Kinetics of nitroxyl radical reactions. A pulse-radiolysis conductivity study.

Absolute rate-constants for the reaction of the nitroxyl free radicals TAN and TMPN with radiation-chemically-formed radicals and ions have been determined. k(TAN + X) (in M(-1) sec(-1)=4-0 X 10(9) (for X = OH-), 2-9 X 10(10) (eaq-), 8-0 X 10(9) (H-), 7-2 X 10(8) (-CH2OH), 4-0 X 10(8) (CH3CHOH), 4-3 X 10(8) ((CH3)2COH) 2-8 X 10(8) (-CH2(CH3)2COH), 5-9 X 10(7) (glucose radical), 4-0 X 10(8) (c-C5H9-), and k(TMPN + X)=3-4 X 10(9) (OH-), 7-8 X 10(9) (eq-), 4-9 X 10(9) (H-), 4-4 X 10(8) (-CH2OH), 4-9 X 10(8) (CH3CHOH), 3-6 X 10(8) ((CH3)2COH), 1-5 X 10(8) (-CH2(CH3)2COH), 4-9 X 10(7) (glucose radical), 4-3 X 10(8) (c-C5H9-). Direct measurements by means of a pulse-radiolysis conductivity technique were based on the formation and destruction of charged species in these reactions within certain pH ranges. It is indicated that the radiosensitizing nitroxyls undergo both redox and addition reactions.     

Studies on the relationship between glycerophosphoglycolipids and lipoteichoic acids. IV. Trigalactosylglycerophospho-acylkojibiosyldiacylglycerol and related compounds from Streptococcus lactis Kiel 42172.

Streptococcus lactis Kiel 42172 contains at least six unusually polar glycerophosphoglycolipids. The predominant one was composed of D-galactose, D-glucose, glycerol, acyl groups and phosphorus in a molar ratio of approx. 3 : 2 : 2 : 3 : 1. By analysis of the breakdown products of HF hydrolysis and Smith-degradation the structure was established to be [Galp (alpha 1 leads to 6)Galp(alpha 1 leads to 3)-sn-glycero(2 comes from 1 alpha Galp)-1-phospho] leads to 6Glcp(alpha 1 leads to 2), acyl leads to Glcp(alpha 1 leads to 3)-acyl2Gro. By HF hydrolysis the other compounds were shown to be in the main also derivatives of GroP leads to 6Glc(alpha 1 leads to 2), acyl leads to 6Glc(alpha 1 leads to 3)acyl2Gro but they released as water-soluble glycosides Gal(alpha 1 leads to 2)Gro, Gal(alpha 1 leads to 3)Gro, Gal(alpha 1 leads to 3)Gro(2 comes from 1 alpha Gal), Gal(alpha 1 leads to 6)Gal(alpha 1 leads to 3)Gro and Gal(alpha 1 leads to 6)Gal-(alpha 1 leads to 6)Gal(alpha 1 leads to 3)Gro(2 comes from 1 alpha Gal), respectively. In the lipid extract Glc(alpha 1 leads to 2), acyl leads to 6Glc(alpha 1 leads to 3)acyl2Gro and GroP leads to 6Glc(alpha 1 leads to 2), acyl leads to 6Glc(alpha 1 leads to 3) acyl2Gro were also observed. This set of compounds is proposed to constitute a biosynthetic series reflecting the individual steps in the synthesis of the lipoteichoic acid of Streptococcus lactis Kiel 42172 which is made up by the same lipid anchor and a non-classical poly(galabiosyl, galactosyl glycerophosphate)-chain (Koch, H.U. and Fischer, W. (1978) Biochemistry 17, 5275--5281).     

Biosynthesis and n.m.r. studies of deuterated poly(3-hydroxybutyrate) produced by Alcaligenes eutrophus H16.

Alcaligenes eutrophus H16 was grown on mixtures of 1H- and 2H-acetate as carbon sources. The accumulation of deuterated poly(3-hydroxybutyrate) (P(3HB)) was observed. The deuterium distributions in the isolated P(3HB)s were determined from 1H and 2H-n.m.r. spectra and confirmed by 13C-n.m.r. spectra. Although one would expect to synthesize P([2,2,4,4,4-2H5]3HB) when the cells were grown on 2H-acetate as the sole carbon source, the methyl, methylene and methine groups of the P(3HB) contained both deuterium and proton. This observation indicates some substitution from 2H to 1H during the P(3HB) synthesis. The 2H content in the methyl groups was larger than that in the methylene groups, which suggests a kinetic isotope effect in the P(3HB) synthesizing process. The deuterium distributions in the two magnetically non-equivalent methylene protons were determined to be different, which indicates stereoselectivity at the C2 site.     

Effects of polyamines and analogs on staphylococcal nuclease.

The promoting activity of polyamine analogs (IV approximately XV) on staphylococcal nuclease with DNA as the substrate was compared with that of natural polyamines (I APPROXIMATELY III): I. NH2(CH2)3NH(CH2)4NH(CH2)3NH2(spermine); II. NH2(CH2)3NH(CH2)3NH(CH2)3NH2(thermine); III. NH2(CH2)4NH2 (putrescine); IV. CN(CH2)2NH(CH2)4NH(CH2)2CN; V. HOOC(CH2)2NH(CH2)4NH(CH2)2COOH; VI. C2H5OOC(CH2)2NH(CH2)4NH(CH2)2COOC2H5; VII. HO(CH2)3NH(CH2)4HH(CH2)3OH; VIII. CH3COHH(CH2)3NH(CH2)4NH(CH2)3NHCOCH3; IX. C2H5NH(CH2)3NH(CH2)4NH(CH2)3NHC2H5; X. NH2(CH2)3S(CH2)4S(CH2)3NH2; XI. NH2(CH2)3NH(CH2)2O(CH2)2NH(CH2)3NH2; XII. NH2(CH2)3NCH3(CH2)4HCH3(CH2)3NH2; XIII. CN(CH2)2NCH3(CH2)4NCH3(CH2)2CN; XIV. (CH3)2N(CH2)3NCH3(CH2)4NCH3(CH2)3N(CH3)2; XV. NH2(CH2)2O(CH2)2NH2 Replacement of the terminal groups by CN, COOH, COOEt, NHAc, NHEt, or N(CH3)2 remarkably decreased the activity. The compound VII with terminal hydroxyl groups had a lower promoting activity at low concentrations, but revealed higher activity at higher concentrations and, in contrast to spermine, no inhibition at all even at very high concentrations. Replacement of both internal amino groups by sulfur or NCH3 decreased the activity. The introduction of an ether bond into the internal methylene groups (compound XI) highly decreased the activity. Based upon these findings the possible relationship between structure and activity is discussed.     

Serum-free culture of Japanese quail kidney cells. Regulation of vitamin D metabolism.

Cells obtained from male quail kidneys by digestion with collagenase and hyaluronidase were plated and maintained in a chemically defined, serum-free medium. Culture dishes (35 mm) were inoculated with 1.5 . 10(6) cells which became confluent in 5 days. The cells maintained an epithelial-like morphology over the entire culture period. During a 2 h incubation the cells metabolized 25--30% of the 10 nM 25-hydroxyvitamin D-3 (25-OH-D-3) provided. Seven metabolites were chromatographically separated on Sephadex LH-20. Three have been identified as 1 alpha, 25-dihydroxyvitamin D-3 (1,25(OH)2D-3), 24,25-dihydroxyvitamin D-3 (24,25(OH)2D-3) and 1 alpha, 24,25-trihhydroxyvitamin D-3 (1,24,25(OH)3D-3). The activities of the 25-OH-D-3:1 alpha- and 24-hydroxylases increased eight times faster than the cell number in 5 days. Preincubation of the cells with 10 nM 25-OH-D-3 or 1,25(OH)2D-3 decreased 1,25(OH)2D-3 synthesis, and increased both 24,25(OH)2D-3 and metabolite IV synthesis. The decrease in 25-OH-D-3:1 alpha-hydroxylase activity required a 2 h preincubation with 25-OH-D-3, while stimulation of 25-OH-D-3:24-hydroxylase activity and metabolite IV production required a 6 h preincubation. Incubations of cells for 1 h with parathyroid hormone resulted in a 30-fold increase in cyclic AMP in the medium. A 6 h preincubation with parathyroid hormone decreased 24,25(OH)2D-3) synthesis 50% relative to control cells. These results demonstrate the amenability of this system for studying the regulation of 25-OH-D-3 metabolism, as well as its use for other in vitro studies on renal cell function in a chemically defined culture system.     

Synthesis of 1,25-dihydroxycholecalciferol and 24,25-dihydroxycholecalciferol by calvarial cells. Characterization of the enzyme systems.

The synthesis of 1,25-dihydroxycholecalciferol [1,25(OH)2D3] and 24,25-dihydroxycholecalciferol [24,25(OH)2D3] from 25-hydroxycholecalciferol [25(OH)D3] has previously been shown to occur in cells isolated from bone. The main findings of the present study are that the enzyme systems which catalyse these syntheses are: (1) active at 'in vitro' substrate concentrations over the range of 2-50 nM; (2) regulatable in a complex way by 1,25(OH)2D3, 24,25(OH)2D3, 25,26-dihydroxycholecalciferol and 25(OH)D3, but not by cholecalciferol ('vitamin D3'); and (3) have relatively short half-lives (approx. 5 h).     

Prolyl endopeptidase inhibitors from Syzygium samarangense (Blume) Merr. & L. M. Perry

Compounds isolated from the hexane extract of the leaves of Syzygium samarangense (Blume) Merr. & L. M. Perry were tested for inhibitory activity against the following serine proteases: trypsin, thrombin and prolyl endopeptidase. The compounds were identified as an intractable mixture of alpha-carotene and beta-carotene (1), lupeol (2), betulin (3), epi-betulinic acid (4), 2',4'-dihydroxy-6'-methoxy-3'-methylchalcone (5), 2'-hydroxy-4',6'-dimethoxy-3'-methylchalcone (6), 2',4'-dihydroxy-6'-methoxy-3',5'-dimethylchalcone (7), 2',4'-dihydroxy-6'-methoxy-3'-methyldihydrochalcone (8) and 7-hydroxy-5-methoxy-6,8-dimethylflavanone (9). Hydrogenation of compounds 5, 6 and 7 yielded compound 8, 2'-hydroxy-4',6'-dimethoxy-3'-methyldihydrochalcone (10) and 2',4'-dihydroxy-6'-methoxy-3',5'-dimethyldihydrochalcone (11), respectively. The hydrogenated products of compounds 6 and 7 were also tested for enzyme inhibitory activity. In addition, beta-sitosterol (12) and beta-D-sitosterylglucoside (13) were also isolated. This is the first report of the isolation of compounds 1-6, 8 and 13 from this plant. Compounds 3-8 and 10 exhibited significant and selective inhibition against prolyl endopeptidase among three serine proteases. This is the first report of this kind of activity for all these compounds.     

Assignment of structures to oligosaccharides produced by enzymic degradation of a beta-D-glucan from barley by 1H- and 13C-n.m.r. spectroscopy.

The structures of one tri-(1), two tetra-(2 and 3), and one hexa-saccharide (4) produced by treatment of barley flour, after removal of the starch components, with a fungal beta-D-glucanase (Finizyme) have been assigned on the basis of 1H- and 13C-n.m.r. data as follows: beta-D-Glcp-(1----3)-beta-D-Glcp-(1----4)-D-Glcp (1), beta-D-Glcp-(1----4)-beta-D-Glcp-(1----3)-beta-D-Glcp-(1----4)-D-Glcp (2), beta-D-Glcp-(1----3)-beta-D-Glcp-(1----4)-beta-D-Glcp-(1----4)-D-Glcp (3), and beta-D-Xylp-(1----4)-[alpha-L-Araf-(1----3)]-[alpha-L-Ara f-(1----2)-beta-D-Xylp-(1----4)-beta-D-Xylp- (1----4)-D-Xylp (4).     

Saponins from Albizzia lucida.

Three main saponins were isolated from the seeds of Albizzia lucida. Their structures were established by spectral analyses and chemical and enzymatic transformations as 3-O-[β- -xylopyranosyl(1→2)-- -arabinopyranosyl (1→6)] [β- -glucopyranosyl (1→2)] β- -glucopyranosyl echinocystic acid; 3-O-[- -arabinopyranosyl (1→6)][β- -glucopyranosyl (1→2)]-β- -glucopyranosyl echinocystic acid and 3-O-[β- -xylopyranosyl (1→2)-β- -fucopyranosyl (1→6)-2-acetamido-2-deoxy-β- -glucopyranosyl echinocystic acid, characterized as its methyl ester.     

Heavy metal binding to heparin disaccharides. II. First evidence for zinc chelation.

To map out the heavy metal binding sites of iduronic acid containing oligosaccharides isolated from human kidneys, we studied Zn(II) binding by nuclear magnetic resonance (NMR) and molecular modeling to two disaccharides isolated after nitrous acid depolymerization of heparin and two synthetic disaccharides representative of the heparin structure, namely, IdopA2S (alpha 1,4)AnManOH, 1 alpha, IdopA2S (alpha 1,4)AnManOH6S, 1b, IdopA2S-(alpha 1,4)GlcNS alpha Me, 2a, and IdopA2S (alpha 1,4)GlcNS6S alpha Me, 2b (see previous article in this series). A conformational analysis of the metal free and metal bound solutions was made by comparing calculated [(NOE)]s, [T1]s, and [J]s to experimental values. The 1C4, 4C1, and 2S0 conformations of the L-idopyranosiduronate ring and the 4E and 4T3 of the anhydro-D-mannitol ring are evaluated as are rotations about the C5-C6 hydroxymethylene of the AnManOH(6S) or GlcNS (6S) residues. The NOE between IdopA2S H1 and H3 and the known NOE between H2 and H5, as well as the T1 of IdopA2S H3, are introduced as NMR observables sensitive to the IdopA2S ring conformation. Similarly, a NOE between IdopA2S H5 and AnManOH(6S) or GlcNS(6S) H3 was observed that directly restricts the allowed interglycosidic conformational space. For all disaccharides, the Zn(II) bound spectral data are consistent with models in which these motions are partially "frozen" such that the 1C4 conformation of the IdopA2S is stabilized along with the 4T3 conformation of the AnManOH(6S) ring. The interglycosidic conformation is also stabilized in one of two minima. Electrostatic potential energy calculations gave the best overall agreement with experiment and suggest metal binding conformations with the carboxylate and ring oxygen of the IdopA2S residues (1C4 conformation) and either O3 of the GlcNS(6S) residues or the sulfate oxygens of the 6-sulphate for 2b providing additional chelating sites. These chelation models concur with the observation of marked 13C and 1H NMR chemical shifts for the IdopA2S resonances and of GlcNS H3 for 2 alpha and GlcNS6S C6 for 2b. This study of model compounds implicates the IdopA2S(alpha 1,4)GlcNS6S group as part of the heavy metal binding site in biologically important acidic oligosaccharides such as heparin.     

On the relationship between glycerophosphoglycolipids and lipoteichoic acids in Gram-positive bacteria. II. Structures of glycerophosphoglycolipids.

1. Eight glycerophosphoglycolipids were isolated from six Gram-positive bacteria. Besides sn-glycero-1-phospho-beta-gentiobiosyldiacylglycerol (i) and sn-glycero-1-phospho-alpha-kojibiosyldiacylglycerol (ii), three novel structures have been established: 1,2-di-O-acyl-3-O-[6-(sn-glycero-1-phospho)-alpha-D-glucopyranosyl-(1 leads to 2)-(6-O-acyl-alpha-D-glucopyranosyl)]glycerol (iii), 1,2-di-O-acyl-3-O-[6-(sn-glycero-1-phospho)-beta-D-glucopyranosyl-(1 leads to 6)-alpha-D-galactopyranosyl-(1 leads to 2)-alpha-D-glucopyranosyl]glycerol (iv), and 1,2-di-O-acyl-3-O-[6-(sn-glycero-1-phospho)-beta-D-glucopyranosyl-(1 leads to 6)-alpha-D-galactopyranosyl-(1 leads to 2)-(6-O-acyl-alpha-D-glucopyranosyl)]glycerol (v). 2. Compound i was isolated from Bacillus licheniformis, Bacillus subtilis and Staphylococcus aureus, compound ii from a group B Streptococcus, compounds ii and iii from Streptococcus lactis, compounds iv and v from Lactobacillus casei. Lactobacillus plantarum contained besides compounds iv and v a glycerophosphate derivative of 1,2-di-O-acyl-3-O-[alpha-D-galactopyranosyl (1 leads to 2)-alpha-D-glucopyranosyl]glycerol. 3. Identical structural features of the described glycerophosphoglycolipids and the corresponding lipoteichoic acids are discussed.     

New types of acetate-oxidizing,sulfate-reducing Desulfobacter species,D. hydrogenophilus sp. nov., D. latus sp. nov., and D. curvatus sp. nov.

Sulfate-reducing bacteria with oval to rod-shaped cells (strains AcRS1, AcRS2) and vibrio-shaped cells (strains AcRM3, AcRM4, AcRM5) differing by size were isolated from anaerobic marine sediment with acetate as the only electron donor. A vibrio-shaped type (strain AcKo) was also isolated from freshwater sediment. Two strains (AcRS1, AcRM3) used ethanol and pyruvate in addition to acetate, and one strain (AcRS1) grew autotrophically with H₂, sulfate and CO₂. Higher fatty acids or lactate were never utilized. All isolates were able to grow in ammonia-free medium in the presence of N₂. Nitrogenase activity under such conditions was demonstrated by the acetylene reduction test. The facultatively lithoautotrophic strain (AcRS1), a strain (AcRS2) with unusually large cells (2×5 m), and a vibrio-shaped strain (AcRM3) are described as new Desulfobacter species, D. hydrogenophilus, D. latus, and D. curvatus, respectively.     

The non-enzymatic hydrolysis of oligoribonucleotides VI. The role of biogenic polyamines.

Single-stranded oligoribonucleotides containing UA and CA phosphodiester bonds can be hydrolyzed specifically under non-enzymatic conditions in the presence of spermidine, a biogenic amine found in a wide variety of organisms. In the present study, the rate of oligonucleotide and tRNA(i)(Met)hydrolysis was measured in the presence of spermidine and other biogenic amines. It was found that spermine [H(3)N(+)(CH(2))(3)(+)NH(2)(CH(2))(4)(+)NH(2)(CH(2))(3)(+)NH(3)] and putrescine [H(3)N(+)(CH(2))(4)(+)NH(3)] can replace spermidine [H(3)N(+)-(CH(2))(4)(+)NH(2)(CH(2))(3)(+)NH(3)] to induce the hydrolysis. For all three polyamines, a bell-shaped cleavage rate versus concentration relationship was observed. The maximum rate of hydrolysis was achieved at 0.1, 1.0 and 10 mM spermine, spermidine and putrescine, respectively. Moreover, we found that the hydrolysis requires at least two linked amino groups since two aminoalcohols, 2-aminoethanol and 3-aminopropanol, were not able to induce the cleavage of the phospho-diester bond. The optimal cleavage rate of the oligo-ribonucleotides was observed when amino groups were separated by tri- or tetramethylene linkers. The methylation of the amino groups reduced the ability of diamines to induce oligoribonucleotide hydrolysis. Non-enzymatic cleavage of tRNA(i)(Met)from Lupinus luteus and tRNA(i)(Met)from Escherichia coli demonstrate that both RNAs hydrolyze as expected from principles derived from oligoribonucleotide models.     

G.L.C.-M.S. of the oxidation products of pentitols and hexitols as O-butyloxime trifluoroacetates

Chemical ionisation (c.i.) mass spectra of trifluoroacetylated O-butyloximes of 2- and 3-pentuloses, 2- and 3-hexuloses, and 2,5-hexodiuloses (products of the oxidation of the pentitols and hexitols with bromine) are reported. Small amounts of 2-pentosuloses and 2,3- and 2,4-pentodiuloses could be detected by using selected ion monitoring at m/z 579 (M + 1). The mass spectra comprise few signals, with those for (M + 1) or (M + 1 ? 2 F₃CCOO) being the most intense.     

Steroidal glycosides from Agave cantala.

Two new steroidal glycosides, agaveside A and B, isolated from the fruits of Agave cantala were characterized as 3 beta-O-[beta-D-xylopyranosyl-(1----2),beta-D-xylopyranosyl-(1----3), beta-D-glucopyranosyl-(1----3)-[beta-D-xylopyranosyl-(1----3)-beta-D- galactopyranosyl-(1----2)]-beta-D-glucopyranosyl]-(25R)-5 alpha-spirostane and 3 beta-O-[beta-D-xylopyranosyl-(1----2), beta-D-xylopyranosyl-(1----3)-beta-D-glucopyranosyl-(1----3)- [beta-D-galactopyranosyl-(1----2)]-beta-D-glucopyranosyl]-(25R)-5 alpha-spirostane. The structures were elucidated by a combination of 13CNMR spectroscopy, chemical degradation and fast atom bombardment mass spectrometry.     

Characterization of methylation of rat liver cytosolic glutathione S-transferases by using reverse-phase h.p.l.c. and chromatofocusing.

Glutathione S-transferase (GST) subunits in rat liver cytosol were separated by reverse-phase h.p.l.c.; five major proteins were isolated and identified as subunits 1, 2, 3, 4 and 8. F.p.l.c. chromatofocusing resolved the affinity-purified GST pool into nine different isoenzymes. The five basic (Alpha class) dimeric peaks of GST activity were 1-1, 1-2a, 1-2b, 2-2a and 2-2b. Reverse-phase h.p.l.c. analysis revealed that subunit 8 was also present in the protein peaks designated 1-1, 1-2a and 1-2b. The four neutral (Mu class) isoenzymes were 3-3, 3-4, 3-6 and 4-4. The GST pool was methylated in vitro before reverse-phase h.p.l.c. or f.p.l.c. chromatofocusing. Chromatofocusing indicated that the Mu class isoforms (3-3, 3-4 and 4-4) were the primary GSTs methylated, and h.p.l.c. analysis confirmed that subunits 3 and 4 were the major methyl-accepting GST subunits. The addition of calmodulin stimulated the methylation in vitro of GST isoenzymes 3-3, 3-4 and 4-4 by 3.0-, 7.5- and 9.9-fold respectively. Reverse-phase h.p.l.c. also indicated that only the methylation of GST subunits 3 and 4 was stimulated by calmodulin. Basic GST isoenzymes were minimally methylated and the methylation was not enhanced by calmodulin. Investigation of the time course of methylation of GST subunits 3 and 4 indicated that at incubation times less than 4 h the methylation of both Mu class subunits was stimulated by calmodulin, and that under such conditions subunit 4 was the preferred substrate. In contrast, there was essentially no calmodulin-stimulated methylation at incubation times of 4 or 6 h, and the methylation of subunit 3 was predominant. Kinetic parameters at 2 h of incubation were determined in the presence and in the absence of calmodulin. The addition of calmodulin doubled the Vmax. for methylation of both subunits 3 and 4 and decreased the Km of subunit 4 for S-adenosyl-L-methionine 3.6-fold. Finally, methylation was substoichiometric and after 6 h of incubation ranged from 2.8 to 7.6% on a mole-to-mole basis for subunits 4 and 3 respectively.     

New generation dopaminergic agents. 7. Heterocyclic bioisosteres that exploit the 3-OH-phenoxyethylamine D2 template.

The synthesis of several bioisosteric analogs based on the 3-OH-phenoxyethylamine dopamine D2 agonist template (i.e., 3) is described. The benzimidazol-2-ones and benzthioimidazol-2-ones (7-10) and 2-trifluoromethyl-benzimidazole (13) were observed to have excellent affinity for the D2 receptor.     

Crystal structure of tert.-butyloxycarbonyl-L-prolyl-D-alanyl-D-alanyl-N-methylamide. Dimeric beta-sheet formation.

The crystal structure of the tripeptide t-Boc-L-Pro-D-Ala-D-Ala-NHCH3, monohydrate, (C17H30N4O5.H2O, molecular weight = 404.44) has been determined by single crystal X-ray diffraction. The crystals are monoclinic, space group P2(1), a = 9.2585(4), b = 9.3541(5), c = 12.4529(4)A, beta = 96.449(3) degrees, Z = 2. The peptide units are in the trans and the tBoc-Pro bond in the cis orientation. The first and third peptide units show significant deviations from planarity (delta omega = 5.2 degrees and delta omega = 3.7 degrees, respectively). The backbone torsion angles are: phi 1 = -60 degrees, psi 1 = 143.3 degrees, omega 1 = -174.8 degrees, phi 2 = 148.4 degrees, psi 2 = -143.1 degrees, omega 2 = -179.7 degrees, phi 3 = 151.4 degrees, psi 3 = -151.9 degrees, omega 3 = -176.3 degrees. The pyrrolidine ring of the proline residue adopts the C2-C gamma conformation. The molecular packing gives rise to an antiparallel beta-sheet structure formed of dimeric repeating units of the peptide. The surface of the dimeric beta-sheet is hydrophobic. Water molecules are found systematically at the edges of the sheets interacting with the urethane oxygen and terminal amino groups. Surface catalysis of an L-Ala to D-Ala epimerization process by water molecules adsorbed on to an incipient beta-sheet is suggested as a mechanism whereby crystals of the title peptide were obtained from a solution of tBoc-Pro-D-Ala-Ala-NHCH3.     

Studies of polysaccharide fractions from the lipopolysaccharide of Pseudomonas aeruginosa N.C.T.C. 1999.

Two polymeric water-soluble fractions were isolated by gel filtration after mild acid hydrolysis of the lipopolysaccharide from Pseudomonas aeruginosa N.C.T.C. 1999. The fraction of higher molecular weight retained the O-antigenic specificity of the lipopolysaccharide and may be 'side-chain' material. This fraction was rich in N (about 10%) and gave several basic amino compounds on acid hydrolysis; fucosamine (at least 2.8% w/w) was the only specifc component identified. The fraction of lower molecular weight was a phosphorylated polysaccharide apparently corresponding to 'core' material. The major components of this fraction and their approximate molar proportions were: glucose (3-4); rhamnose (1); heptose (2); 3-deoxy-2-octulonic acid (1); galactosamine (1); alanine (1-1.5); phosphorus (6-7). In the intact lipopolysaccharide this fraction was probably linked to lipid A via a second residue of 3-deoxy-2-octulonic acid, and probably also contained additional phosphate residues and ethanolamine. The residues of 3-deoxy-2-octulonic acid were apparently substituted in the C-4 or C-5 position, and the phosphorylated heptose residues in the C-3 position. The rhamnose was mainly 2-substituted, though a little 3-substitution was detected. The glucose residues were either unsubstituted or 6-substituted. Four neutral oligosaccharides were produced by partial acid hydrolysis and were characterized by chemical, enzymic, chromatographic and mass-spectrometric methods of analysis. The structures assigned were: Glcpalpha1-6Glc; Glcpbeta1-2Rha; Rhapalpha1-6Glc; Glcpbeta1-2Rhapalpha1-6Glc. The galactosamine was substituted in the C-3 or C-4 position, the attachment of alanine was indicated, and evidence that the amino sugar linked the glucose-rhamnose region to the 'inner core' was obtained.     

Vibrational analysis of nucleic acids. V. Force field and conformation-dependent modes of the phosphodiester backbone modeled by diethyl phosphate.

A generalized valence force field is derived for the diethyl phosphate anion [(CH3CH2O)2PO2-] and its deuterium [(CH3CD2O)2PO2-, (CD3CH2O)2PO2- and (CD3CD2O)2PO2-] and carbon-13 [(CH3 13CH2O)2PO2-] derivatives in the stable trans-gauche-gauche-trans conformation. Normal coordinate analysis of the trans-gauche-gauche-trans conformer, which serves as a structural analog of the nucleic acid phosphodiester group, is based on comprehensive infrared and Raman spectroscopic data and vibrational assignments obtained for the diethyl phosphate anion. The generalized valence force field is in good agreement with the scaled ab initio force field of diethyl phosphate and represents significant improvement over earlier modeling of the phosphodiester moiety with dimethyl phosphate. The conformational dependence of skeletal C-C-O-P(O2-)-O-C-C stretching vibrations is also explored. Starting with the trans-gauche-gauche-trans conformation, the frequency dependence of skeletal stretching modes has been obtained by stepwise rotation of the torsion angles of the P-O and C-O bonds corresponding to nucleic acid torsions alpha (P-O5'), beta (O5'-C5'), epsilon (C3'-O3'), and zeta (O3'-P). Both symmetric and antisymmetric phosphoester stretching modes are highly sensitive to P-O and C-O torsions, whereas symmetric and antisymmetric phosphodioxy (PO2-) stretching modes are less sensitive. The present results provide an improved structural basis for understanding previously developed empirical correlations between vibrational marker bands and nucleic acid backbone conformation.