Crystal structure and conformation of polypeptides: L-leucylglycylglycylglycine

Crystals of L-leucylglycylglycylglycine, LGGG (C₁₂H₂₂N₄O₅), grown from an ethanol-water solution, are orthorhombic, space groups P2₁2₁2₁, with unit cell dimensions (at 22 ± 3°) a = 9.337(1), b = 10.995(1), c = 15.235(1)Å, v = 1563.4 ų, Z = 4 with a density of D_obs= 1.29 g-cm^-3 and D_calc= 1.279 g°cm^-3. The crystal structure was solved by the application of direct methods and refined to an R value of 0.029 for 1018 reflections with I ± 2s?. The molecule exists as a zwitterion in the crystal. The trans peptide backbone takes up a folded conformation at the middle glycylglycyl link accompanied by a significant nonplanarity up to Δω of 8° at the middle peptide and is relatively more extended at the two ends. The molecules are linked together intermolecularly in an infinite sequence of head to tail 1–4′ hydrogen bonds, as is typical of charged peptides. It is interesting to note that while glycylglycylglycine takes up an extended β-sheet conformation, addition of Leu to the N-terminal results in a bent conformation.     

Ring conformation of cyclic octapeptide cyclo (L -Pro-Sar)4 in the crystalline state

A single-crystal X-ray diffraction analysis has been made of the structure of the cyclic octapeptide cyclo(l -Pro-Sar)₄. The material [C₃₂H₄₈O₈N₈ · (21/4) H₂O° (1/2) CH₃OH, M_r = 799.43] crystallizes in the monoclinic space group C2 with cell dimensions a = 14.544 (3), b = 11.902 (2), c = 14.064 (3), and β = 122.26 (2)° (Λ = 1.54178 Å, T = 293 K). The final R value for the 1980 observed reflections is 0.079. The ring conformation has the peptide bond sequences of cis-cis-trans-trans-cis-cis-trans-trans (Pro-Sar-Pro peptide bond linkages are cis-cis -or trans-trans). The pyrrolidine rings in the four proline residues take an envelope form in which the γ-carbon atom deviates from the plane of the remaining four atoms in the ring.     

The crystal structure of Boc-Pro-Val-Gly-NH2, with a remark on the conformation of the valyl residues in peptides

The crystal structure of Boc-Pro-Val-Gly-NH₂ has been determined: monoclinic; P2₁; a = 9.331 (3) Å, b = 9.532 (4), c = 23.080 (9), β= 91.33 (3)R, Z = 4; R = 0.053 for 3400 reflections with ˙Fo˙,>α(Fo). There are two independent but very similar molecules in the crystal. The peptide main chains are in an extended form, and packed in two kinds of antiparallel β sheets, the (φ, Φ) angles of the central Val residues are (-156°, 146°) and (-139°, 155°), and the mean length of the N- H . 0 hydrogen bonds in the sheets is 2.965 Å. A detailed study of the conformations of the Val residues in oligopeptide crystals shows that the preferred conformation of Val in peptides is: the (φ, Φ) angles close to those of the antiparallel β sheet, and C^γ1 and C^γ2, against N with respect to the C^α– C^β bond, at either (trans, gauche) or (-gauche, gauche). The mean π(NC^αC') angle of such Val residues is 107.9(9)°. A twisting in the β sheets is also discussed.     

Conformation of a cyclic decapeptide analog of a repeat pentapeptide sequence of elastin: cyclo-bis(valyl-prolyl-alanyl-valyl-glycyl)

The conformation of a cyclic decapeptide analog of a repeat sequence of elastin has been determined in the crystalline state using X-ray crystallographic techniques. Tetragonal crystals were grown from a solution of the decapeptide in water; space group P4₂2₁2, a = 19.439(2) & c = 13.602(1) Å, with four formula units (C₄₀H₆₆N₁₀O₁₀·4H₂O) per unit cell. The cyclic decapeptide in the crystal exhibits exact twofold symmetry. The asymmetric unit contains one pentapeptide and two water molecules for a total of 32 nonhydrogen atoms. The structure has been determined by the application of direct methods and refined by full-matrix least squares to an R index of 0.053 for 2272 reflections with intensities greater than 2σ(I). The backbone conformation of the asymmetric pentapeptide can be described as consisting of a double β bend of Type III-I. The Type III turn has Pro (ρ= -59.3°, Ψ= -26.8°) and Ala (ρ= -65.9°, Ψ= -23.1°) at the corners while Type I turn has Ala (ρ= -65.9°, Ψ= -23.1°) and Val (ρ= -98.9°, Ψ= 8.3°) as the corner residues. The cyclic decapeptide has two such double bends linked together by Gly-Val bridges.     

The preferred solid-state conformation of (αMe)Trp peptides

The two Z-l -Ala-d l -(xMe)Trp-NH₂ diastereomeric dipeptides were synthesized from (Z-l -Ala)₂O and H-dl -(xMe)Trp-NH₂. The latter racemate, prepared by phase-transfer catalyzed alkylation of the N^α-benzylidene derivative of alanine amide followed by acidic hydrolysis of the resulting Schiff base, was characterized by X-ray diffraction. The molecular and crystal structure of Z-l -Ala-l -(αMe)Trp-NH², separated from its diastereomer by silica-gel column chromatography, was determined by X-ray diffraction analysis. Both independent molecules in the asymmetric unit of the dipeptide adopt a type-II β-bend conformation. However, only the more regularly folded conformation of molecule B is stabilized by a 1←4 C=O…H—N intramolecular H bond. The present results indicate that: (i) the Cα-methylated (αMe)Trp residue is a strong β-bend and helix former, and (ii) the relationship between (αMe)Trp chirality and helix screw sense tends to be opposite to that of protein amino acids. The implications for the use of the (αMe)Trp residue in designing conformationally restricted analogs of bioactive peptides are briefly discussed. ©Munksgaard 1995.     

Variability in the backbone conformation of cyclic pentapeptides

All the peptide bonds in cyclic(Gly-LPro-DPhe-Gly-LAla) are in the trans conformation; however, the peptide bond C₅'-N₁ is twisted by 19° from planarity (ω₅= - 161°). A Type II β-turn encompasses the LPro-DPhe residues. Carbonyl oxygens O₂, O₄ and O₅ are directed to the same side of the average plane through the backbone ring and they form hydrogen bonds with N₃, N₅ and N₁, respectively, in adjacent molecules in a stacked column where the adjacent molecules are related by one translational unit. The conformation of the backbone is different from that established in other molecules with the DLDDL chirality sequence. The P2₁ cell contains two molecules of C₂₁H₂₆N₅O₅ with a = 4.836(2) A, b = 18.346(8) A, c = 12.464(5) A and β= 100.05(4)°. The R factor for 1382 data with ¶F₀¶ > 1 ¶ is 7.0%.     

Synthesis,crystal structure and molecular conformation of Nα‐Boc‐l‐leucyl‐(Z)‐β‐(3‐pyridyl)‐α,β‐ dehydroalanyl‐l‐leucine methyl ester*

Abstract: A protected tridehydropeptide containing (Z)‐β‐(3‐pyridyl)‐α,β‐dehydroalanine (Δ^Z3Pal) residue, Boc‐Leu‐Δ^Z3Pal‐Leu‐OMe ( 1 ), was synthesized via Erlenmeyer azlactone method. X‐ray crystallographic analysis revealed that the peptide 1 adopts an extended conformation, which is similar to that of a Δ^ZPhe analog, Boc‐Leu‐Δ^ZPhe‐Leu‐OMe ( 2 ).     

Unusual intramolecular hydrogen bonding in cycloamanide A,cyclic (LPro-LVal-LPhe-LPhe-LAla-Gly)

The naturally occurring cyclic hexapeptide, cycloamanide A, has only one intramolecular hydrogen bond. It is a 4 ← 1 type that encompasses the LPhe-LAla sequence in which the experimentally determined ø, ← values are + 54d?, – 118d? and – 88d?, – 4d?, respectively. Even though the chirality is L, L, the ø, ← values are characteristic for a D, L β-bend, Type II°. The conformation of the molecule was established by a crystal structure determination using X-ray diffraction analysis. Cycloamanide A (C₃₃H₄₂N₆O₆←. 4H₂O) crystallizes in space group P2₁2₁2₁ with cell parameters a = 13.307(2) Å, b = 24.820(4) Å and c = 11.231(1) Å.     

Force-field dependence of Boltzmann weighting factors on predicted π-π*circular dichroic spectra of cyclo(Gly-Pro-Gly)2

Semi-empirical energy calculations were performed for published conformations of cyclo(Gly-Pro-Gly)₂ using different force fields (DISCOVER cvff and cff91, AMBER, and CHARMM). The resulting potential energies were then used to create Boltzmann weighting factors for an ensemble of cyclo(Gly-Pro-Gly)₂ structures. The dipole interaction model was used to predict π-π* circular dichroic spectra (CD) for the individual structures of cyclo(Gly-Pro-Gly)₂. The Boltzmann weighting factors were applied to the individual spectra so that a composite spectrum was constructed to represent a CD arising from a collection of different structures in solution. Weighting factors determined from different force fields were compared. Boltzmann-weighted spectra better resembled the experimental CD than any calculated spectrum using only a single conformation of cyclo(Gly-Pro-Gly)₂. The structures most heavily weighted contained at least one type I β-turn.     

Crystal structures of a D-residue containing tetrapeptides 1. tert-Boc-D-Valyl-alanyl-leucyl-alanyl methoxide,butanol solvate

The crystal structure of a heterochiral peptide, viz. Boc-D-Val-Ala-Leu-Ala-OMe, with a D-residue in the beginning of the sequence has been determined (a= 9.464(5), b= 35.615(5), c= 9.703(2) Å, space group P2₁2₁2, Z= 4, R= 0.09). The peptide is in the extended β-conformation and the packing is stabilised by four N—H. O hydrogen bonds in an antiparallel β-sheet arrangement. The solvent molecule is disordered and does not have any specific interactions with the peptide.     

Crystal and molecular structure of the dehydropeptide Ac-ΔPhe-Val-ΔPhe-NH-Me

The dehydropeptide Ac-ΔPhe-l -Val-ΔPhe-NH-Me, containing two dehydrophenylalanine (ΔPhe) residues, crystallizes from methanol/water in space group P2₁2₁2₁ with a= 12.622 (1), b= 12.979 (1), and c= 15.733 (1) Å. In the solid state, the molecular structure is characterized by the presence of two intramolecular hydrogen bonds which form two consecutive β-bends. The (φ,Ψ) torsion angles of the three residues are very similar and close to the standard values of type III β-bends, so the molecular conformation corresponds to an incipient right-handed 3₁₀ -helix, only slightly distorted. In the crystal, the molecules are linked by head-to-tail hydrogen bonds, thus forming continuous helical columns packed in antiparallel mode. There are no lateral hydrogen bonds; the only interactions are hydrophobic contacts between the apolar side chains of neighboring helical columns.     

Hydration and distortion of peptide helices in crystals. α-Helical structure of a dodecapeptide,Boc-(Ala-Leu-Aib)4-OMe

The dodecapeptide Boc-(Ala-Leu-Aib)₄-OMe crystallized with two independent helical molecules in a triclinic cell. The two molecules are very similar in conformation, with a 3₁₀-helix turn at the N-terminus followed by an α-helix, except for an elongated N(7)…O(3) distance in both molecules. All the helices in the crystal pack in a parallel motif. Eleven water sites have been found in the head-to-tail region between the apolar helices that participate in peptide-water hydrogen bonds and a network of water-water hydrogen bonds. The crystal parameters are as follows: 2(C₅₈H₁₀₄N₁₂O₁₅) +ca. 10H₂O, space group P1 with a= 12.946(2), b= 17.321(3), c= 20.465(4)Å, α=103.12(2), β= 105.63(2), γ= 107.50(2)?, Z= 2, R= 10.9% for 5152 data observed > 3σ(F), resolution 1.0 Å. In contrast to the shorter sequences [Karle et al. (1988) Proc. Natl. Acad. Sci. USA 85 , 299–3031 and Boc-(Ala-Leu-Aib)₂-OMe [Karle et al. (1989) Biopolymers 28 , 773–781], no insertion of a water molecule into the helix is observed. However, the elongated N—O distance between Ala⁷ NH and Aib³ CO in both molecules (molecule A, 3.40 Å; molecule B, 3.42 Å) is indicative of an incipient break in the helices.     

Synthesis,crystal structure and molecular conformation of the tBuCO-D,L-Ala-Δz-Phe-NhiPr α,β-unsaturated dipeptide

The crystal structure of the tBuCO-d,l -Ala-Δ^z-Phe-NHiPr dipeptide has been solved by X-ray diffraction. The peptide crystallizes in monoclinic space group P2JC with a = 13.445 (3) Å, b = 35.088 (4) Å, c = 14.755(3) Å, β= 116.73(1)°, Z = 12 and d_c= 1.151 g.cm^?3. The three independent molecules per asymmetric unit accommodate a βII-folded conformation, but only one of them contains the typical i + 3 → i interaction characterizing a β-turn. In the other two molecules, the N…O distance exceeds 3.2 Å, a value generally considered the upper limit for hydrogen bonds in peptides. In solution, the βII-turn conformation is largely predominant.     

Effect of aromatic amino acid substitutions in the 3-position of cyclic β-casomorphin analogues on μ-opioid agonist/δ-opioid antagonist properties*

The β-casomorphin-5 analog H-Tyr-c[-D-Orn-2-Nal-D-Pro-Gly-] (2-Nal = 2-naphthylalanine) was the first reported cyclic opioid peptide with mixed μ agonist/δ antagonist properties [R. Schmidt et al. (1994) J. Med. Chem. 37 , 1136-1144]. The 2-Na1³ residue in this peptide was replaced with benzothienylalanine (Bta) (3), His(Bz1) (4), Tyr(Bz1) (5), 4′-benzoylphenylalanine (Bpa) (6), 4′-benzylphenylalanine (Bzp) (7), thyrnine (Thy) (8), thyroxine (Thx) (9), 4′-biphenylalanine (Bip) (10), 4′-biphenylglycine (Bpg) (12) and 3,3-diphenylalanine (Dip) (14), and the in vitro opioid activity profiles of the resulting compounds were determined in μ and δ receptor-representative binding assays and bioassays. Analogues 3, 12 and 14 were full agonists in the μ receptor-representative guinea-pig ileum (GPI) assay and also were agonists in the δ receptor-representative mouse vas deferens (MVD) assay. The agonist effects of the latter compounds in the MVD assay were antagonized by the highly selective δ antagonist H-Tyr-Tic-Phe-Phe-OH (TIPP), indicating that they were triggered by δ receptor activation. The Bzp³- and Bip³-containing peptides 7 and 10 turned out to be μ antagonists against the μ selective agonist H-Tyr-D-Ala-Phe-Phe-NH₂, in the GPI assay. The other analogues were weak partial μ agonists which displayed remarkably decreased μ receptor affinity as compared to parent peptide 1. Compounds 4-10 were found to be δ antagonists in the MVD assay. Analogues 4 and 9 exhibited δ antagonist potency similar to that of parent peptide 1, while compounds 5-8 and 10 showed 3-12-fold higher δ antagonist potency against DPDPE and deltorphin I and, in most cases, increased δ receptor affinity. These results indicate that the & delta; receptor tolerates bulky aromatic side chains in the 3-position of cyclic β-casomorphin analogs with either δ agonist or δ antagonist properties. However, these compounds displayed drastically reduced μ receptor affinity in nearly all cases. © Munksgaard 1996.     

Structure of cyclic peptides: the crystal and solution conformation of cyclo(Phe-Phe-Aib-Leu-Pro)

A solid-state and solution conformation analyses of the cyclopentapeptide cyclo(Phe-Phe-Aib-Leu-Pro) has been carried out by X-ray diffraction and nuclear magnetic resonance techniques. The structure of the hexagonal crystals, grown from a methanol solution [a=b= 16.530(4) Å, c= 21.356(9) Å, space group P6₅, Z = 6], shows the presence of one intramolecular N-H?O=C hydrogen bond with the formation of a γ-turn (C₇). The Aib³ residue, at the center of the γ-turn, presents unexpected values of the torsion angles [φ= 70.5° and ψ= -73.8°], which have been observed only once before for this helicogenic residue. A cis peptide bond occurs between Leu⁴ and Pro⁵; all other peptide bonds are trans. The overall conformation for the cyclopentapeptide with one cis-peptide bond on one side and an intramolecular γ-turn on the opposite side results in an equatorial topology of the side-chains of the Phe¹, Phe² and Leu⁴ residues. Indeed, the C^α-C^βand C^β-C^γ bonds of these residues lie approximately in the mean plane of the cyclic ring system. The structure is compared with data in the literature on cyclic pentapeptides. In addition the Pro-Phe-Phe moiety shows a conformation similar to that observed in other larger cyclic bioactive peptides, which indicates a reduced number of conformations for this sequence. The solution study was carried out in three different solvent systems: chloroform, acetonitrile and methanol in the temperature interval 220–300 K. In all three solvents the room temperature spectra show that the peptide is conformationally nonhomogeneous. In acetonitrile at low temperatures it is possible to reduce the conformational equilibrium to two predominant conformers which differ for the cis-trans isomerism of the Leu⁴-Pro⁵ peptide bond.     

Synthesis of N-(4-aminobenzoyl)-γ-oligo (L-glutamic acid)s*

N-(4-aminobenzoyl)-γ-oligo (l -glutamic acid)s (6) containing from two to six glutamic residues have been prepared in solution using Nα-Boc-α-Bzl protections and isobutyl-chlorocarbonate activation. Key steps in the synthesis were the coupling of γ-oligo(α-benzyl l -glutamate) benzyl esters (1) with N-(4-benzyl-oxycarbonylaminobenzoyl)-l -glutamic acid α-benzyl ester (4) to blocked precursors of N-(4-aminobenzoyl)-γ-oligo (l -glutamic acid)s (5) and catalytic hydrogenolysis of 5 to 6. Elaboration of the required oligo γ-l -glutamate chains (1) was achieved step by step beginning with the coupling of glutamic acid dibenzylester with N-(t-butoxycarbonyl)-l -glutamic acid α-benzyl ester (2) to 3 followed by selective removal of the Boc from 3 with HCl-dioxane followed by coupling with 2.     

Multiple conformational equilibria of cyclic octapeptide,cyclo (L-Pro-Sar)4 in solution

Cyclo (l -Pro-Sar)₄ is asynthetic cyclic octapeptide composed of only N -substituted amino acids. The conformation of this peptide in different solvents was examined by ¹H-and ¹³C-n.m.r. spectroscopy, ¹H-n.m.r. data of this cyclic peptide demonstrated that multiple conformational equilibria take place in solution and they vary with solvent polarity. Three conformers are interconverting with each other in the nonpolar chloroform (CDCl₃); one C₄-symmetric conformer (49%) and two C₂-symmetric conformers (37% and 14%). While three C₂-symmetric (59%, 19%, and 18%) and one asymmetric conformer (4%) are detected to coexist in acetonitrile (CD₃CN), one largely populated C₂-symmetric one (97%) is favored in the polar dimethyl sulfoxide (Me₂SO-d ₆). N.m.r. measurement employing various strategy predicted the occurrence of trans-cis-cis-cis-trans-cis-cis-cis (two Sar-Pro bonds: trans) (tccctccc) peptide bond sequence in a predominant C₂-symmetric conformer of Me₂SO-d ₆ solution. In the same way, tccctccc and ctttcttt (two Sar-Pro bonds: cis) peptide unit arrangement was proposed for the first and the secondly populated conformer in CD₃CN, respectively. In CDCl₃ a conformer having tctctctc (four Sar-Pro bonds: trans) C₄-symmetric peptide bond sequence was deduced.     

Cystine peptides Antiparallel β-sheet conformation of the cyclic biscystine pep tide [Boc-Cys-Ala-Cys-NHCH3]2

The crystal structure analysis of the cyclic biscystine peptide [Boc-Cys¹-Ala²-Cys³-NHCH₃]₂ with two disulfide bridges confirms the antiparallel β-sheet conformation for the molecule as proposed for the conformation in solution. The molecule has exact twofold rotation symmetry. The 22-membered ring contains two transannular NH ? OC hydrogen bonds and two additional NH ? OC bonds are formed at both ends of the molecule between the terminal (CH₃)₃COCO and NHCH₃ groups. The antiparallel peptide strands are distorted from a regularly pleated sheet, caused mainly by the L-Ala residue in which φ=– 155° and ψ= 162°. In the disulfide bridge C^α (1)-C^β (1)-S(1)-(3′)-C^β(3′)-C^α(3′), S—S = 2.030 Å, angles C^β SS = 107° and 105°, and the torsional angles are –49, –104, +99, –81, –61°, respectively. The biscystine peptide crystallizes in space group C2 with a = 14.555(2) Å, b = 10.854(2) Å, c = 16.512(2)Å, and β= 101.34(1) with one-half formula unit of C₃₀H₅₂N₈O₁₀S₄· 2(CH₃)₂SO per asymmetric unit. Least-squares refinement of 1375 reflections observed with |F| > 3σ(F) yielded an R factor of 7.2%.     

Conformational versatility of the Nα-acylated tripeptide amide tail of oxytocin

The synthesis, physical and analytical characterization, and crystal-state structural analysis by X-ray diffraction of three analogues of the N^α-acylated tripeptide amide tail of oxytocin, each containing a cyclic C^{α, α-} disubstituted glycine at position 2, have been performed. The peptides arc Boc-L-Pro-Ac₃c-Gly-NH₂, Z-L-Pro-Ac₅c-Gly-NH₂ and Z-L-Pro-Ac₅c-Gly-NH₂. While the former is folded in a type-II β-turn conformation at the -L-Pro-Ac₃c- sequence, the two latter tripeptides form two consecutive (type-II, type-I′) β-turns. The Ac₅c- and Ac₆c-tripeptides are the first examples of such a highly folded structural combination in a position-2 analogue of the N^α-acylated -L-Pro-L-Leu-GIy-NH² sequence.