On the side-chain conformation of N-acetylneuraminic acid and its epimers at C-7, C-8, and C-7,8

The side-chain conformation of N-acetylneuraminic acid and analogs has been studied by n.m.r. spectroscopy. The results of the 1H-, 13C-n.m.r.-, and 1H-nuclear-Overhauser-enhancement measurements were used to distinguish between different local-minima conformations suggested by hard-sphere calculations. Attempts were made to correlate the major conformation determined for each compound with the behavior towards activation with N-acetylneuraminic acid-CMP-synthetase.     

Synthesis of C-22, C-23-3H-labeled 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestane

This report describes an efficient synthesis of C-22, C-23-(3)H-labeled 3alpha,7alpha,12alpha-trihydroxy-5beta-cholestane. - Somanathan, R., and S. Krisans. Synthesis of C-22, C-23-(3)H-labeled 3alpha,7alpha,12alpha-trihydroxy-5beta-cholestane.     

Measurement of methyl 13C-1H cross-correlation in uniformly 13C-, 15N-, labeled proteins

An understanding of side chain motions in protein is of great interest since side chains often play an important role in protein folding and intermolecular interactions. A novel method for measuring the dynamics of methyl groups in uniformly ¹³C-, ¹⁵N-labeled proteins has been developed by our group. The method relies on the difference in peak intensities of ¹³C quartet components of methyl groups, in a spectrum recording the free evolution of ¹³C under proton coupling in a constant-time period. Cross-correlated relaxation rates between ¹³C-¹H dipoles can be easily measured from the intensities of the multiplet components. The degree of the methyl restrictions (S ²) can be estimated from the cross-correlated relaxation rate. The method is demonstrated on a sample of human fatty acid binding protein in the absence of fatty acid. We obtained relaxation data for 33 out of 46 residues having methyl groups in apo-IFABP. It has been found that the magnitude of the CSA tensor of spin ¹³C in a methyl group could be estimated from the intensities of the ¹³C multiplet components.     

Structure-activity relationships of estrogens. Effects of 14-dehydrogenation and axial methyl groups at C-7, C-9 and C-11

Thirty compounds were evaluated in the rat for uterotropic effects, inhibition of gonadotropin release, and competitive displacement of (3H) estradiol-17 beta from uterine cytosolic preparations. 7 alpha-Methylestradiol-17 beta was 150% as active as estradiol-17 beta as an uterotropic agent. Estradiol-17 beta was the most active inhibitor of gonadotropin release. 11 beta-Methylestradiol-17 beta had 124% of the activity of estradiol-17 beta in displacing (3H) estradiol-17 beta from the "estrogen receptor." The 9 alpha-methyl group considerably decreased the potency of estrogens in any of the three assays. The 14-dehydro modification was advantageous only in the estradiol-17 beta 3-methyl ether series. Uterotropic activities and inhibition of gonadotropin release did not parallel. The best compound for inhibiting gonadotropin release, as compared to uterotropic activity, was estrone. The "estrogen receptor" assay data correlated fairly well with uterotropic assay data, but only for compounds having free 3-hydroxyl groups; even so, some exceptions were noted.     

Probing the vitamin D sterol-binding pocket of human vitamin D-binding protein with bromoacetate affinity labeling reagents containing the affinity probe at C-3, C-6, C-11, and C-19 positions of parent vitamin D sterols

The multiple physiological properties of vitamin D-binding protein (DBP) include organ-specific transportation of vitamin D(3) and its metabolites, manifested by its ability to bind vitamin D sterols with high affinity. In the present investigation we probed the vitamin D sterol-binding pocket of human DBP with affinity labeling analogs of 25-hydroxyvitamin D(3) ?25-OH-D(3) and 1, 25-dihydroxyvitamin D(3) ?1,25(OH)(2)D(3) containing bromoacetate alkylating probe at C-3 (A-ring), C-6 (triene), C-11 (C-ring), and C-19 (exocyclic methylene) of the parent sterol. Competitive binding assays with DBP showed approximately 22-, 68-, and 2000-fold decrease in the binding of 1,25(OH)(2)-D(3)-11-BE, 25-OH-D(3)-3-BE, and 25-OH-D(3)-6-BE, respectively, compared to that seen with 25-OH-D(3), while there was no significant difference in the DBP-binding affinity of 25-OH-D(3)-19-BE and 25-OH-D(3). Surprisingly, ?(14)C25-OH-D(3)-11-BE and ?(14)C1, 25(OH)(2)-D(3)-19-BE failed to label DBP despite high-affinity DBP-binding, indicating the absence of any nucleophilic amino acid in the vicinity of their bromoacetate moiety to form a covalent bond, while these analogs are inside the binding pocket. In contrast, ?(14)C25-OH-D(3)-6-BE and ?(14)C25-OH-D(3)-3-BE specifically labeled DBP. BNPS-skatole digestion of DBP labeled with ?(14)C25-OH-D(3)-3-BE or ?(14)C25-OH-D(3)-6-BE produced two peptides (M(r) 17,400 and 33,840), with radioactivity associated with the N- and C-terminal peptides, respectively, raising the possibility that either different areas of the same vitamin D sterol-binding pocket, or different domains of DBP might be labeled by these analogs. Successful affinity labeling of recombinant domain I (1-203) of DBP with both reagents indicated that different areas of the same vitamin D-binding pocket (domain I) were labeled. These affinity analogs are potentially useful for "mapping" the vitamin D sterol-binding pocket and developing a functional model.     

Synthesis and biological evaluation of C-3'NH/C-10 and C-2/C-10 modified paclitaxel analogues

Concurrent modifications on the C-3'NH/C-10, and C-2/C-10 positions on paclitaxel were carried out as a way of investigating possible synergistic effects. The biological activities of these analogues were evaluated in both a microtubule assembly assay and human ovarian cancer (A2780) and prostate cancer (PC3) cytotoxicity assay. In some cases the doubly modified analogues were more active than would have been predicted based on the activity of the singly modified analogues, indicating probable synergistic effects.     

The development of novel C-2, C-8, and N-9 trisubstituted purines as inhibitors of TNF-alpha production

A series of C-2, C-8, and N-9 trisubstituted purine based inhibitors of TNF-alpha production are described. The most potent analogs showed low nanomolar activity against LPS-induced TNF-alpha production in a THP-1 cell based assay. The SAR of the series was optimized with the aid of X-ray co-crystal structures of these inhibitors bound with mutated p38 (mp38).     

Characterization of lyso-galactolipids, C-2 and C-3 O-acyl trigalactosylglycerol isomers, obtained from the lichenized fungus Dictyonema glabratum

A mixture of two lyso isomers of a galactolipid was obtained from Dictyonema glabratum. Aqueous hydrolysis gave rise to galactose and glycerol in a 3:1 molar ratio. ESI-MS spectroscopy gave, in the positive-ion mode, a pseudomolecular ion at m/z 839 and daughter ions with m/z 677, 600, 515 and 353, suggesting three galactosyl units linked to a glycerol moiety, substituted by one O-acyl group. 1D and 2D NMR experiments were used to characterize the glycolipid, and HMQC examination showed three anomeric signals, corresponding to two alpha-Galp and one beta-Galp residue liked to glycerol. The glycolipid structure was shown to be O-alpha-D-Galp-(1-->6)-O-alpha-D-Galp-(1-->6)-O-beta-D-Galp-(1<-->1)-2- and -3-monoacyl-D-glycerol, the latter structures not having been previously found in nature. The fatty acid composition was determined by GC-MS of derived methyl esters: that of palmitic acid C(16:0) was the most abundant, although the presence of C(12:0), C(14:0), C(16:1) and C(18:0) esters was observed.     

Biosynthesis of ent-kauranes: Evidence for a C-17, C-16 hydrogen 1,2-shift

The incorporation of ent-kaurenoic acid into the derived hydroxy acid and dihydroxy acid by Beyeria calycina has been studied. The biosynthesis of the hydroxy acid involves a hydrogen 1,2-shift from the C-17 position of ent-kaurenoic acid.     

Simultaneous regioselective protection of phenyl 1-thioglucosides at the C-3 and C-6 or at the C-2 and C-6 hydroxy groups

Simultaneous regioselective 3,6- or 2,6-selective protection of 1-thio-beta- or alpha-D-glucopyranosides is described. The C-3 and C-6 hydroxy groups of the beta-thioglucoside were selectively protected with triisopropylsilyl or tert-butyldiphenylsilyl trifluoromethanesulfonate. The C-2 and C-6 hydroxy groups of the alpha-thioglucoside were selectively protected with tert-butyldiphenylsilyl trifluoromethanesulfonate.     

Multifunctionalized alpha,beta-cyclopentenones from C-2 and C-4-ulopyranosyl compounds: a stereospecific rearrangement initiated by base

Base treatment of O-benzyl protected C-2- or C-4-ulopyranosyl compounds (4 alpha, 4 beta, and 11) by either 10% Et(3)N or 1% K(2)CO(3) in MeOH initiated a beta elimination to afford alpha,beta-unsaturated C-ulopyranosyl compounds (5 alpha, 5 beta, and 12), which further rearranged in a stereocontrolled manner to multifuctionalized alpha,beta-cyclopentenones (6 and 14) in 70-80% yield. Both C-alpha- and C-beta-2-ulosides (5 alpha and 5 beta) produced the same cyclopentenone 6, indicating that a 1,2-enolate is formed prior to the cleavage of the C-5--O bond. Because 6 is racemic, it was probably formed by the intramolecular cycloaldolization of two equally populated enantiomeric intermediates. When treated with 90% Et(3)N in MeOH, 5 alpha yielded almost exclusively 15 (isomer of 6), which was formed by a migration of the double bond in 5 alpha during the previously described rearrangement. Thus either 6 or 15 was the major product, depending on the base used.     

Biosynthesis of thiamin. Precursor of C-5, C-6, and hydroxymethyl carbon atoms of the pyrimidine moiety in a eucaryote

We studied the incorporation of radioactive glucose into the pyrimidine moiety of thiamin in the eucaryote Candida utilis. Three carbons of glucose were incorporated into the pyrimidine, and the C-2 of glucose into the C-6 of the pyrimidine. We concluded that the C-5, -6, and hydroxymethyl carbon atoms of the pyrimidine in this eucaryote originate from the C-2, -3 and -4 of glucose via ribose.     

Synthesis of C-2 and C-4 deuterium-labeled estradiol-17 beta

Estradiol-17 beta labeled with deuterium in the positions 2 or 4 can be prepared from 2-chloromercurio-1,3,5(10)-estratriene-3,17 beta-diol 3-methyl ether 17-acetate or 4-chloromercurio-1,3,5(10)-estratriene-3,17 beta-diol, respectively, in refluxing CH3COO(2)H/(2)H2O. The same reaction performed on 4-acetoxymercurio-1,3,5(10)-estratriene-3,17 beta-diol afforded 2,4-dideuterio-estradiol-17 beta in good yields.     

Interaction of uridine diphosphate glucose with calf liver uridine diphosphate glucose dehydrogenase. Significance of hydroxyl groups at C-3, C-4 and C-6 of hexosyl residue.

Analogs of uridine diphosphate glucose (UDPGlc) with a modified hexosyl residue which contained a deoxy-unit at C-3 or C-4 were tested as substrates of calf liver UDPGlc dehydrogenase (EC 1.1.1.22). The 3-deoxyglucose derivative was found not to serve as a substrate for the enzyme whereas the 4-deoxyglucose analog was able to participate in the reaction. The apparent Km of the latter was 5.3 times that of UDPGlc and the relative V was 0.04. The reaction product was identified as uridine diphosphate deoxyhexuronic acid. UDP-deoxyhexoses were non-competitive inhibitors of UDPGlc enzymic oxidation, inhibition increased in the sequence: 2-deoxy-less than 3-and 6-deoxy-less than 4-deoxyglucose derivative. The significance of different HO-groups in hexosyl residue for interaction of UDPGlc with the enzyme is discussed.     

磷脂酶C—γ2对鼠成纤维细胞株迁移能力的影响

通过对敲除磷脂酶C－γ1基因的鼠成纤维细胞株及其转染磷脂酶C-γ2的迁移指标的统计学分析,揭示出磷脂酶C－γ1和γ2在PDGF引起的细胞迁移信号传导中均有正调控作用,前者作用显著而后者作用微小,并且仅在磷脂酶C-γ1功能缺失的条件下方可发挥出来,同时进一步证明敲除磷脂酶C－γ1基因的细胞株中,未见出现磷脂酶C－γ2的代偿,说明磷脂酶C－γ2与在功能上不可相互取代,反映了一种信号可由多种途径传导即细胞信号传导的交互性,沉余性。