Cα-Methyl proline: A unique example of split personality

Abstract

Methylation at the C^α-position of a Pro residue was expected to lock the preceding tertiary amide (ω) torsion angle of the resulting (αMe)Pro to the trans disposition and to restrict the ϕ,ψ surface to the single region where the 3₁₀/α-helices are found (in this five-membered ring residue ϕ is severely constrained to about ±65° by its cyclic nature). The results of the present X-ray diffraction work on a selected set of four N^α-blocked, (αMe)Pro-containing, dipeptide N′-alkylamides clearly show that, although the region of the conformational map largely preferred by (αMe)Pro would indeed be that typical of 3₁₀/α-helices, the semi-extended [type-II poly(Pro)_n helix] region can also be explored by this extremely sterically demanding C^α-tetrasubstituted α-amino acid. In addition, the known high propensity for β-turn formation of the Pro residue is further enhanced in peptides based on its C^α-methylated derivative. © 2007 Wiley Periodicals, Inc. Biopolymers 89: 465–470, 2008.

This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at [e-mail]

INTRODUCTION

L-Proline is conformationally unique among coded amino acids in that its ϕ torsion angle is severely restricted (−65 ± 10°) by its characteristic five-membered pyrrolidine ring structure and the preceding ω torsion angle can undergo a cis (0°) ⇌ trans (180°) equilibrium, thus generating a significant population of cis isomers for tertiary amides when compared with the negligible populations for the secondary amides of the usual peptide bonds (for extensive review articles, see Refs.1–9). In addition, its ψ torsion angle is commonly found either in the right-handed 3₁₀-/α-helical region (−30 to −50°, or cis' conformation) or in the left-handed, semi-extended, region [−150 ± 10°, or trans', or poly-(L-Pro)_n conformation].

Poly-(L-Pro)_n is known to exhibit dimorphism. This phenomenon is related to the ω cis ⇌ trans isomerism: in the poly-(L-Pro)_n I conformation all ω torsion angles are cis, butin the more stable type-II conformation they are all trans. Furthermore, Pro is an amino acid with a marked preference for the i + 1 corner position of either type-I or type-II β-turn.10, 11 A Pro residue internal to a peptide sequence cannot offer the classical H-bonding donor NH group for helix stabilization. It is for this reason that Pro residues are normally found near the N-terminus of peptide helices. However, if a Pro residue is internal to a helical sequence, it usually generates a kink, thereby interrupting the regular continuation of the helix (in that segment the peptide backbone is often solvated). Interestingly, if combined with a strongly helicogenic residue such as α-aminoisobutyric acid (Aib),12–14 Pro can still be part of an intramolecular H-bonded helical structure. Indeed, the -(Aib-L-Pro)_n- sequence was shown to fold into a variant of the right-handed 3₁₀-helix, known as the β-bend ribbon.15, 16 This helix is stabilized by 50% of the intramolecular H-bonds occurring in a regular 3₁₀-helix. An additional property of Pro is that it occurs extensively in the collagen triple helix. The typical -(Pro-Xxx-Gly)_n- triplet of this fibrous protein forms a left-handed, type-II poly-(L-Pro)_n conformation. In this structure the intrachain N…O distances are definitely too long for NH…OC H-bond formation. Indeed, the collagen superhelical arrangement is stabilized by a set of interchain H-bonds, in which the Pro residues behave as H-bonding acceptors.

Despite its great potential interest, only few, scattered, and nonsystematic studies have been so far reported on the conformational preferences of (αMe)Pro. Indeed, among the C^α-methylated derivatives of the coded amino acids, (αMe)Pro is by far the most conformationally restricted (ϕ is blocked; in linear peptides the preceding tertiary amide torsion angle ω is expected to adopt only the trans conformation; the side-chain χⁿ torsion angles are also rigidified).

Conformational potential energy calculations suggested that poly-[L-(αMe)Pro]_n is locked in the type-II poly-(L-Pro)_n conformation.17 Poly-[L-(αMe)Pro]_n of low average molecular weight was synthesized via N-carboxyanhydride polymerization.18 Its CD spectrum in alcohol solution resembles that of type-II poly-(L-Pro)_n. From ¹³C NMR, IR absorption, and CD studies and from conformational energy computations it turns out that the amino acid derivative Ac-L-(αMe)Pro-NHMe (Ac, acetyl; NHMe, methylamino) strongly prefers the C₇′ (inverse γ-turn) conformation, irrespective of the solvent used19, 20; however, it should be recalled here that any study on a compound as short as the amino acid derivative tend to overestimate the γ-turn conformation over other, usually more stable, conformations. Therefore, not surprisingly, an X-ray diffraction analysis showed that Ac-DL-(αMe)Pro-NHMe is folded in a helical, but not intramolecularly H-bonded, conformation (ϕ = ±60.5° and ψ = ±24.9°) in the crystal state.20 The preferred conformations of the heterochiral dipeptides Z-L-Pro-D-(αMe)Pro-NHMe (Z, benzyloxycarbonyl) and Z-D-(αMe) Pro-L-Pro-NHMe were examined by IR absorption, and ¹H and ¹³C NMR techniques.21 The former adopts a type-II β-turn conformation [where D-(αMe)Pro is left-handed helical], while in the latter the coexistence of at least four conformers was observed.

L-(αMe)Pro, incorporated in a peptide antigen,22, 23 or at position 3 or 7 of the nonapeptide hormone bradykinin,24–26 or in the NPNA-repeat motif of the Plasmodium falciparium protein,27, 28 was shown by 2D-NMR experiments to strongly stabilize β-turn conformations. Similar results [β-turn formation and transL-Xxx-L-(αMe)Pro peptide bonds] were reported for L-(αMe)Pro-containing analogues of an antigen mimotope peptide29 and the antimicrobial peptide buforin 2.30 Interestingly, in contrast to the results from molecular mechanics simulations, it was experimentally found that the sequence -L-(αMe)Pro-L-Pro is not tightly folded.31 Finally, a recent X-ray diffraction analysis of two terminally blocked dipeptides of general formula Ac-L-Val-L-Xxx-NHR [where Xxx is 4-methylene-(αMe)Pro] clearly indicates the absence of any intramolecular H-bond. In these structures the 4-substituted L-(αMe)Pro residue is right-handed helical.32

In our ongoing, systematic, investigation on the conformational propensities of (αMe)Pro we have decided to focus on the: (i) cis ⇌ trans isomerization about the ω torsion angle preceding (αMe)Pro, (ii) formation, type (I or II), and stability of the β-turn structures formed, and (iii) type of helical structure generated by the homo-oligomers. In the present study, we have synthesized and investigated by X-ray diffraction four selected, N^α-blocked, (αMe)Pro dipeptides N′-alkylamides. We have chosen NHiPr (isopropylamino) as the C-terminal blocking group because it best mimicks the continuation of the peptide chain. The three Ala/(αMe)Pro dipeptides are either homo- or heterochiral, with the (αMe)Pro residue is either at position 1 or 2 in the sequence. The fourth peptide combines (αMe)Pro with the helically biased Aib residue.12–14

MATERIALS AND METHODS

The N^α-blocked dipeptide N′-alkylamides described in this work were synthesized by solution methods. In particular, couplings involving the severely sterically hindered (αMe)Pro residue were achieved by C-activation with N-ethyl, N′-[3-(dimethylamino)propyl] carbodiimide and as additive either 1-hydroxy-1,2,3-benzotriazole33 or 7-aza-1-hydroxy-1,2,3-benzotriazole.34 Yields were from good to excellent, with the single exception of that of the Aib-D-(αMe)Pro bond (24%).35 Details of syntheses and chemical characterizations will be reported elsewhere.

Colorless single crystals of the N^α-blocked dipeptide N′-alkylamides Ac-D-(αMe)Pro-D-Ala-NHiPr, Ac-D-(αMe)Pro-L-Ala-NHiPr, iBu-L-Ala-D-(αMe)Pro-NHiPr (iBu, isobutyryl), and Ac-D-(αMe) Pro-Aib-NHiPr were grown at room temperature from the solvents reported in Table I. Intensity data collections were performed using a Philips PW1100 four-circle diffractometer. Graphite-monochromated CuKα radiation (λ = 1.54178 Å) and θ–2θ scan mode up to θ = 60° [54.2° for iBu-L-Ala-D-(αMe)Pro-NHiPr, as the crystal did not significantly diffract at higher resolution] were employed. Intensities were corrected for Lorentz and polarization effects. Cell parameters were obtained by least-squares refinements of the angular settings of 48 carefully centered reflections in the 12–20° θ range. The structures were solved by direct methods using the SIR 2002 program.36 Refinements were carried out by least-squares procedures on F², using all data, by application of the SHELX 97 program.37 Hydrogen atoms were calculated at idealized positions and refined using a riding model. Crystallographic parameters are listed in Table I.

Table I. Crystallographic Data and Diffraction Parameters for the (αMe)Pro-Containing Dipeptides Studied in this Work

Parameter	Ac-D-(αMe)Pro-D-Ala-NHiPr	Ac-D-(αMe)Pro-L-Ala-NHiPr	iBu-L-Ala-D-(αMe)Pro-NHiPr	Ac-D-(αMe)Pro-Aib-NHiPr
EtOAc, ethyl acetate; PE, petroleum ether.
Empirical formula	C14H25N3O3	C14H25N3O3	C16H29N3O3	C15H27N3O3
Formula weight (amu)	283.4	283.4	311.4	297.4
Temperature (K)	293(2)	293(2)	293(2)	293(2)
Wavelength (λ)	CuKα (1.54178 Å)	CuKα (1.54178 Å)	CuKα (1.54178 Å)	CuKα (1.54178 Å)
Crystal system	Orthorhombic	Monoclinic	Monoclinic	Orthorhombic
Space group	P212121	P21	P21	P212121
a (Å)	10.324(2)	8.749(2)	9.316(2)	10.758(2)
b (Å)	10.962(3)	9.047(3)	10.747(3)	10.890(2)
c (Å)	14.343(3)	10.801(3)	9.961(2)	14.907(3)
α (°)	90	90	90	90
β (°)	90	104.13(4)	110.81(6)	90
γ (°)	90	90	90	90
V (Å^3)	1623.2(6)	829.1(4)	932.2(4)	1746.4(6)
Z (molecules/unit cell)	4	2	2	4
Density (calc.) (g/cm^3)	1.160	1.135	1.109	1.131
Absorption coeff. (mm^−1)	0.667	0.653	0.620	0.641
F(000)	616	308	340	648
Data collection method	θ −2θ	θ −2θ	θ −2θ	θ −2θ
Solvent of crystallization	CHCl3	EtOAc	EtOAc/PE	Acetone
Crystal size (mm)	0.40 × 0.15 × 0.10	0.45 × 0.25 × 0.10	0.35 × 0.15 × 0.10	0.35 × 0.30 × 0.10
θ Range (°)	5.08 – 60.00	4.22 – 59.96	4.75 – 54.18	5.03 – 59.95
Index ranges	−1 ≤ h ≤ 11	−9 ≤ h ≤ 9	−9 ≤ h ≤ 9	−1 ≤ h ≤ 12
	0 ≤ k ≤ 12	−1 ≤ k ≤ 10	−1 ≤ k ≤ 11	0 ≤ k ≤ 12
	0 ≤ l ≤ 16	0 ≤ l ≤ 12	0 ≤ l ≤ 10	0 ≤ l ≤ 16
Reflections collected	1579	1564	1417	1668
Independent reflections	1543 [R(int) = 0.009]	1489 [R(int) = 0.043]	1209 [R(int) = 0.073]	1640 [R(int) = 0.026]
Data/restraints/parameters	1543/0/182	1489/1/187	1209/1/200	1640/0/197
Goodness of fit (on F^2)	1.082	1.090	1.081	1.063
Final R indices [I > 2(σ)I]	R1 = 0.049, wR2 = 0.128	R1 = 0.050, wR2 = 0.144	R1 = 0.075, wR2 = 0.192	R1 = 0.058, wR2 = 0.141
R indices (all data)	R1 = 0.051, wR2 = 0.129	R1 = 0.053, wR2 = 0.159	R1 = 0.076, wR2 = 0.194	R1 = 0.061, wR2 = 0.145
Δρ (e/Å^3)	0.280/−0.254	0.202/−0.159	0.304/−0.369	0.211/−0.271

CCDC 652059–652062 contain the supplementary crystallographic data for the four structures described in this article. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.

RESULTS AND DISCUSSION

The molecular and crystal structures of the four, terminally blocked, (αMe)Pro-based, dipeptide N′-alkylamides Ac-D-(αMe)Pro-D-Ala-NHiPr, Ac-D-(αMe)Pro-L-Ala-NHiPr, iBu-L-Ala-D-(αMe)Pro-NHiPr, and Ac-D-(αMe)Pro-Aib-NHiPr were solved by X-ray diffraction. The molecular structures are illustrated in Figures 1–4, respectively.

Figure 1. X-ray diffraction structure of Ac-D-(αMe)Pro-D-Ala-NHiPr with atom numbering. The CO…HN intramolecular H-bond is indicated by a dashed line.

Figure 2. X-ray diffraction structure of Ac-D-(αMe)Pro-L-Ala-NHiPr with atom numbering. The CO…HN intramolecular H-bond is indicated by a dashed line.

Figure 3. X-Ray diffraction structure of iBu-L-Ala-D-(αMe)Pro-NHiPr with atom numbering. The CO…HN intramolecular H-bond is indicated by a dashed line.

Figure 4. X-ray diffraction structure of Ac-D-(αMe)Pro-Aib-NHiPr with atom numbering. The CO…HN intramolecular H-bond is indicated by a dashed line.

Table II compares some of their relevant 3D-structural parameters with the published data for the corresponding Pro-based analogues.38–44 In addition, the four (αMe)Pro peptides share the following features: (i) all (secondary and tertiary) amide and peptide bonds (ω torsion angles) are in the trans conformation with modest deviations from the (180°) planarity (|Δω| ≤ 8.2°), and (ii) all of them are folded in a β-turn conformation, which is stabilized by an i ← i + 3 (C0O0…HNT) H-bond of medium strength45 [the O…N separations are in the range 2.922(4)–3.088(5) Å, and the O…HN angles are between 141 and 162°].

Table II. Structural Parameters for the β-Turn Forming Ala/Pro, Ala/(αMe)Pro, Aib/Pro, and Aib/(αMe)Pro Dipeptide Sequences in the Crystal State

Peptide Sequence	Type of β-Turn	Backbone Torsion Angles				Refs.
		ϕ1	ψ1	ϕ2	ψ2
Piv, pivaloyl; NHtBu, tert-butylamino.
Ac-D-(αMe)Pro-D-Ala-NHiPr	III′	53	32	66	25	This work
Ac-L-Pro-L-Ala-NHtBu	—	−66	166	−71	154	38
iBu-L-Pro-L-Ala-NHiPr	II	−59	136	66	14	39
Ac-D-(αMe)Pro-L-Ala-NHiPr	II′	53	−129	−77	−12	This work
Z-D-Pro-L-Ala-NHtBu	II′	58	−137	−76	−14	38
iBu-L-Pro-D-Ala-NHiPr	II	−62	137	96	3	40,41
iBu-L-Pro-D-Ala-NHtBu	II	−60	133	82	15	42
iBu-L-Ala-D-(αMe)Pro-NHiPr	II	−55	133	78	0	This work
Piv-D-Ala-L-Pro-NHiPr	II′	60	−140	−89	9	43
Ac-D-(αMe)Pro-Aib-NHiPr	III′	53	37	61	28	This work
Piv-L-Pro-Aib-NHMe	II	−58	139	61	25	44

The β-turns formed by the Xxx-Ala or Ala-Xxx [Xxx = (αMe)Pro or Pro] L-D or D-L heterochiral sequences compare very well. All of them adopt either the type-II or type-II′ β-turn, respectively, depending upon the sequence chirality, as expected.10, 11 It is worth pointing out here that in the D-(αMe)Pro-L-Ala sequence D-(αMe)Pro is forced to be semi-extended [type-II poly(Pro)_n helical] to fold the peptide in the β-turn conformation without compelling L-Ala to behave like a D-residue.

Also when the strong turn former Aib residue12–14 occurs in the sequence, as in the (αMe)Pro-Aib and Pro-Aib dipeptides, a β-turn conformation is seen in the crystal state. However, the different types of β-turn observed in the two compounds (III′ versus II) should be attributed to the higher propensity of (αMe)Pro versus Pro to explore the 3₁₀/α-helical region of the ϕ,ψ map.

A similar, but even more stringent, proof for the intriguing conformational preference of (αMe)Pro stems from a comparison of the homochiral (αMe)Pro-Ala versus Pro-Ala dipeptides. Although the former sequence adopts a β-turn conformation, only one of the two known examples of the latter is regularly folded. In addition, as in the case of the (αMe)Pro (Pro)-Aib sequence discussed above, the helical (III′) β-turn is observed only for the (αMe)Pro-Ala dipeptide.

The pyrrolidine rings of the (αMe)Pro residues are found in a conformation close to the ³T₄ (twist; C^β-exo, C^γ-endo)46 disposition in the D-(αMe)Pro-D-Ala, D-(αMe)Pro-L-Ala, and D-(αMe)Pro-Aib dipeptides, whereas a conformation intermediate between the ⁴T₃ (twist) and the E₃ (envelope) is seen in the L-Ala-D-(αMe)Pro dipeptide. More specifically, the puckering parameters47 are as follows: q₂ = 0.373(3) Å and φ₂ = 85.6(4)° for the homochiral (αMe)Pro-Ala dipeptide; q₂ = 0.396(9) Å and φ₂ = 96.5(8)° for the heterochiral (αMe)Pro-Ala dipeptide; q₂ = 0.370(7) Å and φ₂ = 260.3(7)° for the heterochiral Ala-(αMe)Pro dipeptide; and q₂ = 0.366(4) Å and φ₂ = 85.0(5)° for the (αMe)Pro-Aib dipeptide.

The packing modes of the four structures are each characterized by the occurrence of a single type of intermolecular H-bond involving the NH group of either the Ala or the Aib residue as the donor, and the O2 carbonyl oxygen atom as the acceptor. In particular, in Ac-D-(αMe)Pro-D-Ala-NHiPr the intermolecular H-bond occurs betweeen the N2H group and the O2 atom of a (1 − x, −½ + y, ½ − z) symmetry related molecule. The same groups are also intermolecularly H-bonded in the packing mode of the heterochiral dipeptide Ac-D-(αMe)Pro-L-Ala-NHiPr, although in this latter case the acceptor atom is defined by the symmetry operator (1−x, −½ + y, 1 − z). In the structure of iBu-L-Ala-D-(αMe)Pro-NHiPr the N1H group is H-bonded to O2 (symmetry equivalence: −1 + x, y, z). In the packing mode of the (αMe)Pro-Aib dipeptide the intermolecular H-bond connects the N2H group with the (−x, −½ + y, −½ − z) symmetry equivalent of O2. All of these intermolecular H-bonds are characterized by N…O separations between 2.917(5) and 2.992(4) Å, and by NH…O angles in the range 137–161°.

CONCLUSIONS

In this study, we demonstrated by an X-ray diffraction analysis that the (αMe)Pro residue, due to its C^α,α-gem-dialkyl effect, largely prefers to explore the 3₁₀/α-helical region of the ϕ, ψ conformational space. However, in partial contradiction with an anticipation put forward when the 3D-structural studies of peptides based on C^α-methylated α-amino acids were still in their infancy,48 we have clearly highlighted the Janus helical nature of (αMe)Pro in that it can also fold in the semi-extended [type-II poly(Pro)_n helix] conformation. Other (αMe)Pro 3D-structural properties disclosed in this work are: (i) an extremely high tendency (remarkably higher than that of Pro itself) to promote a β-turn conformation, and (ii) the dramatic restriction of the preceding tertiary amide bond (ω torsion angle) to the trans (180°) rotamer disposition. We are currently completing the picture of the preferred conformations of the terminally blocked (αMe)Pro dipeptide amides by expanding the results reported in Table II to the Aib-(αMe)Pro and homochiral Ala-(αMe)Pro sequences.