Synthesis of fluorinated mucin core 2 branched oligosaccharides with the potential of novel substrates and enzyme inhibitors for glycosyltransferases and sulfotransferases.

Syntheses of fluorinated mucin core 2 tri- and tetrasaccharides modified at the C-3 or C-4 position of the pertinent galactose residue are reported. These compounds were used for the study of sialyltransferases and 3-O-sulfotransferases involved in the biosynthesis of O-glycans. Our acceptor substrate specificity studies on three cloned sialyltransferases (Sia-Ts) revealed that a 3- or 4-fluoro substituent in beta1,4Gal resulted in poor acceptors for alpha2,6(N)Sia-T and alpha2,3(N)Sia-T, whereas 4-fluoro-Galbeta1,3GalNAcalpha was a good acceptor for alpha2,3(O)Sia-T. Uniquely, 4-F-Galbeta1,4GlcNAcbeta1,6(Galbeta1,3)GalNAcalpha-OBn was an inhibitor of alpha2,6(N)Sia-T activity but not alpha2,3(N)Sia-T activity. Further we found that the activities of only Gal 3-O-sulfotransferases and not sialyltransferases were adversely affected by a C-3 fluoro substituent at the other Gal terminal of mucin core 2. The strategy of building branched mucin core 2 structures by three glycosidation sequence coupling three classes of glycosyl donors with the reactivity-matching acceptors proved to be successful in syntheses of modified mucin-type core structures of O-glycan. The relative poor yields of the glycosylations using fluorinated galactosyl donors indicated that the fluorine modification dramatically decreased the donor reactivity due to electron-withdrawing effect.

Chemical synthesis of sulfated oligosaccharides with a beta-D-Gal-(1-->3).

The syntheses of two sulfated pentasaccharides: beta-D-Gal6SO3Na-(1-->3)-[beta-D-Gal-(1-->4)-alpha-L-Fuc-(1-->3)-beta-D-Glc-NAc-(1-->6)]-alpha-D-GalNAc-->OMe (1) and beta-D-Gal6SO3Na-(1-->3)-[beta-D-Gal-(1-->4)-alpha-L-Fuc-(1-->3)-beta-D-Glc-NAc6SO3Na-(1-->6)]-alpha-D-GalNAc-->OMe (2) by using Lewisx trisaccharides 12 and 16 as glycosyl donors are described. Sulfated oligosaccharides 1-2 and intermediate compounds are fully characterized by 2D 1H-1H DQF-COSY and 2D ROESY experiments.

Effect of substituents in directing the formation of benzochlorins and isobacteriochlorins in porphyrin and chlorin systems.

[formula: see text] A first synthesis of free-base fluorinated benzochlorins by acid-catalyzed cyclization of 20-(2-trisiloxy-trifluoromethylvinyl)octaethylporphyrin++ + is achieved. Under similar reaction conditions, the purpurin-18-N-hexylimide analogues produced the corresponding fluorinated and nonfluorinated ethylidene-substituted isobacteriochlorins and fluorinated chlorin, respectively. The structure of the porphyrin based fluorinated benzochlorin was also confirmed by X-ray analysis.

Conformation and hydrogen bonding of N-formylpeptides: crystal and molecular structure of N-formyl-L-alanyl-L-aspartic acid.

Crystals of N-formyl-L-alanyl-L-aspartic acid (C8H11N2O6) grown from aqueous methanol solution are orthorhombic, space group, P2(1)2(1)2(1) with cell parameters at 294K of a = 13.619(2), b = 8.567(2), c = 9.583(3)A, V = 1118.1A3, M.W. = 232.2, Z = 4, Dm = 1.38 g/cm3 and Dx = 1.378 g/cm3. The crystal structure was solved by the application of direct methods and refined to an R value of 0.075 for 1244 reflections with I greater than or equal to 3 sigma collected on a CAD-4 diffractometer. The structure contains two short intermolecular hydrogen bonds: (i) between the C-terminal carboxyl OH and the N-acyl oxygen (2.624(3)A), a characteristic feature found in many N-acyl peptides and (ii) between the aspartic carboxyl OH. and the peptide oxygen OP1 (2.623(3)A). The peptide is nonplanar (omega = 165.5(6) degrees). The molecule takes up a folded conformation in contrast to N-formyl peptides which form extended beta-sheets; the values of phi 1, psi 1, phi 2, psi 2(1), and psi 2(2) are, respectively -65.7(6), 152.0(5), -107.2(5), 30.9(5), and -150.3(6). The aspartic acid side chain conformation is g- with chi 1 = 73.1(5). The formyl group, as expected, is transplanar [OF-CF-N1-CA1 = -4.0(8) degrees]. The presence of the short O-H ... O hydrogen bond emerges as a structural feature common to this peptide and several other N-formyl peptides. There are no C-H ... O hydrogen bonds in this structure.

Structure of N-methylnicotinamide.

C7H8N2O, Mr = 136.2, monoclinic, P21/a, a = 7.055 (1), b = 9.849 (6), c = 10.066 (4) A, beta = 100.47 (2) degrees, V = 687.5 (5) A3, Z = 4, Dm = 1.32, Dx = 1.315 g cm-3, Cu K alpha, lambda = 1.5418 A, mu = 7.09 cm-1, F(000) = 288, T = 294 K, R = 0.048 for 1134 reflections [I greater than 3 sigma(I)]. The N-methylcarboxamide group is extended with the keto O(7) transoid to C(2) [C(2)--C(3)--C(7)--O(7) +/- 158.9 (3), C(3)--C(7)--N(7)--C(8) +/- 177.1 (3), C(2)--C(3)--C(7)--N(7) +/- 23.2 (3) and C(4)--C(3)--C(7)--N(7) +/- 158.2 (3) degrees]. The dihedral angle between the planes of the pyridine ring and the carboxamide plane is 22 degrees. The molecules are linked together by N--H...O hydrogen bonds involving the amino N(7) and the carbonyl O(7) atoms.

Structural studies of immunomodulators. Part 2: Crystal structure and conformation of azimexon (BM 12.531) an immunostimulant and an anti-tumor drug.

Azimexon [2-cyanaziridinyl-2-carbamoyl-aziridinyl-1-propane] is a derivative of 2-cyanaziridine. Azimexon is an immunostimulant which shows therapeutic effects in tumor models and experimental infections in mice in vitro, enhances T lymphocyte transformation in vitro and increases phagocytosis of latex particles by mouse peritoneal cells. In cancer patients it increases blood active T rosettes, increases T4/T8 ratio and is used in the treatment of melanomas. The crystal structure analysis of azimexon has been undertaken to study the conformation of the molecule in the solid state as a first step in the investigation of a possible structure-function relationship of immunomodulators. Crystals of azimexon are triclinic, space group P1, with a = 6.342(2), b = 6.804(1), c = 13.106(2) A, alpha = 75.17(1), beta = 89.17(2), gamma = 83.26(2) degrees, V = 542.8 A3, Z = 2, D0 = 1.18 g cm-3 and Dc = 1.189 g cm-3. The structure was solved using CAD-4 data by multisolution techniques and refined to a final R value of 0.057. There are two independent molecules in the asymmetric unit with very different conformations relating the two aziridine rings in the molecule. The mean lengths of C-N and C-C in the aziridine rings are 1.461 and 1.494 A and the configuration of the ring nitrogen is pyramidal. The mean C-C-N and C-N-C bond angles in the ring are 59.2 and 60.6 degrees respectively. The two molecules, which differ significantly in the relative orientation of their aziridine rings, are linked by hydrogen bonding involving the amino group of one molecule as the donor and the cyano nitrogen and carbamoyl oxygen of the other molecule as the acceptor. There are a number of C-H...O and C-H...N interactions in the structure. The mode of action of azimexon is unknown. It has been suggested that azimexon may alkylate DNA. The knowledge of the three-dimensional structure should facilitate distance geometry calculations that would determine which modes of crosslinking would be possible. The end-to-end distances between the two ends of the molecule is 9.0 A which is of the same order as the intrastrand distances in the DNA double helix. The present structural work of azimexon strongly indicates that azimexon crosslinks with DNA using its two reactive groups at its ends. Further study is necessary in order to determine which modes of crosslinking would be possible.

Conformation and hydrogen bonding of N-formylmethionyl peptides. II. Crystal and molecular structure of N-formyl-L-methionyl-L-phenylalanine.

Crystals of N-formyl-L-methionyl-L-phenylalanine (C15H20N2O4S), grown from aqueous methanol solution are orthorhombic, space group, P2(1)2(1)2(1), with cell parameters at 294K of a = 4.900(2), b = 17.947(4), c = 18.726(4)A, V = 1646.8A3, M.W. = 324.4, Z = 4 and Dm = 1.308 g/cc, and as expected, all nearly identical to that of N-f-D-Met-D-Phe studied by Jeffs, Heald, Chodosh & Eggleston (Int. J. Peptide Protein Res. 24, 442-446, 1984). The crystal structure was solved and refined using CAD-4 data (1095 reflections greater than or equal to 3 sigma) to a final R value of 0.042. Molecules related by the alpha-translation form a parallel beta-sheet rather than anti-parallel sheet as stated in the earlier study of Jeffs et al. The formation of the parallel rather than the anti-parallel beta-sheet structure, the use of the C-H ...O hydrogen bonds to stabilize the beta-sheet and the very short O-H ...O hydrogen bond between the carboxyl OH and the N-acyl oxygen atom emerge as the main structural features of the chemotactic N-formyl methionyl peptides.

Crystal and molecular structure of methyl O-alpha-D-manno-pyranosyl-(1----2)-alpha-D-mannopyranoside.

The crystal and molecular structure of a synthetic mannosyl disaccharide, methyl O-alpha-D-mannopyranosyl-(1----2)-alpha-D-mannopyranoside, has been determined from X-ray diffractometer data by direct methods by use of the Multan programs. The crystals are monoclinic, space group P2 with unit cell dimensions, a 8.086(1), b 9.775(1), c 9.975(2) A, beta 104.58(1) degrees, Z 2, and Dm 1.54 g/cm3. The structure was refined to an R-value of 0.033 for 1359 reflections measured with CuK alpha radiation. The mannopyranose units have the chair conformations 4C(D) with C-5' and C-2' deviating from the best plane through the other four atoms of the ring by -0.68 and +0.53 A in the nonreducing group, and C-3 and O-5 deviating from the mean plane through the other four atoms by +0.57 and -0.66 A, respectively, in the "potentially" reducing residue. The ring-to-ring conformation can be described as (phi, psi) = (-64.5, 105.5 degrees). The conformation across the C-5--C-6 bond is gauche-gauche in both the sugars. The crystal structure is stabilized by a network of intermolecular O-H...O hydrogen bonds.

Studies on modified nucleic acid bases: structure of 3-isobutyl-1-methylxanthine.

C10H14O2, M = 222.3, monoclinic, P2/c, alpha = 4.882 (3), b = 8.715 (1), c = 25.955 (3) A, beta = 92.28 (1) degree, V = 1103.4 (9) A, Z = 4, Dm = 1.34, Dx = 1.338 g cm 3, Cu K alpha, lamba = 1.5418 A, mu = 7.62 cm 1, F(000) = 472, T = 294 k, R = 0.058 for 984 reflections I greater than 3 sigma (1). The isobutyl chain is oriented almost perpendicular to the xanthine ring (C(2)-N(3)-C(31)-C(32) +/- 99.8 (4)degree]. The isobutyl chain torsion angles are N(3)-C(31)-C(32)-C(33) +/- 62.2 (4) and N(3)-C(31) C(32)-C(34) +/- 174.1 (3) degree. The structure forms self-paired dimers of xanthine bases with a pair of N-H...O and a pair of weaker C-H...N hydrogen bonds across centers of inversion. There is a partial stacking of the xanthine bases.

Conformation of methylated amino acids: structure of 3,4-dimethoxy-alpha-methyl-DL-phenylalanine sesquihydrate.

C12H17NO4.1.5H2O, Mr = 266.3, triclinic, P1, a = 5.872 (1), b = 11.437 (2), c = 20.434 (1) A, alpha = 95.74 (1), beta = 96.91 (1), gamma = 89.18 (1) degrees, V = 1355.5 A3, Z = 4, Dm = 1.29, D chi = 1.305 g cm-3, lambda(Cu K alpha) = 1.5418 A, mu = 8.3 cm-1, F(000) = 572, T = 294 K, R = 0.038 for 4006 reflections, I greater than or equal to 3 sigma(I). Both the molecules A and B in the asymmetric unit exist as zwitterions. With respect to the D enantiomer, the torsion angles psi 1 and psi 2 are +47.2 and -134.4 degrees in molecule A and +33.3 and -147.5 degrees in molecule B respectively. The torsion angles of the alpha-methyl group, NH3+ and COO- groups with respect to Cv are in molecules A and B respectively +67.2, +66.8, -174.3, -175.6, and -59.2 and -59.5 degrees. The hydrogen-bonding environment of water OW1 is trigonal nonplanar; OW2 is trigonal planar and OW3 is tetrahedral. The crystal structure is stabilized by a number of hydrogen bonds involving the amino and carboxylate groups of both molecules A and B and the water molecules.

Crystal structure and conformation of polypeptides: L-leucylglycylglycylglycine.

Crystals of L-leucylglycylglycylglycine, LGGG (C12H22N4O5), grown from an ethanol-water solution, are orthorhombic, space groups P2(1)2(1)2(1), with unit cell dimensions (at 22 +/- 3 degrees) a = 9.337(1), b = 10.995(1), c = 15.235(1)A, v = 1563.4 A3, Z = 4 with a density of Dobs = 1.29 g.cm-3 and Dcalc = 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 greater than or equal to 2 sigma. 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 delta omega of 8 degrees 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 beta-sheet conformation, addition of Leu to the N-terminal results in a bent conformation.

Intercalation of water molecules between nucleic acid bases in the crystal structures of 6-azathymine hemihydrate and 5-amino-2-thiocytosine dihydrochloride dihydrate.

The crystal structure of 6-azathymine hemihydrate (6AzTH) exhibits a novel intercalation of water molecules interposed half-way between the modified bases 6.3 to 6.7 A apart. The crystal contains four molecules of 6-azathymine (6AzT) and two water molecules as the independent repeating unit. These two water molecules together with the four bases form two separate water sandwiches. In the crystal structure these sandwiches form two sets of local clusters. The anhydrous crystalline form of 6AzT, on the other hand, is stabilized by base stacking interactions. Both the water molecules in 6AzTH that are involved in sandwich formation have trigonal coordination around them. A reexamination of the crystal structure of 5-amino-2-thiocytosine (5A2TC) revealed that one of the water molecules in this structure also forms a water sandwich and has trigonal coordination whereas the other water molecule with tetrahedral coordination does not form a sandwich. The environment and the characteristics of the intercalated water molecule in these structures suggest a possible role for such water intercalations in the dynamics of DNA. Crystals of 6AzTH are monoclinic, space group P21/n, with unit cell parameters a = 8.861 (1), b = 13.177 (3), c = 20.662 (2) A, beta = 93.35 (1) degrees, and Z = 16. From diffractometer data (2503 reflections, greater than or equal to 3 sigma), the crystal structure was solved and refined to an R of 0.056.

New patterns of hydrogen bonded interactions between polypeptide chains. Crystal and molecular structure of glycylglycylglycine.

Crystals of glycylglycylglycine (C6H-11N3O4), grown from an aqueous methanol solution, are triclinic, space group P1, with the unit cell dimensions (at 22 +/- 3 degrees) a = 11.656(3), b = 14.817(3), c = 4.823(2) A, alpha = 88.45(3), beta = 95.96(3), gamma = 105.42(3) degrees, Z = 4 (with two molecules in the asymmetric unit) with a density of Dobs = 1.58 g X cm -3 and Dcalc = 1.572 g X cm -3. The crystal structure was solved by a combination of multisolution and trial and error methods and refined with full-matrix least-squares method to a final R value of 0.036 for the observed 3021 reflections (I greater than or equal to 2 sigma). The conformation of the two molecules I and II in the asymmetric unit is very similar (except around the N-terminal end); they have the fully extended trans-planar conformation, and have omega values ranging from 2 to 4 degrees. The peptide chain repeating distances (C1 alpha - C3 alpha) are 7.27 A and 7.18 A in the two molecules as compared with the value of 6.68 A for extended beta-sheets with beta-carbons. There are four different interactions between these two molecules characterized by different hydrogen bonding. Molecule I is hydrogen bonded to a neighboring molecule I using four hydrogen bonds. Molecule II is hydrogen bonded to another II, using bifurcated interactions involving the peptide nitrogen. Molecule I is hydrogen bonded to two different molecules II forming distinctly different hydrogen bonding patterns from the two mentioned above. The molecules are packed in rows, in a head-to-tail fashion (C-terminal opposite N-terminal) and are held together in sheets by hydrogen bonds between carbonyl and amide groups, corresponding to the very familiar anti-parallel pleated sheet arrangement for polypeptides. The hydrogen bonds involving the amino nitrogens as donors are significantly longer and presumably weaker compared to those involving the NH+3 group. The C=O distances show variations that correlated with hydrogen bonding. The N-H...O angle varies from 152 to 174 degrees and the bent N-H...O hydrogen bonds show bifurcated interactions.

Conformation of L-cystathionine, a carba analog of cystine, and stereochemistry of hormone-receptor interactions.

The conformation of L-cystathionine, a carba analog of L-cystine, has been studied in the solid state using X-ray diffraction techniques. Crystal of L-cystathionine are tetragonal, space group P41 with cell constants a = 6.691(1) A, c = 21.998(3) A and Z = 4. From diffractometer data to the limit of 2theta = 162 degrees for Cukalpha, the structure was refined using full-matrix least-squares to an R value of 0.061. L-Cystathionine is isostructural chemically to L-cystine and its crystal structure is isomorphous to tetragonal L-cystine (Chaney, M.O. and Steinrauf, L.K. (1974) Acta Crystallogr. 1330, 711--716). The crystal structure of L-cystathionine is disordered, leading to two slightly differing conformers of L-cystathionine (each with half occupancy) with same helical sense but running in opposite directions and occupying the locations of L-cystine molecules in tetragonal L-cystine structure. Their conformational similarity, even when no sterical constraints such as cyclization are present, offers an explanation of the activities of the carba analogs of neuro-hypophysial hormones in terms of the structural integrity of the disulfide-like bridges.