Sugar Portion Research Articles

Targeting DNA with Anthracyclines: The Importance of the Sugar Moiety

Waldemar PriebeThe University of Texas M. D. Anderson Cancer Center, Houston, TX 77030Our studies focusing on the role of the sugar portion in anthracycline-DNA interaction laid thefoundation for the design of novel DNA interactive agents. We have designed and synthesized twonew classes of such agents which: (1) bind with high affinity to specific sequences of DNA and (2)form cross-links with DNA.The design of agents binding with high affinity to specific sequences of DNA was based on studiesof (1) the effects of a sugar portion’s charge and its orientation on drug-DNA binding (daunosamine),(2) the assessment of energetic contribution to DNA binding (for daunosamine and other selectedstructural fragments), and (3) analysis of the crystallographic structure of the daunorubicin-DNA com-plex. Analysis of the structure of daunorubicin-DNA complex revealed that two daunorubicins faceeach other with sugar moieties and position their 3’-NH2 groups within a distance of 6-7A.H

Investigation into the Acyl Chain Packing of Endotoxins and Phospholipids under Near Physiological Conditions by WAXS and FTIR Spectroscopy

The acyl chain packing of various endotoxins and phospholipids was monitored via the main wideangle reflection between 0.410 and 0.460 nm by wide-angle X-ray scattering (WAXS) and via the absorption band of the symmetric stretching vibration of the methylene groups vs(CH2) around 2849 to 2853 cm−1 by Fourier-transform infrared spectroscopy. The lipids investigated included various rough mutant (R) and smooth form (S) lipopolysaccharides (LPS) differing in the length of the sugar portion, lipid A, the “endotoxic principle” of LPS, and various saturated and unsaturated phospholipids with different head groups under a near physiological (≥85%) water content. The packing density of the saturated endotoxin acyl chains is lower than those of saturated phospholipids but similar to those of monounsaturated phospholipids, each in the gel phase. The hydrophobic moiety of endotoxins thus exhibits significant conformational disorder already in the gel phase. The acyl chain packing of the endotoxins decreases with increasing length of the sugar chain lengths, which seems to be relevant to the observed differences in biological activity. For Re-LPS with different counterions (salt forms), in the presence of externally added cations or at reduced water content (50%), no change of the acyl chain packing density is deduced in the X-ray data, although the FT-IR data indicate its increase. The position of the vs(CH2) vibration is, thus, only a relative measure of lipid order, in particular when lipids with different head groups and in the presence of external agents are compared.

Isolation and structural characterization of rhamnogalacturonan II-borate complex fromPinus densiflora

A boron-containing pectic polysaccharide was isolated from Driselase digests of akamatsu (Pinus densiflora) cell wall. The boron-containing polysaccharide was purified by successive column chromatography of Cosmosil C18 OPN, DEAE Sepharose FF, and Superdex 75. The complex had 0.12% (w/w) boron (B) and11B-NMR spectroscopy showed the complex to be the tetrahedral diester form of boron. The sugar composition and glycosyl linkage analyses showed that the sugar portion of the complex was rhamnogalacturonan-II (RG-11). The boron-attached glycosyl residue was identified to be 2-O-Me-xylose-containing apiosyl side chain in RG-II. These results established that the location of borate in the cell wall is conserved among dicots, gramineous monocots, and coniferous gymnosperm.

Synthesis and biological evaluation of coumarincarboxylic acids as inhibitors of gyrase B. L-rhamnose as an effective substitute for L-noviose

A series of novobiocin-like coumarincarboxylic acids has been prepared bearing the L-rhamnosyl moiety as the sugar portion of the molecule. The similar DNA gyrase inhibitory activity of the novel class of coumarins to that of novobiocin demonstrates that L-rhamnose can effectively replace L-noviose. Introduction of alkyl side-chains at C-5 of coumarin leads to improved in vitro antibacterial properties in the novel series.

Structural identification of a novel degradant of the antibiotic pirlimycin formed under thermal stress conditions

The structure of a novel, thermally-induced degradant of the antibiotic pirlimycin is presented. The degradant was discovered during thermal stress stability testing of pirlimycin. The structure was determined using high resolution mass spectrometry, ms/ms fragmentation, and extensive nmr studies including a combination of homonuclear TOCSY and gradient, inverse-detected 2-D nmr experiments which included GHSQC and GHMBC. All nmr, high resolution ms and ms/ms fragmentation data are consistent with loss of a molecule of HCl during a ring closure involving one of the hydroxyl moieties from the sugar portion of the molecule forming a tetrahydrofurano[3,2-b]perhydropyran ring structure. The X-ray crystal structure is reported for pirlimycin. Using this structure molecular modelling studies are performed which demonstrate the feasibility of the formation of the new structure.

Phase and Surface Properties of Lipid Bilayers Containing Neoglycolipids

The physical properties conferred to DPPC bilayers by including neoglycolipids composed by two different trisaccharides: mannose-mannose-mannose (3M) and glucose-mannose-glucose (GMG) attached to a cholesterol (cho) and a distearylglycerol (diC18) lipid moiety by a spacer were evaluated by means of the measurement of the electrokinetic potential and interfacial fluorescent probes. The phase properties measured with diphenylhexatriene (DPH) were correlated with the surface properties measured with merocyanine 540, dansyl, and Laurdan probes. The results show that the surface properties of large unilamellar vesicles depend on the sugar exposure to the water phase and also on the hydrocarbon moiety by which it is anchored to the bilayer. The combination of the cholesterol moiety with the saccharide attenuates the cooperativity decrease induced by the cholesterol moiety without the sugar portion. The neoglycolipid GMG-diC18 promotes opposite effects affecting slightly the cooperativity at the hydrocarbon core of DPPC and displacing the phase transition temperature to higher values. The presence of neoglycolipid with diC18 introduces defects in the packing at the interface of the membrane in the gel state. It is concluded that a relatively low proportion of neoglycolipids affects significantly the interfacial properties of DPPC bilayers in large unilamellar vesicles in the absence of changes at the membrane bulk at 25°C.

ChemInform Abstract: Stannyl Migration from the Base to the Sugar Portion of 1′,2′‐Unsaturated Uridine: The First Example of Substitution at the 2′‐Position.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Combined Small-Angle X-ray and Neutron Scattering Experiments for Thickness Characterization of Ganglioside Bilayers

Combined small-angle neutron and X-ray scattering experiments on dilute solutions of the ganglioside GM3 in water are presented. The contrast profiles along the lipid and sugar portions of the ganglioside molecule for the two types of radiations are different, a feature which, added to the extension of the scattering q range to q ∼ 0.4 Å-1, allows an accurate determination of the cross section of the bilayer in dilute solution. The electronic density profile of the bilayer cross section can be interpreted only by assuming thickness fluctuations, which have already been theoretically predicted for the GM3 bilayers, due to the peculiar packing properties of the monomer. Increasing temperature from room temperature to 50 °C has the effect of flattening the GM3 bilayer, as the thickness and the water content of the outer hydrophilic layer decrease, while the thickness of the inner hydrophobic region is almost unchanged.

N-glycosylation by transfer of GlcNAc2 from dolichol-PP-GlcNAc2 to the protein moiety of the major yeast exoglucanase.

Transfer of truncated oligosaccharides to yeast exoglucanase (Exg) in Saccharomyces cerevisiae alg1 has been investigated. When incubated at the non-permissive temperature, alg1 cells secreted into the culture medium, in addition to the exoglucanase glycoforms secreted by wild type, underglycosylated forms as well as material with ionic properties of the non-glycosylated enzyme. As expected, none of the latter had affinity towards concanavalin A, but part of it bound to wheat germ agglutinin (WGA), suggesting that it contained, in addition to non-glycosylated Exg, glycoforms carrying non-reducing terminal GlcNAc. Only the WGA-bound material could be labelled with galactosyltransferase; furthermore, the label could be released by treatment with peptide-N4-N-acetyl-beta-glucosamine asparagine amidase. These results unambiguously demonstrate that GlcNAc2 can be transferred from dolichol-PP-GlcNAc2 to one or both sequons of yeast Exg. Accordingly, they support previous observations suggesting that this early intermediate is able to translocate in vivo in order to make its sugar portion accessible to the oligosaccharyltransferase in the lumen of the endoplasmic reticulum.

Stannyl migration from the base to the sugar portion of 1′,2′-unsaturated uridine: the first example of substitution at the 2′-position

Abstract We report that TBDMS-protected 1-(2-deoxy-D- erythro -pent-1-enofuranosyl)-6-(tributyl-stannyl)uracil, when treated with LDA or LTMP, undergoes an anionic stannyl migration to yield the 2′-stannylated product. Optimization of the reaction conditions has disclosed an efficient entry to compounds variously substituted at the 2′-position. Desilylation of these compounds caused no further elimination, and furnished a hitherto unknown series of nucleoside analogues.

How E. coli DNA polymerase I (klenow fragment) distinguishes between deoxy- and dideoxynucleotides

Deoxy- and dideoxynucleotides differ only in whether they have a hydroxyl substituent at C-3′ of the ribose moiety, and yet the Klenow fragment DNA polymerase prefers the natural (dNTP) substrate by several thousandfold. We have used this preference in order to investigate how Klenow fragment interacts with the sugar portion of an incoming dNTP. We screened mutant derivatives of Klenow fragment so as to identify those amino acid residues that play important roles in distinguishing between dNTPs and ddNTPs. Substitution of Phe762 with Ala or Tyr caused a dramatic decrease in the discrimination against ddNTPs, while mutations in Tyr766 and Glu710 had a smaller effect, suggesting that these two side-chains play secondary roles in the selection of dNTPs over ddNTPs. In order to understand the interactions in the enzyme—DNA—dNTP ternary complex, pre-steady-state kinetic parameters for the incorporation of dNTPs and ddNTPs were determined for wild-type Klenow fragment and for mutant derivatives that showed changes in dNTP/ddNTP discrimination. From elemental effect measurements we infer that selection against dideoxynucleotides takes place in the transition state for the conformational change that precedes phosphoryl transfer. The crucial role of the Phe762 side-chain appears to be to constrain the dNTP molecule so that the 3′-OH can make an interaction with another group within the ternary complex. When Tyr is substituted at position 762, the same interactions can take place to position the dNTP, but specificity against the ddNTP is lost because the phenolic OH can compensate for the missing 3′-OH of the nucleotide. Substitution of the smaller Ala side-chain results in a loss in specificity because the dNTP is no longer appropriately constrained. Measurement of reaction rates as a function of magnesium ion concentration suggests that the interaction made with the dNTP 3′-OH may involve a metal ion and the Glu710 side-chain, the simplest scenario being that both the 3′-OH and the carboxylate of Glu710 are ligands to the same metal ion.

Synthesis of pavoninin-1, a shark repellent substance, and its structural analogues toward mechanistic studies on their membrane perturbation

Pavoninin-1 ( 1), which was isolated from a defense secretion of the sole Pardachirus spp. as an ichthyotoxic and a shark repellent principle, and its structural analogue 2 were synthesized, where glycosylation using an 2-azidoglycosyl sulfoxide ( 10) afforded the corresponding β-glycoside exclusively in high yield. Introduction of the α,β-unsaturated ketone system in the ring A of 1 was achieved by phenylselenenylation of dihydropavoninin-1 ( 3) and subsequent oxidative elimination without protection of the hydroxyl groups in the sugar portion. The mode of action of these glycosides was evaluated for their perturbation on phosphatidylcholine liposomal membrane, using the fluorescent dye leakage method. The results revealed that membrane affinity does not parallel membrane perturbation but rather compensates it, and the spatial arrangement of hydrophobic and hydrophilic regions within a molecule is likely to reflect on the difference in potency of action among them.

Characterization of Streptomyces nogalater genes encoding enzymes involved in glycosylation steps in nogalamycin biosynthesis.

The sno gene cluster in Streptomyces nogalater ATCC 27451 contains the nogalamycin biosynthesis genes. A set of plasmid constructions carrying fragments of the sno cluster that lie downstream of snoD were used to complement the S. galilaeus mutant H039, which is blocked in rhodosamine and 2-deoxyfucose biosynthesis in the aclacinomycin pathway. Sequence analysis of this cluster revealed three contiguous open reading frames (ORFs) that were designated snoF, snoG, and snoH. Only those plasmid constructs that expressed SnoG were able to complement H039. SnoG shows similarity to GalE, a UDP-glucose-4-epimerase catalyzing the epimerization of UDP-glucose to UDP-galactose. The putative SnoF protein is similar to 3,5-epimerases involved in rhamnose biosynthesis. The deduced product of snoH is a 489-amino acid polypeptide. It is similar to the product of dau ORF3 found in the daunomycin cluster. However its function is still unclear. Based on the complementation experiments and sequence analysis, this part of the sno cluster is suggested to be involved in the biosynthesis of the sugar portion of nogalamycin. Interestingly, SnoA, a transcriptional activator for the sno minimal polyketide synthase, is also needed to express this cluster.

Selectin-ligand interactions revealed by molecular dynamics simulation in solution.

Through a computer modeling and simulation technique, we investigated the binding mode of a complex of E-selectin-GSC-150, which is a novel selectin blocker. GSC-150 is the 3'-sulfated Lewis X derivative with a long, branched alkyl chain. Initial attempts to construct a model for E-selectin-GSC-150 complex were performed based on a previously reported model of E-selectin-sialyl Lewis X (sLex) complex [Kogan, T.P.; Revelle, B.M.; Tapp, S.; Scott, D.; Beck, P.J.J. Biol. Chem. 1995, 270, 14047-14055]. In our model, the carbohydrate portion of GSC-150 interacted with the protein in a similar manner as that of sLex reported previously. Interestingly, each of the branched alkyl chains extended on the surface of E-selectin and interacted with two different hydrophobic portions. One of these hydrophobic portions consists of Tyr44, Pro46, and Tyr48. Another portion forms a shallow cavity, and it consists of Ala9, Leu114, and the alkyl moieties of the side chains of Lys111, Lys112, and Lys113. A subsequent 200-ps molecular dynamics simulation in solution revealed that the interactions involved in the sugar portion of the ligand were relatively weak, whereas the hydrophobic interactions involved in the branched alkyl chains were fairly stable in solution. These results suggest that the branched alkyl chain serves as an "anchor" for the tight binding of GSC-150 on the surface of E-Selectin. This is the first attempt to evaluate the dynamics of E-Selectin-ligand interactions in solution, and it sheds light on the nature of ligand recognition by selectins.

Glycoproteins: Characterization in a Hybrid Grape Variety (Muscat Bailey A) Juice, Fermenting Must, and Resultant Red Wine

Elucidation of the differences in properties between unstable and soluble proteins will assist in developing effective fining of wine and in breeding of grape varieties with fewer unstable proteins. Soluble proteins from juice, must pressed just after fermentation on skins, and from red wines were isolated by ammonium sulfate precipitation and fractionated by Sephadex G-100 followed by high performance liquid chromatography. At least 17, 18, and 22 protein fractions were obtained from the juice, fermenting must, and red wine samples, respectively. All of the fractions had sugars in the range of 0.3% to 33.3% of total glycoproteins (the sum of the total sugars and amino acids found in the hydrosolates). Most of the glycoproteins had less than 10% sugars. The glycoproteins differed from grape proteins, generally being rich in the amino acids aspartic acid, glycine, threonine, glutamic acid, and alanine and in the sugars arabinose, rhamnose, galactose, and glucose. Their isoelectric points and molecular weights ranged from 3.7 to 4.9 and form 12.1 to 30.8 K daltons, respectively. Differences in the HPLC patterns and in the chemical and physical properties of the protein fractions obtained revealed the presence of several individual glycoproteins, their concentrations, and occurrence of specific glycoproteins at each stage of vinification. The proportion of glycoprotein sugars decreased in the order juice proteins &gt; must proteins &gt; wine proteins — <i>i.e.</i>, degradation or modification of the sugar portions of the glycoproteins occurred during vinification. The origin of wine glycoproteins suggests roles in aging reactions and other wine characteristics.

The Cardenolides ofSpeirantha convallarioides1

The cardenolide pattern of Speirantha convallarioides has been investigated and compared with Convallaria majalis, a close relative with active cardenolides and used extensively as a cardiac remedy. Five cardenolides were isolated from small amounts (25 g) of plant material by application of an efficient isolation procedure. Their identification was achieved by an easy concept mainly based on detailed analysis of 1H-NMR and DCI-NH3-mass spectral data. Rhodexin A(1) and lokundjoside (2) are the major cardenolides (42% and 31% of the total cardenolides in the plant), while digitoxigenin-glucoside (3), periplorhamnoside (4), and periplogenin-glucoside (5) are minor constituents of the cardenolide complex (3 to 4% each). Similarities in cardenolide patterns (structures of the aglycones, variability of the sugar portion) along with morphological and biogenetical characteristics support the classification of S. convallarioides as belonging in the same tribe Convallarieae as C. majalis within the family Convallariaceae.

Synthesis of trehazolin analogues containing modified sugar moieties

In order to elucidate the biological roles of the sugar moiety of the trehalase inhibitor trehazolin 1, the sugar portion was first replaced with hydrophobic aromatic functions, providing the N-phenyl 3 and N-benzyl derivatives 4 of trehazolin 1. Then the six analogues with the sugar moiety being replaced with D-mannopyranose 5, 3-deoxy-ribo-hexopyranose 6, D-galactopyranose 7, 6-deoxy-D-glucopyranose 8, 5a-carba-α-D-glucopyranose 9 and 5a-carba-α-D-xylo-hex-5(5a)-enopyranose residues 10 were synthesized.In the hope of improving the fungicidal activity of trehazolin 1, we prepared two disaccharide analogues, 11 and 12, containing maltose and cellobiose residues. A remarkable decrease in potency was observed in the analogues 5–8 and 10, but not for 5a′-carbatrehazolin 9, suggesting an essential role for the D-gluco configuration of the hexopyranose portion. The β-D-glucosyl analogue 12 showed an increase in antifungal activity against Rhizoctonia solani, as compared with that of trehazolin 1. The analogues 3 and 4 were not trehalase inhibitors, but rather were moderate α-glucosidase inhibitors.

Liquid secondary ionization, tandem and matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometric characterization of glycosphingolipid derivatives

AbstractPermethylated, peracetylated and perbenzoylated derivatives of glycosphingolipids (GSLs) were prepared to compare their liquid secondary ionization mass spectrometric (LSIMS) and collision‐induced dissociation tandem mass spectrometric (CID/MS/MS) fragmentation patterns and also to determine sensitivity improvement in LSIMS and matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOFMS) relative to the native species. Permethylation was carried out in the liquid phase, whereas peracetylation and perbenzoylation could be effected using either liquid (bulk)‐phase or gas‐phase procedures. Lower amounts of starting material were required for the gas‐phase derivatization (⩽ 100 pmol) compared with the bulk phase (⩽1 nmol), because the former method permits more efficient sample handling. All three types of derivatives yielded sensitivity improvements of at least two orders of magnitude over the native species in both LSIMS and MALDI‐TOFMS. The behavior of the permethylated compounds was used as the benchmark for GSL structural information content in normal and tandem mass spectra. Fragments present in spectra of the three types of derivatives generated complementary information. Permethylated GSLs favored the formation of ions related to the ceramide moieties, whereas peracetylation enhanced the production of carbohydrate‐related ions. The LSI mass spectra of perbenzoylated GSLs contained information on both ceramide and sugar portions of the molecules. Each of the LSIMS, MS/MS and MALDI‐TOFMS techniques proved to be complementary to the others in this study; the use of all three is recommended for the generation of complete structural information.

Photocycloaddition of Deoxyuridines to 2,3-Dimethyl-2-butene

Abstract Direct and ketone (acetone or acetophenone)-sensitized photocycloaddition of 2,3-dimethyl-2-butene to 2′-deoxyuridine (1a), thymidine (1b), 5-fluoro-2′-deoxyuridine (1c), and their 3′,5′-di-O-acetyl derivatives 1d—1f by near-UV irradiation have been studied. The triplet excited state of the nucleosides was found to be the major intermeidate by the triplet quenching technique. From the respective reactants, a pair of diastereomeric products having a cyclobutane ring were isolated. The absolute configuration of the bridgehead carbon atoms was identified by X-ray crystallographic analyses with two of them as (1R, 6R)-isomer 3a and (1S, 6S)-isomer 2b and by 1H NMR. The conformations of the glycosyl bond, the pyrimidine ring, and the sugar portion in solution were identified by 1H NMR and compared with the ones in the crystalline state.

The cell envelope structure of the lipopolysaccharide-lacking gram-negative bacterium Sphingomonas paucimobilis.

From the cell envelope preparation of Sphingomonas paucimobilis two membrane fractions with different densities were separated by sucrose density gradient ultracentrifugation. The high-density fraction contained several major proteins, phospholipids, and glycosphingolipids, which are the only glycolipids of this lipopolysaccharide-lacking gram-negative bacterium. The low-density fraction showed many minor bands of proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and NADH oxidase activity was localized in this fraction. Combined with morphological data of vesicles formed by these membrane fractions, the high-density and low-density fractions were proposed to be an outer membrane and a cytoplasmic membrane, respectively. The localization of the glycosphingolipid was investigated also by means of immunoelectron microscopic analysis using a glycosphingolipid-specific antibody. The glycosphingolipid was shown to localize at the cell envelope, and the antigenic sugar portion was exposed to the bacterial cell surface. From these results the glycosphingolipid was assumed to have a function similar to that of the lipopolysaccharide of other gram-negative bacteria.