Acid-base properties of 4-nitro-l-histidine and related compounds

Acid-base properties of 4-nitro-l-histidine (3), N^α-acetyl-4-nitro-l-histidine (2), and N^α-acetyl-4-nitro-l-histidine methyl ester (1) are studied. Their pK_a^II values can be conveniently determined by ultraviolet spectroscopy. Potentiometric titration and ¹H-nuclear magnetic resonance (nmr) titration can also be used. Introduction of a nitro group strongly enhances the acidity of all the compounds. pK_a¹ of compound (3) has been also spectrophotometrically determined. Observed differences in acidity, in those cases where solvation does not play a major role, can be explained by assessing the influence of electrostatic charges on pK_a following Bjerrum's general theory with the aid of data from ¹H-nmr conformational analysis.     

l-Proline transport in Saccharomyces cerevisiae

Transport of l-proline into Saccharomyces cerevisiae K is mediated by two systems, one with a K_T of 31 μM and J_max of 40 nmol · s^?1 · (g dry wt.)^?1, the other with K_T > 2.5 mM and J_max of 150–165 nmol · s^?1 · (g dry wt.)^?1, The kinetic properties of the high-affinity system were studied in detail. It proved to be highly specific, the only potent competitive inhibitors being (i) l-proline and its analogs l-azetidine-2-carboxylic acid, sarcosine, d-proline and 3,4-dehydro-dl-proline, and (ii) l-alanine. The other amino acids tested behaved as noncompetitive inhibitors. The high-affinity system is active, has a sharp pH optimum at 5.8–5.9 and, in an Arrhenius plot, exhibits two inflection points at 15°C and 20–21°C. It is trans-inhibited by most amino acids (but probably only the natural substrates act in a trans-noncompetitive manner) and its activity depends to a considerable extent on growth conditions. In cells grown in a rich medium with yeast extract maximum activity is attained during the stationary phase, on a poor medium it is maximal during the early exponential phase. Some 50–60% of accumulated l-proline can leave cells in 90 min (and more if washing is done repeatedly), the efflux being insensitive to 0.5 mM 2,4-dinitrophenol and uranyl ions, to pH between 3 and 7.3, as well as to the presence of 10–100 mM unlabeled l-proline in the outside medium. Its rate and extent are increased by 1% d-glucose and by 10 μg nystatin per ml.     

Synthesis and biological evaluation of novel analogues of Gly-l-Pro-l-Glu (GPE) as neuroprotective agents

This study investigated the anti-inflammatory effects of novel pseudotripeptides (GPE 1–3) as potential candidates to counteract neuroinflammation processes in Alzheimer’s disease.GPE 1–3 pseudotripeptides are synthetic derivatives of Gly-l-Pro-l-Glu (GPE), the N-terminal tripeptide of IGF-1, obtained through the introduction of isosteres of the amidic bond (aminomethylene unit) to increase the metabolic stability of the native tripeptide. The results showed that all synthetic derivatives possessed higher half-lives (t_1/2?>?4?h) than GPE (t_1/2?=?30?min) in human plasma and had good water solubility. The biological results demonstrated that GPE 1–3 had protective properties in several experimental models of treated THP-1 cells. Notably, the novel pseudotripeptides influenced inflammatory cytokine expression (IL-1β, IL-18, and TNF-α) in Aβ_25–35-, PMA-, and LPS-treated THP-1 cells. In PMA-differentiated THP-1 macrophages, both GPE 1 and GPE 3 reduced the expression levels of all selected cyto-chemokines, even though GPE 3 showed the best neuroprotective properties.     

Enzymatic epimerization of d-erythro-dihydroneopterin triphosphate to l-threo-dihydroneopterin triphosphate

An enzyme has been discovered in Escherichia coli that catalyzes the conversion of the triphosphate ester of 2-amino-4-hydroxy-6-(d-erythro-1′,2′,3′-trihydroxypropyl)-7,8-dihydropteridine, (i.e. d-erythro-dihydroneopterin triphosphate) to an epimer of this compound, l-threo-dihydroneopterin triphophate. The enzyme, which is here named “d-erythro-dihydroneopterin triphosphate 2′-epimerase,” needs a divalent cation (Mg²⁺ or Mn²⁺ is most effective) for maximal activity. Its molecular weight is estimated at 87 000–89 000. Little or no activity can be detected if either the monophosphate or the phosphate-free form of the substrate is incubated with the enzyme. Evidence is presented to establish that all three phosphate residues of the substrate are retained in the product and that the product is of the l-threo configuration.     

Chain elongation by a seemingly stereospecific cyanohydrin synthesis: the preparation and configurational assignment of 3,7-anhydro-d-threo-l-talo- and -l-galacto-octose

2,6-Anhydro-d-glycero-l-manno-heptose (1) is converted by the cyanohydrin reaction into crystalline d-threo-l-talo-octononitrile (3), which shows mutarotation in water. The equilibrium mixture, as measured by ¹³C-n.m.r. spectroscopy, contains about equal amounts of 3 and its epimer, d-threo-l-galacto-octononitrile. On evaporation of the aqueous mixture, pure, crystalline 3 is again obtained. Labelling experiments in ³H₂O proved that epimerization proceeds through reversible deprotonation. Stabilization of 3 in the solid state is explained by intramolecular hydrogen-bonding. In pyridine, rapid isomerization of 3 occurs. When acetylation of 3 is conducted in this solvent, the yield of 2,4,5,6,8-penta-O-acetyl-3,7-anhydro-d-threo-l-talo-octono-nitrile (4) depends strongly on the conditions of acetylation. Acetylation after equilibration produces an equimolar mixture of 4 and its isomer 2,4,5,6,8-penta-O-acetyl-3,7-anhydro-d-threo-l-galacto-octononitrile. Structural assignment for both was achieved by 360-Mhz, ¹H- and ¹³C-n.m.r. spectroscopy. Reduction of 4 in pyridine-acetic acid-water in the presence of N,N-diphenylethylenediamine yields a 1:2.36 mixture of 2,4,5,6,8-penta-O-acetyl-3,7-anhydro-d-threo-l-talo-octose N,N-diphenylimidazolidine (6) and 2,4,5,6,8-penta-O-acetyl-3,7-anhydro-d-threo-l-galacto-octose N,N-diphenylimidazolidine (8). Compounds 6 and 8 could be separated and obtained as crystalline solids, and their structure proved by ¹H- and ¹³C-n.m.r. spectroscopy. Hydrolysis of 6 and 8 gave 2,4,5,6,8-penta-O-acetyl-3,7-anhydro-d-threo-l-galacto-octose and -d-threo-l-talo-octose.     

Solvolysis of charged active esters of carboxylic acids with cyclo (l- or d-Glu-l-His)

Cyclic dipeptide cyclo(l- or d-Glu-l-His) carrying an anionic site and a nucleophilic site has been synthesized and used as a catalyst for the solvolysis of cationic esters in aqueous alcohols. In the solvolysis of 3-acyloxy-N-trimethylanilinium iodide (S⁺_n, n = 2 and 10) and Cl^?H₃N⁺(CH₂)₁₁COOPh(NO₂), no efficient nucleophilic catalysis was observed. On the other hand, in the solvolysis of Gly-OPh(NO₂)·HCl, Val-OPh(NO₂)·HCl and Leu-OPh(NO₂)·HCl a very efficient general base-type catalysis by cyclo(l-Glu-l-His) was observed. In particular, with the latter two substrates the catalysis by cyclo(l-Glul-His) was more efficient than that by imidazole, although the catalysis was not enantiomer-selective. The diastereomeric cyclic dipeptide cyclo(d-Glu-l-His) was almost inactive under the same conditions. Confomation of cyclo(l- or d-Glu-l-His) in aqueous solution was investigated and the structure/catalysis relationship is discussed.     

Synthesis of acetylenic sugars and uronic acids via two-carbon chain extension of d-ribose

Treatment of methyl β-d-ribofuranoside with acetone gave methyl 2,3-O-isopropylidene-β-d-ribofuranoside (1, 90%), whereas methyl α-d-ribofuranoside gave a mixture (30%) of 1 and methyl 2,3-O-isopropylidene-α-d-ribofuranoside (1a). On oxidation, 1 gave methyl 2,3-O-isopropylidene-β-d-ribo-pentodialdo-1,4-furanoside (2), whereas no similar product was obtained on oxidation of 1a. Ethynylmagnesium bromide reacted with 2 in dry tetrahydrofuran to give a 1:1 mixture (95%) of methyl 6,7-dideoxy-2,3-O-isopropylidene-β-d-allo- (3) and -α-l-talo-hept-6-ynofuranoside (4). Ozonolysis of 3 and 4 in dichloromethane gave the corresponding d-allo- and l-talo-uronic acids, characterized as their methyl esters (5 and 6) and 5-O-formyl methyl esters (5a and 6a). Ozonolysis in methanol gave a mixture of the free uronic acid and the methyl ester, and only a small proportion of the 5-O-formyl methyl ester. Malonic acid reacted with 2 to give methyl 5,6-dideoxy-2,3-O-isopropylidene-β-d-ribo-trans-hept-5-enofuranosiduronic acid (7).     

Structure of l-arabino-d-galactan-containing glycoproteins from radish leaves

Two l-arabino-d-galactan-containing glycoproteins having a potent inhibitory activity against eel anti-H agglutinin were isolated from the hot saline extracts of mature radish leaves and characterized to have a similar monosaccharide composition that consists of l-arabinose, d-galactose, l-fucose, 4-O-methyl-d-glucuronic acid, and d-glucuronic acid residues. The chemical structure features of the carbohydrate components were investigated by carboxyl group reduction, methylation, periodate oxidation, partial acid hydrolysis, and digestion with exo- and endo-glycosidases, which indicated a backbone chain of (1→3)-linked β-d-galactosyl residues, to which side chains consisting of α-(1→6)-linked d-galactosyl residues were attached. The α-l-arabinofuranosyl residues were attached as single nonreducing groups and as O-2- or O-3-linked residues to O-3 of the β-d-galactosyl residues of the side chains. Single α-l-fucopyranosyl end groups were linked to O-2 of the l-arabinofuranosyl residues, and the 4-O-methyl-β-d-glucopyranosyluronic acid end groups were linked to d-galactosyl residues. The O-α-l-fucopyranosyl-(1→2)-α-l-arabinofuranosyl end-groups were shown to be responsible for the serological, H-like activity of the l-arabino-d-galactan glycoproteins. Reductive alkaline degradation of the glycoconjugates showed that a large proportion of the polysaccharide chains is conjugated with the polypeptide backbone through a 3-O-d-galactosylserine linkage.     

l-Arginine uptake into renal brush border membrane vesicles

The uphill uptake of l-arginine by renal brush border membrane vesicles was found to be energized by a Na⁺ gradient (extravesicular > intravesicular) in the presence of a membrane potential (inside negative). The uptake was specific for Na⁺. Either a K⁺-diffusion potential, generated by valinomycin, or a H⁺-diffusion potential, generated by the mitochondrial uncoupler, carbonyl cyanide p-trifluoromethoxyphenylhydrazone, provided the electrical driving force. The Na⁺ gradient-dependent l-arginine transport system was shared by specific basic amino acids and l-cystine, but not by d-arginine nor other classes of amino acids. The molecular structure of the basic amino acid recognized by the carrier was postulated.     

Synthesis of crystalline derivatives of 6-deoxy-d-allo- and -l-talo-furanosyl bromide suitable for nucleoside synthesis

6-Deoxy-2,3,5-tri-O-(p-nitrobenzoyl)-β-d-allo- and -α-l-talo-furanosyl bromide (6 and 11) have been synthesized from methyl 2,3-O-isopropylidene-β-d-ribo-pentodialdo-1,4-furanoside (1). Treatment of 1 with methyl Grignard reagent, followed by (p-nitrobenzoyl)ation, afforded two 5-epimers, methyl 6-deoxy-2,3-O-isopropylidene-5-O-(p-nitrobenzoyl)-β-d-allo- and -α-l-talo-furanosides (3 and 8) which were fractionally recrystallized. The l-talo isomer (8) separated first, and was treated with acid to remove the isopropylidene group, the product (p-nitrobenzoyl)ated, and the ester reacted with hydrogen bromide in acetic acid, to afford crystalline compound 11. The mother liquor from the fractional recrystallization was treated with acid, whereby methyl 6-deoxy-5-O-p-nitrobenzoyl)-d-allofuranoside was isolated. It was (p-nitrobenzoyl)ated, and the ester treated with hydrogen bromide in acetic acid, to afford crystalline bromide 6.     

Methanolysis and aminolysis of N-acetylmuramic acid lactones: Evidence for the retention of the d-gluco configuration

Methanolysis of benzyl α-glycosides of N-acetylmuramic acid lactones with HO-6 free (2) and substituted (4, 7, 10, and 12) is catalysed by small amounts of silica gel to give, exclusively, the corresponding methyl esters with HO-4 unsubstituted (3, 5, 8, 11, 13); opening of the lactone ring proceeds with retention of the d-gluco configuration and can be followed by ¹H-n.m.r. spectroscopy. Condensation of 2 with 2-methyl-(3,4,6-tri-O-acetyl-1,2-dideoxy-α-d-glucopyrano)-[2,1-d]-2-oxazoline (15) gave the β-(1→6)-linked disaccharide lactone 16 which, on methanolysis, yielded the disaccharide methyl ester 17, also obtained by condensation of 3 and 15. In the presence of imidazole, the lactones 2 and 4 underwent aminolysis with amino acid and peptide esters as nucleophiles to give the N-acetylmuramoylamide derivatives 19–24. The structures of methanolysis and aminolysis products were established by ¹H-n.m.r. spectroscopy and independent syntheses.     

Synthesis and diazomethane-catalysed 1→2 acyl migration of the l-arabinosyl esters of N-acylamino acids

The fully benzylated α- and β-l-arabino-pyranosyl (1 and 2) and -furanosyl esters (3 and 4) of N-acetyl-d-alanine and N-tert-butoxycarbonyl-l-phenylalanine have been synthesised. Catalytic hydrogenation of 3 and 4 gave both anomers of 1-O-(N-tert-butoxycarbonyl-l-phenylalanyl)-l-arabino-pyranose (5) and -furanose (6), which were characterised as the triacetates 7 and 8, respectively. Treatment of the cis-oriented β-anomers of 5 and 6 with 0.5 equiv. of diazomethane at 0° for 1 h led to the 1→2 acyl rearrangement, with pyranose—furanose interconversion and anomerisation, to give, upon acetylation, a mixture of 1,3,4- and 1,3,5-tri-O-acetyl-2-O-(N-tert-butoxycarbonyl-l-phenylalanyl)-α,β-l-arabino-pyranose and -furanose, the structures of which were determined by ¹H- and ¹³C-n.m.r. spectroscopy. The 1→2 acyl-migration step in the l-arabino series is immediately followed by isomerisation into the four possible forms.     

Synthesis of derivatives of 3-amino-2,3-dideoxy-l-hexoses related to daunosamine (3-amino-2,3,6-trideoxy-l-lyxo-hexose)

The synthesis is described of 3-amino-2,3-dideoxy-l-arabino-hexose (10), methyl 2,3-dideoxy-3-trifluoroacetamido-α-l-lyxo-hexopyranoside (17), methyl 3-amino-2,3-dideoxy-α-l-ribo-hexopyranoside (21), methyl 2,3-dideoxy-3-trifluoroacetamido-α-l-xylo-hexopyranoside (26), and certain derivatives from methyl 4,6-O-benzylidene-2-deoxy-α-l-arabino-hexopyranoside (3). Conversion of 2-deoxy-l-arabino-hexose into 3 by modified, standard procedures, and on a large scale, gave a 75% yield.     

A stereospecific synthesis of l-dendroketose derivatives

The 3,4-O- and 1,2:3,4-di-O-isopropylidene derivatives (7 and 8) of l-dendroketose [4-C-(hydroxymethyl)-l-glycero-pentulose] (1) have been synthesized stereo-specifically from 4-C-(hydroxymethyl)-1,2:3,4-di-O-isopropylidene-l-erythro-pentitol (2).     

Stereosensitive formation and interconversion of sulfur-bridged cobalt(III)-mercury(II) polynuclear complexes with d- and/or l-penicillaminates

The reaction of trans(N)-[Co(d-pen)₂]⁻ (pen = penicillaminate) with HgCl₂ or HgBr₂ in the molar ratios of 1:1 gave the sulfur-bridged heterodinuclear complex, [HgX(OH₂){Co(d-pen)₂}] (X = Cl (1a) or Br (1b)). A similar reaction in the ratio of 2:1 produced the trinuclear complex, [Hg{Co(d-pen)₂}₂] (1c). The enantiomers of 1a and 1c, [HgCl(OH₂){Co(l-pen)₂}] (1a′) and [Hg{Co(l-pen)₂}₂] (1c′), were also obtained by using trans(N)-[Co(l-pen)₂]⁻ instead of trans(N)-[Co(d-pen)₂]⁻. Further, the reaction of cis · cis · cis-[Co(d-pen)(l-pen)]⁻ with HgCl₂ in the molar ratio of 1:1 resulted in the formation of [HgCl(OH₂){Co(d-pen)(l-pen)}] (2a). During the formations of the above six complexes, 1a, 1b, 1c, 1a′, 1c′, and 2a, the octahedral Co(III) units retain their configurations. On the other hand, the reaction of cis · cis · cis-[Co(d-pen)(l-pen)]⁻ with HgCl₂ in the molar ratio of 2:1 gave not [Hg{Co(d-pen)(l-pen}₂] but [Hg{Co(d-pen)₂}{Co(l-pen)₂}] (2c), accompanied by the ligand-exchange on the terminal Co(III) units. The X-ray crystal structural analyses show that the central Hg(II) atom in 1c takes a considerably distorted tetrahedral geometry, whereas that in 2c is of an ideal tetrahedron. The interconversion between the complexes is also examined. The electronic absorption, CD, and NMR spectral behavior of the complexes is discussed in relation to the crystal structures of 1c and 2c.     

Preparation of some acylated D-arabino-hexopyranosuloses and 4-deoxy-D-glycero-hex-3-enopyranosuloses

Oxidation of 1,3,4,6-tetra-O-benzoyl-α- and β-D-glucopyranose gave the tetra-O-benzoyl-α- and -β-D-arabino-hexopyranosuloses (3α and β), from which benzoic acid was readily eliminated to give the anomeric tri-O-benzoyl-4-deoxy-D-glycero-hex-3-enopyranosuloses (4α and β). The anomeric 1-O-acetyl-tri-O-benzoyl-D-arabino-hexopyranosuloses (7α and β) were obtained as very unstable syrups which readily lost benzoic acid. Treatment of tetra-O-benzoyl-2-O-benzyl-D-glucopyranose (1) with hydrogen bromide gave 3,4,6-tri-O-benzoyl-α-D-glucopyranosyl bromide (5) in one step.     

Silk fibroin model,poly(l-alanylglycyl-l-alanylglycyl-l-alanylglycyl-l-seryglycine)

l-Alanylglycyl-l-alanylglycyl-l-alanylglycyl-l-serylglycine and its pentachlorophenyl ester methanesulphonate have been synthesized as monomers for the preparation of silk fibroin model polypeptide. The former octapeptide was polymerized with diphenylphosphorylazide (DPPA) and triethylamine in DMSO or in HMPA—pyridine, and the latter octapeptide pentachlorophenylester was polymerized by adding triethylamine in DMSO to give poly(l-alanylglycyl-l-alanylglycyl-l-alanylglycyl-l-serylglycine). This sequential polypeptide gave a similar i.r. pattern to the crystalline part of Bombyx mori silk fibroin, which indicated antiparallel β-conformation. Dialysis of the solution of this polymer in 60%, aqueous LiBr against water gave mainly the polymer of α-form. O.r.d. measurements suggest that this polypeptide exists as a random structure in dichloroacetic acid on in 60% aqueous LiBr.     

Non-pyridine nucleotide dependent l-(+)-glutamate oxidoreductase in Azotobacter vinelandii

l-(+)-Glutamate oxidation that is non-pyridine nucleotide dependent is readily carried out by a membrane-bound enzyme in Azotobacter vinelandii strain O. Enzyme activity concentrates in a membranous fraction that is associated with the Azotobacter electron transport system. This l-glutamate oxidation is not dependent on externally added NAD⁺, NADP⁺, FAD, or FMN for activity. O₂, phenazine methosulfate and ferricyanide all served as relatively good electron acceptors for this reaction; while cytochrome c and nitrotetrazolium blue function poorly in this capacity. Paper chromatographic analyses revealed that the 2,4-dinitrophenylhydrazine derivative formed from the enzymatic oxidation of l-glutamate was α-ketoglutarate, while microdiffusion studies indicated that ammonia was also a key end product. These findings suggest that the overall reaction is an oxidative deamination. Ammonia formation was found to be stoichiometric with the amount of oxygen consumed (2 : 1 respectively, on a molar basis). The oxidation of glutamate was limited to the l-(+)-enantiomer indicating that this reaction is not the generalized type carried out by the l-amino acid oxidase. This oxidoreductase is functionally related to the Azotobacter electron transport system: (a) the activity concentrates almost exclusively in the electron transport fraction; (b) the l-glutamate oxidase activity is markedly sensitive to electron transport inhibitors, i.e. 2-n-heptyl-4-hydroxyquinoline-N-oxide, cyanide, and 4,4,4-trifluoro-1-(2-thienyl)-1,3-butanedione; and (c) spectral studies on the Azotobacter R₃ fraction revealed that a substantial amount of the flavoprotein (non-heme iron) and cytochrome (a₂, a₁, b₁, c₄ and c₅) are reduced by the addition of l-glutamate.     

Preparation of NG-monoethyl-l-arginine

N^G-Monoethyl-l-arginine, a putative in vivo product after administration of the potent hepatocarcinogen l-ethionine to rats, has been chemically synthesized by coupling N-ethyl, S-methylthiopseudouronium iodide with α-amino-blocked l-ornithine. The structure of the compound as N^G-monoethyl-l-arginine was confirmed by ¹³C NMR. Its elution time on an automatic amino acid analyzer, R_f values using thin-layer chromatography, and isoelectric point have been compared with those of N^G-monomethyl-l-arginine.     

An application of the palladium-catalyzed,allylic amination of unsaturated sugars: a new synthesis of d-forosamine

Methyl 4-O-benzoyl-6-bromo-6-deoxy-α-d-glucopyranoside, obtainable from methyl 4,6-O-benzylidene-α-d-glucopyranoside (1), was converted into the 2,3-unsaturated 4-benzoate (3) by application of the triiodoimidazole method. Debenzoylation of 3, followed by acetylation, afforded crystalline methyl 4-O-acetyl-6-bromo-2,3,6-trideoxy-α-d-erythro-hex-2-enopyranoside (5). Treatment of 5 with benzylmethylamine under conditions of palladium-catalyzed, allylic substitution gave a separable mixture of the corresponding 4-(N-benzyl)methylamino-6-bromo-2-enoside (37%) and the 4,6-di-[(N-benzyl)methylamino]-2-enoside (55%). Debromination of 5 with lithium triethylborohydride, proceeding with simultaneous deacetylation, readily yielded methyl 2,3,6-trideoxy-α-d-erythro-hex-2-enopyranoside (8). The 4-acetate of 8 (obtained by reacetylation), and also its 4-benzoate (prepared by a different synthetic route), furnished high yields (～80%) of methyl 4-[(N-benzyl)-methylamino]-2,3,4,6-tetradeoxy-α-d-erythro-hex-2-enopyranoside (13) upon palladium-catalyzed animation with benzylmethylamine. Catalytic hydrogenation of 13 effected saturation of the alkenic double bond and removal of the N-benzyl group, to afford methyl 2,3,4,6-tetradeoxy-4-methylamino-α-d-erythro-hexopyranoside, which was subsequently N-methylated with formaldehyde and sodium borohydride, to give its N,N-dimethyl analog, methyl α-d-forosaminide (15). The overall yield of 15 from 1 was 24%. Hydrolysis of 15 to the free sugar has been described previously.