Solvent and side-chain contributions to the two-cis ⇄ all-trans equilibria of cyclic hexapeptides,cyclo(Xxx-Pro-D-Yyy)2

Cyclic hexapeptides of the type cyclo(L -Xxx-L -Pro-D -Yyy)₂ or cyclo(L -Xxx-L -Pro-Gly)₂ exist in solution predominantly in two forms of C₂ average symmetry, one with all-trans peptide bonds and generally well-established conformation, and another with both Xxx-Pro peptide bonds cis. We have been measuring the thermodynamic parameters of this equilibrium using carbon and proton nmr spectroscopy. Data have been obtained for peptides in which Yyy = Gly, D -Ala, or D -Phe, and Xxx = Gly, L -Ala, L -Leu, and L -Val. In a given solvent, stability of the all-trans form decreases (ΔG⁰ increases) as Xxx is changed through the series Gly, L -Ala-, L -Leu, and L -Val, consistent with expected increasing repulsion between the Xxx side chain and the proline δ methylene across the trnas Xxx-Pro bond. Also, for a given set of side chains, the stability of the all-trnas form increases as the polarity of the solvent decreases, consistent with models in which all C?O and N? H groups are accessible for solvation in the two-cis form, but two C?O and two N? H groups are somewhat sequestered in the all-trans form. With the available data it is not possible to identify pure intramolecular (solvent-independent) or pure peptide-bond solvation (side chain-independent) terms in ΔH° or ΔS°, although trends are discernible.     

Experimental investigations on the backbone folding of proline-containing model tripeptides

Some proline-containing tripeptides with the general formulas R⁰CO-L -Pro-X-NHR³ (X = Gly,Sar,L -Ala,D -Ala) and R⁰CO-X-L -Pro-NHR³ (X = Gly,L -Ala,D -Ala) have been investigated in solution by ir and ¹H-nmr spectroscopies. Their favored conformational states depend mainly on both the primary structure and the chiral sequence of the molecules. In inert solvents the βII-folding mode is the most favored conformation for the L -Pro-D -Ala and L -Pro-Gly tripeptides, while the βII′-turn is largely preferred by D -Ala-L -Pro derivatives. Under the same conditions only about one-third of the whole conformers of L -Pro-L -Ala molecules adopts the βI-folding mode. Semiopened C₇C₅ and C₅C₇ conformations are appreciably populated in the L -Pro-L -Ala sequence, on the one hand, and in the Gly-L -Pro and L -Ala-L -Pro derivatives, on the other hand. In L -Pro-Sar and X-L -Pro models, the cis–trans isomerism around the middle tertiary amide function is observed. Thus cis L -Pro-Sar and L -Ala-L -Pro conformers are folded by an intramolecular i + 3 → i hydrogen bond, whereas cis D -Ala-L -Pro and Gly-L -Pro molecules accommodate an open conformation. In dimethylsulfoxide the βII- and βII′-folding modes are not essentially destabilized, as contrasted with the βI conformation, which is less populated. In water solution all the above-mentioned conformations, with the possible exception of the βII′-folding mode for D -Ala-L -Pro molecules, seem to vanish. Solute conformations are also compared with the crystal structures of four proline-containing tripeptides.     

Structural investigation of poly-L,D-proline, a synthetic ion channel across bilayer membranes: crystal structure and molecular conformation of two tetra-D,L-proline derivatives

The crystal structures and molecular conformations of two tetraproline derivatives with alternating configurations Boc(D -Pro,L -Pro)₂OH and Boc(D -Pro,L -Pro)₂OCH₃ are investigated in connection with the ability of the homologous polymer to selectively increase (as an ion channel) the ion permeability across bilayer membranes. Both tetramers are characterized by the cis-trans alternating conformation of the peptide bonds, which formally transforms in a turn of the poly-D ,L -proline channel after a cis-trans change of the central peptide residue.     

Cyclic peptides. XVIII. 13C spin-lattice relaxation times of (X-pro-Y)2 cyclic hexapeptides

¹³C spin-lattice relaxation times (T₁'s) of four cyclic hexapeptides of sequence, (X-L -Pro-Y)₂, are reported. The T₁'s of the protonated carbons, which undergo dipolar relaxation, are interpreted qualitatively in terms of the overall tumbling motion of the molecule and in terms of internal motion. It is found that three of the cyclic hexapeptides, those which adopt all-trans β-conformers, tumble isotropically and appear to lack internal motion in the peptide backbone. The method of Torchia and Lyerla was applied to these compounds in order to compare the mobility of the proline rings. The results show that the sequence and particular type of β-turn present affect the internal motion of the Pro ring. Data on a fourth cyclic hexapeptide, which occurs in a conformation with two-cis X-Pro bonds, suggests that internal motion of the backbone contributes an additional frequency component to the motion of the Y residue α-carbons. A consideration of the mobility of the proline rings in the conformer with two-cis peptide bonds revealed that they are significantly more rigid in the two-cis structure than in the all-trans.     

Conformations of oligoprolines with different configurational sequences and their association complexes with alkali and alkali-earth ions

To clarify the possible molecular mechanism of cation unit conductance of poly(D ,L -proline) across lipidic membranes, the structure and conformation of tetraproline derivatives with different configurational sequences in solid state, as well as in solution and in the presence of alkali and alkali-earth ions, were investigated using x-ray analysis, CD, and nmr spectroscopy. The tetraproline derivatives Boc(D -Pro-L -Pro-L -Pro-D -Pro)OBg and Boc(L -Pro)₄OBg show very different conformational versatilities in solutions as well as different propensities to form complexes with Ca²⁺ ion. This is interpreted, based on previous evidence, as due to different abilities to form ion complexing cavities. © 1998 John Wiley & Sons, Inc. Biopoly 45: 257–267, 1998     

Cation binding cyclic peptides composed of imino acid residues

Cyclo(L -Pro-Sar)_n (n = 2–4) with moderate flexibility and hydrophobicity of molecular structure was synthesized, and the characteristics of these cyclic peptides and their metal complexes in acetonitrile were investigated in connection with the residual properties using ¹³C-nmr measurements. The cyclic tetrapeptide cyclo(L -Pro-Sar)₂ showed a sterically hindered phenomenon in acetonitrile in which the amide backbone adopted a cis-trans-cis-trans sequence. The cyclic hexapeptide cyclo(L -Pro-Sar)₃ existed as a mixture of several conformers whose interconversion is slow on the nmr time scale, including cis-cis-trans and/or cis-trans-trans arrangement of the Sar-Pro bond. Finally, it was demonstrated that the cyclic octapeptide cyclo(L -Pro-Sar)₄ behaved as a mixture of multiple conformers which allowed for cis-trans isomerism about the Pro-Sar peptide bond, of which 20–30% had the all-cis Sar-Pro bond isomer and the remaining 70–80% had one (or more) cis Sar-Pro bond isomer. ¹³C-nmr spectra also demonstrated that cyclo(L -Pro-Sar)_n (n = 3,4) formed a 1:1 ion complex whose conformation was characterized by an all-trans peptide bond in the presence of excess metal salt. Cation binding studies, using CD measurements, established that the ion selectivity of cyclo(L -Pro-Sar)₄ in acetonitrile decreased in the order, Ba²⁺ > Ca²⁺ > Na⁺ > Mg²⁺ > Li⁺.     

Conformation of the cyclic tetrapeptide dihydrochlamydocin. Iabu-L-Phe D-Pro-LX, and experimental values for 3 leads to 1 intramolecular hydrogen bonds by X-ray diffraction.

Chlamydocin, Iabu-L -Phe-D -Pro-L X, is a naturally occurring cyclic tetrapeptide that exhibits high cytostatic activity. The conformation of the peptide ring, as well as the stereo configuration in the vicinity of the epoxide ring, have been established by a single-crystal X-ray study of dihydrochlamydocin: C₂₈H₄₀N₄O₆·H₂O. It crystallizes in the monoclinic space group P2₁ with a = 12.616(6) Å, b = 12.355(6) Å, c = 9.442(5) Å, and β = 99.5(1)°. The structure was solved by the symbolic addition procedure for phase determination followed by the tangent formula method of phase refinement. This structure represents the first cyclic tetrapeptide in which all four peptide units have been found in the trans conformation; however, each peptide unit is significantly nonplanar with ω angles deviating by 14–24° from the ideal value of 180°. This molecule contains two intramolecular 3 → 1 hydrogen bonds and experimentally determined parameters for these seven-membered turns are presented.     

The X-Pro peptide bond as an nmr probe for conformational studies of flexible linear peptides

The equilibrium between the cis and trans forms of X-Pro peptide bonds can readily be measured in the ¹³C nmr spectra. In the present paper we investigate how observation of this equilibrium could be used as an nmr probe for conformational studies of flexible polypeptide chains. The experiments include studies by ¹³C nmr of a series of linear oligopeptides containing different X-L -Pro peptide bonds, with X = Gly, L -Ala, L -Leu, L -Phe, D -Ala, D -Leu, and D -Phe. Overall the study confirms that X-Pro peptide bonds can generally be useful as ¹³C nmr probes reporting the formation of nonrandom conformation in flexible polypeptide chains. It was found that the cis–trans equilibrium of X-Pro is greatly affected by the side chain of X and the configuration of the α-carbon atom of X. On the basis of these observations some general rules are suggested for a practical applications of the X-Pro nmr probes in conformational studies of polypeptide chains.     

Influence of D and L amino-acid residues on the conformation of peptides in solution: A carbon-13 nuclear magnetic resonance study of cyclo(prolyl-leucyl)

The ¹³C chemical shifts and spin-lattice relaxation times are reported for cyclo(L -Pro-L -Leu) and cyclo(L -Pro-D -Leu). The chemical shifts of the D and L leucyl residues in the cyclic peptides differ from each other by 1.8 and 3.6 parts per million for the α and β carbons, respectively. The α-carbons of the prolyl residues differ by 1.0 ppm as a consequence of proximity to a D or an L leucyl residue. The ¹³C spin-lattic relaxation time(T₁) of the prolyl residues, but not the leucyl residues, in both compounds are indicative of difference in conformational equilibria within the pyrrolidine ring in the L -L isomer as compared to the L -D isomer. Anisotropic overall molecular reorientation is not responsible for the differences observed in the T₁ values. The differences in T₁ values and chemical shifts between cyclo(L -Pro-L -Leu) and cyclo(L -Pro-D -Leu) appear to result from a difference in conformations of the two diketopiperazine rings.     

13C- and 1H-nmr evidence for all-cis peptide bonds in cyclic tetrapeptide cyclo(L-Pro-Sar)2

Conformations of the cyclic tetrapeptide cyclo(L -Pro-Sar)₂ in solution were studied by ¹H- and ¹³C-nmr spectrometry and model building. The nmr data provide definite evidence that this cyclic peptide exists chiefly in two conformations, namely, a C₂-symmetric conformation and an asymmetric structure. The former was demonstrated to be predominant in polar solvents (100% in Me₂SO-d₆). This structure contains all cis-peptide bond linkages and all trans′ Pro C^α?CO bonds. It represents the first cyclic tetrapeptide in which all four peptide bonds have been found in the cis-conformation. As the polarity of the solvent decreases, the population of C₂-symmetric conformers decreases (88% in CD₃CN and 65% in CDCl₃). At the same time, a minor asymmetric conformer, characterized by cis-cis-cis-trans peptide bond sequences (two cis Sar-Pro bonds, one cis Pro-Sar bond, and one trans Pro-Sar bond), is seen to increase (9% in CD₃CN and 30% in CDCl₃). A proposed predominant conformation in solution for cyclo(L -Pro-Sar)₂ was compared with a crystal structure, as reported in an accompanying paper. Both structures show striking overall similarities.     

β-Alanine containing cyclic peptides with turned structure: The “pseudo type II β-turn.” VI

In the present paper we describe the synthesis, purification, single crystal x-ray analysis, and nmr solution characterization, combined with restrained molecular dynamic simulations, of the cyclic hexapeptide cyclo-(L -Pro-L -Phe-β-Ala)₂. The peptide was synthesized by classical solution methods and the cyclization of the free hexapeptide was accomplished in good yields in diluted methylene chloride solution using N,N-dicyclohexyl-carbodiimide. The compound crystallizes in the monoclinic space group P2₁ from methanol-dichloro-methane solution. The two identical halves of the molecule adopt in the solid state two different conformations. One β-Ala-L -Pro peptide bond is trans, while the second is cis. The molecule is present in dimethylsulfoxide d₆ solutions as a mixture of conformational families. One of these corresponds to a C₂ symmetrical molecule with both β-Ala-Pro cis peptide bonds, while the second major conformation is very similar to that observed in the solid state. All Pro-Phe segments, both in the solid state and the symmetrical and unsym-metrical solution conformations, display ?,ψ angles close to that of position i + 1 and i + 2 of type II β-turns. In addition, the segments preceeded by a trans β-Ala-Pro peptide bond are characterized by a typical i ← i + 3 hydrogen bond, which is absent in the conformer containing a cis β-Ala-Pro peptide bond. The latter conformation corresponds to a new structural domain we define as the “pseudo type II β-turn.” © 1994 John Wiley & Sons, Inc.     

Conformational properties of Pro-Pro sequences. I. Crystal structures of two dipeptides with L-Pro-L-Pro and L-Pro-D-Pro sequences

The crystal structures of Bu^tCO-L -Pro-L -Pro-NHMe, H₂O (1: monoclinic; P2₁; a = 6.662, b = 11.067, c = 12.205 Å; β = 96.28°) and Bu^tCO-L -Pro-D -Pro-NHMe (2: monoclinic; P2₁; a = 10.770, b = 15.039, c = 11.325 Å; β = 110.00°) have been solved by x-ray diffraction. Structure 1 accommodates an open disposition with intermolecular interactions involving the water molecule, while 2 is βII-folded by an intramolecular i + 3 → i hydrogen bond. In both derivatives, small thermal parameters are indicative of fairly fixed conformations for the proline rings. Comparison between conformations of either isolated or adjacent L -Pro residues in the crystal structures of unstrained oligopeptides shows that the conformational properties of L -Pro-L -Pro sequences are probably a simple combination of those found for isolated L -Pro residues.     

Nmr studies of the rates of proline cis–trans isomerization in oligopeptides

¹H-Nmr was used to measure the rate of cis–trans interconversion of X-Pro bonds in linear and cyclic oligopeptides. k(cis → trans) = 2.5 × 10^?3 s^?1 at 25°C was found for the zwitterionic form of H-Ala-Pro-OH, in good agreement with earlier measurements. Replacement of Ala by Phe, Tyr, or Trp resulted in a 10-fold slower interconversion rate, whereas after substitution of Ala by His or Glu, the rate decreased only slightly. Independent of the residues X, the interconversion rate was increased by a factor of ca. 20 when the peptide chain was elongated by addition of Ala to the C-terminal Pro. An additional increase by a factor of 6 was observed when going from the protected linear peptide CF₃CO-Gly-Gly-Pro-Ala-OCH₃ to the closely related cyclic compound c[-Gly-Gly-Pro-Gly-Ala-]. These data are evaluated with regard to their possible use in future studies on the role of X-Pro cis–trans isomerization in the kinetics of protein folding.     

Crystal structure and conformation of a cyclic tetrapeptide cyclo(L-Pro-Sar)2 containing all-cis peptide units

The crystal structure and conformation of the synthetic cyclic tetrapeptide, cyclo(L -Pro-Sar)₂, was determined by x-ray analysis. The peptide crystallizes in the orthorhombic space group P2₁2₁2₁ with cell parameters a = 9.277(1), b = 12.884(1), and c = 15.581(2) Å. The crystal structure was solved by the symbolic addition procedure for direct phase determination and least-squares refinement using 1796 reflections, which led to the final R value of 0.043. This structure provides the first example observed in a crystal of a cyclic tetrapeptide in which all four peptide units have been found in the cis conformation with ω angles deviating slightly by 2°–10° from the ideal value of 0°. It was also found that the two Pro C^α-CO single bonds assumed a trans′ (ψ = 159.6° and 158.4°) conformation. Adjoining average planes of the peptide groups fall at nearly right angles to each other. The pyrrolidine ring conformations of the two prolyl residues are in the envelope form, with C^γ carbon out of the least-squares planes for the remaining four atoms.     

Conformational properties of the sequential polyproline analogs poly(Pro-Aze-Pro) and poly(Aze-Pro-Aze)

The lack of the positive band at around 226 nm in the CD spectra of poly(prolyl-azetidine-2-carbonyl-proline) in trifluoroethanol and of poly(azetidine-2-carbonyl-prolyl-azetidine-2-carboxylic) acid in F₃EtOH and water, the hyperchromism of the absorption maximum at about 202 nm, and the extremely small intensity of the C^β-Pro, C^γ-Pro, and C^β-Aze signals for the cis peptide bonds in the ¹³C nmr spectrum of poly(Pro-Aze-Pro) in F₃EtOH indicate that both polyproline analogs exist as disordered chains in this solvent, the trans peptide group being maintained. The disordering of the chains is attributed to an increase in the accessible range of ψ due to the reduced dimensions of the square ring of L -azetidine-2-carboxylic acid residue relative to the pyrrolidine ring of proline and to strong interactions of the haloalcohol with the peptide groups of the chains.     

Synthesis and conformation of the cyclic octapeptides cyclo(Phe-Pro)4, cyclo(Leu-Pro)4, and cyclo[Lys(Z)-Pro]4

Cyclic octapeptides, cyclo(X-Pro)₄, where X represents Phe, Leu, or Lys(Z), were synthesized and their conformations investigated. A C₂-symmetric conformer containing two cis peptide bonds was found in all of these cyclic octapeptides. The numbers of available conformations due to the cis–trans isomerization of Pro peptide bonds depended on the nature of the solvent and X residue: they decreased in the following order: cyclo[Lys(Z)-Pro]₄ > cyclo(Leu-Pro)₄ > cyclo(Phe-Pro)₄ in CDCl₃. ¹³C spin-lattice relaxation times (T₁) of these cyclic octapeptides were measured, and the contribution of segmental mobility to T₁ was found to vary with the nature of the X residue.     

Cis–trans peptide-bond equilibria in cyclic hexapeptides

Data are presented on the position of the equilibria of cyclo(Xxx-Pro-Yyy)₂ backbones between forms with two cis Xxx-Pro peptide bonds and forms with only trans peptide bonds. These data are interpreted in terms of two factors: a solvent-independent steric interaction between the Xxx and Pro side chains, and the ability of solvent to influence the transannular electrostatic interaction between N? H and C?O groups of the Xxx units in the all-trans form.     

β-Alanine containing peptides: γ-Turns in cyclotetrapeptides

In the present paper we describe the synthesis, purification, single-crystal x-ray analysis, solution conformational characterization, and conformational energy calculations of the cyclic tetrapeptide cyclo- (β-Ala-L -Pro-β-Ala-L -Val). The peptide was synthesized by classical solution methods and the cyclization of the free tetrapeptide was accomplished in good yields in diluted methylene chloride solution using N,N-dicyclohexyl-carbodiimide. The compound crystallizes in the monoclinic space group P2₁ from ethanol with two independent molecules in the unit cell. All peptide bonds are trans. The nmr molecular conformation in the acetonitrile solution as well as that derived from the molecular dynamic simulation in vacuo is quite different from those observed in the solid state and is very similar to that previously observed for the parent compound cyclo-(β-Ala-L -Pro-β-Ala-L -Pro). © 1993 John Wiley & Sons, Inc.     

Nmr and computer-aided studies of the three interchanging stereoisomers of the cyclic hexapeptide cyclo[-Pro1-Gly2-Glu3(OBzl)-Pro4-Phe5-Leu6-]: Unexpected observation of a cis isomer of a secondary amide peptide bond in the presence of two trans proline peptide bonds

The cyclic hexapeptide cyclo[-Pro¹-Gly²-Glu³(OBzl)-Pro⁴-Phe⁵-Leu⁶-] ( 1 ; OBzl: benzyl ester) was modeled and synthesized to be used as a chiral site for the separation of enantiomers. Total correlation spectroscopy and nuclear Ovehauser effect spectroscopy spectra of the peptide in CDCl₃ showed the presence of three stereoisomers. The two dominant stereoisomers 1a and 1b exchanged chemically with each other, while the minor stereoisomer 1c exchanged exclusively with the stereoisomer 1b . Stereoisomer 1a had two cis proline peptide bonds while stereoisomer 1b had all-trans peptide bonds. The stereoisomer 1c had, for nonstrained peptides, an unusual cis phenylalanine peptide bond while both proline peptide bonds were trans. © 1993 John Wiley & Sons, Inc.     

Studies of peptide conformation: Backbone folding of tetrapeptide derivatives in methanol and water

Derivatives of tetrapeptide sequences considered likely to form β-turns were investigated by the study of their proton magnetic resonances in methanol and in water. Differential broadening of N—H resonances by an added nitroxyl was used to indicate the presence of the sequestered N—H proton expected in β-turn conformations. Transfer of magnetic saturation from solvent water protons to N—H protons was also examined. The evidence is consistent with significant contributions by β-turn-like backbones to the conformational averages in methanol of the sequences Gly-L -Pro-D -Val-Gly, D (or L )-Val-L -Pro-Gly-Gly, and Gly-L -Pro-L -Asn-Gly, but not the sequence Gly-D -Ala-L -Val-Gly. It is suggested that a Type I turn, Likely in Gly-L -Pro-L -Asn-Gly derivatives, is characterized by sequestered N—H protons of both the third and fourth residues. For all of the peptide derivatives, save possibly Ac-L -Val-L -Pro-Gly-Gly-NHNH₂, contributions from folded structures in water are not detectable by line-broadening experiments. However, the transfer of saturation experiments may be interpreted as indicating some degree of chain folding in water.