Diversity in short β-peptide 12-helices: High-resolution structural analysis in aqueous solution of a hexamer containing sulfonylated pyrrolidine residues [2]

Full text of DOI:10.1021/ja010734r

J. Am. Chem. Soc. 2001, 123, 7721-7722
7721
Diversity in Short â-Peptide 12-Helices:
High-Resolution Structural Analysis in Aqueous
Solution of a Hexamer Containing Sulfonylated
Pyrrolidine Residues
Hee-Seung Lee, Faisal A. Syud, Xifang Wang, and
Samuel H. Gellman*
Department of Chemistry, UniVersity of Wisconsin
Fmoc-protected S-APC residues were prepared via straight-
forward methods6 and used for solid-phase synthesis of hexamers
1-3. The two S-APC residues in 1-3 illustrate that alkyl and
aromatic side chains can be introduced in this way; a variety of
polar S-APC residues should also be readily available (for
example, we have prepared a taurine-derived S-APC residue, with
which it should be possible to replace the cationic APC residues
of 1-3). Hexamer 1 was analyzed by 2D NMR in 9:1 H2O:D2O
to determine whether a backbone comprised solely of APC and
S-APC residues adopts the 12-helical conformation. 1H chemical
shifts of 1 did not change significantly upon dilution from 2.5
mM to 0.3 mM, which suggests that the hexamer does not self-
Madison, Wisconsin 53706
ReceiVed March 20, 2001
Oligomers that adopt well-defined conformations in solution
(“foldamers”) have been a subject of increasing interest.1 Results
from many laboratories demonstrate that shape control can be
achieved with a wide variety of backbones, and recent efforts
have shown that foldamers can be endowed with useful activities.2
Many applications require placement of specific functional groups
at defined positions along the foldamer backbone, so that folding
brings these groups into the arrangement necessary for activity.
Described here is a strategy for functionalizing the 12-helix, a
secondary structure defined by 12-membered-ring hydrogen bonds
[CdO(i) f N-H(i+3)] that is formed by â-amino acid oligomers
in which the residues are constrained by five-membered rings.3
Two appropriately constrained residues have been identified,
trans-2-aminocyclopentanecarboxylic acid (ACPC)3a and trans-
3-aminopyrrolidine-4-carboxylic acid (APC),3d each of which can
be prepared efficiently in large quantities in either enantiomeric
form.4 We have now explored sulfonylated APC (S-APC) residues
for functionalization of water-soluble 12-helices. Attachment of
side chains via N-sulfonylation should be advantageous relative
to N-alkylation, which would introduce cationic charge, or N-
acylation, which would interfere with structural characterization
because of cis-trans rotamer equilibria of the resulting tertiary
amide groups. However, sulfonylation introduces a non-sp3 atom5
into the five-membered ring of the â-amino acid residue and might
therefore adversely affect 12-helix stability by altering the
CR-Câ torsional preference of S-APC residues relative to APC
and ACPC residues.
1
associate in this concentration range. The H NMR resonances
of 1 in water are more dispersed than those of previously
examined3d APC/ACPC hexamer 4, presumably because of the
greater residue diversity of 1 relative to 4 and ring current effects
from the aromatic side chain. Seven NOEs (present in both
NOESY7a and ROESY7b spectra) between protons on nonadjacent
residues were observed along the backbone of 1 (Figure 1a). All
three types of nonadjacent NOEs, CâHi f NHi+2, CâHi f CRHi+2
,
and CâHi f NHi+3, are characteristic of the 12-helical folding
pattern.3a,d The lack of NOEs in 1 between residues 1 and 3
(numbered from N-terminus) suggests that the N-terminus of the
12-helix is frayed; terminal fraying is observed among R-helices
formed by conventional peptides in aqueous solution.8
* To whom correspondence should be addressed (e-mail: gellman@
chem.wisc.edu).
(1) Reviews: (a) Seebach, D.; Matthews, J. L. J. Chem. Soc., Chem.
Commun. 1997, 2015-2022. (b) Gellman, S. H. Acc. Chem. Res. 1998, 31,
173. (c) DeGrado, W. F.; Schneider, J. P.; Hamuro, Y. J. Pept. Res. 1999,
54, 206. (d) Kirshenbaum, K.; Zuckermann, R. N.; Dill, K. A. Curr. Opin.
Struct. Biol. 1999, 9, 530. (e) Stigers, K. D.; Soth, M. J.; Nowick, J. S. Curr.
Opin. Chem. Biol. 1999, 3, 714. (f) Barron, A. E.; Zuckermann, R. N. Curr.
Opin. Chem. Biol. 1999, 3, 681. (g) Gademann, K.; Hintermann, T.; Schreiber,
J. V. Curr. Med. Chem. 1999, 6, 905.
Comparison of NOE data for hexamers 1 and 4 suggests that
incorporating S-APC residues (in 1) in place of ACPC residues
(in 4) leads to subtle differences in 12-helix geometry. Previous
data3d showed that 4 in water displayed a set of four weak CâHi
f CRHi+1 NOEs (only CâH5 f CRH6 was missing). In contrast,
no CâHi f CRHi+1 NOEs were observed for 1, which suggests
that the 12-helix formed by 1 is slightly more tightly wound than
the 12-helix formed by 4 (Figure 1b). The subtle differences
between the 12-helical conformations of 1 and 4 are comparable
to differences among R-helices formed by conventional peptides.9
NOE data obtained for 1 in 9:1 H2O:D2O were used for NOE-
restrained dynamics simulations with the program DYANA.10 This
approach was used to generate 400 structures, the best 10 of which
(all 12-helical) were used as starting points for NOE-restrained
(2) (a) Biologically active â-peptides: Werder, M.; Hausre, H.; Abele, S.;
Seebach, D. HelV. Chim. Acta 1999, 82, 1774. Hamuro, Y.; Schneider, J. P.;
DeGrado, W. F. J. Am. Chem. Soc. 1999, 121, 12200. Porter, E. A.; Wang,
X.; Lee, H.-S.; Weisblum, B.; Gellman, S. H. Nature 2000, 404, 565. (b) A
carbohydrate oligomer that binds to DNA: Xuereb, H.; Maletic, M.;
Gildersleeve, J.; Pelczer, I.; Kahne, D. J. Am. Chem. Soc. 2000, 122, 1883.
(c) m-Phenylene ethynylene oligomers that bind specific guests: Prince, R.
B.; Barnes, S. A.; Moore, J. S. J. Am. Chem. Soc. 2000, 122, 2758. Tanatani,
A.; Mio, M. J.; Moore, J. S. J. Am. Chem. Soc. 2001, 123, 1792.
(3) (a) Appella, D. H.; Christianson, L. A.; Klein, D. A.; Powell, D. R.;
Huang, X.; Barchi, J. J.; Gellman, S. H. Nature 1997, 387, 381. (b) Appella,
D. H.; Christianson, L. A.; Klein, D. A.; Richards, M. R.; Powell, D. R.;
Gellman, S. H. J. Am. Chem. Soc. 1999, 121, 7574. (c) Barchi, J. J.; Huang,
X.; Appella, D. H.; Christianson, L. A.; Durell, S. R.; Gellman, S. H. J. Am.
Chem. Soc. 2000, 122, 2711. (d) Wang, X.; Espinosa, J. F.; Gellman, S. H. J.
Am. Chem. Soc. 2000, 122, 4821. (e) Christianson, L. A.; Lucero, M. J.;
Appella, D. H.; Klein, D. A.; Gellman, S. H. J. Comput. Chem. 2000, 21,
763. (f) Alternative residues for the 12-helix: Winkler, J. D.; Piatnitski, E.
L.; Mehlmann, J.; Kasparec, J.; Axelsen, P. H. Angew. Chem., Int. Ed. 2001,
40, 743.
(6) Please see the Supporting Information.
(7) (a) Macura, S.; Ernst, R. R. Mol. Phys. 1980, 41, 95. (b) Bothner-By,
A. A.; Stephens, R. L.; Lee, J.; Warren, C. D.; Jeanloz, R. W. J. Am. Chem.
Soc. 1984, 106, 811.
(8) Rohl, C. A.; Baldwin, R. L. Biochemistry 1994, 31, 7760. Scholtz, J.
M.; Baldwin, R. L. Annu. ReV. Biophys. Biomol. Struct. 1992, 21, 95.
(9) For example, ideal R-helices have 3.6 residues per turn while R-helices
that participate in coiled-coil structures are slightly unwound, with 3.5 residues
per turn: Creighton, T. E. Proteins: Structures and Molecular Properties,
2nd ed.; W. H. Freeman and Company: New York, 1993.
(10) Guntert, P.; Mumenthaler, C.; Wu¨thrich, K. J. Mol. Biol. 1997, 273,
283. The structure library in DYANA was modified to include cyclopentane
and pyrrolidine rings for these calculations.
(4) (a) Lee, H.-S.; LePlae, P. L.; Porter, E. A.; Gellman, S. H. J. Org.
Chem. 2001, 66, 3597. (b) LePlae, P. L.; Umezawa, N.; Lee, H.-S.; Gellman,
S. H. J. Org. Chem., in press.
(5) Radkiewicz, J. L.; McAllister, M. A.; Goldstein, E.; Houk, K. N. J.
Org. Chem. 1998, 63, 1419 and references therein.
10.1021/ja010734r CCC: $20.00 © 2001 American Chemical Society
Published on Web 07/11/2001

7722 J. Am. Chem. Soc., Vol. 123, No. 31, 2001
Communications to the Editor
mers.3a,b It was not possible to determine a structure of 4 in water
because of overlap among crucial proton resonances and a general
decrease in the number of characteristic NOEs, relative to
methanol.3d In contrast, the proton resonance dispersion of 1 was
comparable in water and methanol, and the number and intensities
of the helix-defining NOEs were comparable in the two solvents.
Circular dichroism is widely used to assess the secondary
structures of conventional peptides,11 and CD has also been useful
for â-peptides12,13 and other unnatural oligomers.14 CD spectra
of 1-3 in H2O and in CH3OH show a characteristic maximum
near 200 nm and a minimum near 220 nm,6 which is consistent
with theoretical predictions12 and previously reported CD data3
for 12-helical â-peptides. In CH3OH the maximum and minimum
are somewhat more intense, and the maximum is slightly shifted
to the red, relative to H2O. The intensity trends suggest that
addition of methanol enhances 12-helix population, which mirrors
well-established trends for alcohol cosolvents among R-helix-
forming conventional peptides;15 however, strong similarities
between NOE data sets for 1 in water and methanol6 suggest that
the solvent effect on 12-helix population is not large.16 The
similarities among CD data for 1-4 indicate that neither the
sulfonamide groups nor the aromatic side chains exert a large
effect on the 12-helical CD signature.6
We have shown that S-APC residues represent a general
strategy for introducing specific side chains at defined positions
along the surface of 12-helical â-peptides. The â-peptide 12-helix
and the R-helix of conventional peptides have similar geometric
features (inner diameter: 3.1 vs 3.2 Å; rise per turn: 5.5 vs 5.4
Å; helix dipole: positive to negative from N-terminus to C-
terminus), which suggests that specifically functionalized 12-
helices may be able to mimic the structure of R-helical segments
of natural proteins. 12-Helical â-peptides may therefore offer a
rational approach to development of specific inhibitors of protein-
protein interactions that depend on R-helix recognition.17 â-Pep-
tides containing S-APC and other five-membered-ring-constrained
residues are especially interesting from this perspective because
of their high conformational stability relative to conventional
peptides, which do not form R-helices with fewer than 10-15
residues.8,18
Figure 1. (a) Graphical summary of NOEs between nonadjacent residues
observed for hexa-â-peptide 1 (2.5 mM, trifluoroacetate salt) in 9:1 H2O:
D2O, 4 °C. Three types of NOE were observed: CâHi f NHi+2, CâHi f
CRHi+2, and CâHi f NHi+3 (solid arrows in part b). All of these NOEs
are consistent with the 12-helical conformation; no NOEs inconsistent
with the 12-helical conformation were observed. (b) Cartoon showing
the spatial relationship between protons on residue i and protons on
residues i+2 and i+3 in the 12-helix; as mentioned in the text, CâHi f
CRHi+1 NOEs (dashed arrow) were observed for 4 (ref 3d) but not for 1,
which suggests that the helix formed by 1 is slightly more tightly wound.
Supporting Information Available: CD data for 1-4 in water and
in MeOH; NOE lists for 1 in water and MeOH; summary of Fmoc-S-
APC residue synthesis (PDF). This material is available free of charge
via the Internet at http://pubs.acs.org.
JA010734R
(11) For leading references, see: Johnson, W. C. Annu. ReV. Biophys.
Biophys. Chem. 1988, 17, 145. Woody, R. W. In The Peptides; Academic
Press: New York, 1985; Vol. 7, Chapter 2.
(12) Applequist, J.; Bode, K. A.; Appella, D. H.; Christianson, L. A.;
Gellman, S. H. J. Am. Chem. Soc. 1998, 120, 4891.
(13) (a) Abele, S.; Guichard, G.; Seebach, D. HelV. Chim. Acta 1998, 81,
2141. (b) Gung, B. W.; Zou, D.; Stalcup, A. M.; Cottrell, C. E. J. Org. Chem.
1999, 64, 2176.
(14) Green, M. M.; Peterson, N. C.; Sato, T.; Teramoto, A.; Cook, R.;
Lifson, S. Science 1995, 268, 1860. Yashima, E.; Maeda, Y.; Okamoto, Y. J.
Am. Chem. Soc. 1998, 120, 8895. Brunsveld, L.; Prince, R. B.; Meijer, E.
W.; Moore, J. S. Org. Lett. 2000, 2, 1525.
(15) (a) Goodman, M.; Verdini, A. S.; Toniolo, C.; Phillips, W. D.; Bovey,
F. A. Proc. Natl. Acad. Sci. U.S.A. 1969, 64, 444. (b) Luo, P.; Baldwin, R. L.
Biochemistry 1997, 36, 8413. (c) Cammers-Goodwin, A.; Allen, T. J.; Oslick,
S. L.; McClure, K. F.; Lee, J. H.; Kemp, D. S. J. Am. Chem. Soc. 1996, 118,
3082. (d) Walgers, R.; Lee, T. C.; Cammers-Goodwin, A. J. Am. Chem. Soc.
1998, 120, 5073.
(16) Switching from water to methanol exerts a profound effect on
â-peptides lacking cyclic backbone constraints, which usually display little
or no secondary structure in water: ref 13a and references therein.
(17) For leading references, see: (a) Zutshi, R.; Brickner, M.; Chmielewski,
J. Curr. Opin. Chem. Biol. 1998, 2, 62. (b) Daugherty, D. L.; Gellman, S. H.
J. Am. Chem. Soc. 1999, 121, 4325. (c) Chin, J. W.; Schepartz, A. J. Am.
Chem. Soc. 2001, 123, 2929.
(18) This work was supported by the National Institutes of Health
(GM56414) and the Leukemia Society of America. NMR measurements were
made in the Department of Chemistry (NIH 1 S10 RR04981) and at the
National Magnetic Resonance Facility at Madison (NIH RR02301, RR02781
and RR08438; NSF DMB-8415048 and BIR-9214394; U.S. Department of
Agriculture). CD measurements were made in the Biophysics Instrumentation
Facility (NSF BIR-9512577).
Figure 2. NOE-restrained dynamics results for hexa-â-peptide 1, based
on NMR data obtained in 9:1 H2O:D2O, 4 °C (details in text). Overlay
of 10 structures, backbone atoms only, with low energy and NOE restraint
violation. The N-termini are at the top.
simulated annealing with the SYBYL 6.4 program (Tripos
Software, St. Louis, MO). Figure 2 shows an overlay of the
resulting 10 structures, backbone atoms only. Fraying, apparent
at both ends, is pronounced at the N-terminus, but the 12-helix
is well-formed in the central portion. Among the 10 structures
shown root-mean-squared deviation is 0.69 ( 0.51 Å for residues
3-6 (backbone atoms). The 12-helical conformation deduced for
1 in water agrees well with the conformation previously deduced
for 4 in methanol3d and with crystal structures of ACPC oligo-