsyn, Proteins: Struct., Funct., Genet., 1991, 10, 287.
3 J. W. Neidigh, R. M. Fesinmeyer and N. H. Andersen, Nat. Struct. Biol.,
2002, 9, 425.
4 (a) M. J. I. Andrews and A. B. Tabor, Tetrahedron, 1999, 55, 11711; (b)
J. Venkatraman, S. C. Shankaramma and P. Balaram, Chem. Rev., 2001,
101, 3131.
5 For the conversion of the ellipticity to the % helix, see: C. E.
Schafmeister, J. Po and G. L. Verdine, J. Am. Chem. Soc., 2000, 122,
5891.
6 (a) D. Y. Jackson, D. S. King, J. Chmielewski, S. Singh and P. G.
Schultz, J. Am. Chem. Soc., 1991, 113, 9391; (b) N. Voyer and B.
Guerin, Tetrahedron, 1994, 50, 989; (c) J. S. Albert and A. D. Hamilton,
Biochemistry, 1995, 34, 984; (d) J. C. Phelan, N. J. Skelton, A. C.
Braisted and R. S. McDowell, J. Am. Chem. Soc., 1997, 119, 455; (e) V.
Semetey, D. Rognan, C. Hemmerlin, R. Graff, J.-P. Briand, M. Marraud
and G. Guichard, Angew. Chem., Int. Ed., 2002, 41, 1893.
7 (a) A. Ravi, B. V. V. Prasad and P. Balaram, J. Am. Chem. Soc., 1983,
105, 105; (b) M. Pellegrini, M. Royo, M. Chorev and D. F. Mierke, J.
Pept. Res., 1997, 49, 404; (c) I. Sayers, S. A. Cain, J. R. M. Swan and
B. A. Helm, Biochemistry, 1998, 37, 16152.
8 (a) M. R. Ghadiri and A. K. Fernholtz, J. Am. Chem. Soc., 1990, 112,
9633; (b) R. J. Todd, M. E. Van Dam, D. Casimiro, B. L. Haymore and
F. H. Arnold, Proteins: Struct., Funct., Genet., 1991, 10, 156.
9 (a) For the use of salt bridges, see: A. Bierzynsky, P. S. Kim and R. L.
Baldwin, Proc. Natl. Acad. Sci. USA, 1982, 79, 2470; (b) J. M. Scholtz,
H. Qian, V. H. Robbins and R. L. Baldwin, Biochemistry, 1993, 32,
9668.
Fig. 3 CD spectra of the native and the cross-linked peptides dissolved in 2.5
3 1023 M phosphate buffer (pH 7.0) at 5 °C, (a) for peptide A and (b) for
peptide B: native (black), 1a (blue), 1b (blue dashed), 2 (red), 3a (red
dashed), 3b (green), 3c (green dashed), 4a (yellow).
10 (a) For the use of cyclic lactams, see: G. Ösapay and J. W. Taylor, J. Am.
Chem. Soc., 1992, 114, 6966; (b) C. Bracken, J. Gulyás, J. W. Taylor
and J. Baum, J. Am. Chem. Soc., 1994, 116, 6431.
11 (a) M. Bouvier and J. W. Taylor, J. Med. Chem., 1992, 35, 1145; (b) M.
Chorev, R. F. Epand, M. Rosenblatt, M. P. Caulfield and R. M. Epand,
Int. J. Pept. Protein Res., 1993, 42, 342; (c) J. R. Kumita, O. S. Smart
and G. A. Woolley, Proc. Natl. Acad. Sci. USA, 2000, 97, 3803.
12 (a) L. Montesano, D. Cawley and H. R. Hershman, Biochem. Biophys.
Res. Commun., 1982, 109, 7; (b) M. Kondo, Y. Shimizu and A. Murata,
Agric. Biol. Chem., 1982, 46, 913; (c) K. Tanizawa, T. Mano and Y.
Kanaoka, Chem. Pharm. Bull., 1990, 38, 464.
Fig. 4 Thermal-profiles of a-helical contents of the native and the cross-
linked peptides at a temperature range from 5 to 70 °C, (a) for peptide A and
(b) for peptide B: native (black closed circle), 1a (blue closed circle), 1b
(blue open circle), 2 (red closed circle), 3a (red open circle), 3b (green
closed circle), 3c (green open circle), 4a (yellow closed circle).
13 V. Wittmann, S. Takayama, K. W. Gong, G. Weitz-Schmidt and C.-H.
Wong, J. Org. Chem., 1998, 63, 5137.
14 N. Sreerama and R. W. Woody, Circular Dichroism: Principles and
Applications, 2nd edn., ed. N. Berova, K. Nakananishi, and R. W.
Woody, Wiley-VCH, New York, 2000, ch. 21.
C h e m . C o m m u n . , 2 0 0 4 , 1 2 8 0 – 1 2 8 1
1281