Stereoselective synthesis of N-galactofuranosyl amides

α- or β-Galactofuranosyl (Galf) amides can be synthesized with high stereoselectivity by traceless Staudinger ligation starting from unprotected β-galactofuranosyl azide or tetra-O-acetyl-β-galactofuranosyl azide, respectively. The resulting Galf amides are hitherto unknown molecules, with interesting potential as inhibitors of mycobacterial growth.     

In situ evaluation of esterase stereoselectivity in two-dimensional electropherograms and tissue sections

Staining with both enantiomers of an α-naphthyl ester plus a diazonium salt and comparing the color intensities given by the two enantiomers is a convenient method to evaluate the esterase stereoselectivity for that ester in two-dimensional electropherograms and tissue sections. Application of this method for rat liver has shown that (1) several esterases, e.g., one of pI 6.4 and M_r 118 kDa, are moderately stereoselective against α-naphthyl (R)-N-acetylalaninate and (R)-N-methoxycarbonylalaninate but strictly stereoselective against α-naphthyl (S)-N-methoxycarbonylvalinate, implying that esterase stereoselectivity may be inverted by changing the ester structure; and (2) these esterases are mainly contained in the hepatocytes around central veins.     

The recombinational analysis of aberrations and the position of the notch locus on the polytene chromosome of Drosophila

Summary The recombinational analysis of heterozygotes for a point-mutant N and a deficiency N suggests that the map region approximated by the interval fa to nd ² is at the right edge of salivary band 3C7 or in the interband to the right. The map region N ^55ell to fa can be anywhere between the left interband and the right edge of 3C7. We discovered that small inversions also can be used in the recombinational analysis, and the inversion data support the conclusions already described.The reactivation of latent mutability in a Notch inversion resulted in reinversion of the original aberration, followed by reversion of N to N ⁺. From the same Notch inversion, we isolated a spontaneous deficiency superimposed upon the original aberration, which supported our hypothesis that two of our w to N deficiencies probably originated as deficiencies superimposed upon inversions.     

Solvent effect on the stereoselective addition reaction of optically active amines to N-methylphenylalanine N-carboxyanhydride

The effect of solvent on stereoselectivity in the nucleophilic addition reaction of various optically active amines to N-methylphenylalanine N-carboxyanhydride has been investigated. In m-dimethoxybenzene as solvent, (S)-valine, (S)-leucine, and (S)-phenylalanine ethyl esters reacted preferentially with (R)-N-methylphenylalanine N-carboxyanhydride, but the stereoselectivity decreased considerably in nitrobenzene and dimethylacetamide as solvents. In the latter solvents, the dipolar interactions between an amine and an N-carboxyanhydride and the orientation of the substituent of N-carboxyanhydride were seriously affected, hence the stereoselectivity decreased. As a consequence, the enantiomer selection by the terminal amine of a growing chain in the nucleophilic addition-type polymerization of α-amino acid N-carboxyanhydride can be controlled by the choice of solvent. (S)-Proline ethyl ester and (S)-α-phenylethylamine reacted preferentially with (S)-N-methylphenylalanine N-carboxyanhydride in m-dimethoxybenzene, and this type of selectivity did not change in nitrobenzene. But in dimethylacetamide the stereoselectivity decreased. In the transition state of the reaction of these amines and N-methylphenylalanine N-carboxyanhydride dipolar interactions are operating, which should be destroyed by dimethylacetamide but may not be affected by nitrobenzene.     

N,N-Diacetylsialyl chloride—a novel readily accessible sialyl donor in reactions with neutral and charged nucleophiles in the absence of a promoter

N,N-Diacetylneuraminic acid glycosyl chloride was prepared for the first time and made to react with various nucleophiles to give the corresponding α-glycosyl phosphate, β-glycosyl dibenzyl phosphate, α-glycosyl azide, α-phenyl thioglycoside and α-glycosyl xanthate in 65-82% yields and high stereoselectivity while its reactions with simple alcohols were not stereoselective. The new sialyl donor made possible the first stereoselective synthesis of sialic acid glycosyl phosphate with α-configuration and highly efficient synthesis of β-configured sialic acid glycosyl dibenzyl phosphate.     

Polymerization of N-carboxyanhydrides of various α-amino acids using secondary amines as initiator

In the polymerizations of alanine, γ-ethyl glutamate, and leucine N-carboxyanhydrides (NCA's) initiated by tertiary amines and some secondary amines such as N-methyl-L -alanine dialkylamide, a stereoselectivity was observed: the polymerization rates of L - and D -NCA's were identical to each other and larger than that of DL -NCA. However, this selectivity was not observed in the polymerizations of valine and isoleucine NCA's initiated by N-methyl-L -alanine dialkylamide. The stereoselective polymerizations of valine and isoleucine NCA's were induced only with tetriary amines such as tri-n-butylamine. N-Methyl-L -alanine di-alkylamide has been shown to initiate the polymerization of usual α-amino acid NCA according to the activated-NCA mechanism, but it initiated the polymerizations of valine and isoleucine NCA's according to the primary amine-type mechanism. This is because in the latter NCA's the N–H group is masked by the adjacent C^β-branched alkyl substituent against the approach of the secondary amine. Poly(DL -alanine)s produced in the stereoselective polymerization had higher viscosities and were more stereoblock-like than those produced without the stereoselectivity. These experimental results indicate that the stereoselective polymerization is possible only when the polymerization proceeds through the activated-NCA mechanism.     

Gram-Schmidt Orthogonalization of Multinormal Variates: Applications in Genetics

The computation of an N-variate normal density function requires the inversion of an N × N co-variance matrix. Furthermore, if each mean depends on u unobservable factors, a mixture of u^N N-variate normal densities must be computed, making likelihood calculations impractical even for moderate N. The Gram-Schmidt orthogonalization process is used to express a multinormal density as a product of univariate normal densities. When the pattern of the correlation matrix is taken into account the formulas may be considerably simplified. In some cases each of the orthogonal variates can be written as a linear combination of only a few of the original variates. Such results are crucial for applications of multinormal distributions and of mixtures of multinormal distributions. An intraclass correlation model and a genetic variance components model applicable to family data are discussed as examples.     

Tunable stereoselectivity during sialylation using an N-acetyl-5-N,4-O-oxazolidinone-protected p-toluene 2-thio-sialoside donor with Tf(2)O/Ph(2)SO/TTBPy

An N-acetyl-5-N,4-O-oxazolidinone-protected p-toluene 2-thio-sialoside donor, promoted by Ph₂SO/Tf₂O/TTBPy, was thoroughly investigated in the coupling to various acceptors. The stereoselectivity of the sialylation was found to be dependent on the various reaction conditions, such as pre-activation time, reaction time, the amount of Ph₂SO, and TTBPy. A detailed Ph₂SO/Tf₂O-promoted glycosylation mechanism was proposed, which contained three crucial reactive species: an oxacarbenium ion, C2-sialyloxosulfonium salts, and oxosulfonium supramers. Our research results indicate that it is possible to tune the stereoselectivity of the sialylation by carefully changing the reaction conditions. For instance, Ph₂SO (2.0–3.0 equiv)/TTBPy (0–1.0 equiv) promotion gives higher α-selective sialylation in dichloromethane, while Ph₂SO (4–5 equiv)/TTBPy (0 equiv) or Ph₂SO (2.0 equiv)/TTBPy (2.0 equiv) affords lower stereoselectivity.     

Metabolic oligosaccharide engineering with N-Acyl functionalized ManNAc analogs: cytotoxicity, metabolic flux, and glycan-display considerations

Metabolic oligosaccharide engineering (MOE) is a maturing technology capable of modifying cell surface sugars in living cells and animals through the biosynthetic installation of non‐natural monosaccharides into the glycocalyx. A particularly robust area of investigation involves the incorporation of azide functional groups onto the cell surface, which can then be further derivatized using “click chemistry.” While considerable effort has gone into optimizing the reagents used for the azide ligation reactions, less optimization of the monosaccharide analogs used in the preceding metabolic incorporation steps has been done. This study fills this void by reporting novel butanoylated ManNAc analogs that are used by cells with greater efficiency and less cytotoxicity than the current “gold standard,” which are peracetylated compounds such as Ac₄ManNAz. In particular, tributanoylated, N‐acetyl, N‐azido, and N‐levulinoyl ManNAc analogs with the high flux 1,3,4‐O‐hydroxyl pattern of butanoylation were compared with their counterparts having the pro‐apoptotic 3,4,6‐O‐butanoylation pattern. The results reveal that the ketone‐bearing N‐levulinoyl analog 3,4,6‐O‐Bu₃ManNLev is highly apoptotic, and thus is a promising anti‐cancer drug candidate. By contrast, the azide‐bearing analog 1,3,4‐O‐Bu₃ManNAz effectively labeled cellular sialoglycans at concentrations ～3‐ to 5‐fold lower (e.g., at 12.5–25 µM) than Ac₄ManNAz (50–150 µM) and exhibited no indications of apoptosis even at concentrations up to 400 µM. In summary, this work extends emerging structure activity relationships that predict the effects of short chain fatty acid modified monosaccharides on mammalian cells and also provides a tangible advance in efforts to make MOE a practical technology for the medical and biotechnology communities. Biotechnol. Bioeng. 2012; 109:992–1006. © 2011 Wiley Periodicals, Inc.     

Substituent effects in the selective reductive opening of epoxides with borohydrides in the presence of β‐cyclodextrin

The opening of ortho‐substituted phenyl oxiranes by borohydrides is described both in aqueous and solid phases. Some remarkable differences in the regioselectivity and inversion of enantioselectivity of oxirane ring‐opening are reported. The results obtained are compared with previous studies and have allowed some factors governing the reactivity, the regio‐ and stereoselectivity of this reaction to be evaluated. Preferential orientations of the substrate included inside the cyclodextrin are proposed from these results. Chirality 11:541–545, 1999. © 1999 Wiley‐Liss, Inc.     

Mutagenicity of nitrofuran derivatives, including furylfuramide, a food preservative.

The effects of sodium azide (NaN₃) in combination with diethyl sulfate (dES) or N-methyl-N′-nitrosourea (MNH) on mutation frequency in barley were studied. It was found that sodium azide produced high frequencies of chlorophyll mutations when used alone and has a synergistic effect on mutation yields following MNH treatments. However, the mutation frequency was decreased whe azide was applied following dES treatment of seeds. The mutagenic efficiency of azide was found to be high, possibly because of low “physiological” damage. The synergistic increase in mutation yields by MNH and azide treatment indicates that azide has unusual promise as a mutagen for both practical and research applications.     

Effects of azide on the S2 state EPR signals from Photosystem II

The anion azide, N₃ ^-, has been previously found to be an inhibitor of oxygen evolution by Photosystem II (PS II) of higher plants. With respect to chloride activation, azide acts primarily as a competitive inhibitor but uncompetitive inhibition also occurs [Haddy A, Hatchell JA, Kimel RA and Thomas R (1999) Biochemistry 38: 6104–6110]. In this study, the effects of azide on PS II-enriched thylakoid membranes were characterized by electron paramagnetic resonance (EPR) spectroscopy. Azide showed two distinguishable effects on the S₂ state EPR signals. In the presence of chloride, which prevented competitive binding, azide suppressed the formation of the multiline and g = 4.1 signals concurrently, indicating that the normal S₂ state was not reached. Signal suppression showed an azide concentration dependence that correlated with the fraction of PS II centers calculated to bind azide at the uncompetitive site, based on the previously determined inhibition constant. No evidence was found for an effect of azide on the Fe(II)Q_A ^- signals at the concentrations used. This result is consistent with placement of the uncompetitive site on the donor side of PS II as suggested in the previous study. In chloride-depleted PS II-enriched membranes azide and fluoride showed similar effects on the S₂ state EPR signals, including a notable increase and narrowing of the g = 4.1 signal. Comparable effects of other anions have been described previously and apparently take place through the chloride-competitive site. The two azide binding sites described here correlate with the results of other studies of Lewis base inhibitors.This revised version was published online in October 2005 with corrections to the Cover Date.     

Ring inversion barrier of diazepam and derivatives: An ab Initio study

Systematic ab initio calculations were performed to investigate the ring inversion process of various 1,4‐diazepines including diazepam, N¹‐desmethyldiazepam, and 3‐methyl‐N¹‐desmethyldiazepam. The diazepine ring adopts a shape of a boat; owing to asymmetric substitution two such boats are possible in mirror image relation to each other. In the present study both structural and solvent effects were investigated on the energetics of ring inversion of nine diazepine derivatives. The calculated ring inversion barriers for diazepam (17.6 kcal/mol) and N¹‐desmethyldiazepam (10.9 kcal/mol) are in good agreement with the corresponding experimental data. In the cases of diazepam and N¹‐desmethyldiazepam, the calculated minimum energy path of the ring inversion is asymmetric contrary to the fact that the terminals (M and P conformers) are equienergetic. Chirality 11:651–658, 1999. © 1999 Wiley‐Liss, Inc.     

Studies on cytochrome c. Part III. Synthesis of the protected heneicosapeptide (sequence 24–44) of Baker's yeast iso-1-cytochrome c

Syntheses are described for two N-benzyloxycarbonylpeptide tert-butoxycarbonylhydrazides which correspond to positions 24–34 and 35–44, respectively, of the primary structure of baker's yeast iso-1-cytochrome c. The two peptide derivatives were coupled via the azide procedure to form the N-benzyloxycarbonylheneicosapeptide tert-butoxycarbonylhydrazide (sequence 24–44).     

Stereoselective hydrolysis of xenobiotic esters by different cell lines from rat liver and hepatoma and its application to chiral prodrugs for designated growth suppression of cancer cells

Stereoselectivity in the hydrolysis of racemic ethyl 2-phenylacetate derivatives by cultured cells of noncancerous cell lines from rat liver (BRL, BRL 3A, Clone 9, and ARLJ301–3), spontaneously or oncogene transformed rat liver cell lines (ARLJ301–3TR1, Anr4, Anr9–1, and Anr13–1), and cancer cell lines from rat hepatoma (H4-II-E, McA-RH7777, and MH₁C₁) and sarcoma (XC) was studied. A strong (R)-enantiomer preference was found in the hydrolysis of ethyl 2-hydroxy- ( 2c ) and 2-methoxy-2-phenylacetate ( 3c ) by the noncancerous and oncogene-transformed cells and an (S)-enantiomer preference for ethyl N-acylphenylalaninates with all the present cell lines. These inclinations were, however, not recognized with ethyl 2-methoxy-2-phenylpropanoate and ethyl N-difluoroacetyl-or N-trifluoroacetylphenylalaninate. Moreover, the R preference for 3c was reversed in the reaction by hepatoma cells. Thus, the stereoselectivity is influenced by both structure of acyl group and species of cell lines. The hepatoma cells were considerably different from the noncancerous or oncogene-transformed cells in stereoselectivity. This fact was consistent with the order of colony formation in soft agar cultures (index of malignancy) and the resemblance in actively stained esterase patterns in gel electrophoresis. The stereoselective hydrolysis leads to cell-specific activation of anticancer prodrugs. This has been confirmed for the first time by the stereoselectivity of Anr4 and H4-II-E cells in the hydrolysis of a chiral mustard ester, bis(2-chloroethyl)aminophenyl 2-methoxy-2-phenylacetate ( 14 ) and by the difference of IC₅₀ values of (R)- and (S)- 14 against the two cell lines. © 1995 Wiley-Liss, Inc.     

Studies on cytochrome c. Part IV. Synthesis of the protected dodecapeptide (sequence 45–56) of Baker's yeast iso-1-cytochrome c

The synthesis is described for the N-benzyloxycarbonyldodecapeptide tert-butoxycarbonylhydrazide corresponding to the sequence 45–56 of baker's yeast iso-1-cytochrome c by fragment condensation using the Rudinger modified azide procedure for the assembly of the small subunits.     

Inhibition of superoxide dismutases by azide.

Iron-containing Superoxide dismutases are more sensitive to inhibition by azide than are the corresponding manganese containing enzymes, while the copper-zinc Superoxide dismutases are least sensitive. Thus, at pH 7.8, 10 mm azide inhibited Cu-Zn, Mn, and Fe enzymes by ~10%, ~30% and ~73%, respectively. Stated differently, the concentrations of N₃^? required to cause 50% inhibition of the Cu-Zn, Mn, and Fe enzymes was ~32 mm, ~20 mm and ~4 mm, respectively. These inhibitions by azide, which were imposed and reversed rapidly, appear to provide a useful criterion for distinguishing among the classes of these enzymes. Sensitivity towards inhibition by N₃^?can be applied to the Superoxide dismutases in crude extracts, for the purpose of deciding to which class they belong.     

The synthesis of a mannosyl-N-acetylglucosamine-l-asparagine compound: 2-acetamido-N-(l-aspart-4-oyl)-2-deoxy-3-O-α-d-mannopyranosyl-β-d-glucopyranosylamine

The title compound, used in the synthesis of glycopeptides and as a reference substance in the structural elucidation of glycoproteins, was synthesized by condensation of 2,3,4,6-tetra-O-acetyl-α-d-mannopyranosyl bromide with 2-acetamido-4,6-O-benzylidene-α-d-glucopyranosyl azide, followed by removal of the benzylidene group to give the disaccharide azide 6 and acetylation. The resulting fully acetylated disaccharide azide 7 was also obtained by treatment of the known 2-acetamido-1,4,6-tri-O-acetyl-2-deoxy-3-O-(2,3,4,6-tetra-O-acetyl-α-d-mannopyranosyl)-α-d-glucopyranose with hydrogen chloride and then with silver azide. The azide 7 was reduced in presence of platinum oxide (Adams' catalyst), and the resulting amine was condensed with 1-benzyl N-benzyloxycarbonyl-l-aspartate in the presence of N,N′-dicyclocarbodiimide. The removal of the protective group was accomplished by hydrogenolysis and O-deacetylation. In a second route, the disaccharide azide 6 was reduced and then condensed with 1-benzyl N-benzyloxycarbonyl-l-aspartate, and the resulting product hydrogenolyzed.     

Microbial asymmetric hydrolysis of N-acetyl-1-methyl-3-phenylpropylamine to optically active 1-methyl-3-phenylpropylamine

Summary The asymmetric hydrolysis of N-acetyl-1-methyl-3-phenypropylamine (MPAc) by microorganisms was investigated. Various bacteria belonging to the genera Bacillus, Agrobacterium, Corynebacterium, Arthrobacter, Brevibacterium, Cellulomonas, Acinetobacter, Nocardia and Rhodococcus showed this hydrolysing activity and yielded (S)-1-methyl-3-phenylpropylamine (MPPA). Using washed cells of N. erythropolis IAM 1440, 15.1 mg/ml of (S)-MPPA was formed, with a 38.8% conversion yield and high stereoselectivity (97.9% enantiomeric excess), in an organic solvent-water diphase system. The same (S)-amine and (S)-1-phenylethylamine were also produced in good yields from the valeryl and isovaleryl derivatives of MPPA, and N-acetyl-1-phenylethylamine, respectively.