Stereochemistry of leukotriene C-1

Leukotriene C-1, a “Slow Reacting Substance” (SRS), has been shown to possess the molecular Structure depicted by V (5(S)-hydroxy-6(R)-$\text{S}$-glutathionyl-7,9-$\text{trans}$-11,14-$\text{cis}$-eicosatetraenoic acid) by its identity with a totally synthetic product of known structure and stereochemistry.     

Bromine oxidation of 1,2-O-isopropylidene-α-d-Glucofuranose and sucrose

Sucrose and $\text{1,2-O-}\text{isopropylidene-α-}\text{d}\text{-glucofuranose}$ (1) were oxidised with bromine in aqueous solution at pH 7 and room temperature. The resulting keto derivatives were converted into their more-stable O-methyloximes, which were characterised by spectroscopic and chromatographic methods. Oxidation of 1 occurred at C-3 and C-5, with a preference for C-5. In the sucrose derivatives isolated after oxidation, those having a keto group in the glucopyranosyl moiety preponderated. The axial fructofuranosyl aglycon protects position 3 in the glucopyranosyl group and oxidation occurs only at C-2 and C-4. Small amounts of sucrose oxidised at C-3 in the fructofuranosyl moiety were also found.     

Enzymatic formation of (20R, 22R)-20,22-dihydroxycholesterol from cholesterol and a mixture of 16O2 and 18O2: random incorporation of oxygen atoms

[4-¹⁴C]Cholesterol was incubated with an adrenocortical preparation in the presence of ¹⁶O₂ and ¹⁸O₂ devoid of significant ¹⁶O¹⁸O. Isolated (20R,22R)-20,22-dihydroxycholesterol was converted to a trimethylsilyl derivative and analyzed by gas chromatography - mass spectrometry to determine the isotope distribution of the oxygen atoms at C-20 and C-22. The ions of $\text{m}\text{e}$ 289, 291, and 293 (comprising the C₈ C-20 to C-27 side-chain and containing, respectively, ¹⁶O₂, ¹⁶O¹⁸O, and ¹⁸O₂) exhibited a binomial distribution indicating that the oxygen atoms of the vicinal glycol were drawn at random from the atomic pool of the oxygen molecules. If both side-chain hydroxyl groups had originated from the atoms of the same oxygen molecule, the ion of $\text{m}\text{e}$ 291 would have been absent.     

Deamination of 2-amino-2-deoxyhexitols and of their per-O-methylated derivatives with nitrous acid

The products of nitrous acid deamination of per-O-methylated 2-amino-2-deoxy-d-glucitol and $\text{2-}\text{amino}\text{-2-}\text{deoxy}\text{-3-O-β-}\text{d}\text{-galactopyranosyl-}\text{d}\text{-glucitol}$ and its per-O-methylated derivative have been characterized by g.l.c.—mass spectrometry after treatment with sodium borodeuteride and further substitution by acetylation, methylation, or (trideuteriomethyl)ation. The results confirm that the most important reaction pathway (1) involves a 1 → 2-hydride shift to give $\text{2-}\text{deoxy-}\text{d}\text{-arabino-}\text{hexoses}$, but that significant side-reactions include (2) solvolytic displacement at C-2, (3) a 3 → 2-hydride shift, to give $\text{2-}\text{deoxy-}\text{d}\text{-erythro-3-}\text{hexuloses}$, and (4) a C-4→C-2 migration to give $\text{2-}\text{deoxy}\text{-2-C-}\text{(hydroxymethyl)-}\text{d}\text{-ribose}$ and -d-arabinose. Reactions (3) and (4) result in elimination of the original 3-O-substituents, with the exposure of new reducing groups, from oligosaccharides terminated by 3-O-substituted 2-amino-2-deoxyhexitols.     

An intramolecular C-2 → C-1 hydrogen transfer during the acid-catalyzed conversion of d-xylose to d-threo-pentulose (d-xylulose)

Aldose-ketose isomerases are known to catalyze a partial and sometimes complete intramolecular hydrogen transfer between C-1 of the ketose and C-2 of the aldose. It was recently shown (Feather, M. S., and Harris, D. W. (1975) J. Amer. Chem. Soc.97, 178–181) that the same type of transfer occurs during the acid-catalyzed interconversion of d-fructose, d-glucose, and d-mannose. A similar transfer is demonstrated herein for the conversion of d-xylose to d-xylulose in acid solution. d-[2-³H]xylose was isomerized in aqueous sulfuric acid and the resulting d-[³H]xylulose was isolated in 6% yield. The ketose had 18.3% the activity of the starting aldose. Chemical degradation showed that all the carbon-bound tritium of the d-[³H]xylulose was located at C-1, thus indicating a C-2 → C-1 intramolecular hydrogen transfer. During the reaction, less than 1.2% of the total radiochemical activity was found in the solvent, and, the unreacted d-[2-³H]xylose was recovered, having an activity nearly the same as the starting material. The differences in activity, therefore, of the d-[2-³H]xylose and the d-[1-³H]xylulose are due to an isotope effect ($\text{K}{}_{\mathrm{<mtext>H</mtext>}}\text{K}{}_{\mathrm{<mtext>T</mtext>}}$) which is indicated to be 5.4. The data are discussed in terms of currently accepted models for isomerase mechanisms.     

Highly active analogs of 1alpha,25-dihydroxyvitamin D(3) that resist metabolism through C-24 oxidation and C-3 epimerization pathways

The secosteroid hormone 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] is metabolized in its target tissues through modifications of both the side chain and the A-ring. The C-24 oxidation pathway, the main side chain modification pathway is initiated by hydroxylation at C-24 of the side chain and leads to the formation of the end product, calcitroic acid. The C-23 and C-26 oxidation pathways, the minor side chain modification pathways are initiated by hydroxylations at C-23 and C-26 of the side chain and lead to the formation of the end product, calcitriol lactone. The C-3 epimerization pathway, the newly discovered A-ring modification pathway is initiated by epimerization of the hydroxyl group at C-3 of the A-ring to form 1alpha,25(OH)(2)-3-epi-D(3). A rational design for the synthesis of potent analogs of 1alpha,25(OH)(2)D(3) is developed based on the knowledge of the various metabolic pathways of 1alpha,25(OH)(2)D(3). Structural modifications around the C-20 position, such as C-20 epimerization or introduction of the 16-double bond affect the configuration of the side chain. This results in the arrest of the C-24 hydroxylation initiated cascade of side chain modifications at the C-24 oxo stage, thus producing the stable C-24 oxo metabolites which are as active as their parent analogs. To prevent C-23 and C-24 hydroxylations, cis or trans double bonds, or a triple bond are incorporated in between C-23 and C-24. To prevent C-26 hydroxylation, the hydrogens on these carbons are replaced with fluorines. Furthermore, testing the metabolic fate of the various analogs with modifications of the A-ring, it was found that the rate of C-3 epimerization of 5,6-trans or 19-nor analogs is decreased to a significant extent. Assembly of all these protective structural modifications in single molecules has then produced the most active vitamin D(3) analogs 1alpha,25(OH)(2)-16,23-E-diene-26,27-hexafluoro-19-nor-D(3) (Ro 25-9022), 1alpha,25(OH)(2)-16,23-Z-diene-26,27-hexafluoro-19-nor-D(3) (Ro 26-2198), and 1alpha,25(OH)(2)-16-ene-23-yne-26,27-hexafluoro-19-nor-D(3) (Ro 25-6760), as indicated by their antiproliferative activities.     

The synthesis of methyl 4-O-(4-O-α-d-galactopyranosyl-β-d-galactopyranosyl)-β-d-glucopyranoside: The methyl β-glycoside of the trisaccharide related to Fabry's disease

As part of a program to synthesize the ceramide trisaccharide (1) related to Fabry's disease, methyl 4-O-(4-O-α-$\text{d}$-galactopyranosyl-β-$\text{d}$-galactopyranosyl)-β-$\text{d}$-glucopyranoside (12) was prepared. Methyl β-lactoside (2) was converted into methyl 4-O-(4,6-O-benzylidene-β-$\text{d}$-galactopyranosyl)-β-$\text{d}$-glucopyranoside (4). Methyl 2,3,6-tri-O-benzoyl-4-O-(2,3,6-tri-O-benzoyl-β-$\text{d}$-galactopyranosyl)-β-$\text{d}$-glucopyranoside (7) was synthesized from 4 through the intermediates methyl 2,3,6-tri-O-benzoyl-4-O-(4,6-O-benzylidene-2,3-di-O-benzoyl-β-$\text{d}$-galactopyranosyl)-β-$\text{d}$-glucopyranoside (5) and methyl 2,3,6-tri-O-benzoyl-4-O-(2,3-di-O-benzoyl-β-$\text{d}$-galactopyranosyl)-β-$\text{d}$-glucopyranoside (6). The halide-catalyzed condensation of 7 with 2,3,4,6-tetra-O-benzyl-$\text{d}$-galactopyranosyl bromide (8) gave methyl 2,3,6-tri-O-benzoyl-4-O-[2,3,6-tri-O-benzoyl-4-O-(2,3,4,6-tetra-O-benzyl-α-$\text{d}$-galactopyranosyl)- β-$\text{d}$-galactopyranosyl]-β-$\text{d}$-glucopyranoside (10). Stepwise deprotection of 10 led to 12, the methyl β-glycoside of the trisaccharide related to Fabry's disease.     

1H NMR spectra of (25s)-steroidal sapogenins. Reassignments of the C-20 and C-25 methyl signals

¹H NMR spectra of ten (25$\text{S}$)-steroidal sapogenins (I–X) and their acetates (Ia–Xa) in deuteriochloroform and pentadeuterio-pyridine were examined. The C-20 and C-25 methyl signal ass?ignments were revised. Some other features including benzene-induced shifts are described.     

11-Trans leukotriene C: A naturally occurring slow reacting substance

A slow reacting substance, produced by murine mastocytoma cells, has been shown to have the structure 5(S)-hydroxy-6(R)-$\text{S}$-glutathionyl-7,9,11-$\text{trans}\text{-14-}\text{cis}\text{-eicosatetraenoic acid (11-}\text{trans}$ leukotriene C, previously referred to as leukotriene C-2) by ultraviolet spectroscopy, amino acid analyses, lipoxygenase conversion and comparisions with a synthetic compound of known structure and stereochemistry.     

Production of a hybrid 16-membered macrolide antibiotic by genetic engineering of Micromonospora sp. TPMA0041

Some polyketide-derived bioactive compounds contain sugars attached to the aglycone core, and these sugars often enhance or impart specific biological activity to the molecule. Mycinamicin II, a 16-member macrolide antibiotic produced by Micromonospora griseorubida A11725, contains a branched lactone and two different deoxyhexose sugars, d-desosamine and d-mycinose, at the C-5 and C-21 positions, respectively. We previously engineered an expression plasmid pSETmycinose containing the d-mycinose biosynthesis genes from M. griseorubida A11725. This plasmid was introduced into Micromonospora sp. FERM BP-1076 cells, which produce the 16-membered macrolide antibiotic izenamicin. The resulting engineered strain TPMA0041 produced 23-O-mycinosyl-20-deoxy-izenamicin B₁ and 22-O-mycinosyl-izenamicin B₂. 23-O-mycinosyl-20-deoxy-izenamicin B₁ has been produced by the engineered strain M. rosaria TPMA0001 containing pSETmycinose as 23-O-mycinosyl-20-deoxo-20-dihydro-12,13-deepoxyrosamicin (=IZI) in our recent study, and 22-O-mycinosyl-izenamicin B₂ has previously been synthesized as a macrolide antibiotic TMC-016 with strong antibacterial activity. The production of 22-O-mycinosyl-izenamicin B₂ (=TMC-016) was increased when propionate, a precursor of methylmalonyl-CoA, was added to the culture broth.     

Intramolecular catalysis. VII: The nature of side chain shielding of the 12alpha-hydroxyl group of steroids

A series of 12α-hydroxy steroids with varying side chains was prepared, and their 24-hour acetylation yields were compared, l2α-Hydroxy-5β-pregnan-20-one ($\text{lb}$) was prepared from 3α, 12α-diacetoxy-5β~pregnan-20-one ($\text{2}$) and also by side chain degradation of 12α-acetoxy-5β-cholanoic acid ($\text{5d}$). 21-Benzyl-5β-pregnan-12α-ol ($\text{1g}$) was synthesized by hydrogenation of the 21-benzylidine derivative of ketone $\text{1b}$. 23-Pheny1-5β-norcholan-12α-ol ($\text{1k}$) was obtained by the Grignard reaction of 2-phenyl-ethylmagnesium bromide and ketone $\text{1b}$, dehydration, hydrogenation and hydride reduction; a similar sequence produced 20-methyl-5β-pregnan-12α-ol ($\text{lm}$). The acetylation results (Table 11) imply that branching at C-20 may be more significant for 12α-hydroxyl reactivity than side chain length or type. An additional compound with an unbranched side chain, 21-nor-5β-cholan-12α-ol ($\text{14}$), was synthesized by a Grignard reaction on the 21-bromo intermediate $\text{11b}$. Acetylation rates determined by glc indicate (Table 111) That compounds with unbranched side chains have 12α-hydroxyl groups about ten times as reactive as their analogs with 20-methyl groups.     

Structural studies on the extracellular polysaccharide of the red alga, Porphyridium cruentum

Partial acid hydrolyzates of the extracellular polysaccharide from Porphyridiunm cruentum yield three disaccharides and two uronic acids. These constitute all of the uronic acid in the polymer. The novel disaccharides are 3-O-(α-$\text{D}$-glucopyranosyl- uronic acid)-$\text{L}$-galactose, 3-O-(2-O-methyl-ca-glucopyranosyluronic acid)-$\text{D}$- galactose, and 3-0-(2-0-methyl-a-$\text{D}$-glucopyranosyluronic acid)-$\text{D}$-glucose. The polyanion of high molecular weight contains $\text{D}$- and $\text{L}$-galactose, xylose, $\text{D}$-glucose, $\text{D}$-glucuronic acid and 2-O-methyl-D-glucuronic acid, and sulfate in molar ratio (relative to $\text{D}$-glucose) of 2.12:2.42:1.00:1.22:2.61. Preliminary periodate-oxidation studies suggest that the hexose and uronic acids are joined to other residues by ( 1→3) glycosidic linkages. About one-half of the xylose residues are (1→3)-linked.     

The stereospecificity of D-glucose-6-phosphate: 1L-myo-inositol-1-phosphate cycloaldolase on the hydrogen atoms at C-6

D-Glucose-6-phosphate: 1L-$\text{myo}$-Inositol-1-phosphate cycloaldolase from rat testis or mammary gland removed stereospecifically the pro-S hydrogen atom at C-6 from D-glucose-6-phosphate. The pro-R hydrogen at C-6 remained in the product, 1L-$\text{myo}$-Inositol-1-phosphate and evidence is given that it is the hydrogen at C-1 of 1L-$\text{myo}$-Inositol-1-phosphate. The possible mechanism of cyclization is discussed.     

Further characterization of the 2-deoxyecdysone C-2 hydroxylase from Locusta migratoria

Additional data are provided on the enzyme 2-deoxyecdysone C-2 hydroxylase which has been shown in a previous study (Kappler et al., 1986) to be a mitochondrial hydroxylase with some classical characteristics of a cytochrome P-450 monooxygenase but which appeared to be insensitive to CO. Using ¹⁸O₂, we have now demonstrated that molecular oxygen is directly incorporated into ecdysone during the process of C-2 hydroxylation. Neither cumene hydroperoxide nor linoleyl hydroperoxide could support C-2 hydroxylation. When the reaction was sustained by α-ketoglutarate, addition of cofactors like Fe²⁺, ascorbate and catalase caused only a slight increase of the enzymatic activity whereas the α-ketoglutarate-dependent hydroxylation was largely decreased in the presence of malonate; these data eliminate the possible existence of a dioxygenase mechanism for C-2 hydroxylation.The paper also provides inhibition kinetics which indicate that 2-deoxy-20-hydroxyecdysone, 2,22-bisdeoxyecdysone and 2,22,25-trideoxyecdysone are competitive inhibitors of the C-2 hydroxylase whereas the 3-epi isomer of 2-deoxyecdysone is a non-competitive inhibitor.     

The Origin of Carbons of Dihydrofuran Ring and C-6 in the Biosynthesis of Rotenone

For the investigation of rotenone biosynthesis, acetate-2-¹⁴C, mevalonic acid-2-¹⁴C lactone and methionine-methyl-¹⁴C were administered to Derris elliptica plants, respectively, and the distribution of carbon-14 in the labeled rotenone was determined by degradation. When mevalonic acid-2-¹⁴C lactone was incorporated into rotenone, the radioactivity was found equally in the carbons at both C-7′ and C-8′, indicating that these carbons are derived from the carbon-2 of mevalonic lactone. In the case of methionine-methyl-¹⁴C about 80% of the total radioactivity was found to enter two methoxyl groups. This result demonstrates that methionine is an efficient precursor of the methoxyl group. Furthermore, it is also suggested that methionine may be a precursor of the carbon at C-6.     

Synthesis of l-idofuranurono-6,3-lactone and its derivatives via hexodialdodifuranoses

1,2-O-Alkylidene-β-l-idofuranurono-6,3-lactones were obtained from the corresponding 5-O-toluene-p-sulphonyl-α-d-glucofuranurono-6,3-lac tones by a sequence involving lactone reduction, benzoylation of HO-6, inversion of configuration at C-5, deacylation, and lactol oxidation. Hydrogenolysis or methanolysis of 1,2-O- benzylidene-β-l-idofuranurono-6,3-lactone gave l-idofuranurono-6,3-lactone and a mixture of its methyl glycosides, respectively.     

Sterol biosynthesis: Establishment of the structure of 3β-p-bromobenzoyloxy-5α-cholest-8(14)-en-15β-ol

Previous studies have established that hydride reduction of 3β-benzoyloxy-5α-cholest-8(14)-en-15-one yields two epimers (at C-15) of 5α-cholest-8(14)-en-3β,15-diol which were designated as diol A and B. Efficient enzymatic conversion of both compounds to cholesterol was observed. To determine the absolute configuration of the 15-OH function in the two compounds, the 3β-p-bromobenzoyl ester of diol B was prepared from 3β-p-bromobenzoyloxy-5α-cholest-8(14)-en-15-one by reduction with sodium borohydride. Crystals of the derivative were found to belong to the space group P1, with unit cell parameters; $\text{a = 9.24}\text{A}\text{?}$, $\text{b = 12.61}\text{A}\text{?}$, $\text{c = 7.03}\text{A}\text{?}$, α = 93.05°, β = 100.27°, γ = 90.82°, and one molecule per unit cell. Least-squares refinement of the structure was carried out to final R value of 0.14. The configuration of the hydroxyl group at the 15 position of diol B has been determined to be β.     

Hydrogen bonding requirements for the insulin-sensitive sugar transport system of rat adipocytes

(1) The $\text{t}\text{1}\text{2}$ for 1.3 mM D-allose uptake and efflux in insulin-stimulated adipocytes is 1.7 ± 0.1 min. In the absence of insulin mediated uptake of D-allose is virtually eliminated and the uptake rate ($\text{t}\text{1}\text{2}\text{= 75.8 ± 4.99}\text{min}$) is near that calculated for nonmediated transport. The kinetic parameters for D-allose zero-trans uptake in insulin-treated cells are K_zt^oi = 271.3 ± 34.2 mM, V_zt^oi = 1.15 ± 0.12 mM · s^?1. (2) A kinetic analysis of the single-gate transporter (carrier) model interacting with two substrates (or substrate plus inhibitor) is presented. The analysis shows that the heteroexchange rates for two substrates interacting with the transporter are not unique and can be calculated from the kinetic parameters for each sugar acting alone with the transporter. This means that the equations for substrate analogue inhibition of the transport of a low affinity substrate such as D-allose can be simplified. It is shown that for the single gate transporter the K_i for a substrate analogue inhibitor should equal the equilibrium exchange K_m for this analogue. (3) Analogues substituted at C-1 show a fused pyranose ring is accepted by the transporter. 1-Deoxy-D-glucose is transported but has low affinity for the transporter. High affinity can be restored by replacing a fluorine in the β-position at C-1. The K_i for $\text{d}\text{-glucose}\text{= 8.62}\text{mM}$; the K_i for $\text{β-}\text{fluoro-}\text{d}\text{-glucose}\text{= 6.87}\text{mM}$. Replacing the ring oxygen also results in a marked reduction in affinity. The K_i for $\text{5-}\text{thio-}\text{d}\text{-glucose}\text{= 42.1}\text{mM}$. (4) A hydroxyl in the gluco configuration at C-2 is not required as 2-deoxy-d-galactose (K_i = 20.75 mM) has a slightly higher affinity than d-galactose (K_i = 24.49 mM). A hydroxyl in the manno configuration at C-2 interferes with transport as d-talose (K_i = 35.4 mM) has a lower affinity than d-galactose. (5) d-Allose (K_m = 271.3 mM) and 3-deoxy-d-glucose (K_i = 40.31 mM) have low affinity but high affinity is restored by substituting a fluorine in the gluco configuration at C-3. The K_i for $\text{3-}\text{fluoro-}\text{d}\text{-glucose}\text{= 7.97}\text{mM}$. (6) Analogues modified at C-4 and C-6 do not show large losses in affinity. However, 6-deoxy-d-glucose (K_i = 11.08 mM) has lower affinity than d-glucose and 6-deoxy-d-galactose K_i = 33.97 mM) has lower affinity than d-galactose. Fluorine substitution at C-6 of d-galactose restores high affinity. The K_i for $\text{6-}\text{fluoro-}\text{d}\text{-galactose}\text{= 6.67}\text{mM}$. Removal of the C-5 hydroxymethyl group results in a large affinity loss. The K_i$\text{d}\text{-xylose}\text{= 45.5}\text{mM}$. The K_i for $\text{l}\text{-arabinose}\text{= 49.69}\text{mM}$. (7) These results indicate that the important hydrogen bonding positions involved in sugar interaction with the insulin-stimulated adipocytes transporter are the ring oxygen, C-1 and C-3. There may be a weaker hydrogen bond to C-6. Sugar hydroxyls in non-gluco configurations may sterically hinder transport.     

Biosynthesis of chondroitin sulfate. Purification of UDP-D-xylose:core protein beta-D-xylosyltransferase by affinity chromatography

Silver carbonate on Celite (the Fetizon reagent) was shown to be selective as an oxidizing agent, and convenient for the preparation of various aldonolactones. Whereas substituted aldoses having the 1-hydroxyl group free were readily converted into the corresponding lactones, partially protected 2-acetamido-2-deoxypyranoses having more than one free hydroxyl group were selectively oxidized at C-1. The oxidation was carrried out in boiling benzene or 1,4-dioxane. A series of partially protected 2-acetamido-2-deoxy-1,5-aldonolactones [2-acetamido-4,6-O-benzylidene-2-deoxy-D-mannono-1,5-lactone (13),2-acetamido-4,6-O-benzylidene-2-deoxy-D-glucono-1,5-lactone (15), 2-acetamido-2-deoxy-4,6-O-isopropylidene-D-glucono-1,5-lactone (18), 2-acetamido-2-deoxy-4,6-O-isopropylidene-D-mannono-1,5-lactone (20), 2-acetamido-2-deoxy-3,4-di-O-methyl-D-mannono-1,5-lactone (24), and 2-acetamido-2-deoxy-3,4-di-O-methyl-D-glucono-1,5-lactone (25)] was thus prepared; for these, the oxidation was accompanied by two side-reactions: (a) an elimination (dehydration) that gave the unsaturated lactones [2-acetamido-4,6-O-benzylidene-2,3-dideoxy-D-erythro-hex-2-enono-1,5-lactone (12), 2-acetamido-2,3-dideoxy-4,6-O-isopropylidene-D-erythro-hex-2-enono-1,5-lactone (17), and 2-acetamido-2,3-dideoxy-4-O-methyl-D-erythro-hex-2-enono-1,5-lactone (23)], and (b) partial gluco-to-manno epimerization occurring during the oxidation of 2-acetamido-4,6-O-benzylidene-2-deoxy-D-glucopyranose (14), 2-acetamido-2-deoxy-4,6-O-isopropylidene-D-glucopyranose (16), and 2-acetamido-2-deoxy-3,4-di-O-methyl-D-glucopyranose (22).The free unsaturated lactone, 2-acetamido-2,3-dideoxy-D-erythro-hex-2-enono-1,5-lactone (26), was obtained on hydrolysis of the isopropylidene group in lactone 17.