Factors governing 3₁₀-helix vs α-helix formation in peptides: Percentage of C^α-tetrasubstituted α-amino acid residues and sequence dependence

INTRODUCTION

C^α-Tetrasubstituted α-amino acids are well known as the most effective peptide 3₁₀/ α-helix formers and stabilizers.1-7 To this aim even Nature takes advantage of some of them (Aib, α-aminoisobutyric acid or C^α,α-dimethylglycine; Iva, isovaline or C^α-ethyl-C^α-methylglycine) in the family of membrane-active peptaibols (for terminology of peptaibols, see Ref. 8; for review articles, Refs. 9-11; for x-ray diffraction analyses of alamethicin, zervamicins, antiamoebins, and trichogin, Refs. 12, 13-16, 17 and 18, and 19, respectively). The Thorpe–Ingold effect20, 21 has been invoked to explain this structural tendency.22 The question of the preferred helix type (3₁₀- vs α-helix) of peptides rich in C^α-tetrasubstituted α-amino acids has been experimentally addressed considering such factors as peptide main-chain length, percentages of C^α-tetrasubstituted α-amino acids, sequence dependence, solvent polarity, temperature, and intermolecular interactions.3-6, 23-27

This work is part of a program aiming at a deeper understanding of the factors mentioned above. In particular, in no peptide system based exclusively on C^α-tetrasubstituted α-amino acids explored so far (with one exception) has the α-helix ever been found, neither in the crystal state nor in solution [the single exception is given by N^α-acylated homo-octapeptide esters based on C^α-methyl-L-valine, L-(αMe)Val, which slowly undergo a 3₁₀-helix to α-helix conformational switch in solvents of high polarity under appropriate temperature and concentration conditions28, 29]. In this connection we decided to better explore the effect of the number and positioning of C^α-trisubstituted α-amino acid residues needed to convert a 3₁₀-helix into an α-helix in a peptide of a given length. The peptide substrate for this study was chosen to be an N^α-acylated heptapeptide alkylamide (equivalent to an N^α-acylated octapeptide ester in terms of intramolecular H-bond potential) because peptides having this main-chain length are known to be at the interface between 3₁₀- and α-helices, and are presumably the most susceptible to this type of helix → helix conformational transformation.3 The only published work on the preferred conformation of N^α-acylated octapeptide esters (or heptapeptide alkylamides) containing a single C^α-trisubstituted α-amino acid deals with a host –(Aib)₈– chain (1, Figure 1) with an Aib → L-Leu guest replacement at position 6 (2) (i.e., in an internal position, near the C-terminus of the chain). Results of x-ray diffraction and spectroscopic analyses clearly indicated that the –C(O)–(Aib)₅–L-Leu–Aib– sequence 2 is folded in a regular 3₁₀-helical structure both in the crystal state and in solution,30-35 as it is the –(Aib)₈– parent sequence.36, 37

More recently, we have expanded our investigation to the analysis of the effect of a single, internal C^α-trisubstituted α-amino acid positioned near the N-terminus of the peptide chain. To this purpose, a standard, fully C^α-methylated, N^α-acylated heptapeptide alkylamide sequence –C(O)–L-Iva–L-(αMe)Val–L-Iva–L-(αMe)Val–L-(αMe)Phe–L-(αMe)Val–L- Iva–NH– (3) [L-(αMe)Phe, C^α-methyl phenylalanine] was first synthesized and conformationally characterized.38 Both x-ray diffraction and bidimensional NMR/molecular dynamics simulation analyses clearly showed that the peptide adopts a fully developed, regular right-handed 3₁₀-helical structure under all experimental conditions tested. In the next step, for an NMR investigation purpose, we designed two singly modified peptide sequences (4 and 5) in which (a) L-(αMe)Val2 was replaced by the C^α-trisubstituted protein L-Val residue (peptide 4), and (b) an L-Abu (Abu, α-aminobutyric acid) residue was substituted for the original L-Iva3 (peptide 5). It is worth noting that in both modified peptides the longest (isopropyl and ethyl, respectively) side chain at the replacement site was preserved. The L-Val2 sequence 4, more soluble in organic solvents, studied by bidimensional NMR/molecular dynamics simulations, was shown to exist in a right-handed 3₁₀/α-helical equilibrium with a slight preference for the α-helical conformation.39 Thus, the missing C^α-methylation of Val2 seems to have introduced some flexibility, which had an impact on the overall structure. Conversely, the sparingly soluble mBrBz–L-Iva–L-(αMe)Val–L-Abu–L-(αMe)Val–L-(αMe)Phe–L-(αMe)Val–L-Iva–NHMe 5 (mBrBz, meta-bromobenzoyl; NHMe, methylamino), nicely crystallized out of an acetone solution. Here, we describe details of the x-ray diffraction structure of this L-Abu3 N^α-acylated heptapeptide alkylamide analog. The mBrBz group was incorporated at the N-terminus to help solve the phase problem in the roentgenographic investigation by virtue of its heavy atom (Br). The results are analyzed in parallel with those of the C-terminal sequence Z–L-(αMe)Val–L-(αMe)Phe–L-(αMe)Val–L-Iva–NHMe (6) (Z, benzyloxycarbonyl), the x-ray diffraction structure of which is also described in the present article. This study allowed us to compare the crystal-state conformational preference of this N^α-acylated tetrapeptide alkylamide in isolation with that when it is inserted into a longer peptide sequence.

MATERIALS AND METHODS

Synthesis and Characterization of Peptides

Melting points were determined using a Leitz (Wetzlar, Germany) model Laborlux 12 apparatus and are not corrected. Optical rotations were measured using a Perkin-Elmer (Norwalk, CT) model 241 polarimeter equipped with a Haake (Karlsruhe, Germany) model D thermostat. Thin-layer chromatography was performed on Merck (Darmstadt, Germany) Kieselgel 60F254 precoated plates with the following solvent systems: (1) chloroform/ethanol, 9:1; (2) 1-butanol/acetic acid/water, 3:1:1; (3) toluene/ethanol, 7:1. The chromatograms were examined using ultraviolet fluorescence or developed by chlorine/starch/potassium iodide or ninhydrin chromatic reaction as appropriate. All compounds were obtained in a chromatographically homogeneous state. Their analytical data and physical properties are listed in Table I. All of the synthetic intermediates and the final compound were also characterized by ¹H-NMR (data not reported). The chemical structure of the final compound was confirmed by amino acid analysis and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (data not reported).

Table I. Analytical Data and Physical Properties for the Newly Synthesized Peptides

Compound	Yield (%)	Melting Point (°C)	Crystalliz. Solventa	[α] (deg)b	Thin Layer Chromatography			IR (cm−1)c
					RF1	RF2	RF3
Z–L-Abu–OH	55	66–68	DE/PE	−13.4	0.45	0.90	0.25	3418, 1743, 1727, 1662, 1546
Z–L-Abu–L-(αMe)Val–L-(αMe)Phe–L-(αMe)Val–L-Iva–NHMe	34	204–205	EtOAc/PE	−3.8d	0.70	0.95	0.35	3330, 1697, 1657, 1524
Z–L-(αMe)Val–L-Abu–L-(αMe)Val–L-(αMe)Phe–L-(αMe)Val–L-Iva–NHMe	48	196–198	CHCl3/PE	−6.4	0.70	0.90	0.30	3327, 1702, 1659, 1524
Z–L-Iva–L-(αMe)Val–L-Abu–L-(αMe)Val–L-(αMe)Phe–L-(αMe)Val–L-Iva–NHMe	66	227–228	EtOAc/PE	15.4	0.65	0.90	0.30	3322, 1696, 1656, 1526
mBrBz–L-Iva–L-(αMe)Val–L-Abu–L-(αMe)Val–L-(αMe)Phe–L-(αMe)Val–L-Iva–NHMe	71	311–312	CH2Cl2/PE	14.0e	0.60	0.95	0.20	3362, 1660, 1522

• a DE: diethyl ether; PE: petroleum ether; EtOAc: ethyl acetate.
• b c = 0.5, MeOH.
• c The ir absorption spectra were obtained in KBr pellets; only bands within the 3500–3200 and 1800–1500 cm⁻¹ regions are reported.
• d [α].
• e c = 0.2, 2,2,2-trifluoroethanol.

For the large-scale production of the optically pure L-Iva, L-(αMe)Val, and L-(αMe)Phe, we exploited an economically attractive, chemoenzymatic synthesis developed by DSM Research a few years ago.40, 41 It involves a combination of organic synthesis for the preparation of the racemic α-amino acids followed by the use of a broadly specific aminopeptidase to achieve optical resolution. The Z N^α-protected, C^α-methylated α-amino acids42-44 were activated by the acid fluoride method.38, 45, 46 The Z group was removed by catalytic hydrogenation in methanol (MeOH) solution. The synthesis of the mBrBz-heptapeptide 5 was achieved by use of mBrBz-OAt38, 47 (OAt, 1-oxy-7-aza-1,2,3-benzotriazole) in a 9:1 CH₂Cl₂/CH₃CN solvent mixture in the presence of N-methylmorpholine.

RESULTS AND DISCUSSION

We determined by x-ray diffraction the molecular and crystal structures of the N^α-acylated heptapeptide alkylamide mBrBz–L-Iva–L-(αMe)Val–L-Abu–L-(αMe)Val–L-(αMe)Phe–L-(αMe)Val–L-Iva–NHMe (5) and its C-terminal tetrapeptide alkylamide sequence Z–L- (αMe)Val–L-(αMe)Phe–L-(αMe)Val–L-Iva–NHMe (6). The molecular structures with the atomic numbering schemes are illustrated in Figures 2 and 3, respectively. Selected backbone and side-chain torsion angles50 are given in Table III. In Table IV the intra- and intermolecular H-bond parameters are listed. For comparison the two tables also report the corresponding data for the parent N^α-acylated heptapeptide alkylamide 3.

The molecules of the Abu3 heptapeptide amide 5 are fully-developed right-handed helices, stabilized by a large set of intramolecular, consecutive, or bifurcated, CO · · · H—N H-bonds. The right-handed helical screw sense is in particular dictated by the known conformational bias of the L-Iva (with a linear side chain)7, 38, 44 and L-(αMe)Val (with a β-branched side chain)7, 38, 46 residues. The average values for the seven sets of backbone ϕ,ψ torsion angles are −56.3°, −45.2°. The average ϕ value is closer to that typical of a 3₁₀-helix (−57°) than to that of an α-helix (−63°).23 However, the opposite holds true for the average ψ value (−30° for a typical 3₁₀-helix and −42° for a typical α-helix). This mixed 3₁₀/α-helical structure, suggested by the backbone torsion angles, is confirmed by the analysis of the intramolecular H-bonds, although some of them are weak,51-53 particularly if part of the bifurcation sites (in any case, intramolecular H-bonds with an N · · · O distance > 3.0 Å are a common observation in 3₁₀- and α-helices). At the N-terminus the molecular conformation displays a short 3₁₀-helix with two consecutive type III β-turn (C₁₀) structures.54-56 The CO group of the mBrBz group forms a H-bond with the L-Abu3 N–H group, while the L-Iva1 CO group is involved in a H-bond with the L-(αMe)Val4 N—H group. However, the L-Iva1 CO group is also involved in a weak interaction with the L-(αMe)Phe5 N—H group giving rise to an α-turn (C₁₃) conformation.57-59 Then, this region of bifurcation is followed by two weakly stabilized, consecutive α-turn structures involving carbonyl oxygens of L-(αMe)Val2 and L-Abu3 and amino groups of L-(αMe)Val6 and L-Iva7. A second site of bifurcated arrangement is seen at the C-terminus, as the L-Iva7 amino group is also involved in the formation of a weakly stabilized β-turn with the L-(αMe)Val4 CO group. The short C-terminal 3₁₀-helical segment ends up with a H-bond between the methylamido N—H and the L-(αMe)Phe5 carbonyl groups.

In the tetrapeptide amide 6 each independent molecule A and B is folded in a right-handed 3₁₀-helix stabilized by three intramolecular CO · · · H—N H-bonds. Two regular type III β-turns are followed by a C-terminal, distorted β-turn in which the values of the ϕ,ψ torsion angles of the L-(αMe)Val and L-Iva residues are intermediate between those of a type I and a type III β-turn (position i+2). The conformational differences, including side-chain dispositions (χ torsion angles),60 between molecules A and B of the tetrapeptide amide are only of minor significance.

For reason of comparison, in Tables III and IV the backbone and side-chain torsion angles, and the intra- and intermolecular H-bond parameters, respectively, for the regular 3₁₀-helical, parent L-Iva3 heptapeptide amide (3) (independent molecules A and B)38 are also listed. Significant backbone conformational differences among the five molecules are observed: (a) in the ψ₄ and ψ₅ torsion angles (the absolute values of which are consistently higher in the L-Abu3 heptapeptide amide 5) and (b) in the related, α-turn type, O2 · · · H6—N6 and O3 · · · H7—N7 intramolecular H-bonds (seen only in the L-Abu3 heptapeptide amide 5) and β-turn type O3 · · · H6—N6 intramolecular H-bond (seen only in the L-Iva3 heptapeptide amide 3 and in the tetrapeptide amide 6). From this comparison, it may be concluded that this N^α-acylated tetrapeptide amide segment may adopt either a mixed α/3₁₀-helical structure (when incorporated at the C-terminus of the L-Abu3 heptapeptide amide 5) or an essentially 3₁₀-helical structure (either when in isolation or when incorporated at the C-terminus of the fully C^α-methylated L-Iva3 heptapeptide amide 3).

The packing mode of the heptapeptide amide 5 is characterized by an intermolecular H-bond between the N1 group and the O6 carbonyl oxygen atom of a (−x − ½, −y, z− ½) symmetry related molecule. Rows of molecules, head-to-tail H-bonded and related through a crystallographic twofold screw axis, are thus formed through the c direction.

In the packing mode of the tetrapeptide amide 6 head-to-tail intermolecular H-bonds are observed between molecules of the same type. More specifically, the N4A—H group is H-bonded to the O7A carbonyl oxygen atom of a (x + 1, y, z + 1) translational equivalent of molecule A, while the N4B—H group is H-bonded to the O7B carbonyl oxygen atom of a (x + 1, y, −z + 1) translational equivalent of molecule B. As a consequence, rows of molecules A are formed along the c direction, while rows of molecules B are observed along the ac direction. Rows of the two kinds alternate along the b direction.

CONCLUSIONS

This paper clearly shows that in the crystal state an N^α-acylated heptapeptide alkylamide, containing six C^α-tetrasubstituted α-amino acids and a single C^α-trisubstituted residue near the N-terminus (L-Abu3) (5), tends to fold in a well-developed α-helical structure in the central region, although this segment is preceded and followed by two short 3₁₀-helical segments. This result is in good agreement with the published NMR data on the L-Val2 analog 439 of the same, fully 3₁₀-helical, parent peptide 3,38 and beautifully parallels that of an NMR study on an Ala-rich peptide, where an α-helical sequence was identified in the middle and short 3₁₀-helical stretches at both termini.61

A previous detailed structural investigation on an N^α-acylated octapeptide ester (equivalent to a heptapeptide alkylamide as far as its capability of intramolecular H-bond formation is concerned), characterized by seven Aib residues and a single C^α-trisubstituted α-amino acid (L-Leu6) near the C-terminus (2), unequivocally established the onset of a fully developed, rigid, regular 3₁₀-helical conformation,30-35 as observed in the –(Aib)₈– homopeptide sequence (1).36, 37 Taken together, these findings support the view that, even in the case of a very high percentage of C^α-tetrasubstituted α-amino acids discussed here, the precise positioning of the single C^α-trisubstituted residue in the sequence may have a dramatic effect on the helical structure of the peptide backbone. More specifically, incorporation of the guest residue near the N-terminus seems critical for the observation of the conformational transition. Even if in a single, specific case (an N^α-tert-butyloxycarbonylated or N^α-acetylated heptapeptide methyl ester characterized by as many as six C^α-trisubstituted amino acids) it has been reported that in the crystal state the nature of the N^α-blocking may play a role on the type of helical structure adopted,62 it is evident that in our heptapeptide methylamides 3–5 such an effect cannot be operative as all three compounds are N^α-acylated with the same moiety (mBrBz).

Other interesting information extracted from this work is that the preferred type of helical structure of even a fully C^α-tetrasubstituted, N^α-acylated tetrapeptide alkylamide, as 6, may be governed by the presence/absence of additional residues in the sequence.