A new method for selective N-acylation of aminoglycoside antibiotics

Per-N-formylation of aminoglycoside (aminocyclitol) antibiotics followed by mild hydrolysis with aqueous ammonia gave mono-N-deformylated derivatives. Each positional isomer of the mono-N-deformylated derivatives thus obtained was separated by column chromatography on Amberlite CG-50 (NH₄⁺ ). Acylation of mono-N-deformylated derivatives gave the corresponding mono-N-acylated derivatives. The N-formyl groups of the mono-N-acylates were removed by the treatment with dilute aqueous hydrazine acetate, whereas the newly introduced N-acyl group was stable under these conditions. The 1-N-formyl group of the deoxystreptamine moiety of per-N-formylated aminoglycoside antibiotics containing neamine (or 3′-deoxyneamine) is more readily deformylated than the 3-N-formyl group. In this report, isolation and structural-elucidation studies, including ¹³C-n.m.r. spectral assignments, of positional isomers of tri-N-formyl derivatives of xylostasin (1), 3′-deoxyxylostasin (2), kanamycin A (3), and neamine (4) are described. This selective N-acylation provides a useful method for the preparation of 1-N-modified derivatives, and the synthesis of 3′-deoxybutirosin A (2f) from 2 is described in detail as an example.     

Synthesis of 4′-methoxyadenosine and related compounds

Addition of iodine and methanol to N⁶,N⁶-dibenzoyl-9(2,3-O-carbonyl-5-deoxy-β-d-erythro-pent-4-enofuranosyl)adenine (4) selectively gives N⁶,N⁶-dibenzoyl-2′,3′-O-carbonyl-5′-deoxy-5′-iodo-4′-methoxyadenosine (5). Compound 5 can be converted into 4′-methoxyadenosine via hydrolysis of the carbonate followed by benzoylation, displacement of the 5′-iodo function by benzoate ion, and hydrolysis with ammonia. Configurational assignments are based upon comparisons of ¹H- and ¹³C-n.m.r. spectra with those of previously characterised analogues in the uracil series and by borate electrophoresis. Intermediates in the above scheme have also been converted into 5′-amino-5′-deoxy-4′-methoxyadenosine, 4′-methoxy-5′-O-sulfamoyladenosine, and ethyl 4′-methoxyadenosine-5′-carboxylate, each of which is a 4′-methoxy analogue of biologically active derivatives of adenosine.     

C-n.m.r. spectroscopic investigation of methylated and charged agarose oligosaccharides and polysaccharides

The ¹³C-n.m.r. signals of agarose oligomers with various substituted repeating units have been assigned. Enzymic hydrolysis of agaroses gave 2¹-O-methylagarobiose, 6²-O-methylagarobiose, the agarobiose biological precursor, agarobiose 4²-sulfate, 2¹-O-methylagarobiose 4²-sulfate, and pyruvylated agarobiose. The chemical shift data of the oligomers and the parent polymers were compared, and indicated the distribution of the substituents in hybrid polymers.     

C-n.m.r. spectral study of C reductively methylated glycopeptides derived from glycophorin A

¹³C-n.m.r. spectral data for ¹³C reductively methylated intact homozygous and heterozygous glycophorins A were compared with the ¹³C-n.m.r. spectral data for the ¹³C reductively methylated homozygous and heterozygous N-terminal glycopeptides derived from the trypsin digest of glycophorin A. The results indicate that pronounced aggregation of this glycoprotein in solution does not affect the structural differences that we have previously observed for glycophorins A^M and A^N at and/or near the N-terminal amino acid. Moreover, the data suggest that two structural states exist for glycophorin A^M.     

Block-synthesis of higher oligosaccharides: Synthesis of hexa- and nona-saccharide fragments of the o-antigenic polysaccharide of Salmonella newington

Successive condensation of derivatives of the trisaccharide, biological repeating-unit of the O-antigenic polysaccharide of Salmonella newington, followed by removal of protecting groups, has given the hexa- and nona-saccharides. The structures of these oligosaccharides were confirmed chemically and by ¹³C-n.m.r. spectroscopy.     

Room temperature, low-field C-n.m.r. spectra of degraded carrageenans. Part II. On the specificity of the autohydrolysis reaction in kappa/iota and mu/nu structures

A kappa/iota carrageenan from Gigartina skottsbergii and a partially cyclized mu/nu carrageenan from Iridaea undulosa were submitted to autohydrolysis. The ¹³C-n.m.r. spectra of the degraded products give structural information on the polysaccharides and show clearly that besides the known splitting of 3,6-anhydrogalactosidic linkages, the linkage between - -galactose 2,6-disulphate and β- -galactose 4-sulphate is cleaved with similar specific reaction rate.     

Benzylidene acetal structural elucidation by N.M.R. Spectroscopy: Application of carbon-13. N.M.R.-Spectral parameters

The ₁₃C-n.m.r. spectra of 19 2-phenyl-1,3-dioxolane, -1,3-dioxane and -1,3-dioxopane derivatives were examined and it was found that both the ₁₃C-n.m.r. chemical shift for the acetal carbon atom and the one-bond coupling constant between the acetal carbon atom and the acetal proton had values that could be used to distinguish between acetals having different ring sizes. In addition, the presence of axial substituents at positions 4 or 6 in substituted 2-phenyl-1,3-dioxane rings and 4 or 7 in substituted 2-phenyl-1,3-dioxepane rings could be readily detected. The structures of a number of carbohydrate examples were determined by using these two parameters and also the chemical shift of the acetal proton from ₁H-n.m.r. spectra. The use of all three parameters made assignment of benzylidene acetal ring-size unambiguous.     

The C-n.m.r. spectra of inositols and cyclohexanepentols: the validity of rules correlating chemical shifts with configuration

The ¹³C-n.m.r. spectra of the eight diastereomeric inositols and the ten diastereomeric cyclohexanepentols have been recorded and all of the signals have been assigned by a variety of methods. The additive empirical constants used to correlate the configuration of hydroxyl groups with the ¹³C-chemical shifts differ, in the latter group, from those derived for the inositols; the removal of one hydroxyl group had altered the “rules” considerably. A better correlation was found between the spectra of the cyclohexanepentols and those of the inositols derived therefrom, in each case, by the addition of an equatorial hydroxyl group.     

Structural studies of gum arabic, the exudate polysaccharide from Acacia senegal

A 25.182-MHz ¹³C-n.m.r. spectrum of gum arabic allows unambiguous characterisation of all the C-1 resonances. These assignments have been confirmed by correlation of the modification of the intensities of these signals after controlled acid hydrolysis and characterisation of the released fragments. The resonances of the other carbons have been assigned through partial relaxed T₁ spectra of the polysaccharides obtained by graded degradation of the gum. These results indicate gum arabic to consist mainly of a (1→3)-β- -galactan core with (1→6)-β- -galacto-pyranosyl branches and with - -arabinofuranosyl-(1→3)-- -arabinofuranosyl and - -rhamnopyranosyl-(1→4)-β- -glucopyranosyluronic acid groups attached to positions 3 and 6, respectively, of the branch units.     

Analysis of a pyruvic acid acetal-containing polysaccharide by the reductive-cleavage method.

The applicability of the reductive-cleavage method to the analysis of polysaccharides bearing pyruvic acid acetals has been demonstrated. Direct reductive cleavage of fully methylated gum xanthan yielded the expected products, including 1,5-anhydro-4,6-O-[(S)-1-methoxycarbonylethylidene]-2,3-di-O-methy l-D- mannitol. The latter product was not observed when reductive cleavage was performed subsequent to reduction of ester groups in the fully methylated polysaccharide and mild hydrolysis to remove pyruvic acid acetal substituents. Instead, the latter experiment yielded 1,5-anhydro-2,3-di-O-methyl-D-mannitol, establishing the presence in the polysaccharide of terminal (nonreducing) D-mannopyranosyl groups bearing 4,6-O-(1-carboxyethylidene) substituents. The products of reductive cleavage were characterized, where appropriate, by comparison of the gas chromatographic retention times and chemical ionization- and electron ionization-mass spectra of their acetates to those of authentic standards. Alternatively, the products of reductive cleavage could be characterized without resort to comparison with authentic standards by analysis of the 1H-n.m.r. spectra of their benzoates, which were obtained in pure form by high-performance liquid chromatography. By either method of product characterization, this two-step procedure of analysis reveals the presence of pyruvic-acetal residues in polysaccharides and establishes both the identity of the sugar residue to which they are attached and their positions of attachment.     

Hydrogenolysis of the isomeric 1,2:4,6-di-O-benzylidene-α-d-glucopyranose derivatives with the LiAlH4AlCl3 reagent

Both isomers of 1,2:4,6-di-O-benzylidene-α-d-glucopyranose (and their 3-O-acetyl and 3-O-benzyl derivatives) have been prepared and their ¹H- and ¹³C-n.m.r. spectra assigned. The mode of hydrogenolysis of the dioxolane ring in these isomers by the LiAlH₄AlCl₃ reagent is determined by the configuration at the acetal carbon and is independent of the electronic character of the two oxygen atoms.     

Reaction of sulfur diimides with organoboranes — studied by multinuclear magnetic resonance in solution

Reactions between sulfur diimides R(NSN)R′ (R=R′=^tBu (1a), SiMe₃ (1b), SnMe₃ (1c); R=^tBu, R′=SnMe₃ (1d); R=SiMez₃, R′=SnMe₃ (1e)) and various organoboranes were studied, and the products were characterized by multinuclear magnetic resonance data (¹H, ¹¹B, ¹³C, ¹⁵N, ²⁹Si and ¹¹⁹Sn NMR). Tetraalkyldiboranes(6) (Et₂BH₂BEt₂ (2), dimeric 9-borobicyclo[3,3.1]nonane (3)) react with 1a and 1b by 1,3-hydroboration to give the N-sulfanyl-dialkylaminoboranes 4 and 5 which are instable with respect to eliminatio of short-lived [R---NS]. Trialkylboranes (Et₃B (8)) react only sluggishly with 1a, but more readily with 1b mainly via S-ethylation, formally a 1,2-ethyloboration, to give the diethylborylamido-imino-ethanesulfinic acid 9b decomposes slowly at room temperature via ethene elimination to give 4b, followed by further decomposition via [R---NS] elimination. The compounds 9 can be prepared independently from the reaction between the N-lithio-imino-ethanesulfinic acid amide 10 and diorganoboron halides. The molecular structure of the lithium amide 10a (R=R′=tBu) was determined by X-ray analysis as a dimer in which the four nitrogen, two sulfur and two lithium atoms adopt a boat conformation, in contrast with other known derivatives of this type. If the sulfur diimides bear at least one trimethylstannyl group (1c-e), their reactions with Et₃B (8), iPr₃B (12) or 9-iso-butyl-9-borabicyclo[3,3,1]nonane (13) lead to the novel aminoboranes 14–16. These are products of a 1,1,-organoboration, since the Me₃Sn group moves from one nitrogen atom to the other, and both the boryl and an alkyl group end up at the same nitrogen atom.     

Amphiphilic derivatives of sodium alginate and hyaluronate: synthesis and physico-chemical properties of aqueous dilute solutions

This paper reports on the synthesis and the physico-chemical characterisation of various amphiphilic derivatives of two natural polysaccharides, sodium alginate and sodium hyaluronate, in which a rather small proportion of the carboxylic groups (≤10% mol) was esterified by long alkyl chains (C₁₂H₂₅ or C₁₈H₃₇).

The derivatives thus prepared were characterised by gas chromatography, ¹H and ¹³C n.m.r. spectroscopy and size exclusion chromatography coupled to a multi-angle laser light scattering detection. The tendency of these water-soluble compounds to hydrophobic association in aqueous solutions was evidenced firstly in dilute regime using capillary viscometry as well as fluorescence spectroscopy in the presence of a molecular probe, 1,1-dicyano-(4′-N,N-dimethylaminophenyl)-1,3-butadiene.     

Properties of C-N.M.R. spectra of O-(1-carboxyethylidene) derivatives of methyl β-d-galactopyranoside: models for determination of pyruvic acid acetal structures in polysaccharides

¹³C-N.m.r. spectra were recorded of compounds containing O-(1-carboxyethylidene) groups linked to galactopyranose and fucopyranose derivatives. These compounds are useful as aids in determination of the positions and configurations of pyruvic acid acetal substituents in polysaccharides. Chemical shifts of non-protonated acetal carbons depend on whether the acetal ring is 5-membered (δ_c 107–109.5) or 6-membered (δ_c 100.5–102.4). The C-3 signals of 3,4-(1-carboxyethylidene) acetals are typical, being at δ_c 81 and in the case of the barium salt of the methyl β-d-galactopyranoside derivative. The exact value depends on the configuration, whether it is as in 6 (δ_c 81.1) or 5 (δ_c 80.4). The CH₃ signals of proton-n.m.r. spectra are also diagnostically useful, falling at δ 1.97 and 2.07 respectively. (The foregoing shift-values are pH-dependent). The pyruvylated galactan from the snail, Pomacea lineata, was shown to contain some residues that could be assigned a structure corresponding, in the positions of acetal substitution and acetal configuration, to structure 6. Compound 6 (barium salt) is of interest as its ¹³C-n.m.r. spectrum lacks non-protonated carbonyl and acetal carbon resonances, when obtained by the usual procedures. While this is principally because of long T₁ values, the non-protonated acetal carbon signals are comparatively broad, possibly through slow conformational interchange. In the case of the carbonyl resonance, the lack of sensitivity is because of a low n.O.e. value of 1.4, approximately one half that of other carbon atoms in the molecule.     

N.m.r. studies of synergistic kappa carrageenan—carob galactomannan gels

¹³C-n.m.r. spectroscopy has been used to investigate the carob galactomannan—kappa carrageenan binary gels. Starting from partially depolymerized carob samples, evidence for interaction and intermolecular binding was found by analysis of the spectra obtained in quantitative conditions and in the absolute intensity mode. From these, a reconstitution of the signals corresponding to C-1, C-4, C-5 and C-6 showed the existence of three kinds of galactomannan chains: the first ones with a low galactose content were strongly connected and as much had completely lost their mobility; but the chains with a high galactose content were still detected and could be divided in two groups according to their mobility.     

C n.m.r. study of the pH behaviour of N-methylated peptides related to the N-terminus of glycophorins

¹³C nuclear magnetic resonance spectroscopy (¹³C n.m.r.) was used to determine the pH titration parameters for the N-terminal N,N-[¹³C]dimethylamino and N,N-[¹³C]monomethylamino groups of glycophorins A^M and A^N, and some 28 related glycoproteins, glycopeptides and peptides. The results show that glycosylation of the Ser and Thr residues at positions 2, 3 and 4 of the glycophorins have a pronounced effect on the titration parameters. Substitution of amino acids 4 and 5 in the glycophorin sequence appears to minimally affect our titration parameters. Internal hydrogen-bonding involving the N-terminal Ser residue may explain some of the unusual pH titration results observed for glycophorin A^M.     

Thermal depolymerization of alginate in the solid state

A new method of introduction carboxyl groups to chitosan sulfate by the acylation reaction between hydroxyethyl chitosan sulfates and butane dioic anhydride in homogeneous solution was used to obtain carboxybutyrylated hydroxyethyl chitosan sulfates. The structures of the derivatives were characterized by element analysis, FT-IR, ¹³C-NMR, and gel permeation chromatography. The content and position of the carboxyl groups could be controlled favorably. Their anticoagulant activity was determined for human plasma with respect to activated partial thromboplastin time (APTT), thrombin time (TT), and prothombin time (PT). The introducing of carboxyl groups to amino groups greatly prolonged the APTT and TT. The best result occurred when the degree of substitution of the carboxyl groups was about 0.4/unit that prolonged APTT and TT with about 5 and 1.5 times compared to that of the uncarboxylated hydroxyethyl chitosan sulfates; another conclusion is that introducing of carboxyl groups into N,O-position gave better results than that just into N-positions. Low S% chitosan sulfate and 6-O-desulfated chitosan sulfate showed little anticoagulant activity but their N,O-carboxybutyrylated derivatives (0.6/unit ds) showed increased APTT or TT, while their N-carboxybutyrylated derivatives (0.6/unit ds) gave no improvement. Generally, the introducing of carboxyl groups could not increase PT in spite of the position introduced.     

Tyrosine attack by free radicals derived from catalytic decomposition of carbon tetrachloride

The interaction between free radicals derived from the catalytic decomposition of carbon tetrachloride and tyrosine (the N-acetyl tyrosine ethyl ester, ATEE) under anaerobic and aerobic conditions was studied. The structure of the reaction products formed was desciphered by the GLC/MS analysis of their trimethylsilyl derivatives. Under anaerobic conditions the formation of the following products was found: (1) an unsaturated derivative of the amino acid; (2) the trimethylsilyl derivative of N-acetyl chloro tyrosine ethyl ester; (3) a hydroxyl adduct of ATEE ; (4) an ATEE adduct having a chlorine and a CCl₃ group in the molecule (it is suggested that CCl₃ is attached to the benzyl carbon and the chlorine located in the benzene ring); (5) an ATEE adduct having only a CCl₃ group tentatively assigned to be located on the benzyl carbon; and (6) and (7) were found to be two isomers of an ATEE having one CCl₃ on the aromatic ring. Under aerobic conditions the following reaction products were identified: Two products which were similar to those numbered (1) and (2) and formed anaerobically; (8) and (11) two isomeric dichlorinated adducts of ATEE; (9) and (10) two isomeric dichlorinated monohydroxylated derivatives of ATEE. Concerning the potential relevance of these findings, we consider that if similar interactions to those here reported occurred during CCl₄ poisoning, the activity of enzymes having tyrosine in their active center might result in impairment. Further, enzymes operating on tyrosine moieties in proteins might be perturbed in their action if tyrosine groups were attacked by the free radicals arising from catalytic decomposition of CCl₄ evidenced here.     

Synthesis and hydrogenolysis of the methylene,ethylidene, isopropylidene,and diastereoisomeric 1-phenylethylidene acetals of β-l-arabino- and α-l-rhamnopyranoside derivatives

Both diastereoisomers of 1-phenylethylidene acetals (acetophenone acetals) of methyl and benzyl β-l-arabinopyranoside and α-l-rhamnopyranoside were prepared. Acetal-exchange reactions gave only the endo-phenyl isomers; their 2-O- and 4-O-acetyl derivatives were isomerised into the exo-phenyl compounds. ¹H-N.m.r. data were used to determine the absolute configuration at the acetal carbon atom in these compounds. The protons of the methyl group of the exo-phenyl isomers resonate at lower field than those of the endo-phenyl isomers. Hydrogenolysis of various methylene, ethylidene, and isopropylidene derivatives gave axial ethers. The endo-phenyl isomers of the acetophenone derivatives also gave axial 1-phenylethyl ethers in two diastereoisomeric forms. The exo-phenyl isomers of the arabinosides were stable towards the reagent (LiAlH₄AlCl₃), whereas the corresponding rhamnopyranosides gave the 2-(1-phenylethyl) ethers, but cleavage required prolonged reaction time and higher temperature.     

Syntheses and molecular structures of 3-N,N-di-n-Propylamino-2-chromanones as new analogues of dopamine

A series of 3-N,N-di-n-propylamino-2-chromanones were synthesized as dopamine analogues. The lactone ring was introduced as a means to reduce their propensity to cross the blood-brain barrier and to avoid central side effects, rendering these compounds potentially useful for the treatment of glaucoma. Pharmacological activities were determined in vitro on rat striatum, by examining their capacity to displace the specific binding of the labeled dopaminergic ligand ³H sulpiride or ³H spiperone and ³H SCH 23390 for D₂ and D₁ sites, respectively. Compound 6a showed a weak dopaminergic activity on D₂-receptors and no affinity for D₁-receptors, which can be explained, at least in part, by a weak pKa and the presence of an internal hydrogen bonding. Furthermore, computer molecular modelling studies showed that the aromatic ring of 6a was negatively charged in contrast to the classical D₂-agonists aminotetralin derivatives, hampering a possible interaction with a negatively charged area of the D₂-receptor. These results, taken together, can account for the moderate dopaminergic activities exhibited by these lactone derivatives.