Convenient synthesis of nucleoside borane diphosphate analogues: deoxy- and ribonucleoside 5'-P(alpha)-boranodiphosphates

The nucleoside boranophosphates, having one of the nonbridging phosphate oxygens substituted with a borane (BH(3)) group, have shown potential therapeutical applications as aptamers, antisense agents, and antiviral prodrugs. An oxathiaphospholane approach, which does not require exocyclic amine protection of the nucleobase, has been successfully developed to efficiently synthesize 5'-P(alpha)-boranodiphosphates of 2'-deoxythymidine, adenosine, guanosine, and uridine. The approach involves a key intermediate, the borane complex of nucleoside 5'-O-1,3,2-oxathiaphospholane 16, that undergoes a ring-opening reaction catalyzed by 1,4-diazabicyclo[5.4.0]-undec-7-ene to form the protected nucleoside 5'-P(alpha)-boranodiphosphate 18. Treatment of 18 with ammonium hydroxide yielded diastereoisomeric mixtures of nucleoside 5'-P(alpha)-boranodiphosphates 5. This oxathiaphospholane approach ensures the availability of nucleoside 5'-P(alpha)-boranodiphosphate analogues needed for antiviral drug research.     

Synthesis and conformational analysis of novel N(OCH3)-linked disaccharide analogues

N(OMe)-linked disaccharide analogues, isosteric to the corresponding natural disaccharides, have been synthesized by chemoselective assembly of unprotected natural monosaccharides with methyl 6-deoxy-6-methoxyamino-alpha-D-glucopyranoside in an aqueous environment. The coupling reactions were found to be chemo- and stereoselective affording beta-(1-->6) disaccharide mimics when using Glc and GlcNAc; in the case of Gal, the beta-anomer was prevalent (beta:alpha=7:1). An iterative method for the synthesis of linear N(OMe) oligosaccharide analogues was demonstrated, based on the use of an unprotected monosaccharide building block in which an oxime functionality at C-6 is converted during the synthesis into the corresponding methoxyamino group. The conformational analysis of these compounds was carried out by using NMR spectroscopy, ab initio, molecular mechanics, and molecular dynamics methods. Optimized geometries and energies of fourteen conformers for each compound have been calculated at the B3LYP/6-31G* level. Predicted conformational equilibria were compared with the results based on NMR experiments and good agreement was found. It appears that N(OMe)-linked disaccharide analogues exhibit a slightly different conformational behavior to their parent natural disaccharides.     

Synthesis of 1‐C‐Glycoside‐Linked Lipid II Analogues Toward Bacterial Transglycosylase Inhibition

Preparation of Lipid II analogues containing an enzymatically uncleavable 1‐C‐glycoside linkage between the disaccharide moiety and the pyrophosphate‐ or pyrophosphonate‐lipid moiety is described. The synthesis of a common 1‐C‐vinyl disaccharide intermediate has been developed that allows easy preparation of both an elongated sugar‐phosphate bond and a sugar‐phosphonate moiety, which are coupled with the polyprenyl phosphate to give the desired molecules. Inhibition studies show how a subtle structural modification results in dramatically different potency toward bacterial transglycosylase (TGase), and the results identify Lipid II‐C‐O‐PP (IC₅₀=25 μM ) as a potential TGase inhibitor.     

Solid-phase synthesis of bleomycin A(5) and three monosaccharide analogues: exploring the role of the carbohydrate moiety in RNA cleavage

The solid-phase synthesis of bleomycin A5 (BLM A5) and three monosaccharide analogues is presented. The monosaccharide analogues incorporated alpha-d-mannose, alpha-l-gulose, and alpha-l-rhamnose moieties in lieu of the disaccharide normally present in BLM A5. Also explored were the abilities of each of the monosaccharide congeners to cleave a 53-nt RNA. The elaboration of these carbohydrate-modified bleomycin analogues helps to define the role of the disaccharide moiety during the RNA cleavage event. The relatively facile solid-phase synthesis of bleomycin A5 and each of the carbohydrate analogues constitutes an important advance in the continuing mechanistic studies of bleomycin.     

Investigation of the surfactant type and concentration effect on the retention factors of glutathione and its analogues by micellar electrokinetic chromatography

In the present study, a micellar electrokinetic chromatographic method was used to determine the retention factors of hydrophilic monomeric and homodimeric forms of glutathione analogues. Ionic‐liquid‐based surfactant, 1‐tetradecyl‐3‐methylimidazolium chloride, as well as cetyltrimethylammonium bromide and phosphate buffer (pH 7.4) were employed in the experiments. Since the studied peptides possess a negative charge under physiological conditions, it is expected that the peptides interact with the oppositely charged 1‐tetradecyl‐3‐methylimidazolium chloride and cetyltrimethylammonium bromide micelles via hydrophobically assisted electrostatic forces. The dependence of the retention factor on the micellar concentration of 1‐tetradecyl‐3‐methylimidazolium chloride and cetyltrimethylammonium bromide is nonlinear and the obtained curves converge to a limiting value. The retention factor values of GSH analogues were in the range of 0.36–2.22 for glutathione analogues and –1.21 to 0.37 for glutathione when 1‐tetradecyl‐3‐methylimidazolium chloride was used. When cetyltrimethylammonium bromide was employed, the retention factor values were in the range of 0.27–2.17 for glutathione analogues and –1.22 to 0.06 for glutathione. If sodium dodecyl sulfate was used, the retention factor values of glutathione analogues with carnosine moiety were in the range of –1.54 to 0.38.     

Bile acids. LXXVI. Analyses of bile acids and sterols by high-performance liquid chromatography with a microbore column

The mobilities of several free and conjugated 5 beta-bile acids, cholesterol and analogues, and alpha, beta-unsaturated sterols and steroidal acids have been investigated with a microbore reversed-phase high-performance liquid chromatographic column (50 cm X 1 mm I.D., 12% C18) with appropriate solvent mixtures at flow-rates of 50-100 microliter/min and a UV monitor set at 193, 198, 212, or 243 nm. With a solvent mixture of 2-propanol-10 mM phosphate buffer, pH 7.0 (160:340) bile acids or their conjugates were separated in a manner similar to those by microBondapak columns (10% C18). The lower detection limit of the conjugates was 20 pmoles with the UV detector set at 193 nm, whereas the lower limit for alpha, beta-unsaturated keto sterols or steroidal acids was 5 pmoles at 243 nm. The detection limit for cholesterol with the UV monitor at 198 nm was 10 pmoles. Contributions of substituent groups of sterols to their time of elution (capacity factor) were calculated for several substituted 4-cholesten-3-ones.     

Ready access to sialylated oligosaccharide donors

[formula: see text] Numerous glycoconjugates contain the disaccharide Neu5Ac alpha (2-->3)DGalp. An efficient way to incorporate this disaccharide into synthetic glycoconjugates is to develop a disaccharide building block. This communication reports a chemoenzymatic route to such a building block which requires as few as four steps. Some examples using more chemical steps are also presented, which increase the flexibility. These disaccharide donors were used to prepare synthetic trisaccharides.     

Synthesis and evaluation of 1-deoxy-D-xylulose 5-phosphate analogues as chelation-based inhibitors of methylerythritol phosphate synthase

[structures: see text] A series of 1-deoxy-D-xylulose 5-phosphate (DXP) analogues were synthesized and evaluated as inhibitors of E. coli methylerythritol phosphate (MEP) synthase. In analogues 1-4, the methyl group in DXP was replaced by hydroxyl, hydroxylamino, methoxy, and amino moieties, respectively. In analogues 5 and 6, the acetyl moiety in DXP was replaced by hydroxymethyl and aminomethyl groups. These compounds were designed to coordinate to the active site divalent metal in MEP synthase. The carboxylate (1), methyl ester (3), amide (4), and alcohol (5) analogues were inhibitors with IC50's ranging from 0.25 to 1.0 mM. The hydroxamic acid (2) and amino (6) analogues did not inhibit the enzyme.     

An Approach to the Synthesis of Four Rhodomicrobium Vannielii Lipid a Analogues

Abstract

The preparation of four Rh. Vannielii Lipid A analogues (i.e. compounds 22, 23, 30 and 33) is described. Non-neighbouring group supported introduction of the β-glycosidic linkages was performed by coupling the mannopyranosyl bromide 2 and the 2-azido-2-deoxy-glucopyranosyl bromides 10 and 13 with the suitably protected glycosyl acceptors 3, 4 and 5 in the presence of a heterogeneous silver catalyst, to give compounds 6, 7, 14 and 24, respectively. Selective removal of the ally1 group and reduction of the azido functions followed by several O,N-acylation steps afforded, after complete deblocking, the tri-and disaccharide Rh. Vannielii Lipid A analogues 22, 23, 30 and 33.     

A stereodivergent,two-directional synthesis of stereoisomeric C-linked disaccharide mimetics

Dipyranones, such as 1,2-bis[(2R,3S,6S)-3-hydroxy-6-methoxy-3-oxo-6H-pyran-2-yl]ethane, were exploited as templates for the synthesis of some novel C-linked disaccharide analogues. Efficient methods, such as stereoselective reduction and dihydroxylation, were developed for two-directional functionalisation of these templates. Peracetylated derivatives of ten stereoisomeric disaccharide analogues [acetic acid 4,5-diacetoxy-6-methoxy-[(3',4',5'-triacetoxy-6'-methoxytetrahydropyran- 2'-yl)ethyl]tetrahydropyran-3-yl esters] were synthesised from a virtual library of 136 compounds; furthermore, an additional eight stereoisomers could have been synthesised simply by using the enantiomeric ligand in the enantioselective step. The ability of (2S,3S,4R,5R,6R)-6-methoxy-2-[2'-((2'R,3'R,4'S, 5'R,6'S)-3',4',5'-trihydroxy-6'-methoxytetrahydropyran-2'-yl) ethyl]tetrahydropyran-3,4,5-triol to bind to the repressor protein, LacI, was estimated to be similar to that of isopropyl-beta-thiogalactoside. The disaccharide mimetics were concluded to be a new and interesting class of C-linked disaccharide mimetics with promising, though largely unstudied, biological activity.     

Synthesis of thioglycoside-based UDP-sugar analogues

Arbuzov reaction of O-acetyl-protected glycosylthiomethyl chlorides with triethyl phosphite and then phosphonate ethyl ester cleavage with trimethylsilyl bromide afforded glycosylthiomethyl phosphonates 13, 18, 22, and 26. These intermediates could be readily transformed into the O-deprotected phosphonates 7-10 and into title compounds 1-4. Similarly, sulfonomethyl phosphonate moieties containing UDP-sugar analogues 5 and 6 were obtained.     

Synthesis of hexasaccharide fragments of pectin

Short syntheses of partially methyl-esterified hexagalacturonates 1-5 are described as part of the development of strategies for the preparation of larger pectic oligosaccharides. The methodology is based on the repeated coupling of galactose mono- and disaccharide donors onto a galactose acceptor until a hexagalactan is obtained. All glycosylations are carried out with n-pentenyl glycosides to provide good yields of the desired alpha anomers. Pentenyl disaccharide donors are prepared by the coupling of two pentenyl galactosides controlled by either the armed-disarmed effect or by converting one pentenyl galactoside into the corresponding galactosyl bromide or fluoride. Two orthogonal protecting groups are employed at C6, which makes it possible to oxidize these positions to either the carboxylic acid or to the methyl ester. Each hexagalactan is therefore able to bifurcate into two different hexagalacturonates with a reverse methyl-esterification pattern. The methyl ester distribution in the hexagalacturonates is confirmed by tandem mass spectrometry.     

Synthesis, structure, and inclusion capabilities of trehalose-based cyclodextrin analogues (cyclotrehalans)

Concise and efficient strategies toward the synthesis of D2h- and D3h-symmetric cyclodextrin analogues alternating alpha,alpha'-trehalose disaccharide subunits and pseudoamide segments (cyclotrehalans, CTs) are reported. The conformational properties of these cyclooligosaccharides are governed by the rigidity of the alpha,alpha'-trehalose disaccharide repeating unit and the partial double-bond character of the N-(C=X) linkages. In contrast to the typical concave-shaped cavity of cyclodextrins (CDs), CTs feature a convex-shaped hydrophobic cavity in which the beta-face of the monosaccharide subunits is oriented toward the inner side, as supported by NMR and modeling (molecular mechanics and dynamics) studies. In the case of cyclodimeric CTs (CT2s), the existence of intramolecular hydrogen bonds results in collapsed cavities, too small to allow the formation of inclusion complexes with organic molecules. Cyclotrimeric CTs (CT3s) display cavity sizes that are intermediate between those of alphaCD and betaCD, ideally suited for the complexation of complementary guests with ternary symmetry such as adamantane 1-carboxylate (AC). The higher flexibility of the pseudoamide bridges as compared with classical glycosidic linkages endow these glyconanocavities with some conformational adaptability properties, making them better suited than CDs for complexation of angular guests, as seen from comparative inclusion capability experiments against the fluorescent probes 6-p-toluidinonaphthalene-2-sulfonate (TNS; linear) and 8-anilinonaphthalene-1-sulfonate (ANS; angular).     

Simultaneous determination of magnesium L-ascorbyl-2-phosphate and kojic acid in cosmetic bleaching products by using a microbore column and ion-pair liquid chromatography

A rapid, sensitive, and reliable method was developed for the simultaneous determination of magnesium L-ascorbyl-2-phosphate (AS-PM) and kojic acid (KA), commonly using bleaching agents in cosmetic products. The method uses a prepacked C18 microbore column for ion-pair liquid chromatography with photodiode array detection. The eluant is 0.5mM tetrabutylammonium bromide and 0.05M phosphate buffer, pH 5, containing 5% methanol, at a flow rate of 70 microL/min. The determinations of AS-PM and KA were linear in the range of 0.5-1,000 and 0.25-500 ng, respectively. The precision of the assay ranged from 0.31 to 2.19%, and the detection limits (signal-to-noise ratio = 3) were 0.04 and 0.02 ng for AS-PM and KA, respectively.     

3-芳基苯并呋喃酮类化合物的合成及抗肿瘤活性

以甲氧基取代苯甲醛和氯仿为起始物, 以四丁基溴化铵(TBAB)为催化剂, 在氢氧化钠溶液中反应制得甲氧基取代扁桃酸钠, 经酸化得到相应的甲氧基取代扁桃酸; 在三氟化硼-乙醚催化下, 甲氧基取代扁桃酸分别与不同的酚类化合物发生串联的酯化-分子内环合反应, 便捷、 高效地合成了13种3-芳基苯并呋喃酮类化合物. 采用噻唑蓝(MTT)法对其中11种化合物进行了肿瘤细胞增殖抑制活性研究, 结果表明化合物4a, 4j及4a, 4i具有较强的抗肿瘤活性.     

Total synthesis and biological evaluation of the protein phosphatase 2A inhibitor cytostatin and analogues

The total synthesis of the natural product cytostatin is described which inhibits protein phosphatase 2A. Cytostatin has anti-metastatic properties and induces apoptosis. On the basis of this synthesis the relative and absolute configuration of cytostatin could be assigned. Key structural elements of cytostatin are an alpha,beta-unsaturated lactone group and a side chain embodying a phosphate and a rather unstable (Z,Z,E)-triene subunit. In addition, the natural product carries six stereocenters. For the construction of the stereocenters reagent-controlled transformations were used in order to ensure maximum stereochemical flexibility. The Evans syn-aldol reaction was chosen to establish the stereochemistry at C-4, C-5, C-9 and C-10; C-6 was introduced by means of the Evans asymmetric alkylation. In all cases the same chiral auxiliary was employed as stereodirecting group. The stereocenter at C-11 was established by an asymmetric reduction using CBS-oxazaborolidine. Temporary protection of the phosphate group was achieved best by using the base-labile 9-fluorenylmethyl group, which could be cleanly cleaved by an excess of triethylamine; this reaction yielded analytically pure phosphates after a simple aqueous work-up. The (Z,Z,E)-triene embodied in cytostatin was synthesized by means of a Stille coupling as key transformation. The synthesis sequence established in this way readily gave access to a series of analogues with simplified structure. Initial biological testing of these analogues proved that the alpha,beta-unsaturated lactone, the C-11-hydroxy group and a fully deprotected phosphate moiety at C-9 are essential for the PP2A-inhibitory activity of cytostatin. The rather unstable triene moiety in the side chain can be replaced by other lipophilic residues with only moderate decrease of biological activity. Other phosphatases, that is, PP1, VHR, PTP1B, CD45, were not inhibited by cytostatin or any of the analogues, demonstrating the high selectivity of this compound. These findings will be useful for the design and synthesis of cytostatin-derived chemical tools for the study of biological processes influenced by PP2A.     

Improved workup for glycosaminoglycan disaccharide analysis using CE with LIF detection

This work describes improved workup and instrumental conditions to enable robust, sensitive glycosaminoglycan (GAG) disaccharide analysis from complex biological samples. In the process of applying CE with LIF to GAG disaccharide analysis in biological samples, we have made improvements to existing methods. These include (i) optimization of reductive amination conditions, (ii) improvement in sensitivity through the use of a cellulose cleanup procedure for the derivatization, and (iii) optimization of separation conditions for robustness and reproducibility. The improved method enables analysis of disaccharide quantities as low as 1 pmol prior to derivatization. Biological GAG samples were exhaustively digested using lyase enzymes, the disaccharide products and standards were derivatized with the fluorophore 2‐aminoacridone and subjected to reversed polarity CE‐LIF detection. These conditions resolved all known chondroitin sulfate (CS) disaccharides or 11 of 12 standard heparin/heparan sulfate disaccharides, using 50 mM phosphate buffer, pH 3.5, and reversed polarity at 30 kV with 0.3 psi pressure. Relative standard deviation in migration times of CS ranged from 0.1 to 2.0% over 60 days, and the relative standard deviations of peak areas were less than 3.2%, suggesting that the method is reproducible and precise. The CS disaccharide compositions are similar to those obtained by our group using tandem MS. The reversed polarity CE‐LIF disaccharide analysis protocol yields baseline resolution and quantification of heparin/heparan sulfate and CS/dermatan sulfate disaccharides from both standard preparations and biologically relevant proteoglycan samples. The improved CE‐LIF method enables disaccharide quantification of biologically relevant proteoglycans from small samples of intact tissue.     

Synthesis of a Single Repeat Unit of Type VIII Group B Streptococcus Capsular Polysaccharide 1

Abstract

We have synthesized a single repeat unit of type VIII Group B Streptococcus capsular polysaccharide, the structure of which is {L-Rhap(β1→4)-D-Glcp(β1→4)[Neu5Ac(α2→3)]-D-Galp(β→4)}_n. The synthesis presented three significant synthetic challenges namely: the L-Rhap(β→4)-D-Glcp bond, the Neu5Ac(α2→3)-D-Galp bond and 3,4-D-Galp branching. The L-Rhap bond was constructed in 60% yield (α:β 1:1.2) using 4-O-acetyl-2,3-di-O-benzoyl-α-L-rhamnopyranosyl bromide 6 as donor, silver silicate as promotor and 6-O-benzyl-2,3-di-O-benzoyl-1-thio-β-D-glucopyranoside as acceptor to yield disaccharide 18. The Neu5Ac(α2→3) linkage was synthesized in 66% yield using methyl [phenyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-thio-D-glycero-D-galacto-nonulopyranosid]onate as donor and triol 2-(trimethylsilyl) ethyl 6-O-benzyl-β-D-galactopyranoside as acceptor to give disaccharide 21. The 3,4-D-Galp branching was achieved by regioselective glycosylation of disaccharide diol 21 by disaccharide 18 in 28% yield to give protected tetrasaccharide 22. Tetrasaccharide 22 was deprotected to give as its 2-(trimethylsilyl)ethyl glycoside the title compound 1a. In addition the 2-(trimethylsilyl)ethyl group was cleaved and the tetrasaccharide coupled by glycosylation (via tetrasaccharide trichloroacetimidate) to a linker suitable for conjugation.

     

Synthesis and evaluation of 1-deoxy-D-xylulose 5-phosphoric acid analogues as alternate substrates for methylerythritol phosphate synthase

[structure: see text] Four deoxyxylulose phosphate (DXP) analogues were synthesized and evaluated as substrates/inhibitors for methylerythritol phosphate (MEP) synthase. In analogues CF(3)-DXP (1), CF(2)-DXP (2), and CF-DXP (3), the three methyl hydrogens at C1 of DXP were sequentially replaced by fluorine. In the fourth analogue, Et-DXP (4), the methyl group in DXP was replaced by an ethyl moiety. Analogues 1, 2, and 4 were not substrates for MEP synthase under normal catalytic conditions and were instead modest inhibitors with IC(50) values of 2.0, 3.4, and 6.2 mM, respectively. In contrast, 3 was a good substrate (k(cat) = 38 s(-)(1), K(m) = 227 muM) with a turnover rate similar to that of the natural substrate. These results are consistent with a retro-aldol/aldol mechanism rather than an alpha-ketol rearrangement for the enzyme-catalyzed conversion of DXP to MEP.     

Syntheses of anomerically phosphodiester-linked oligomers of the repeating units of the Haemophilus influenzae types c and f capsular polysaccharides

Spacer-equipped dimers and trimers of the repeating units of the capsular polysaccharide of Haemophilus influenzae type c, -4)-3-O-Ac-beta-D-GlcpNAc-(1-->3)-alpha-D-Galp-(1-OPO(3-)-, and type f, -3)-beta-D-GalpNAc-(1-->4)-3-O-Ac-alpha-D-GalpNAc-(1-OPO(3-)-, have been synthesized for use in immunological studies. H-Phosphonate chemistry was used for the formation of the interglycosidic phosphate diester linkages. Two types of building blocks, a spacer glycoside disaccharide starting monomer (15 and 22) and an anomeric monoester alpha-H-phosphonate disaccharide elongating monomer (12 and 27), were built up for each serotype structure from properly protected monosaccharide precursors using mainly thioglycosides as glycosyl donors. Stereospecificity in the formation of the alpha-linked monoester H-phosphonate was possible in type c through crystallization of the pure alpha-anomer of the precursor hemiacetal from an alpha/beta-mixture, whereas in type f, the hemiacetal was isolated directly as exclusively the alpha-anomer. Subsequent phosphonylation using triimidazolylphosphine was performed without anomerization. Formation of the anomeric phosphate diester linkages was performed using pivaloyl chloride as coupling reagent followed by I(2)/H(2)O oxidation of the formed diester H-phosphonates. Original experiments afforded no diester product at all, but optimization of the oxidation conditions (lowering the temperature and dilution with pyridine prior to I(2) addition) gave the dimers in good yields (71% and 81%) and, subsequently, after removal of a temporary silyl protecting group in the dimers, the trimers in fair yields (36% and 37%), accompanied by hydrolysis of the dimer phosphate linkage. One-step deprotection through catalytic hydrogenolysis efficiently afforded the target dimer (30 and 36) and trimer structures (32 and 39). The synthetic scheme allows for further elongation to give higher oligomers.