Characterization of bent helical conformations in polymorphic forms of a designed 18-residue peptide containing a central Gly-Pro segment.

An 18-residue sequence Boc-Aib-Val-Ala-Leu-Aib-Val-Ala-Leu-Gly-Pro-Val-Ala-Leu-Aib-Val-Ala-Leu-Aib-OMe (UK18) was designed to examine the effect of introducing a Gly-Pro segment into the middle of a potentially helical peptide. The crystal structures of two polymorphic forms yielded a view of the conformation of three independent molecules. Form 1 (space group P2(1)2(1)2(1,) a = 14.620Å; b = 26.506Å, c = 28.858Å, Z = 4) has one molecule in the asymmetric unit, with one cocrystallized water molecule. Form 2 (space group P2(1)2(1)2(1,) a = 9.696Å; b = 19.641Å, c = 114.31Å, Z = 8) has two molecules in the asymmetric unit with four cocrystallized water molecules. In Form 1, residues 1 to 18 adopt ϕ,ψ values that lie in the right-handed helical (α(R) ) region of the Ramachandran map. Two residues, Leu (8) (ϕ = -92.0°, ψ = -7.5°) and Leu (17) (ϕ = -94.7°, ψ = -1.7°) adopt conformations that deviate significantly from helical values. In Form 2, molecule A, residues 2 to 16 lie in the α(R) region of ϕ,ψ space, with Leu (8) (ϕ = -94.9°, ψ = -2.9°) deviating significantly from helical values. Aib (1) and Aib (18) adopt left-handed (α(L)) helical conformation. Significant distortion is observed at Leu (17) (ϕ = -121.3°, ψ = -31.3°). Molecule B, Form 2, adopts a right-handed helix over residues 1 to 17. In all three molecules, a distinct bend in the helix is observed, with the bend angle values varying from 40.8° to 58.9°.

SUBTLE CONTROL IN SOLUTION AND CRYSTAL STRUCTURES WITH WEAK HYDROGEN BONDS: THE UNUSUAL PROFILE OF DIMETHYL 3, 12-DIOXO-7, 8 DITHIA 4, 11-DIAZABICYCLO[12.2.2]OCTADECA-1(16), 14, 17-TRIENE 5, 10-DICARBOXYLATE (TDA1).

The structural features of the title compound were determined or examined by three diverse procedures: single crystal X-ray diffraction analysis, solution spectroscopic procedures and Quantum mechanical theoretical calculations. The conformational asymmetry of the macrocycle provides the opportunity to form one strong NH···OC intermolecular hydrogen bond, as well as, a number of weak CH···OC bonds. The interior of the macrocycle has short approaches for NH(...)π and NH···S. The many weak hydrogen bonds cooperate to form a very hard, robust crystal. Crystal parameters: C(18)H(22)N(2)O(6)S(2), P2(1)2(1)2(1), a = 5.108(1) Å, b = 18.948(4) Å, c = 21.029(3) Å, α = ß = γ = 90°. Quantum chemical calculations have provided a strong foundation for weak hydrogen bonds. Contrary to popular belief the present work has conclusively proved that the importance of weak hydrogen bonds are perhaps underestimated since calculations show that the energy of duplex are significantly lower then estimated from the identified hydrogen bonding.

Design, synthesis, conformational and membrane ion transport studies of proline-adamantane hybrid cyclic depsipeptides.

The design, synthesis, conformational, crystallographic, and ion transport studies of 30-membered, proline containing depsipeptides that incorporate the rigid low molecular weight lipophilic adamantane (Adm) building blocks are reported. The adamantyl groups provide the desired membrane permeability and conformational constraint for efficient transport in lipid membranes. The novel cyclic depsipeptides are: c[--Adm--C(O)--Pro-- O--CH(2)-- CHR--NH--C(O)--Pro--C(O)-- Adm--C(O)--Pro--C(O)--NH--CHR--CH(2)-- O--Pro--C(O)--] where R==H for A and R==CONH--Adm for B. Crystal structure analysis of A established that the two peptide segments are identical in formula and in conformation and that the peptides are bonded to the interleaving Adm at the 1 and 3 positions. However, the complete ring is highly asymmetric in shape since bonds for both Peptide-Adm-Peptide segments have the syn-anti motif. Torsional angles for the connecting bonds to Adm are -162 degrees , +71 degrees and -169 degrees , -48 degrees . The irregular clamshell shape of the molecule has three internal C==O moieties directed in a manner that could provide three Na(+)--O ligands. While A exhibited negligible transport of Na(+) ions across membranes, peptide B endowed with two additional adamantanes in the periphery did transport Na(+) ions from outside to inside.

Diphenic acid as a general conformational lock in the design of bihelical structures.

The bihelical (figure of "infinity") topology was examined from vantages of design, crystal structures, chirality, circular dichroism (CD) studies and molecular-orbital calculations. The minimalistic design envisaged the sequential linking of cystine to the anchor diphenic acid, which proved to be a general conformational lock. The bihelical compound 4 was obtained in two steps from diphenic anhydride 1 and cystine di-OMe. The chirality of 4 arises largely from the L-cystine. The bihelical compound 5 obtained from D-cystine di-OMe was found, by X-ray crystallography, CD studies, and optical rotation, to be the perfect mirror image of 4 prepared from L-cystine. The crystal structure of prototype 8, prepared by protocols used for 4 from the achiral cystine analogue cystamine, had a "U"-shaped conformation held together by intramolecular hydrogen bonds. Analysis of 4 and 5 show that the pairs of nine-membered beta-turn-like constructs made compact through hydrogen bonding with DMSO hold the key for the bihelical conformation. Another factor is the need for the presence of a ligand at the Calpha position. The absence of this, as in 8, allows major flexibility in the torsional angles around this critical region, promoting flexible alternatives. The CD analysis of 4, confirmed to be bihelical by X-ray crystallography, showed a typical negative band at about 210 A attributed to the beta-turn-like motif, and in the positive-band region a peak at about 227 A, generally related to the twist of the biphenyl unit. The cystamine analogue 8, which showed a "U"-type structure, presented a CD spectrum with no typical features. The total energy, derived from theoretical calculations by using the X-ray structure data, support the bihelical structure for 4 and a "U"-shaped one for 8. The limited utility of such calculations was tested with composite 9. Composite 9, in which the anchor diphenic acid is linked to cystamine on the one hand and to cystine on the other, showed a CD spectrum similar to that of 4, and this coupled with molecular-orbital calculations, using data from 4 and 8, predict a bihelical structure for this compound.

Hybrid peptides: expanding the beta turn in peptide hairpins by the insertion of beta-, gamma-, and delta-residues.

The beta turn segment in designed peptide hairpins has been expanded by the insertion of beta-, gamma- and delta-amino acids at the i+2 position. The model octapeptides Boc-Leu-Phe-Val-DPro-Ac6c-Leu-Phe-Val-OMe (1), Boc-Leu-Phe-Val-DPro-beta3-Ac6c-Leu-Phe-Val-OMe (2), and Boc-Leu-Phe-Val-DPro-Gpn-Leu-Phe-Val-OMe (3) have been shown to adopt beta hairpin conformations in methanol by the observation of key diagnostic nuclear Overhauser effects. Boc-Leu-Val-Val-DPro-delta-Ava-Leu-Val-Val-OMe (4) adopts a beta hairpin conformation in crystals; this is stabilized by three cross-strand hydrogen bonds as demonstrated by X-ray diffraction. The canonical C10 turn in an alpha-alpha segment is expanded to C11, C12, and C13 turns in alpha-beta, alpha-gamma, and alpha-delta segments, respectively. The crystal structures of Piv-LPro-beta3-Ac6c-NHMe (5) and Boc-Ac6c-Gpn-Ac6c-OMe (6) reveal intramolecularly hydrogen-bonded C11 and C12 conformations, respectively. Computer modeling of octapeptide sequences that contain centrally positioned hybrid-turn segments, by using turn parameters derived from the structures of peptides 5 and 6, establishes the stereochemical acceptability of the beta hairpins in the cases of peptides 2 and 3. Accommodation of omega-amino acids into the turn segments is achieved by the adoption of gauche conformations around the backbone C--C bonds.

A robust hybrid peptide crystal formed with weak hydrogen bonds.

In the course of our work relating to the design of a bihelical structure (I) from diphenic anhydride by tethering with cystine di-OMe, stable, hard, and rigid crystals, mp 215-218 degrees C were isolated in low yields ( approximately 2%). The crystal structure established that it was a bis amide (II) arising from diphenic acid and cystine di-OMe [(II), C(22)H(22)N(2)O(6)S(2) (a = 9.897 (1) A, b = 12.210 (1) A, c = 18.192 (1) A, sp. gr. P2(1)2(1)2(1))]. An authentic sample of (II) was subsequently prepared in 47% yields by condensation of diphenic acid dichloride with cystine di-OMe. A most surprising feature of II was, despite its high density, rigidity, and hardness, it did not exhibit any normal hydrogen bonds. The nearest approximation to a "usual" hydrogen bond was the single NH...OC linkage that occurred between molecules along a twofold screw axis. In this linkage, N...O = 3.265 A and H...O = 2.43 A, values that are at least 10% longer than those usually observed in peptides. The rigidity of the crystals appears to depend upon many weak hydrogen bonds of the type CH...O, CH...pi, CH...S, and NH...S working in concert. Even these attractions have separations that are at the high end of the range of previously observed values, although some of the weak hydrogen bonds have been rarely reported and have poorly defined ranges. The attractive effect of each of these weak bonds may be enhanced by the occurrence of a number of them in a parallel fashion like rungs in a ladder.

Hybrid peptide hairpins containing alpha- and omega-amino acids: conformational analysis of decapeptides with unsubstituted beta-, gamma-, and delta-residues at positions 3 and 8.

The effects of inserting unsubstituted omega-amino acids into the strand segments of model beta-hairpin peptides was investigated by using four synthetic decapeptides, Boc-Leu-Val-Xxx-Val-D-Pro-Gly-Leu-Xxx-Val-Val-OMe: peptide 1 (Xxx=Gly), peptide 2 (Xxx=betaGly=betahGly=homoglycine, beta-glycine), peptide 3 (Xxx=gammaAbu=gamma-aminobutyric acid), peptide 4 (Xxx=deltaAva=delta-aminovaleric acid). 1H NMR studies (500 MHz, methanol) reveal several critical cross-strand NOEs, providing evidence for beta-hairpin conformations in peptides 2-4. In peptide 3, the NMR results support the formation of the nucleating turn, however, evidence for cross-strand registry is not detected. Single-crystal X-ray diffraction studies of peptide 3 reveal a beta-hairpin conformation for both molecules in the crystallographic asymmetric unit, stabilized by four cross-strand hydrogen bonds, with the gammaAbu residues accommodated within the strands. The D-Pro-Gly segment in both molecules (A,B) adopts a type II' beta-turn conformation. The circular dichroism spectrum for peptide 3 is characterized by a negative CD band at 229 nm, whereas for peptides 2 and 4, the negative band is centered at 225 nm, suggesting a correlation between the orientation of the amide units in the strand segments and the observed CD pattern.

Peptide hairpins with strand segments containing alpha- and beta-amino acid residues: cross-strand aromatic interactions of facing Phe residues.

The incporation of beta-amino acid residues into the strand segments of designed beta-hairpin leads to the formation of polar sheets, since in the case of beta-peptide strands, all adjacent carbonyl groups point in one direction and the amide groups orient in the opposite direction. The conformational analysis of two designed peptide hairpins composed of alpha/beta-hybrid segments are described: Boc-Leu-betaPhe-Val-(D)-Pro-Gly-Leu-betaPhe-Val-OMe (1) and Boc-betaLeu-Phe-betaVal-D-Pro-Gly-betaLeu-Phe-betaVal-OMe (2). A 500-MHz 1H-NMR (nuclear magnetic resonance) analysis in methanol supports a significant population of hairpin conformations in both peptides. Diagnostic nuclear Overhauser effects (NOEs) are observed in both cases. X-ray diffraction studies on single crystals of peptide 1 reveal a beta-hairpin conformation in both the molecules, which constitute the crystallographic asymmetric unit. Three cross-strand hydrogen bonds and a nucleating type II' beta-turn at the D-Pro-Gly segment are observed in the two independent molecules. In peptide 1, the betaPhe residues at positions 2 and 7 occur at the nonhydrogen-bonding position, with the benzyl side chains pointing on opposite faces of the beta-sheet. The observed aromatic centroid-to-centroid distances are 8.92 A (molecule A) and 8.94 A (molecule B). In peptide 2, the aromatic rings must occupy facing positions in antiparallel strands, in the NMR-derived structure. Peptide 1 yields a normal "hairpin-like" CD spectrum in methanol with a minimum at 224 nm. The CD spectrum of peptide 2 reveals a negative band at 234 nm and a positive band at 221 nm, suggestive of an exciton split doublet. Modeling of the facing Phe side chains at the hydrogen-bonding position of a canonical beta-hairpin suggests that interring separation is approximately 4.78 A for the gauche+ gauche- (g+ g-) rotamer. A previously reported peptide beta-hairpin composed of only alpha-amino acids, Boc-Leu-Phe-Val-D-Pro-Gly-Leu-Phe-Val-OMe also exhibited an anomalous far-UV (ultraviolet) CD (circular dichroism) spectrum, which was interpreted in terms of interactions between facing aromatic chromophores, Phe 2 and Phe 7 (C. Zhao, P. L. Polavarapu, C. Das, and P. Balaram, Journal of the American Chemical Society, 2000, Vol 122, pp. 8228-8231).

Crystal structure of cyclic (APGVGV)2, an analog of elastin, and a suggested mechanism for elongation/contraction of the molecule.

Tropoelastin is a complex polymeric protein composed primarily of repeating segments of Val-Pro-Gly-Gly, Val-Pro-Gly-Val-Gly, and Ala-Pro-Gly-Val-Gly-Val that occurs in connective tissue and arteries. It has rubber-like extensible properties. A synthetic cyclic dodecapeptide, with a double repeat of the hexapeptide sequence, has been shown to undergo a reversible inverse temperature transition; that is, crystals grow at 60 degrees C and dissolve in the mother liquor upon cooling. An x-ray crystal structure analysis established that the cyclic backbone formed an elongated loop with a Pro-Gly, type II beta turn at both ends. Six internal cross strand NH...OC hydrogen bonds form between six NH donors and four O=C acceptors where two of the carbonyl O atoms are bifurcated acceptors. As a result, the molecule is pulled up into a corrugated profile. The corrugated loops form extended beta-sheets by additional intermolecular hydrogen bonds. An analysis of the dome region in a corrugated sheet suggests a reversible mechanism for extending and contracting the length of the whole molecule, akin to the motion of opening and closing an umbrella, caused by the motion of a water molecule with its associated hydrogen bonds acting as spokes. Crystal parameters: C44H72N12O12.3H2O, sp. gr. P2(1)2(1)2(1), a = 9.212 angstroms, b = 19.055 angstroms, c = 32.247 angstroms, d = 1.157 g/cm3.

Alpha,beta hybrid peptides: a polypeptide helix with a central segment containing two consecutive beta-amino acid residues.

Conformational studies on the synthetic 11-aa peptide t-butoxycarbonyl (Boc)-Val-Ala-Phe-alpha-aminoisobutyric acid (Aib)-(R)-beta3-homovaline (betaVal)-(S)-beta3-homophenylalanine (betaPhe)-Aib-Val-Ala-Phe-Aib-methyl ester (OMe) (peptide 1; betaVal and betaPhe are beta amino acids generated by homologation of the corresponding l-residues) establish that insertion of two consecutive beta residues into a polypeptide helix can be accomplished without significant structural distortion. Crystal-structure analysis reveals a continuous helical conformation encompassing the segment of residues 2-10 of peptide 1. At the site of insertion of the betabeta segment, helical hydrogen-bonded rings are expanded. A C15 hydrogen bond for the alphabetabeta segment and two C14 hydrogen bonds for the alphaalphabeta or betaalphaalpha segments have been characterized. The following conformational angles were determined from the crystal structure for the beta residues: betaVal-5 (= -126 degrees, = 76 degrees, and psi = -124) and betaPhe-6 (=-88 degrees, = 80 degrees, and psi =-118). The N terminus of the peptide is partially unfolded in crystals. The 500-MHz 1H-NMR studies establish a continuous helix over the entire length of the peptide in CDCl3 solution, as evidenced by diagnostic nuclear Overhauser effects. The presence of seven intramolecular hydrogen bonds is also established by using solvent dependence of NH chemical shifts.

Crystal and molecular structure of a benzo[a]pyrene 7,8-diol 9,10-epoxide N2-deoxyguanosine adduct: absolute configuration and conformation.

Benzo[a]pyrene 7,8-diol 9,10-epoxide adducts in DNA are implicated in mutagenesis, and their formation from the diol epoxides and subsequent incorrect replication by human DNA polymerases provide an attractive mechanism for the induction of cancer by this highly carcinogenic hydrocarbon and its diol epoxide metabolites. Here, we describe the crystal structure of such an adduct at the exocyclic amino group of a purine nucleoside. The present adduct derives from trans opening at C10 of the (-)-(7S,8R)-diol (9R,10S)-epoxide enantiomer by the exocyclic N(2)-amino group of deoxyguanosine. In the crystal, the pyrene rings of adjacent molecules stack with each other, but the guanine bases do not stack either intermolecularly with each other or intramolecularly with the pyrene. The most notable features of the molecular structure are (i) independent and unambiguous proof of the absolute configuration of the adduct based on the spatial relationship between the known chiral carbon atoms of the deoxyribose and the four asymmetric centers in the hydrocarbon moiety; (ii) visualization of the relative orientations of the pyrene and guanine ring systems as well as the conformation of the partially saturated hydrocarbon ring (comprising carbon atoms 7, 8, 9, and 10), both of which conformational features in the crystal are in good agreement with deductions from NMR and CD measurements in solution; and (iii) the presence in the crystal of a syn glycosidic torsion angle, a conformation that is unusual in B-DNA but that may be involved in error-prone replication of these benzo[a]pyrene 7,8-diol 9,10-epoxide deoxyguanosine adducts by DNA polymerases.

Two novel hexadepsipeptides with several modified amino acid residues isolated from the fungus Isaria.

Two new cyclohexadepsipeptides have been isolated from the fungus Isaria. Fungal growth in solid media yielded hyphal strands from which peptide fractions were readily isolable by organic-solvent extraction. Two novel cyclodepsipeptides, isaridin A and isaridin B, have been isolated by reverse-phase HPLC, and characterized by ESI-MS and 1H-NMR. Single crystals of both peptides have been obtained, and their 3D structures were elucidated by X-ray diffraction. The isaridins contain several unusual amino acid residues. The sequences are cyclo(beta-Gly-HyLeu-Pro-Phe-NMeVal-NMePhe) and cyclo(beta-Gly-HyLeu-beta-MePro-Phe-NMeVal-NMePhe), where NMeVal is N-methylvaline, NMePhe N-methylphenylalanine, and HyLeu hydroxyleucine (= 2-hydroxy-4-methylpentanoic acid). The two peptides differ from one another at residue 3, isaridin A having an (S)-proline at this position, while beta-methyl-(S)-proline (= (2S,3S)-2,3,4,5-tetrahydro-3-methyl-1H-pyrrole-2-carboxylic acid) is found in isaridin B. The solid-state conformations of both cyclic depsipeptides are characterized by the presence of two cis peptide bonds at HyLeu(2)-Pro(3)/HyLeu(2)-beta-MePro(3) and NMeVal(5)-NMePhe(6), respectively. In isaridin A, a strong intramolecular H-bond is observed between Phe(4)CO...HNbeta-Gly(1), and a similar, but weaker, interaction is observed between beta-Gly(1)CO...HNPhe(4). In contrast, in isaridin B, only a single intramolecular H-bond is observed between beta-Gly(1)CO...HNPhe(4).

Crystal structure of a hydrophobic 19-residue peptide helix containing three centrally located D amino acids.

The design of the synthetic 19-residue peptide Boc-Leu-Aib-Val-Ala-Leu5-Aib-Val-D-Ala-d-Leu-Leu10-Val-Phe-Val-Aib-D-Val15-Leu-Phe-Val-Val-OMe (Aib, alpha-aminoisobutyric acid; OMe, methyl ester) was intended to produce a crystalline peptide with independent helical and hairpin domains. The design was partially based on an octapeptide with the same sequence as residues 11-18 above, which was shown to fold into a beta-hairpin in the crystal. However, the crystal structure of the present peptide provided a surprising result. The conformation is the longest characterized right-handed alpha-helix, with as many as three internal d residues in the sequence. The completely helical structure was also unexpected, because beta-branched residues such as Val have a low propensity for helix formation in proteins. The helical peptides in the present structure assemble to form hydrophobic channels that accommodate five toluene molecules per peptide along the length of the channel. The structural results illustrate the similarity in energetics between helical and beta-hairpin conformations for peptides containing Aib residues. The crystallographic parameters for C107H179N19O22.3H2O.2.5 toluenes are: space group C2, a = 34.679(3) A, b = 12.866(1) A, c = 31.915(3) A, beta = 96.511(8) degrees, V = 14,148 A3, Z = 4, dcalc = 1.099 g/cm3, and agreement factor R1 = 10.2%.

Aromatic-aromatic interactions in crystal structures of helical peptide scaffolds containing projecting phenylalanine residues.

Aromatic-aromatic interactions between phenylalanine side chains in peptides have been probed by the structure determination in crystals of three peptides: Boc-Val-Ala-Phe-Aib-Val-Ala-Phe-Aib-OMe, I; Boc-Val-Ala-Phe-Aib-Val-Ala-Phe-Aib-Val-Ala-Phe-Aib-OMe, II; Boc-Aib-Ala-Phe-Aib-Phe-Ala-Val-Aib-OMe, III. X-ray diffraction studies reveal that all three peptides adopt helical conformations in the solid state with the Phe side chains projecting outward. Interhelix association in the crystals is promoted by Phe-Phe interactions. A total of 15 unique aromatic pairs have been characterized in the three independent crystal structures. In peptides I and II, the aromatic side chains lie on the same face of the helix at i/i + 4 positions resulting in both intrahelix and interhelix aromatic interactions. In peptide III, the Phe side chains are placed on the opposite faces of the helix, resulting in exclusive intermolecular aromatic interactions. The distances between the centroids of aromatic pair ranges from 5.11 to 6.86 A, while the distance of closest approach of ring carbon atoms ranges from 3.27 to 4.59 A. Examples of T-shaped and parallel-displaced arrangements of aromatic pairs are observed, in addition to several examples of inclined arrangements. The results support the view that the interaction potential for a pair of aromatic rings is relatively broad and rugged with several minima of similar energies, separated by small activation barriers.

Crystal structure of (-)-mefloquine hydrochloride reveals consistency of configuration with biological activity.

The absolute configuration of (-)-mefloquine has been established as 11R,12S by X-ray crystallography of the hydrochloride salt, thus allowing comparison of the configuration of mefloquine's optical isomers to those of quinine and quinidine. (-)-Mefloquine has the same stereochemistry as quinine, and (+)-mefloquine has the same stereochemistry as quinidine. Since (+)-mefloquine is more potent than (-)-mefloquine in vitro against the D6 and W2 strains of Plasmodium falciparum and quinidine is more potent than quinine, a common stereochemical component for antimalarial activity is implicated. The crystal of (-)-mefloquine hydrochloride contained four different conformations which mainly differ in a small rotation of the piperidine ring. These conformations are essentially the same as the crystalline conformations of racemic mefloquine methylsulfonate monohydrate, mefloquine hydrochloride, and mefloquine free base. The crystallographic parameters for (-)-mefloquine hydrochloride hydrate were as follows: C17H17F (6)N(2)O(+)Cl(-) .0.25 H2O; M(r), 419.3; symmetry of unit cell, orthorhombic; space group, P2(1)2(1)2(1); parameters of unit cell, a = 12.6890 +/- 0.0006 A (1 A = 0.1 nm), b = 18.9720 +/- 0.0009 A, c = 32.189 +/- 0.017 A; volume of unit cell, 7,749 +/- 4 A(3); number of molecules per unit cell, 16; calculated density, 1.44 g cm(-3); source of radiation, Cu Kalpha (lambda = 1.54178 A); mu (absorption coefficient), 2.373 mm(-1); room temperature was used; final R(1) (residual index), 0.0874 for 3,692 reflections with intensities greater than 2sigma. All of the hydroxyl and amine hydrogen atoms participate in intermolecular hydrogen bonds with chloride ions. The orientation of the amine and hydroxyl groups in (+)-mefloquine may define the optimal geometry for hydrogen bonding with cellular constituents.