Synthesis of 4,5-dideoxy-4-C-[(R,S)-phenylphosphinyl]-d-ribo- and l-lyxo-furanose and their 1,2,3-triacetates

2,3-O-Isopropylidene-d-ribose diethyl dithioacetal, prepared from d-ribose, was converted in three steps into the corresponding dimethyl acetal, which was monotosylated at O-5, and the ester oxidized at C-4 with pyridinium chlorochromate; addition of methyl phenylphosphinate to the resulting pentos-4-ulose derivative then provided (4R,S)-4,5-anhydro-2,3-O-isopropylidene-4-C-[(R,S)-(methoxy)phenylphosphinyl]-d-erythro-pentose dimethyl acetal. Hydrogenation of this compound in the presence of Raney Ni, followed by reduction with SDMA, hydrolysis, and acetylation, yielded the title compounds (seven kinds), the structures of which were established on the basis of their 400-MHz, ¹H-n.m.r. and mass spectra. A general dependence of the ²J_PH and ³J_PH values on the OPCH and PCCH dihedral angles provided an effective method for the assignment of the configurations and conformations of these 4-deoxy-4-phosphinyl-pentofuranoses.     

Synthesis of 3-O Substituted D-Mannoses

1,2,4,6-Tetra-O-acetyl-3-O-benzyl-α-D-mannopyranose (7) was obtained in good yield from 3,4,6-tri-O-benzyl-1,2-O-(1-methoxyethylidene)-β-D-mannopyranose (1) by acetolysis. Hydrogenolysis of 7 afforded 1,2,4,6-tetra-O-acetyl-α-D-mannopyranose which is a versatile intermediate for the preparation of other 3-O-substituted D-mannoses, such as 3-O-methyl-D-mannose and 3-O-α-D-mannopyranosyl-D-mannose. 3,4-Di-O-methyl-D-mannose was readily prepared from 1,2,6-tri-O-acetyl-3,4-di-O-benzyl-α-D-mannopyranose, which was also obtained from 1 by controlled acetolysis.     

Synthesis of aminocyclitols from l-quinic acid

A variety of 1,3-diamino and 1,4-diaminocyclitols, manoaminocyclitols, and triaminocyclohexanol have been synthesized starting with the chiral ketone intermediate, 2, derived from l-quinic acid. Reduction of 2 with lithium borohydride afforded two epimeric diols (4 and 5), both of which were transformed by straight-forward but distinctly different chemical procedures into potentially useful aglycons for preparing novel tupes of bioactive, aminocyclitol glycoside antibiotics. The disposition of the substituents at C-1, C-3, C-4, and C-5 in 19 and 37 is identical with that present in the 2-deoxystreptamine nucleus in the naturally occurring antibiotics     

Quantitation of l-glycero-d-manno-heptose and 3-deoxy-d-manno-octulosonic acid in rough core lipopolysaccharides by partition chromatography

A partition chromatographic procedure utilizing a cationic exchange resin column in the Li⁺ form and 90% ethanol as the mobile phase was employed to quantify 3-deoxy-d-manno-octulosonic acid (KDO) and l-glycero-d-manno-heptose in the lipopolysaccharides (LPS) of R_e and R_dP^? rough mutants of Salmonella minnesota. In a standard mixture of monosaccharides, KDO eluted shortly after the void volume and heptose eluted after the neutral hexoses. Mild acid treatment of either the R_e or R_dP^? LPS with 0.16 n methanesulfonic acid in the presence of Dowex 50-X8 resin (H⁺ form) released more than 80% of the KDO residues within 15 min. The heptose of the R_dP^? LPS, first detected after 90 min of hydrolysis, increased gradually to a maximum level at 12 h. A secondary gradual increase in KDO became apparent during the heptose release. The weight contents of these two monosaccharides based upon aheir maximum values detected during hydrolysis were 20.3 ± 0.6% KDO, for the R_e LPS, and 13.8 ± 0.4% KDO and 12.0 ± 0.4% heptose, for the R_dP^? LPS. The relationship between the kinetics of release of KDO and heptose and the nature of the linkages involving these two monosaccharides are discussed.     

A kinetic analysis of d-xylose transport in rhodotorula glutinis

The kinetics of D-xylose transport were studied in Rhodotorula glutinis. Analysis of the saturation isotherm revealed the presence of at least two carriers for d-xylose in the Rhodotorula plasma membrane. These two carriers exhibited Km values differing by more than an order of magnitude. The low K_m carrier was repressed in rapidly growing cells and depressed by starvation of the cells.Several hexoses were observed to inhibit d-xylose transport. In the studies reported here, the inhibitions produced by d-galactose and 2-deoxy-d-glucose were examined in some detail in order to define the interactions of these sugars with the d-xylose carriers. 2-Deoxy-d-glucose competitively inhibited both of the d-xylose carriers. In contrast, only the low-K_m carrier was competitively inhibited by d-galactose.     

An enzymic synthesis of purine d-Arabinonucleosides

A method is described for the synthesis of purine d-arabinonucleosides that uses purine bases and 2,2′-anhydro-(1-β-d-arabinofuranosylcytosine), AraC-an, as the starting materials. AraC-an was chosen as the precursor to the d-arabinosyl donor, because it is more readily available than any of the products that may be sequentially derived from it, namely, 1-β-d-arabinofuranosylcytosine (AraC), 1-β-d-arabinofuranosyluracil (AraU), and α-d-arabinofuranosyl-1-phosphate (Araf 1-P), a d-arabinofuranosyl donor. Four reactions were involved in the overall process; (a) AraC-an was nonenzymically hydrolyzed at alkaline pH to AraC which was then (b) deaminated by cytidine deaminase to AraU, a nucleoside, (c) phosphorylyzed by uridine phosphorylase to Araf 1-P, and (d) this ester caused to react with a purine base to afford a purine d-arabinonucleoside, the reaction being catalyzed by purine nucleoside phosphorylase. All four reactions occurred in situ, the first and second being performed sequentially, whereas the third and fourth were combined in a single step. The three enzyme catalysts were purified from Escherichia coli. The efficiency of the method is exemplified by the synthesis of the d-arabinonucleosides of 2,6-diaminopurine and adenine; the overall yields, based on AraC-an, were 60 and 80%, respectively.     

The structure of d-threo-2,5-hexodiulosonic acid and derivatives in solution

The structure of d-threo-2,5-hexodiulosonic acid (1) and various derivatives in solution was determined by ¹³C-n.m.r. spectroscopy to be a hydrated, pyranose form. The structures of the methyl ester of 1 and of its 5-(dimethyl acetal) were confirmed by chemical means and by X-ray structure analysis.     

The bioadhesive of Mytilus byssus: A protein containing L-DOPA

L-DOPA was identified in hydrolysates of $\text{Mytilus}$ byssal adhesive discs and is present at about $\text{10}\text{res}\text{1000}$. The compound was isolated and purified by ion exchange on cellulose phosphate and Biogel P-2 gel filtration. Identity with standard DOPA was demonstrated using thin-layer chromatography, the effect of pH on UV absorbance, fluorescence spectrophotometry, amino acid analysis, and the preparation of ethylenediamine derivatives. Contrary to earlier reports, dityrosine was not detected. A sodium dodecylsulfate-insoluble protein containing $\text{48}\text{res}\text{1000}$ of DOPA was isolated from the gland that secretes the disc adhesive. This protein is presumed to be a precursor of the adhesive.     

Aromatic and heterocyclic 1-C-substituted derivatives of 1,5-anhydro-d-glucitol

Reaction of anomeric 1-O-acyl and 1-halide derivatives of 2,3,4,6-tetra-O-benzyl-d-glucose with anisole, ferrocene, thiophene, furan, and 1,3,5-tri-methoxybenzene in the presence of a Lewis acid gives the corresponding C-β-d-glucopyranosyl derivatives.     

Gonadotropin releasing hormone (GnRH) is not degraded by intact pituitary tissue in vitro

The possible degradation of GnRH by intact pituitary tissue was investigated. Trypsin-, collagenase- and mechanically dispersed pituitary cells in culture and fresh pituitaries cut into eight segments were incubated with specifically tritiated GnRH or unlabeled GnRH which were detected by HPLC and RIA in incubation media. Degradation of GnRH could not be detected by either method during incubations with any of the pituitary cell cultures or fresh tissue. The results suggest that pituitary degradation is not involved in the regulation of circulating levels of GnRH.     

An oxygen-18 tracer investigation of the mechanism of myo-inositol oxygenase

In the conversion of $\text{myo}$-inositol to D-glucuronic acid catalyzed by $\text{myo}$-inositol oxygenase only one atom of ¹⁸O from ¹⁸O₂ is incorporated into the product, and it is found exclusively in the carboxyl group. Control experiments indicate that under the reaction conditions no exchange of solvent oxygens with D-glucuronate occurs. To avoid exchange during isolation and analysis the oxygenase product was enzymically reduced to L-gulonate and isolated in that form. The results eliminate one possible mechanism for the oxygenase reaction, but are consistent with two others which seem chemically reasonable.     

Amino acid sequence homologies in alfa-sarcin, restrictocin and mitogillin

The NH₂-terminal amino acid sequence of the three anti-tumor proteins, alfa-sarcin, mitogillin and restrictocine, has been determined for 20 cycles by automated sequencing procedure. A high degree of sequence homology was observed in this region of the molecule. In addition, extensive sequence homology, ranging from 65 to 100% was found in three other carboxymethylcysteine-containing peptides isolated and sequenced from each molecule.     

Sequence homology of bacterial and mitochondrial cytochrome c oxidases: Partial sequence data of cytochrome c oxidase from Paracoccus denitrificans

The aerobic electron transport chain of $\text{Paracoccus denitrificans}$ is very similar to that of mitochondria. It has therefore been suggested that this bacterium might be evolutionarily related to mitochondria. The two subunits (Mr 45.000 and 28.000) of the $\text{Paracoccus}$ cytochrome c oxidase were isolated and partially sequenced. The sequences were found to be surprisingly homologous to sequences of the subunits I and II of mitochondrial cytochrome c oxidases. The data provide a molecular basis for the symbiotic origin of mitochondria and strongly support the notion that in eucaryotic oxidases subunits I and/or II carry the redox centers, heme and copper.     

Primary structure of heat-stable enterotoxin produced by Yersinia enterocolitica

A heat-stable enterotoxin was isolated and purified from the culture supernatant of $\text{Yersinia enterocolitica}$ by reversed-phase high-performance liquid chromatography. The amino acid sequence of the purified toxin was determined to be as follows: Gln-Ala-Cys(X)-Asp-Pro-Pro-Ser-Pro-Pro-Ala-Glu-Val-Ser-Ser-Asp-Trp-Asp-Cys-Cys-Asp-Val-Cys-Cys-Asn-Pro-Ala-Cys-Ala-Gly-Cys (X: not determined). The C-terminal sequence containing 6 half-cystine residues was highly homologous to that of heat-stable enterotoxin of enterotoxigenic $\text{Escherichia coli.}$     

Identification of a new glutathione S-transferase from rat liver cytosol

A new glutathione $\text{S}$-transferase has been purified to homogeneity from 105,000 × g supernatant of Sprague-Dawley rat liver homogenates. The purified enzyme exhibited specific activities of approximately 1.8, and 0.12 μmoles. min^?1. mg^?1 toward 1-chloro 2,4-dinitrobenzene and cumene hydroperoxide respectively. The SDS gel electrophoresis data on subunit composition revealed that the new transferase is composed of two subunits with an identical M_r of 24,400 (Y_α Family). Our $\text{in}\text{vitro}$ translation experiments with rat liver poly(A) RNAs and substrate specificity data suggest that this subunit is different from the previously reported Ya, Yb and Yc subunits of rat liver glutathione $\text{S}$-transferases. Comparatively, the new isozyme showed significant activity toward 1,2 epoxy-3-(P-nitrophenoxy)-propane, ethacrynic acid and P-nitrophenyl acetate, 0.4, 0.34 and 0.18 μ moles. min^?1. mg^?1 respectively.     

Structure of the D-fructan isolated from garlic (Allium sativum) bulbs

Extraction of defatted garlic bulbs with hot water yielded a mixture of polysaccharides containing pectic acid, a D-galactan, and a fructan component. The pectic acid was partially removed as calcium pectate, and the galactan-enriched portion was separated by fractional precipitation with alcohol; on concentration and several fractionations, the supernatant liquor furnished the fructan component, which contained fructose (94.4%) and glucose (4.3 %). Methanolysis and hydrolysis of the permethylated fructan gave (a) 1,3,4,6-tetra-O-methyl-D-fructose, (b) 2,3,4,6-tetra-O-methyl-D-glucose, (c) 2,4,6-tri-O-methyl-D-glucose, and (d) 3.4,6-tri-O-methyl-D-fructose in the ratio (a + b): (d) = 1:20.3. On periodate oxidation, the fructan reduced one molar equivalent of the oxidant per hexosyl residue, and liberated one molar equivalent of formic acid per 51 hexosyl residues. On Smith degradation, the major product was glycerol, and ～2 % of the glucose survived. From these results, and from the fact that the fructan is hydrolyzed by β-D-fructofuranosidase, a linear, inulin-type of structure is suggested for it.     

Proton and carbon-13 nuclear magnetic resonance studies on methyl (methyl d-galactosid)uronates and their per-O-acetyl derivatives

The ¹H- and ¹³C-n.m.r. spectra of the anomeric methyl (methyl d-galactosid)uronates, as well as the ¹H-n.m.r. spectra of their acetyl derivatives, were analyzed. The spectra of the unacetylated d-galactopyranosiduronates showed good correlation with those of the corresponding anomeric d-galactopyranuronic acids and their methyl esters, and with those of the anomeric methyl d-galactopyranosides. From the values of the chemical shifts and coupling constants, it was concluded that the anomeric methyl (methyl d-galactopyranosid)uronates and their corresponding peracetates are in the ⁴C₁(d) conformation. The chemical shifts in the ¹³C-n.m.r. spectra show good correlation with those of the methyl d-galactosides. The signals of the furanose derivatives appear at fields lower than those of the corresponding pyranose compounds.     

l-Myoinositol-1-phosphate synthase from Neurospora crassa: Purification to homogeneity and partial characterization

The purification procedure of milligram quantities of stable myoinositol-1-phosphate synthase (EC 5.5.1.4) from Neurospora crassa is reported. The procedure includes: (a) (NH₄)₂SO₄ and protamine sulfate precipitations, (b) gel filtration in Ultrogel AcA-34 (LKB), (c) DEAE-cellulose chromatography, (d) AH-Sepharose 4B chromatography, and (e) calcium phosphate gel chromatography. The enzyme is considered pure according to the following criteria: (a) gel filtration, (b) sucrose density gradient centrifugation, (c) polyacrylamide gel electrophoresis, and (d) isoelectric focusing technique. The molecular weight estimated by gel filtration chromatography and sucrose density gradient centrifugation is 345,000. The subunit molecular weight is 59,000. The active enzyme seems to posses an hexameric structure. The isoelectric point estimated for the pure enzyme is 5.2. The enzyme was optimally stimulated by 10 mm (NH₄)₂SO₄ and by 50 mm KCl, while NaCl had a minor inhibitory effect at higher concentrations. The divalent cations Mg²⁺ and Mn²⁺ were inhibitory only at nonphysiological concentrations. The enzymatic activity after the salt fractionation steps was about 33% NAD⁺ independent; but with purification the resulting homogeneous enzyme showed less than 5% NAD⁺-independent activity.     

The formation of N-alkylprotoporphyrin IX and destruction of cytochrome P-450 in the liver of rats after treatment with 3,5-diethoxycarbonyl-1,4-dihydrocollidine and its 4-ethyl analog

The effects of two porphyrogenic agents, 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) and 3,5-diethoxycarbonyl-2,6-dimethyl-4-ethyl-1,4-dihydropyridine (DDEP), have been studied in rats. The administration of these compounds leads to the formation and accumulation in the liver of N-methylprotoporphyrin IX and N-ethylprotoporphyrin IX, respectively. In each case, the alkyl group of the porphyrin is derived from the 4-alkyl group of the porphyrogenic chemical. Each N-alkylporphyrin is a potent inhibitor of protoheme ferrolyase (EC 4.99.1.1) (ferrochelatase) activity. N-Methylprotoporphyrin IX is somewhat more potent than N-ethylprotoporphyrin IX as an inhibitor of ferrochelatase activity in vitro. However, more N-ethylprotoporphyrin IX accumulates in rat liver than does the N-methyl analog. Since alkylporphyrins are formed during the catabolism of heme (or hemoprotein), the effects of DDC and DDEP on hepatic microsomal cytochrome P-450 were also studied. Whereas DDC treatment led to only a slight decrease in cytochrome P-450 levels (25%), DDEP administration led to a marked decrease (75%) in the total cytochrome P-450 level. In phenobarbital- and 3-methylcholanthrene-treated rats, DDC administration did not alter the hepatic microsomal cytochrome P-450 content, while administration of DDEP to either phenobarbital-treated or 3-methylcholanthrene-treated rats led to marked reduction of levels in cytochrome P-450. Although the N-methylprotoporphyrin IX level was not increased following DDC administration to either phenobarbital- or 3-methylcholanthrene-treated rats, there was a marked increase in N-ethylprotoporphyrin IX accumulation in both phenobarbital- and 3-methylcholanthrene-treated rats after the administration of DDEP. These results suggest that DDC and DDEP react with different forms of rat hepatic microsomal cytochrome P-450.