Synthesis of 3-methylpseudouridine and 2′-deoxy-3-methyl-pseudouridine

The first chemical synthesis of 3-methyl-ψ-uridine (5) and its 2′-deoxy analogue (9) has been achieved. ψ-Uridine was trimethylsilylated and the crude product was treated with acetyl chloride, to give the 1-acetyl derivative (3). Crude 3 was methylated with dimethoxymethyldimethylamine and then saponified, to give crystalline 5 in 82% overall yield. Treatment of 5 with 1,3-dichloro-1,1,3,3-tetraiso-propyldisiloxane afforded the 3′,5′-protected product, which was converted into the 2′-O-[(imidazol-1-yl)thiocarbonyl] derivative 7. Reduction of 7 with tributyltin hydride followed by deblocking of the product gave crystalline 2′-deoxy-3-methyl-ψ-uridine (9) in 35% yield from 5.     

Synthesis of 24S and 24R-hydroxy-[24-3H]vitamin D3 and their metabolism in rachitic rats.

An epimeric mixture of 24-hydroxy-[24-₃H]vitamin D₃ was synthesized by the reduction of 24-ketovitamin D₃ by sodium borotritide. The epimeric mixture was converted to the trimethylsilylether derivatives and subjected to high-pressure liquid chromatography using silica gel columns to separate the 24-hydroxy-[24-₃H]vitamin D₃ isomers. The 24R-hydroxy-[24-₃H] vitamin D₃ induced calcification in rachitic rats while the 24S-hydroxy-[24-₃H] vitamin D₃ had little or no such activity. As both isomers of 24-hydroxy-vitamin D₃ are metabolized to 24,25-dihydroxyvitamin D³, it appears that the 24-hydroxyvitamin D₃-25-hydroxylase does not discriminate between the isomers. Only the R-isomer of 24-hydroxyvitamin D₃ is metabolized to 1,24-dihydroxyvitamin D₃, although only trace amounts of this compound were found 2 days after the administration of 24-hydroxyvitamin D₃. The striking difference in the metabolism of the isomers is the high selectivity of the 1-hydroxylase for R-isomer. It is suggested that the high specificity of biological activity for the R-isomer of 24-hydroxyvitamin D₃ is because of the specificity of the 1-hydroxylation of 24,25-dihydroxyvitamin D₃ for the R configuration.     

Synthesis of vitamin D5: its biological activity relative to vitamins D3 and D2.

The chemical synthesis, spectral characterization, and biological activity of vitamin D₅ in vitamin D-deficient rats is reported. Vitamin D₅ is about 180-fold less active than vitamin D₃ in calcification of rachitic cartilage and about 100- to 200-fold less active in induction of bone-calcium mobilization. In stimulation of intestinal-calcium transport, vitamin D₅ is about 80-fold less active than vitamin D₃. Vitamins D₂ and D₃ appear to be equiactive in all three responses when low doses are administered.     

Properties of epinephrine-induced activation of cardiac adenosine 3′,5′-monophosphate-dependent protein kinase

The effects of epinephrine on cyclic AMP content and protein kinase activity were examined in an in situ rat heart preparation. Bolus injection of epinephrine into the superior vena cava caused an increase in the activity ratio (—cyclic AMP/+cyclic AMP) of 12 000 × g supernatant protein kinase. The increase was significant within 5 s and maximal in 10 s. Epinephrine produced a dose-dependent increase in both protein kinase activity ratio and cyclic AMP content. The increases in both parameters exhibited a high degree of correlation. The increase in protein kinase activity ratio observed with low doses of epinephrine (less than or equal to 1 μg/kg) resulted from an increase in independent protein kinase activity (—cyclic 2 AMP) without a change in total protein observ activity (+cyclic AMP). However, the increase in the activity ratio observed with higher doses of epinephrine (greater than 1 μg/kg) was due mainly to a decrease in total protein kinase activity rather than a further increase in independent protein kinase activity. The loss of supernatant total protein kinase activity could be accounted for by an increase in activity associated with particulate fractions obtained from the homogenates. A similar redistribution of protein kinase could be demonstrated by the addition of cyclic AMP to homogenates prepared from hearts not stimulated with epinephrine. These results demonstrate that epinephrine over a wide dose range produces a parallel increase in the content of cyclic AMP and the activation of soluble protein kinase. The findings also suggest that protein kinase translocation to particulate material may depend on the degree of epinephrine-induced enzyme activation.     

Corpus luteum susceptibility to prostaglandin F2α (PGF2α) luteolysis in hysterectomized prepuberal and mature gilts

The susceptibility of induced corpora lutea (CL) of prepuberal gilts and spontaneously formed CL of mature gilts to prostaglandin F_2α (PGF_2α) luteolysis was studied. Prepuberal gilts (120 to 130 days of age) were induced to ovulate with Pregnant Mare Serum Gonadotropin and Human Chorionic Gonadotropin (HCG). The day following HCG was designated as Day 0. Mature gilts which had displayed two or more estrous cycles of 18 to 22 days were used (onset of estrus = Day 0). Gilts were laparotomized on Day 6 to 9, their CL marked with sterile charcoal and totally hysterectomized. On Day 20, gilts were injected IM with either distilled water (DW), 2.5 mg PGF_2α or 5.0 mg PGF_2α. An additional group of prepuberal gilts was injected with 1.25 mg PGF_2α, a dose of PGF_2α equivalent, on a per kilogram body weight basis, to the 2.5 mg PGF_2α dose given to the mature gilts. The percentages of luteal regression on Day 27 to 30 for mature and prepuberal gilts given DW, 2.5 mg PGF_2α and 5.0 mg PGF_2α were 0.0 vs 4.4, 43.5 vs 96.8 and 47.7 vs 91.6, respectively; the percentage of luteal regression for the prepuberal gilts given 1.25 mg PGF_2α was 75.1. These results indicate that induced CL of the prepuberal gilt were more susceptible to PGF_2α luteolysis than spontaneously formed CL of the mature gilt and that pregnancy failure in the prepuberal gilt could be due to increased susceptibility of induced CL to the natural luteolysin.     

Methyl β-lactoside: 600-MHz H- and 75-MHz C-n.m.r. studies of H- and C-enriched compounds

Using UDP-d-galactose : 2-acetamido-2-deoxy-d-glucose 4-β-d-galactosyltransferase (EC 2.4.1.22), several methyl β-lactosides have been prepared with ²H- and/or ¹³C-enrichment at specific sites to facilitate study by ¹³C (75 MHz) and ¹H (600 MHz) n.m.r. spectroscopy. ¹³C-Chemical shift assignments were verified and the ¹H-spectrum of β-lactoside was fully assigned. Sites of enrichment were selected to permit all of the potential three-bond C-C and C-H couplings through the glycosidic bond to be obtained. Replacement of H-3 of the d-glucose residue of methyl β-lactoside with ²H allowed resolution of C-1–H-4′ coupling in the 600-MHz ¹H-spectrum. Single or multiple ¹³C-enrichment at C-1, C-2, C-3, C-1′, C-3′, and/or C-4′ in the disaccharide allowed observation of intra- and inter-residue couplings. ¹³C-Spin-lattice relaxation-times (T₁) are interpreted in terms of molecular motion in solution. The data suggest that methyl β-lactoside has an extended conformation with little rotation about the glycosidic bond. Inter-residue couplings are best explained by tortion angles of φ ~ 40° and ψ ~ 15°, indicating that the conformations of β-lactoside in solution and in the crystal are similar.     

Evidence for the metabolism of 24R-hydroxy-25-fluorovitamin D3 and 1alpha-hydroxy-25-fluorovitamin D3 to 1alpha,24R-dihydroxy-25-fluorovitamin D3.

High-pressure liquid chromatography capable of resolving all known vitamin D metabolites and a sensitive competitive binding protein assay specific for 1α,25-dihydroxyvitamin D₃ were used to assay the blood of rats dosed with ethanol, 1α-hydroxyvitamin D₃, 24R-hydroxy-25-fluorovitamin D₃, or 1α-hydroxy-25-fluorovitamin D₃. Compared to the ethanoldosed animals, the blood of rats dosed with 1α-hydroxyvitamin D₃ had increased levels of 1α,25-dihydroxyvitamin D₃; but those dosed with the fluorinated vitamins did not. Instead, their blood contained a compound that cochromatographs with 1α,24R-dihydroxyvitamin D₃ on high-pressure liquid chromatography and binds to the 1,25-dihydroxyvitamin D₃ receptor proteins. 1α,24R-Dihydroxyvitamin D₃ binds as well as 1α, 25-dihydroxyvitamin D₃ to the chick-intestinal cytosol receptor protein for 1α,25-dihydroxyvitamin D₃; whereas 1α,24S-dihydroxyvitamin D₃ binds only one-tenth as well as 1α,25-dihydroxyvitamin D₃. Thus it appears that in vivo, the fluorinated vitamin D compounds are converted to a compound likely to be 1α,24R-dihydroxy-25-fluorovitamin D₃ and that may rival the potency of 1α,25-dihydroxyvitamin D₃.     

Characterization of the progesterone receptor of rabbit uterus with the synthetic progestin 16α-ethyl-21-hydroxy-19-norpregn-4-ene-3,20-dione

The synthetic progestin 16α-ethyl-21-hydroxy-19-norpregn-4-ene-3,20-dione (Org 2058) was used to characterize the progesterone receptor in the uterine cytosol of the rabbit. [³H] Org 2058 binds to a homogeneous population of protin binding sites with an apparent association equilibrium constant of 7.7· 10⁸ M⁻¹ at 0°C. The concentration of protein-bound steroid at saturation is 2.3 pmol per mg of cytosol protein. [³H] Progesterone binds to the same set of binding sites but exhibits a 4–5 fold lower apparent association constant. The difference in affinity is mainly due to a 13-fold slower rate of dissociation of the synthetic progestin compared with progesterone. Org 2058 competes very efficiently for the binding of [³H] progesterone to the uterine cytosol, and progesterone also competes, although less efficiently, for the binding of [³H]-Org 2058. There is a good correlation between the progestational activity of various steroids and their ability to compete with [³H] Org 2058 binding to the cytosol. At 0°C, there is no metabolic transformation of either Org 2058 or progesterone in the uterine cytosol.When filled with the steroid, the progesterone receptor is stable, but in the absence of the steroid the receptor binding sites are thermolabile and show a rapid decay at 20°C . Org 2058 is more effective than progesterone in protecting the receptor against thermal inactivation. The rate constant of association and dissociation of [³H] Org 2058 and the cytosol receptor are strongly dependent on temperature and the activation energy of the dissociation reaction is 17.8 kcal/mol. The equilibrium association constant is less dependent on temperature and exhibits ΔH° of −4.7 kcal/mol. The binding reaction shows a positive entropy change of 23 cal · K⁻¹ · mol⁻¹.At low ionic strength the complex of Org 2058 and the progesterone receptor tends ot aggregate. It sediments as a broad peak on sucrose gradients (4–6 S), and is excluded from columns of Sephadex G-100 and G-200. At concentrations of NaCl above 0.15 M, the receptor sediments in sucrose gradients as an homogeneous peak at 3.6 S, but upon gel filtration it aggregates and a complex elution pattern is observed, that prevents a precise estimation of the molecular weight.     

Uptake of alpha-ketoisocaproic acid in lymphoblast line WI-L2.

The uptake of α-ketoisocaproate by the cultured human lymphoblast line WI-L2 appears to be mediated by a transport system which has an apparent Km of 125 μM. The rate of uptake of α-ketoisocaproate decreases with increasing pH values, i.e., pH 6 > 7 > 8 and is stimulated by sodium at all pH values. Closely related branched chain α-ketoacids, α-keto-β-methylvaleric and α-ketoisovaleric exhibited the greatest inhibition of α-ketoisocaproate transport. Straight chain α-keto acids inhibited α-ketoisocaproic acid uptake to a lesser degree as did the α-hydroxy analogs of the branched chain α-keto acids. Inhibitors of the general anion transport system of erythrocytes, 1-anilino-8-napthalene sulfonic acid and 4-acetamido-4-isothiocyanostilbene-2-1′-disulfonic acid did not affect α-ketoisocaproate transport. A reduced sulfhydryl group is critical for α-ketoisocaproate acid uptake; transport is partially or completely inhibited by sulfhydryl reagents such as dithio-bis-nitrobenzoate, iodoacetamide, and p-chloromercuribenzoate. Inhibition by the sulfhydryl reagents is reversed with β-mercaptoethanol or partially with dithiothreitol.     

Morphogenesis in the slime mold Dictyostelium discoideum. 1. The accumulation and erasure of "morphogenetic information".

When developing cultures of the cellular slime mold Dictyostelium discoideum are disaggregated and morphogenesis immediately reinitiated, they recapitulate the morphogenetic scheme, but at an increased rate. Employing this feature of the system, we have identified time periods when “information” accumulates for specific morphogenetic events. In this case, we have defined “morphogenetic information” as the reduction in time for the appearance of a particular morphology during morphogenetic recapitulation. Accumulated information can be erased by disaggregating developing cultures and reinoculating them into liquid growth medium. Erasure occurs as a discrete event and can be blocked by inhibiting protein synthesis.By comparing morphogenesis in log-phase and stationary-phase cultures, at least two parallel timers have been distinguished, one specific for aggregation and one or more specific for morphogenetic events following aggregation. Approaches to the molecular identity of morphogenetic information and morphogenetic timers are discussed.     

Synthesis of 1-chloroestradiol

The synthesis of the previously unknown 1-chloroestradiol (1), is reported. Addition of N-chlorosuccinimide/NaI in dioxane to 4-amino-estradiol 3-methyl ether (2), resulted in exclusive chlorination of the 1-position. Subsequent deamination and demethylation afforded 1 in good yields.     

Isolation and identification of 24-hydroxyvitamin D2 and 24,25-dihydroxyvitamin D2

The isolation and identification of two metabolites of vitamin D₂ found in mammalian and avian species are reported. They are 24-hydroxyvitamin D₂ and 24,25-dihydroxyvitamin D₂. Their existence suggests that 24-hydroxylation occurs in a sterospecific manner in the 24R position and adds further support to the theory that vitamin D₂ metabolism qualitatively parallels that of vitamin D₃.     

Synthesis, properties and reactions of 3 beta-benzoyloxy-7 alpha-15 beta-dichloro-5 alpha-cholest-8(14)-ene.

Treatment of 3 beta-benzoyloxy-14 alpha,15 alpha-epoxy-5 alpha-cholest-7-ene (I) with gaseous HCl in chloroform at -40 degrees C gave, in 87% yield, 3 beta-benzoyloxy-7 alpha,15 beta-dichloro-5 alpha cholest-8(14)-ene (III). Reduction of the latter compound with lithium aluminum hydride in ether at room temperature for 20 min gave, in 86% yield, 7 alpha-15 beta-dichloro-5 alpha-cholest-8(14)-en-3 beta-ol (IV). The latter compound was fully characterized and assignments of the individual carbon peaks in the 13C nuclear magnetic resonance spectra of this sterol have been completed. Reduction of III with excess lithium aluminum hydride in refluxing ether for 4 days gave, in 74% yield, 5 alpha-cholesta-7,14-dien-3 beta-ol (VI). Reduction of the dichloro-steryl benzoate III with lithium triethylborohydride in tetrahydrofuran gave, in 88% yield, 5 alpha-cholest-8(14)-en-3 beta-ol (VII). A similar reduction using lithium triethylborodeuteride led to the formation of [7 beta, 15 xi-2 H2]-VIIa. Treatment of III with concentrated HCl in a mixture of chloroform and methanol gave, in 79% yield, 3 beta-benzoyloxy-5 alpha-cholest-8(14)-en-15-one (II) which was characterized as such and as the corresponding free sterol.     

Addition of 1-nitroalkanes to methyl 2,3-O-isopropylidene-β-d-ribo-pentodialdo-1,4-furanoside and N6-benzoyl-2′,3′-O-isopropylideneadenosine-5′-aldehyde

Various 1-nitroalkanes reacted with methyl 2,3-O-isopropylidene-β-d-ribo-pentodialdo-1,4-furanoside to yield methyl 6-alkyl-6-deoxy-2,3-O-isopropylidene-6-nitro-β-d-ribofuranosides in 64–79% yield. Similarly, nitromethane and 1-nitropentane reacted with N⁶-benzoyl-2′,3′-O-isopropylideneadenosine-5′-aldehyde, to yield the corresponding 9-[6-alkyl-6-deoxy-2,3-O-isopropylidene-6-nitro-α-l-talo(β-d-allo)furanosyl]-N⁶-benzoyladenines in 74 and 44% yield, respectively. The potential utility of this nitroalkane addition for the synthesis of nucleosides having a C-5′C-6′ bond is discussed.     

Azo dye-α-cyclodextrin adduct formation

Association constants for the complexation of a series of azo dyes with α-cyclodextrin have been determined by means of changes in uv-visible spectra in aqueous solution at pH 7.0, an ionic strength of 0.15 M and at 25 and 35°C. Association constants for some of the systems were also determined in water-dimethyl sulfoxide mixtures. All dyes were azobenzenes with a hydroxy group at the 4′ position of one ring and a carboxy group at the 4 position of the other ring. The results were interpreted as demonstrating that the carboxy end of the dye entered the cavity of the α-cyclodextrin. An electrostatic model failed to account for all of the trends in the data. A view of the binding process involving a hydration shell in the region of the primary hydroxy groups of the cyclodextrin is presented.     

Perinatal exposure to 19-Nor-17α-ethynyltestosterone (norethindrone) influences morphology and aggressive behavior of female mice

Pregnant mice were injected with 50 or 100 μg of the synthetic progestin 19-nor-17α-ethynyltestosterone (NET) on Days 14 through 17 of gestation. Others received only the vehicle or were left undisturbed. Exposure to NET during the prenatal period increased anogenital distance of the female offspring as measured on Day 60 but did not influence the duration of adult testosterone (T) exposure required to activate male-like intraspecific fighting behavior. In a second experiment, female mice were exposed to NET either prenatally, postnatally (Day 1), or pre- and postnatally. Exposure during both pre- and postnatal periods increased anogenital distance but only the exposure during the postnatal period enhanced later behavioral sensitivity to the aggression-activating property of T. Thus, NET is similar to testosterone in its ability to virilize morphology and behavior, although, at the dosages administered, behavioral alternation only occurred as a result of treatment during the neonatal period.