Structures of ascaroside aglycones

Free and derivatized aglycones and parent ascarosides of Ascaris lumbricoides and A. columnaris (Nematoda) were isolated by thin-layer and gas chromatography and analyzed by mass spectrometry and polarimetry. Results for monol aglycones confirmed previous chain-length assignments and the location of the l-hydroxyl group at C-2. The major monols of even carbon number possessed a methyl branch on the penultimate carbon (ω ? 1). Diol alycones were entirely unbranched homologues, mainly 31 and 33 carbons long, and had hydroxyl groups on C-2 and C-(ω ? 1). These aglycones were truly symmetrical, for they were optically inactive with the center bearing the glycone in diol ascaroside (C-2), being mostly or entirely the l configuration. Three major features of ascaroside structure—chain length composition of the aglycones, molecular weight of the unusual glycone (a dideoxyhexose), the kind and position of acyl groups—were clearly discernible in the spectra of intact ascarosides.     

Induction of monocytic differentiation of HL-60 cells by 1,25-dihydroxyvitamin D analogs

1,25-Dihydroxyvitamin D3, the hormonal form of vitamin D, induces differentiation of HL-60 human promyelocytes into monocyte-like cells in vitro. We assessed the relative activity of 30 analogs of 1,25-dihydroxyvitamin D3 in inducing development of monocytic markers in HL-60 cells. The three differentiation markers assayed were nonspecific acid esterase activity, nitro blue tetrazolium reducing activity, and phagocytic capacity. Of the known metabolites of vitamin D, 1,25-dihydroxyvitamin D3 is the most active; 50% of the cells exhibit the mature phenotype following a 4-day treatment with 10(-8) M 1,25-dihydroxyvitamin D3. Removal of either the C-1 or C-25-hydroxyl group reduces activity by 2 orders of magnitude, while epimerization of the 1 alpha- to 1 beta-hydroxyl group virtually abolishes activity. Elongation of the steroidal side chain of 1,25-dihydroxyvitamin D3 by addition of one carbon at C-24 or C-26 improves the potency by an order of magnitude. Truncation of the steroidal side chain leads to a 10-fold reduction in activity for each carbon removed. Elimination of the C-26 and C-27 methyl groups reduces activity 100-fold. Analogs with short aliphatic side chains as 1 alpha-hydroxyhomo- and bishomopregnacholecalciferol have surprisingly high activity, being only 20-fold less potent than the natural hormone. The activity of most analogs in the HL-60 system parallels their known relative affinities for the well characterized 1,25-dihydroxyvitamin D3 receptor in chick intestine, providing further evidence that this function of 1,25-dihydroxyvitamin D3 is receptor mediated.     

1H nuclear magnetic resonance studies of the conformations of vitamin D compounds in various solvents

Using proton NMR, the solution conformation of the A ring of vitamin D3 and its analogs has been studied by application of the Karplus relation to the observed coupling constants. The A-ring conformations of vitamins D3, D2, and 25-hydroxyvitamin D3 were found to be solvent dependent, with a clear preference for an equatorial hydroxyl group in polar solvents such as methanol and dimethyl sulfoxide. Conversion of the hydroxyl group to an acetate did not affect solution conformation appreciably, but the corresponding t-butyl-dimethylsilyl ether derivative of vitamin D3 showed a strong preference for the 3 beta-equatorial conformer. The A-ring conformation of the active hormone, 1,25-dihydroxyvitamin D3, which has two hydroxyl groups competing for the equatorial position, was found not to be solvent-dependent.     

On the structure and biological properties of decarbamoylsaxitoxin

The acid hydrolysis product of saxitoxin is shown to be decarbamoylsaxitoxin by spectral characterization and its reconversion to saxitoxin by carbamoylation. Natural and resynthesized saxitoxin are identical in chromatographic and spectral properties and in their potencies in blocking the sodium channel in squid giant axon. The hydrolysis product, decarbamoylsaxitoxin, exhibits 20% of the potency of saxitoxin in the squid axon system. These results confirm the structure of the hydrolysis product and its biological activity relative to saxitoxin.     

In vivo and in vitro inhibition of rat liver vitamin D3-25-hydroxylase activity by 19-hydroxy-10(S),19-dihydrovitamin D3

Rats treated with varying amounts of 19-hydroxy-10(S),19-dihydrovitamin D3 prior to administration of physiologic doses of vitamin D3 exhibit normal intestinal calcium transport but are unable to mobilize bone calcium. In contrast, 19-hydroxy-10(R),19-dihydrovitamin D3 had no inhibitory activity. Circulating serum levels of 25-hydroxy[3H]vitamin D3 and 1 alpha, 25-dihydroxy[3H]vitamin D3 are markedly suppressed but not totally eliminated in animals predosed with 19-hydroxy-10(S),19-dihydrovitamin D3 before [3H]vitamin D3. Hepatic 25-hydroxy[3H]vitamin D3 levels were approximately equal in both 19-hydroxy-10(S),19-dihydroviotamin D3 treated and untreated rats. However, the rate of conversion of [3H]vitamin D3 to 25-hydroxyvitamin D3 in vivo is greatly reduced in the treated rats. The inhibitory vitamin analogue was also show to block hepatic microsomal 25-hydroxylation in vitro. These results indicate that 19-hydroxy-10(S),19-dihydrovitamin D3 is a specific inhibitor for a hepatic microsomal vitamin D3-25-hydroxylase system.     

Isolation and identification of 23,25-dihydroxyvitamin D3, an in vivo metabolite of vitamin D3

Vitamin D supplemented rats produce a metabolite of 25-hydroxy[3 alpha-3H]vitamin D3 that is easily separated from known metabolites by using high-performance liquid chromatography. The production of this metabolite in vivo as well as 1,25-dihydroxyvitamin D3, 24(R),25-dihydroxyvitamin D3, and 25-hydroxyvitamin D3 26,23-lactone is largely if not totally eliminated by nephrectomy. Kidney homogenates from vitamin D supplemented chickens incubated with 25-hydroxyvitamin D3 produce significant quantities of the new, unknown metabolite. This metabolite was isolated in pure form from such incubation mixtures by using both straight-phase and reversed-phase high-performance liquid chromatography. This metabolite has been positively identified as 23,25-dihydroxyvitamin D3 by ultraviolet absorption spectrophotometry, mass spectrometry, and derivatization. This structure was confirmed by chemical synthesis of both C-23 stereoisomers. Although the natural product exactly comigrates with one of the synthetic isomers, the exact stereochemistry of the natural product remains unknown. It is possible that this new metabolite is an intermediate in the biosynthesis of 25-hydroxyvitamin D3 26,23-lactone.