Identification of partially deuterated exometabolites during culture of tea fungi in heavy water

Exometabolites of tea fungus during the cultivation on heavy water were studied by methods of high-resolution proton and deuterium exchange. Is was shown that, depending on the degree of exchange of proton for deuterium in the methyl group of acetate, three isotopomers exist: -CH3, -CH2D, and -CHD2. Ethanol is represented by six isotopomers: -CHD-CH3, -CD2-CH3, -CD2-CH2D, -CD2-CHD2, -CHD-CH2D, and -CHD-CHD2. Relative concentration of these isotopomers and the percent content of deuterium were determined. Based on these data, the kinetics of metabolism of the object cultivated on heavy water was analyzed. It was show that the efficiency of glucose utilization is the same as during cultivation on light water with the exception that the time of utilization is significantly greater. At the same time, the parameters of utilization of ethanol and its conversion into acetic acid in the life activity of the tea fungus in heavy and light water are very similar.     

Quantitation of positional isomers of deuterium-labeled glucose by gas chromatography/mass spectrometry.

A method for determining the site and extent of deuterium (D) labeling of glucose by GC/MS and mass fragmentography was developed. Under chemical and electron impact ionization, ion clusters m/z 328, 242, 217, 212, and 187 of glucose aldonitrile pentaacetate and m/z 331 and 169 of pentaacetate derivative were produced. From the mass spectra of 13C- and D-labeled reference compounds, glucose carbon and hydrogen (C-H) positions included in these fragments were deduced to be m/z 328 = C1-C6, 2,3,4,5,6,6-H6; m/z 331 = C1-C6, 1,2,3,4,5,6,6-H7; m/z 169 = C1-C6, 1,3,4,5,6,6-H6; m/z 187 = C3-C6, 3,4,5,6,6-H5; m/z 212 = C1-C5, 2,3,4,5-H4; m/z 217 = C4-C6, 4,5,6,6-H4; and m/z 242 = C1-C4, 2,3,4-H3. After correction for isotope discrimination and deuterium-hydrogen exchange, the D enrichment of these fragments can be quantitated using selective ion monitoring, and the D enrichment of all C-H positions can be obtained by the difference in enrichment of the corresponding ion pairs. The validity of this approach was tested by examining D enrichment of known mixtures of 1-d1-, 2-d1-, 3-d1-, and 5,6,6-d3-glucose with unlabeled glucose and D enrichment of perdeuterated glucose using these fragments. This method was used to determine deuterium incorporation in C1 through C6 of blood glucose in fasted (24 h) rats infused with deuterated water. The distribution of deuterium was similar to that found by Postle and Bloxham (1980, Biochem. J. 192, 65-73). Approximately one deuterium atom was incorporated into C5 and only 75% deuterium atom was incorporated into C2. The enrichment of C2 and C6 of glucose relative to that of water indicated that 74 +/- 9% of plasma glucose was newly formed 4 h after the onset of deuterium infusion, and gluconeogenesis accounted for about 76 +/- 7% of the glucose 6-phosphate flux.     

Biosynthesis of acyl groups on molecular species of biliary phosphatidylcholines during metabolism of [2,2,2-2H3]ethanol.

Incorporation of deuterium into different positions of individual molecular species of biliary phosphatidylcholines was determined in bile fistula rats given [2,2,2-2H3]ethanol under conditions ensuring maximal rate of oxidation for 24 h. The deuterium-labelling of the glycerol moiety of the major molecular species was about 6-8 atom% at the end of ethanol administration. The deuterium excess at each of the different positions of the glycerol moiety of 1-palmitoyl-2-linoleoyl phosphatidylcholine was less than 3 atom%. From the isotopic composition of the palmitoyl residues of the phosphatidylcholines, it was calculated that [2,2,2-2H3]ethanol supplied about 35-40% of the acetyl-CoA forming the terminal methyl group and about 25-30% of the other C2 units of the palmitic acid chain. This difference in deuterium incorporation was interpreted as being due to an isotope effect, probably in the rate-limiting carboxylation step of acetyl-CoA. Most or perhaps all of the acetyl groups derived from ethanol were introduced into the terminal methyl group without loss of deuterium. This indicates that citrate is not an important carrier of acetyl-CoA in the biosynthesis of fatty acids from ethanol.     

Mechanism of anaerobic ether cleavage: conversion of 2-phenoxyethanol to phenol and acetaldehyde by Acetobacterium sp.

2-Phenoxyethanol is converted into phenol and acetate by a strictly anaerobic Gram-positive bacterium, Acetobacterium strain LuPhet1. Acetate results from oxidation of acetaldehyde that is the early product of the biodegradation process (Frings, J., and Schink, B. (1994) Arch. Microbiol. 162, 199-204). Feeding experiments with resting cell suspensions and 2-phenoxyethanol bearing two deuterium atoms at either carbon of the glycolic moiety as substrate demonstrated that the carbonyl group of the acetate derives from the alcoholic function and the methyl group derives from the adjacent carbon. A concomitant migration of a deuterium atom from C-1 to C-2 was observed. These findings were confirmed by NMR analysis of the acetate obtained by fermentation of 2-phenoxy-[2-(13)C,1-(2)H(2)]ethanol, 2-phenoxy-[1-(13)C,1-(2)H(2)]ethanol, and 2-phenoxy-[1,2-(13)C(2),1-(2)H(2)]ethanol. During the course of the biotransformation process, the molecular integrity of the glycolic unit was completely retained, no loss of the migrating deuterium occurred by exchange with the medium, and the 1,2-deuterium shift was intramolecular. A diol dehydratase-like mechanism could explain the enzymatic cleavage of the ether bond of 2-phenoxyethanol, provided that an intramolecular H/OC(6)H(5) exchange is assumed, giving rise to the hemiacetal precursor of acetaldehyde. However, an alternative mechanism is proposed that is supported by the well recognized propensity of alpha-hydroxyradical and of its conjugate base (ketyl anion) to eliminate a beta-positioned leaving group.     

Exchange of methyl hydrogens in ethanol during incorporation in bile acids in vivo

[2,2,2-2H]Ethanol was administered continuously to bile fistula rats for 72 h, with or without (--)-hydroxycitrate. The deuterium labelling of biliary bile acids was determined by GC-MS and 13C NMR. Difference spectra between 2H,1H- and 1H-decoupled 13C NMR spectra showed the presence of partly deuterated methyl and methylene groups in methyl cholate, indicating exchange of deuterium in [2,2,2-2H]ethanol for protium prior to or during incorporation of acetate into the bile acid. The extent of exchange was 20--30% as calculated from the isotopic composition of a fragment ion containing one methyl and one methylene group derived from C-2 of acetate. The exchange was unaffected by (--)-hydroxycitrate, indicating that it was not due to reversible incorporation of deuterated acetate into citrate. About 60% of the acetyl-CoA serving as precursor of cholic and chenodeoxycholic acids were derived from ethanol. This value was not changed by administration of (--)-hydroxycitrate. The half-life time of cholesterol molecules acting as precursors of both bile acids was about 50 h in the presence of (--)-hydroxycitrate, which is about the same as previously found in the absence of the inhibitor.     

Biosynthesis of theobroxide and its related compounds, metabolites of Lasiodiplodia theobromae

Administration of (13)C labeled acetates ([1-(13)C], [2-(13)C] and [1,2-(13)C(2)] to Lasiodiplodia theobromae showed the tetraketide origins of both theobroxide, a potato-tuber inducing substance [1, (1S, 2R, 5S, 6R)-3-methyl-7-oxa-bicyclo[4.1.0]hept-3-en-2,5-diol]) and its carbonyldioxy derivative [2, (1S, 4R, 5S, 6R)-7,9-dioxa-3-methyl-8-oxobicyclo [4.3.0]-2-nonene-4,5-diol]. The incorporation of acetate-derived hydrogen into 1 and 2 was studied using [2-(2)H(3), 2-(13)C]acetate. Three and one deuterium atoms were incorporated at one methyl and epoxy carbons, respectively. The observed loss of deuterium atoms from the methyl group suggests a considerable amount of exchange from the methyl group of [2-(2)H(3), 2-(13)C]acetate during biosynthesis of 1 and 2. Incorporation of [1-(13)C]- and [1,2-(13)C(2)]acetates indicates the carbonyl carbon of the carbonyldioxy derivative is derived from the carboxy carbon of the precursor.     

Isotopic exchange during derivatization of platelet activating factor for gas chromatography-mass spectrometry

One approach to the quantitative analysis of platelet activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycerol-3-phosphocholine; also referred to as AGEPC, alkyl glyceryl ether phosphocholine) is hydrolytic removal of the phosphocholine group and conversion to an electron-capturing derivative for gas chromatography-negative ion mass spectrometry. [2H3]Acetyl-AGEPC has been commonly employed as an internal standard. When 1-hexadecyl-2-[2H3]acetyl glycerol (obtained by enzymatic hydrolysis of [2H3]-C16:0 AGEPC) is treated with pentafluorobenzoyl chloride at 120 degrees C, the resulting 3-pentafluorobenzoate derivative shows extensive loss of the deuterium label. This exchange is evidently acid-catalyzed since derivatization of 1-hexadecyl-2-acetyl glycerol under the same conditions in the presence of a trace of 2HCl results in the incorporation of up to three deuterium atoms. Isotope exchange can be avoided if the reaction is carried out at low temperature in the presence of base. Direct derivatization of [2H3]-C16:0 AGEPC by treatment with pentafluorobenzoyl chloride or heptafluorobutyric anhydride also results in loss of the deuterium label. The use of [13C2]-C16:0 AGEPC as an internal standard is recommended for rigorous quantitative analysis.     

The crystal structure of mouse phosphoglucose isomerase at 1.6A resolution and its complex with glucose 6-phosphate reveals the catalytic mechanism of sugar ring opening

Phosphoglucose isomerase (PGI) is an enzyme of glycolysis that interconverts glucose 6-phosphate (G6P) and fructose 6-phosphate (F6P) but, outside the cell, is a multifunctional cytokine. High-resolution crystal structures of the enzyme from mouse have been determined in native form and in complex with the inhibitor erythrose 4-phosphate, and with the substrate glucose 6-phosphate. In the substrate-bound structure, the glucose sugar is observed in both straight-chain and ring forms. This structure supports a specific role for Lys518 in enzyme-catalyzed ring opening and we present a "push-pull" mechanism in which His388 breaks the O5-C1 bond by donating a proton to the ring oxygen atom and, simultaneously, Lys518 abstracts a proton from the C1 hydroxyl group. The reverse occurs in ring closure. The transition from ring form to straight-chain substrate is achieved through rotation of the C3-C4 bond, which brings the C1-C2 region into close proximity to Glu357, the base catalyst for the isomerization step. The structure with G6P also explains the specificity of PGI for glucose 6-phosphate over mannose 6-isomerase (M6P). To isomerize M6P to F6P requires a rotation of its C2-C3 bond but in PGI this is sterically blocked by Gln511.     

Carbohydrate fermentation by three species of polycentric ruminal fungi from cattle and water buffalo in tropical Australia

Fructose, glucose and xylose were the only monosaccharides to be fermented by the polycentric fungi, Orpinomyces joyonii (three cattle isolates) and O. intercalaris (two cattle isolates) and Anaeromyces spp. (four cattle isolates and two water buffalo isolates). Both Orpinomyces spp. utilised a similar range of oligosaccharides and polysaccharides by fermenting cellobiose, gentiobiose, lactose, maltose, sucrose, cellulose, glycogen, starch and xylan. In contrast, there was considerable variation in carbohydrate fermentation amongst Anaeromyces spp., with only cellobiose, gentiobiose and cellulose being fermented by all strains. Formate, acetate and ethanol were the major fermentation end-products formed from glucose by all polycentric fungi. In addition, Anaeromyces spp. produced considerable amounts of lactate, although only small amounts were formed by Orpinomyces spp. This difference was explained by the low specific activity for lactate dehydrogenase in Orpinomyces spp. Several Anaeromyces spp. also produced malate as a significant end-product of glucose fermentation. Fermentation of specifically-labelled Z14C]glucose molecules by polycentric fungi showed that hexose was catabolised by both polycentric and monocentric fungi via the glycolysis pathway with end-products being derived from the following carbon atoms: lactate and malate (C1-C3; C4-C6), acetate and ethanol (C1-C2; C5-C6), CO2 and formate (C3; C4). The results were compared to those obtained for monocentric and polycentric fungi isolated from temperate climate ruminants.     

Effect of heavy water on metabolism of a symbiotic organism

The metabolism of the symbiotic organism medusomycete (tea fungus) and the influence of D2O on its development was studied by high-resolution NMR methods using isotopically enriched (by 13C and 2H) metabolites. The results demonstrate that D2O influences the selective utilization of certain protonated substrates during the formation of triose phosphates. It was found that protonated isotopomers derived from the first glucose fragment C1-C2-C3 are predominantly utilized. This explains why the metabolism slows down by a factor of 2 to 3 if D2O concentration in the medium increases. It was also shown that approximately 10% of the organisms are in the state of dynamic extracellular endosymbiosis. This state is characterized by the ability to exchange the metabolic products through close intercellular contacts. As a result of the metabolic exchange, a multicellular organism is formed, with metabolic elements localized in different partners. A distinguishing feature of this organism is the ability to accumulate the internal resources of carbon, thus making it better adapted to the unfavorable environment.     

Deuterium labeling of diethylstilbestrol and analogues

E-2,2,3',3″,5,5,5',5″-octadeuteriodiethylstilbestrol (DES-d8) and Z-2,3',3″,4,5,5,5',5″-octadeuterio-3,4-bis(p-hydroxyphenyl)-2-hexene (ψ-DES-d8) were synthesized from E-diethylstilbestrol (DES) by hydrogen/ deuterium exchange in a mixture of methanol-d and deuterium chloride in deuterium oxide. The structures, isotopic purity, and positions of up-take of deuterium were determined by nuclear magnetic resonance (NMR) and mass spectrometry (MS). Additional confirmation of the positions of deuterium exchange in stilbestrols was obtained from an analysis of the oxidation of DES-d8 to Z,Z-2,3',3″,5,5',5″-hexadeuteriodienestrol (β-DIES-d6) and of the hydrogen/deuterium exchange reaction of hexestrol (HEX) to 3',3″,5',5″-hexestrol (HEX-d4). Structural analysis and the determination of isotopic purity of the latter two compounds were also carried out by NMR and MS. The uptake of eight deuterium atoms by DES is postulated to proceed via two different reactions occurring simultaneously: 1. acid catalyzed deuteration of all four phenolic ortho-positions (3',3″,5',5″); 2. acid catalyzed deuteration of the olefin bridge with subsequent formation of deuterated ψ-DES (3 or 4). Due to the equilibration between DES, ψ-DES, and Z-diethylstilbestrol (cis-DES) in the acidic reaction mixture at 85°C, the deuterated ψ-DES is thought to rapidly rearrange to deuterated DES. Repeated deuteration will eventually form DES-d8 fully labeled in the 2,2,5,5 methylene positions.     

X-ray structural and n.m.r.-spectral studies of methyl alpha-L-evalopyranoside: reassignment of anomeric configuration for the methanolysis product of methyl 6-deoxy-3-C-methyl-alpha-L-mannofuranoside

The methanolysis product of methyl 6-deoxy-3-C-methyl-alpha-L-mannofuranoside has been reassigned as methyl 6-deoxy-3-C-methyl-alpha-L-mannopyranoside by X-ray crystallographic and n.m.r.-spectral analyses. The crystals of methyl alpha-L-evalopyranoside are monoclinic, space group C2, with cell dimensions: a = 12.913(2), b = 8.052(1), c = 9.766(2) A, B = 105.13(2) degrees. The pyranoside ring exists in the 1C4 conformation, with the methoxyl and 3-C-methyl groups axial. Nuclear Overhauser effects were measured for selected proton resonances in the 1H-n.m.r. spectrum. Irradiation of the 3-C-methyl and 5-C-methyl group proton signals resulted in enhancements for H-2, H-4, H-5, and the methoxyl group hydrogen atoms, but not for H-1.     

Chemospecific deuteration of prostaglandin silyl ethers

Complete chemical selectivity (i.e., chemospecificity) has been achieved in the homogeneous deuteration of C5-C6 and endocyclic C10-C11 prostaglandin double bonds without arrangement or partial reduction of C13-C14- or C8-C12 double bonds. The homogeneous deuteration reaction utilizes protection of the C13-C14 double bond as the C15O-silyl ether and protection of the carboxyl group as the methyl ester prior to reduction under molecular deuterium with tris(triphenylphosphine)chlorohodium (I) (Wilkinson's catalyst) in 60:40 acetone:benzene at 25 degrees C. The reaction has been used to prepare six specifically deuterated prostaglandin: 5,6-dideuterio-PGE1 alpha 5,6-dideuterio-PGE1, 5,6-dideuterio-PGB1, 3,3,4,4,5,6-hexadeuterio-PGF1 alpha, 5,6,10,11-tetradeuterio-11-deoxy-PGE1, and 10, 11-dideuterio-11-deoxy-PGE1.     

Proton exchange and internal motions in two chromomycin dimer-DNA oligomer complexes.

Previous structural studies on the complexes of the chromomycin (CHR) dimer with duplexes of d(A1-A2-G3-G4-C5-C6-T7-T8) and of d(A1-G2-G3-A4-T5-C6-C7-T8) in solution [one Mg(II) and two drugs per duplex] are extended to hydrogen exchange measurements. Exchange of the OH8 proton of chromomycin, measured by real time proton-deuterium exchange, is very slow and requires dissociation of the complex, whose lifetime is thus determined. The lifetimes and apparent dissociation constants of base pairs are deduced from the catalysis of imino proton exchange by ammonia. The four central base pairs, which interact with the CHR chromophores in the minor groove (Gao & Patel, 1990), may open within the complex, but the opening rate is less than in the free duplex by one to two orders of magnitude. The activation energy for base-pair opening and the differences between the lifetimes of adjacent pairs suggest that single base-pair opening is the predominant imino proton exchange pathway in all cases. In the symmetrical complex of chromomycin with the first duplex, the lifetimes of the central base pairs (G3.C6 and G4.C5) are in the same range (52 and 29 ms, respectively, at 38 degrees C). In the asymmetrical complex formed with the second duplex, the base-pair lifetimes in the G2-G3-A4-T5 segment that interacts with the chromophore moiety are strongly increased. That of G3.C6 is particularly long. Above 50 degrees C, exchange of the G3 imino proton is opening limited.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hydrogen and hydration in proteins

Neutron diffraction provides an experimental method of directly locating hydrogen atoms in proteins. High-resolution neutron diffractometers dedicated to biological macromolecules (BIX-type diffractometer) have been constructed at the Japan Atomic Energy Research Institute and they have been used in the 1.5A-resolution crystal structure analyses of several proteins. Interesting topics relevant to hydrogen and hydration in proteins, such as (1) the detailed geometry of hydrogen bonds; (2) information regarding hydrogen/deuterium exchange behavior; (3) the acidities of certain H atoms; (4) the role of hydrogen atoms in enzyme mechanisms and thermostability; (5) the location methyl hydrogen atoms; and (6) dynamical behavior of hydration structures that include H positions have been extracted from these structural results. In addition, a method for the systematic growth of large single crystals based on phase diagrams has been introduced and will be briefly described in this article.     

Syntheses of stable isotope-labeled 6 beta-hydroxycortisol, 6 beta-hydroxycortisone,and 6 beta-hydroxytestosterone

A method is described for the preparation of two types of multi-labeled 6 beta-hydroxycortisol containing either five deuterium atoms at C-19 methyl and C-1 methylene or four 13C atoms at C-1, C-2, C-4, and C-19 in addition to the five deuterium atoms for use as analytical internal standards for gas chromatography-mass spectrometry (GC-MS). BMD derivatives of [1,1,19,19,19-2H(5)]cortisone and [1,2,4,19-13C(4),1,1,19,19,19-2H(5)]cortisone (cortisone-2H(5)-BMD and cortisone-13C(4),2H(5)-BMD) were first synthesized via indan synthon method starting from optical active 11-oxoindanylpropionic acid and labeled isopropenyl anion ([1,1,3,3,3-2H(5)]- or [1,3-13C(2),1,1,3,3,3-2H(5)]isopropenyl anion). The labeled isopropenyl anion was prepared from commercially available [1,1,1,3,3,3-2H(6)]- or [1,3-13C(2),1,1,1,3,3,3-2H(6)]acetone. Ultraviolet (UV) irradiated autoxidation at C-6 position of 3-ethyl-3,5-dienol ether derivatives of the labeled cortisone-BMDs gave 6 beta-hydroxy-[1,1,19,19,19-2H(5)]cortisone-BMD and 6 beta-hydroxy-[1,2,4,19-13C(4),1,1,19,19,19-2H(5)]cortisone-BMD, respectively, as a mixture of 6 beta- and 6 alpha-epimers in a ratio of 4:1. Separation of 6 beta- and 6 alpha-epimers by thin-layer chromatography (TLC) and subsequent hydrolysis of the BMD group at C-17 gave pure labeled 6 beta-hydroxycortisone. After protecting the keto group at C-3 of the labeled 6 beta-hydroxycortisone-BMD as semicarbazone, reduction of 11-keto group with NaBH(4) and subsequent removal of the C-3 and C-17 protecting groups gave 6beta-hydroxy-[1,1,19,19,19-2H(5)]cortisol (6 beta-hydroxycortisol-2H(5)) and 6 beta-hydroxy-[1,2,4,19-13C(4),1,1,19,19,19-2H(5)]cortisol (6 beta-hydroxycortisol-13C(4),2H(5)), respectively, as a mixture of 6 beta- and 6 alpha-epimers (6 beta:6 alpha=4.4:1). The isotopic compositions of 6 beta-hydroxycortisol-2H(5) and 6 beta-hydroxycortisol-13C(4),2H(5) were 90.9 and 92.1 at.%, respectively. Furthermore, 6 beta-hydroxy-[1 alpha,16,16,17 alpha-2H(4)]testosterone was synthesized by the UV irradiated autoxidation at C-6 position of 3-ethyl-3,5-dienol ether derivative of deuterium-labeled testosterone ([1 alpha,16,16,17 alpha-2H(4)]testosterone) obtained by using catalytic deuteration and hydrogen-deuterium exchange reactions.     

Non-equivalence of hydrogen transfer from glucose to the pro-R and pro-S methylene positions of ethanol during fermentation by Leuconostoc mesenteroides quantified by 2H NMR at natural abundance

The anaerobic fermentation of glucose by Leuconostoc mesenteroides via the reductive pentose phosphate pathway leads to the accumulation of lactic acid and ethanol. The isotope redistribution coefficients (a(ij)) that characterize the specific derivation of each hydrogen atom in ethanol in relation to the non-exchangeable hydrogen atoms in glucose and the medium water have been determined using quantitative (2)H NMR. First, it is confirmed that the hydrogens of the methylene group are related only to the 1 and 3 positions of glucose via the NAD(P)H pool and not to the 4 position, in contrast to ethanol produced by Saccharomyces cerevisiae. Second, it is found that the conversion factors (C(f)) for the transfer of hydrogen to the pro-S and pro-R positions of the methylene group are not equivalent: the C(f)-1-R:C(f)-1-S ratio is 2.1, whereas the C(f)-3-R:C(f)-3-S ratio is 0.8. It is shown that this non-equivalence is not determined by the stereochemistry of the terminal NADH- and NADPH-dependent alcohol dehydrogenases, but is dependent on the cofactor selectivities of the reductive and oxidative steps of the reduced nucleotide cycle.     

Shapes of benz[a]anthracenes: the crystal and molecular structure of 2-methylbenz[a]anthracene

The crystal structure of 2-methylbenz[a]anthracene (2-MBA), the least carcinogenically active of the monomethylbenz[a]anthracenes, has been determined by application of direct methods to single-crystal X-ray diffractometric data and refined by least squares to R = 0.033 (Rw = 0.035). Deviations of the carbon atoms from the mean molecular plane are much smaller than in the rather more active 1-MBA; in 2-MBA, the benzo-ring A is inclined at about 2 degrees to each of the three rings in the anthracene moiety and even the methyl carbon atom is displaced by only 0.07 A from the ring-carbon atom plane of 2-MBA (and by 0.01 A from the ring-A plane). As in other MBA, the shortest C-C bond in this accurately determined structure is at the K-region (C(5)-C(6) = 1.330(3) A) but three other bonds are short; C(8)-C(9) = 1.347(4), C(10)-C(11) = 1.353(3) and the M-region bond C(3)-C(4) = 1.359(4) A (0.003 A longer if corrected for rigid-body librations). The 2-methyl group appears to take up two orientations with one trio of hydrogen positions more favored than the other.     

The molecular and crystal structures of 4-N-(2-acetamido-2-deoxy-β-d-glucopyranosyl)-l-asparagine trihydrate and 4-N-(β-d-glucopyranosyl)-l-asparagine monohydrate. The X-ray analysis of a carbohydrate–peptide linkage

X-ray analyses have shown that the glucopyranose rings of GlcNAc-Asn [4-N-(2-acetamido-2-deoxy-beta-d-glucopyranosyl)-l-asparagine] and Glc-Asn [4-N-(beta-d-glucopyranosyl)-l-asparagine] both have the C-1 chair conformation and also that the glucose-asparagine linkage of each molecule is present in the beta-anomeric configuration. The dimensions (the estimated standard deviations of the last digit are in parentheses) of the glycosidic bond in GlcNAc-Asn and Glc-Asn are, respectively, C((1))-N((1)) 0.1441(6)nm, 0.146(2)nm; angle O((5))-C((1))-N((1)) 106.8(3) degrees , 105.7(8) degrees ; angle C((2))-C((1))-N((1)) 111.1(4) degrees , 110.4(9) degrees ; angle C((1))-N((1))-C((9)) 121.4(4) degrees , 120.5(9) degrees . The glycosidic torsion angle C((9))-N((1))-C((1))-C((2)) is 141.0 degrees and 157.6 degrees in GlcNAc-Asn and Glc-Asn respectively. Hydrogen-bonding is extensive in these two crystal structures and does affect one torsion angle in particular. Two very different values of chi(1)(N-C(alpha)-C(beta)-C(gamma)) occur for the asparagine residue of the two different molecules; the values of chi(1), -69.0 degrees in GlcNAc-Asn and 61.9 degrees in Glc-Asn, correspond to two different staggered conformations about the C(alpha)-C(beta) bond as the NH(3) (+) group is adjusted to different hydrogen-bonding patterns. The two trans-peptide groups in GlcNAc-Asn show small distortions in planarity whereas that in Glc-Asn is more non-planar. The mean plane through the atoms of the amide group at C((2)) in GlcNAc-Asn is approximately perpendicular (69 degrees ) to the mean plane through the C((2)), C((3)), C((5)) and O((5)) atoms of the glucose ring and that at C((1)) is less perpendicular (65 degrees ). The mean plane through the atoms of the amide group in Glc-Asn makes an angle of only 55 degrees with the mean plane through these same four atoms of the glucose ring. The N((1))-H bond of the amide at C((1)) is trans to the C((1))-H bond in these two compounds; the N((2))-H bond of the amide at C((2)) is trans to the C((2))-H bond in GlcNAc-Asn. The values of the observed and final calculated structure amplitudes have been deposited as Supplementary Publication SUP 50035 (26 pages) at the British Library (Lending Division), (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies may be obtained on the terms given in Biochem. J. (1973) 131, 5.     

Deoxyguanosine-deoxyadenosine pairing in the d(C-G-A-G-A-A-T-T-C-G-C-G) duplex: conformation and dynamics at and adjacent to the dG X dA mismatch site

Nuclear magnetic resonance (NMR) has been used to monitor the conformation and dynamics of the d-(C1-G2-A3-G4-A5-A6-T6-T5-C4-G3-C2-G1) self-complementary dodecanucleotide (henceforth called 12-mer GA) that contains a dG X dA purine-purine mismatch at position 3 in the sequence. These results are compared with the corresponding d(C-G-C-G-A-A-T-T-C-G-C-G) dodecamer duplex (henceforth called 12-mer) containing standard Watson-Crick base pairs at position 3 [Patel, D.J., Kozlowski, S.A., Marky, L.A., Broka, C., Rice, J.A., Itakura, K., & Breslauer, K.J. (1982) Biochemistry 21, 428-436]. The dG X dA interaction at position 3 was monitored at the guanosine exchangeable H-1 and nonexchangeable H-8 protons and the nonexchangeable adenosine H-2 proton. We demonstrate base-pair formation between anti orientations of the guanosine and adenosine rings on the basis of nuclear Overhauser effects (NOE) observed between the H-2 proton of adenosine 3 and the imino protons of guanosine 3 (intra base pair) and guanosines 2 and 4 (inter base pair). The dG(anti) X dA(anti) pairing should result in hydrogen-bond formation between the guanosine imino H-1 and carbonyl O-6 groups and the adenosine N-1 and NH2-6 groups, respectively. The base pairing on either side of the dG X dA pair remains intact at low temperature, but these dG X dC pairs at positions 2 and 4 are kinetically destabilized in the 12-mer GA compared to the 12-mer duplex. We have estimated the hydrogen exchange kinetics at positions 4-6 from saturation-recovery measurements on the imino protons of the 12-mer GA duplex between 5 and 40 degrees C. The measured activation energies for imino proton exchange in the 12-mer GA are larger by a factor of approximately 2 compared to the corresponding values in the 12-mer duplex. This implies that hydrogen exchange in the 12-mer GA duplex results from a cooperative transition involving exchange of several base pairs as was previously reported for the 12-mer containing a G X T wobble pair at position 3 [Pardi, A., Morden, K.M., Patel, D.J., & Tinoco, I., Jr. (1982) Biochemistry 21, 6567-6574]. We have assigned the nonexchangeable base protons by intra and inter base pair NOE experiments and monitored these assigned markers through the 12-mer GA duplex to strand transition.(ABSTRACT TRUNCATED AT 400 WORDS)