Conformationally homogeneous cyclic tetrapeptides: Useful new three-dimensional scaffolds

Article abstract of DOI:10.1021/ja029205t

The most commonly recognized motifs in protein-protein interactions are γ and β turns, which are defined by three to four contiguous amino acids in a peptide sequence. Cyclic tetrapeptides thus represent minimalist turn mimetics, but their usefulness is c

Full text of DOI:10.1021/ja029205t

Published on Web 12/18/2002
Conformationally Homogeneous Cyclic Tetrapeptides: Useful New
Three-Dimensional Scaffolds
Matthew P. Glenn, Michael J. Kelso, Joel D. A. Tyndall, and David P. Fairlie*
Centre for Drug Design and DeVelopment, Institute for Molecular Bioscience, UniVersity of Queensland,
Brisbane Qld 4072, Australia
Received November 4, 2002 ; E-mail: d.fairlie@imb.uq.edu.au
â-turns in proteins are defined by four consecutive amino acids.
Cyclic tetrapeptides (CTPs) derived therefrom as minimalist turn
mimetics have limited uses as biological probes or drug leads due
to inefficient synthesis, instability to hydrolysis/metabolism, and
conformational heterogeneity in water.1 These disadvantages stem
from unfavorable strain in a 12-membered ring containing four
trans-amide bond restraints. The few known naturally occurring
CTPs usually possess turn-inducing constraints (e.g., Pro, D-amino
acids) that facilitate cyclization and enable access to a cis-amide.1b,2
Some CTPs exhibit one conformation in nonpolar solvents or when
a cis-amide is present, but usually CTPs exist as multiple conforma-
tions in polar solvents (H2O, DMSO).3 We demonstrate here that
Figure 1. 1H NMR spectra (amide NH region) for (a) 4R in d6-DMSO,
CTPs, with 13-membered rings and a â-amino acid, are easier to
(b) 6R in d6-DMSO, and (c) 6R in 90% H2O:10% CD3CN.
synthesize, chemically more stable, conformationally homogeneous,
and are novel three-dimensional scaffolds.
To investigate the influence of a â-amino acid (e.g., âhPhe, â-S-
homophenylalanine) in CTPs, we synthesized the tetrapeptides H2N-
Phe-(R)Pro-Asu-Phe-OH (1), H2N-âhPhe-(R)Pro-Asu-Phe-OH (2),
and H2N-Phe-(R)Pro-Asu-âhPhe-OH (3) featuring the racemic
amino acid Asu (R,S-amino suberic acid methyl ester) present in
some known CTPs.2 Cyclization of 1 by slow addition (syringe
pump) to BOP (10-3 M) in DMF over 24 h gives ∼50% 4 as two
diastereomers, plus octapeptide cyclodimer. By contrast 2 and 3
with a â-amino acid cyclized efficiently (>95%) under the same
conditions to 5 and 6, respectively, each as two separable (rp-HPLC)
diastereomers.
Figure 2. Summary of ROESY, 3JNHCHR and variable-temperature 1H NMR
data for 6R in d6-DMSO and 90% H2O:10% CD3CN. Bars represent
observed ROEs with intensities (strong, e2.5 Å; medium, e3.5 Å; weak,
e5.0 Å) proportional to thickness. â* represents âhPhe backbone-CH2.
#Data acquired using the more soluble free acid.
1H NMR spectra for 4R and 5R (R-Asu, Figure 1a) and 4S and
5S (S-Asu) in d6-DMSO were consistent with multiple (g3) solution
conformations that interconvert slowly on the NMR time scale.
However each diastereomer of 6, where âhPhe was inserted between
Phe and Asu, displayed only one set of sharp resonances (6R in
Figure 1b,c; 6R and 6S, Figure S2) in DMSO or H2O (+10%
MeCN), indicating just one stable solution conformation for 6R or
6S. The contrast between conformational homogeneity for 6 and
heterogeneity for 5 reflects the cooperative effect of separating two
turn-inducing constraints (R-Pro and âhPhe) in 6 by an amino acid.
6R and 6S were also chemically more stable to hydrolysis in 0.3
M LiOH (t1/2 ≈ 50 h, 20 °C) than the 12-membered 4R and 4S
(t1/2 < 10 h, 20 °C), as assessed by rp-HPLC (Supporting
Information).
1H NMR resonances for 6R and 6S in d6-DMSO were assigned
from 2D TOCSY and ROESY spectra. ROEs (dRR(i,i+1)) character-
istic of cis peptide bonds were not observed, but strong ROEs
between Phe CHR and Pro CHδ indicated a trans-amide bond at
Pro. Multiple interresidue ROEs for both 6R (Figure 2) and 6S
suggested a well populated, stable conformation defined by (i, i +
2) correlations. A temperature-independent chemical shift for Asu
NH (∆δ/∆T e 3 ppb)4 in both 6R (Figure 2) and 6S supported its
involvement in a structure-stabilizing H-bond. Large JNHCHa > 9
Hz for residues in 6R (âhPhe, Asu) and 6S (âhPhe) also provided
strong evidence for a stable conformation in DMSO.
Two-dimensional 1H NMR data (Figure 2) were similar for 6R
in DMSO and water (+ 10% MeCN), suggesting similar structures.
Structures were calculated from DMSO, where there were more
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10.1021/ja029205t CCC: $25.00 © 2003 American Chemical Society

C O M M U N I C A T I O N S
Figure 5. Sugar analogue 10 and spirocyclic compound 11 showing bonds
(boxed) that match side chain CR-Câ vectors in 6S and 6R, respectively.
Figure 3. Superimposition of the 30 lowest-energy calculated NMR
structures for 6S (left, backbone RMSD 0.053 Å) and 6R (right, RMSD
0.047 Å) in DMSO. White dashes indicate hydrogen bonds.
tidic templates, including sugars and spirocyclic compounds (Figure
5), found in natural products. For example, CR-Câ atoms of 6S
superimposed well (RMSD ) 0.40 Å over eight atom pairs) on
the indicated bonds (boxed) of carbohydrate 10, which did not
match nearly so well to 6R (1.07 Å) or 7 (0.59 Å). On the otherhand
6R superimposed well on the indicated bonds (boxed) of the
tricyclic spiro-compound 11 (RMSD 0.51 Å), which did not match
so well to 6S or 7 (RMSDs 1.08, 1.20 Å). Thus CTPs with a
â-amino acid could provide a useful link between protein archi-
tecture and nonpeptidic molecules. Libraries of such CTPs may be
useful screening tools to rapidly identify pharmacophore space that
can then be computer-matched to nonpeptidic structures.
Thus selective incorporation of a â-amino acid can make CTPs
easier to synthesize, more resistant to hydrolysis, conformationally
homogeneous, and more useful as chiral scaffolds that can be
derived from protein sequence alone. These advantages may expand
the utility of CTPs as pharmacophoric probes which mimic both
protein turns and natural products.
Figure 4. Superposition of X-ray structure of 7 (yellow)7 on (a, left):
modeled 8 (orange) and 9 (pink); (b, right): 6S from Figure 3 (blue, RMSD:
0.55 Å (4 CR-Câ vectors), 0.48 Å (12 common backbone atoms)).
ROEs, for 6R (30 ROE distance restraints: 14 intraresidue, 14
sequential, 2 medium range, and 2 φ-angle restraints: âhPhe φ
-120 ( 30°, Asu φ -120 ( 30°) and for 6S (32 ROE distance
restraints: 20 intraresidue, 10 sequential, 2 medium range, and 1
φ-restraint: âhPhe φ -120 ( 30°) using a dynamic simulated
annealing and energy minimization protocol in XPLOR.5 Initial
structures indicated a likely H-bond in both 6R (Asu NH‚‚‚OC
âhPhe) and 6S (Asu NH‚‚‚OC Phe), consistent with VT-NMR data
(Figure 2).
Acknowledgment. We thank ARC for financial support.
Supporting Information Available: Syntheses, structures, char-
acterization data for 4-6 (PDF). This material is available free of charge
via the Internet at http://pubs.acs.org.
The H-bond restraints improved structural convergence (Figure
3), well-defined backbones being consistent with stabilization by
four trans-amides, D-proline, a trans-annular H-bond and steric
constraints imposed by the 13-membered ring. The backbones and
three side-chain projections (âhPhe, Phe, R-Pro) were similar for
6R and 6S, the epimeric Asu side chain being directed into or above
the plane of the cycle, respectively. φ-Angles agreed with measured
3JNHCHR constants. (Supporting Information).
6S is homologous to bioactive CTPs such as chlamydocin (7),
HC toxin (8), and trapoxin B (9).2,3 Modeled6 structures for 7-9
(Figure 4a) and structures in CDCl33 for 7 and 8 have two γ-turns
and a backbone conformation very similar to those of 6S (Figure
4b). Inserting a methylene into the 12-membered ring rotates the
Asu-âhPhe amide bond in 6, preventing formation of one of the
γ-turn H-bonds and redirecting the CR-Câ vector at âhPhe.
Inversion of stereochemistry at Asu causes the Pro CO to rotate
69° in 6R relative to 6S (Figure 3), restoring a gauche relationship
between the Pro carbonyl and the CR-Câ vector of Asu, permitting
a â turn-defining Asu NH‚‚‚OC âhPhe H-bond.
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