Syntheses of stable isotope-labeled 6 beta-hydroxycortisol, 6 beta-hydroxycortisone,and 6 beta-hydroxytestosterone

A method is described for the preparation of two types of multi-labeled 6 beta-hydroxycortisol containing either five deuterium atoms at C-19 methyl and C-1 methylene or four 13C atoms at C-1, C-2, C-4, and C-19 in addition to the five deuterium atoms for use as analytical internal standards for gas chromatography-mass spectrometry (GC-MS). BMD derivatives of [1,1,19,19,19-2H(5)]cortisone and [1,2,4,19-13C(4),1,1,19,19,19-2H(5)]cortisone (cortisone-2H(5)-BMD and cortisone-13C(4),2H(5)-BMD) were first synthesized via indan synthon method starting from optical active 11-oxoindanylpropionic acid and labeled isopropenyl anion ([1,1,3,3,3-2H(5)]- or [1,3-13C(2),1,1,3,3,3-2H(5)]isopropenyl anion). The labeled isopropenyl anion was prepared from commercially available [1,1,1,3,3,3-2H(6)]- or [1,3-13C(2),1,1,1,3,3,3-2H(6)]acetone. Ultraviolet (UV) irradiated autoxidation at C-6 position of 3-ethyl-3,5-dienol ether derivatives of the labeled cortisone-BMDs gave 6 beta-hydroxy-[1,1,19,19,19-2H(5)]cortisone-BMD and 6 beta-hydroxy-[1,2,4,19-13C(4),1,1,19,19,19-2H(5)]cortisone-BMD, respectively, as a mixture of 6 beta- and 6 alpha-epimers in a ratio of 4:1. Separation of 6 beta- and 6 alpha-epimers by thin-layer chromatography (TLC) and subsequent hydrolysis of the BMD group at C-17 gave pure labeled 6 beta-hydroxycortisone. After protecting the keto group at C-3 of the labeled 6 beta-hydroxycortisone-BMD as semicarbazone, reduction of 11-keto group with NaBH(4) and subsequent removal of the C-3 and C-17 protecting groups gave 6beta-hydroxy-[1,1,19,19,19-2H(5)]cortisol (6 beta-hydroxycortisol-2H(5)) and 6 beta-hydroxy-[1,2,4,19-13C(4),1,1,19,19,19-2H(5)]cortisol (6 beta-hydroxycortisol-13C(4),2H(5)), respectively, as a mixture of 6 beta- and 6 alpha-epimers (6 beta:6 alpha=4.4:1). The isotopic compositions of 6 beta-hydroxycortisol-2H(5) and 6 beta-hydroxycortisol-13C(4),2H(5) were 90.9 and 92.1 at.%, respectively. Furthermore, 6 beta-hydroxy-[1 alpha,16,16,17 alpha-2H(4)]testosterone was synthesized by the UV irradiated autoxidation at C-6 position of 3-ethyl-3,5-dienol ether derivative of deuterium-labeled testosterone ([1 alpha,16,16,17 alpha-2H(4)]testosterone) obtained by using catalytic deuteration and hydrogen-deuterium exchange reactions.     

Regioselective synthesis of 1I,1II,5I,5II,6I,6I,6II,6II-2H8-cellobiose

Partially deuteriated 1,5,6,6-(2)H(4)-d-glucose and 1(I),1(II),5(I),5(II),6(I),6(I),6(II),6(II)-(2)H(8)-d-cellobiose were synthesized in high yields and on a large scale from d-glucose. (2)H enrichment at C-5 and C-6 of each glucopyranosyl unit in excess of 85% and 90%, respectively, was realized by (1)H-(2)H exchange in (2)H(2)O containing deuteriated Raney Ni. Nucleophilic addition of LiAlD(4) to 5,6,6-(2)H(3)-2,3,4,6-tetra-O-benzyl-d-gluconolactone led to a 98% (2)H enrichment at C-1. Deuteriated cellobiose is of interest as building block for the synthesis of a model compound of cellulose I.     

6-Methylcryptoacetalide, 6-methyl-epicryptoacetalide and 6-methylcryptotanshinone from Salvia aegyptiaca

From the whole plant of Salvia aegyptiaca, 6-methylcryptoacetalide, 6-methyl-epicryptoacetalide and 6-methylcryptotanshinone have been isolated and characterized, mainly by spectroscopic means. In addition to these novel diterpenoids, the known compounds 3beta-hydroxy-olean-12-en-28-oic acid, 3beta-hydroxy-oleana-11,13(18)-dien-28-oic acid, sitosterol-3beta-glucoside, sitosterol, stigmasterol, 5-hydroxy-7,3',4'-trimethoxyflavone and 5, 6-dihydroxy-7,3',4'-trimethoxyflavone were isolated.     

Mechanisms of inactivation of human S-adenosylhomocysteine hydrolase by 5',5',6',6'-tetradehydro-6'-deoxy-6'-halohomoadenosines

In an effort to design more specific and potent inhibitors of S-adenosylhomocysteine (AdoHcy) hydrolase, we investigated the mechanisms by which 5',5',6', 6'-tetradehydro-6'-deoxy-6'-halohomoadenosines (X = Cl, Br, I) inactivated this enzyme. The 6'-chloro (a) and 6'-bromo (b) acetylenic nucleoside analogues produced partial ( approximately 50%) loss of enzyme activity with a concomitant ( approximately 50%) reduction of E-NAD(+) to E-NADH. In addition, Ade and halide ions were released from the inhibitors in amounts suggestive of a process involving enzyme catalysis. AdoHcy hydrolase, which was inactivated with compound a, was shown to contain 2 mol of the inhibitor covalently bound to Lys318 of two subunits of the homotetramer. These data suggest that the enzyme-mediated water addition at the 5' position of compound a or b produces an alpha-halomethyl ketone intermediate, which is then attacked by a proximal nucleophile (i.e., Lys318) to form the enzyme-inhibitor covalent adduct (lethal event); in a parallel pathway (nonlethal event), addition of water at the 6' position produces an acyl halide, which is released into solution and chemically degrades into Ade, halide ion, and sugar-derived products. In contrast, compound c completely inactivated AdoHcy hydrolase by converting 2 equiv of E-NAD(+) to E-NADH and causing the release of 2 equiv of E-NAD(+) into solution. Four moles of the inhibitor was shown to be tightly bound to the tetrameric enzyme. These data suggest that compound c inactivates AdoHcy hydrolase by a mechanism similar to the acetylenic analogue of Ado described previously by Parry et al. [(1991) Biochemistry 30, 9988-9997].     

A synthesis of N6,N6,N6-trimethyl-L-lysine dioxalate in gram amounts

A procedure is described for the synthesis of crystalline N6,N6,N6-trimethyl-L-lysine dioxalate in gram amounts starting from the commercially available N2-tert-butoxycarbonyl-N6-benzyloxycarbonyl-L-lysine, which is reacted with methyl iodide in methanol in the presence of potassium hydrogen carbonate after deprotection of the side-chain amino group by catalytic hydrogenation. The work-up involves only filtrations and evaporations.     

Preparation of 6,6,1',1',6',6'-hexadeutero sucrose

The preparation of 6,6,1',1',6',6'-hexadeutero sucrose is reported. The synthesis is based on a triple oxidation of a protected sucrose 6,1',6'-triol to the corresponding 6,1',6'-tricarboxylic acid or ester, followed by reduction with lithium aluminium deuteride. This triple oxidation could be achieved either using cat. TEMPO-NaOCl (to the acid) or PDC-Ac(2)O-t-BuOH (to the t-butyl carboxylic ester).     

Mutagenic and cytotoxic properties of 6-thioguanine, S6-methylthioguanine, and guanine-S6-sulfonic acid

Thiopurine drugs, including 6-thioguanine ((S)G), 6-mercaptopurine, and azathioprine, are widely employed anticancer agents and immunosuppressants. The formation of (S)G nucleotides from the thiopurine prodrugs and their subsequent incorporation into nucleic acids are important for the drugs to exert their cytotoxic effects. (S)G in DNA can be methylated by S-adenosyl-l-methionine to give S(6)-methylthioguanine (S(6)mG) and oxidized by UVA light to render guanine-S(6)-sulfonic acid ((SO3H)G). Here, we constructed single-stranded M13 shuttle vectors carrying a (S)G, S(6)mG, or (SO3H)G at a unique site and allowed the vectors to propagate in wild-type and bypass polymerase-deficient Escherichia coli cells. Analysis of the replication products by using the competitive replication and adduct bypass and a slightly modified restriction enzyme digestion and post-labeling assays revealed that, although none of the three thionucleosides considerably blocked DNA replication in all transfected E. coli cells, both S(6)mG and (SO3H)G were highly mutagenic, which resulted in G-->A mutation at frequencies of 94 and 77%, respectively, in wild-type E. coli cells. Deficiency in bypass polymerases does not result in alteration of mutation frequencies of these two lesions. In contrast to what was found from previous steady-state kinetic analysis, our data demonstrated that 6-thioguanine is mutagenic, with G-->A transition occurring at a frequency of approximately 10%. The mutagenic properties of 6-thioguanine and its derivatives revealed in the present study offered important knowledge about the biological implications of these thionucleosides.     

Coordination features of difunctionalized beta-cyclodextrins with carnosine: ESI-MS and spectroscopic investigations on 6A,6D-di-(beta-alanyl-L-histidine)-6A,6D-dideoxy-beta-cyclodextrin and 6A,6C-di-(beta-alanyl-L-histidine)-6A,6C-dideoxy-beta-cyclodextrin and their copper(II) complexes

The synthesis and characterization of two beta-cyclodextrins (beta-CD) functionalized with two units of carnosine (beta-alanyl-L-histidine) through the amino group, 6A,6C-(beta-alanyl-L-histidine)-6A,6C-dideoxy-beta-cyclodextrin (ACCDAH) and 6A,6D-(beta-alanyl-L-histidine)-6A,6D-dideoxy-beta-cyclodextrin (ADCDAH), are reported. NMR and C.D. data of the ligands indicate a different interaction of dipeptide chains with upper rim and cavity of beta-CD. Analogously, spectroscopic and electrospray ionization mass spectrometry data show different copper(II) complex species formed by the two regioisomers. The ability of carnosine-cyclodextrin derivatives to bind copper ions in a head-to-tail fashion induces the formation of oligomeric species (up to hexamers) in the case of ACCDAH, where the two carnosine moieties are adjacent, while in the ADCDAH case the mutual interaction between the peptidic chains of two ADCDAH molecules allows the almost exclusive formation of a copper-assisted self-assembled dimeric species.     

Site-specific mutagenesis induced by single O6-alkylguanines (O6-n-propyl, O6-n-butyl, O6-n-octyl) in vivo.

The mutagenic activity of a series of longer chain O6-n-alkylguanine residues (O6-n-propyl, O6-n-butyl, O6-n-octyl) has been analyzed using a plasmid molecule (pUC 9) in which single O6-alkylguanines were positioned in the unique Pstl recognition site by shot gun ligation (Nucleic Acids Res. 13, 3305-3316 (1985)) of overlapping synthetic oligonucleotides. After transfection of these vectors into E. coli cells having normal DNA repair systems, progeny plasmids were produced, of which 2.6%, 2.8% and 4.3% were mutated in their Pstl site when containing O6-n-propylguanine, O6-n-butylguanine, O6-n-octylguanine, respectively. DNA sequence analysis of mutant plasmid genomes revealed that O6-n-propylguanine and O6-n-butylguanine induced exclusively G-->A transitions located specifically at the preselected site. O6-n-octylguanine induced apart from G-->A transitions (70%) also targeted G-->T transversions (30%). These results indicate that the mutation frequency of longer chain O6-alkylguanines can be substantial in cells with normal repair systems and that the mutation pattern depends on the nature of the alkyl group.     

Purin-6-yl 6-deoxy-1-thio-beta-D-glucopyranoside. Enzymology, chemistry, and cytotoxicity

Purin-6-yl 6-deoxy-1-thio-beta-D-glucopyranoside (4) is a substrate for almond beta-glucosidase and a weak competitive inhibitor of bovine liver beta-D-glucuronidase (Ki approximately 20mM). Both 4 and purine-protonated 4 undergo hydrolysis catalyzed by dilute acid in the pH range 0.17-2.59. These results are compared with those previously obtained with ammonium (purin-6-yl 1-thio-beta-D-glucopyranosid)uronate, (purin-6-yl 1-thio-beta-D-glucopyranosid)uronamide, purin-6-yl 1-thio-beta-D-glucopyranoside, and purin-6-yl 2-deoxy-1-thio-beta-D-glucopyranoside, and it is concluded that the data support an involvement of substituents at C-5 in producing productive Michaelis-complex conformers. The 6-deoxyglucoside is more active than the D-glucosiduronic acid in an L1210 mouse screen.     

The Rab6-binding kinesin, Rab6-KIFL, is required for cytokinesis

The Rab6-binding kinesin, Rab6-KIFL, was identified in a two-hybrid screen for proteins that interact with Rab6, a small GTPase involved in membrane traffic through the Golgi apparatus. We find that Rab6-KIFL accumulates in mitotic cells where it localizes to the midzone of the spindle during anaphase, and to the cleavage furrow and midbody during telophase. Overexpression of Rab6-KIFL causes a cell division defect resulting in cell death. Microinjection of antibodies to Rab6-KIFL results in the cells becoming binucleate after one cell cycle, and time-lapse microscopy reveals that this is due to a defect in cleavage furrow formation and thus cytokinesis. These data show that endogenous Rab6-KIFL functions in cell division during cleavage furrow formation and cytokinesis, in addition to its previously described role in membrane traffic.     

Enzymatic glycosylation using 6-O-acylated sugar donors and acceptors: beta-N-acetylhexosaminidase-catalysed synthesis of 6-O,N,N'-triacetylchitobiose and 6'-O,N,N'-triacetylchitobiose

p-Nitrophenyl 6-O-acetyl-2-acetamido-2-deoxy-beta-D-glucopyranoside (5a) was used as the glycosyl donor in a beta-N-acetylhexosaminidase-catalysed (from Penicillium brasilianum) glycosylation of GlcNAc yielding 6'-O,N,N'-triacetylchitobiose (6), while 6-O-acetyl-2-acetamido-2-deoxy-beta-D-glucopyranose (3a) served as a selectively protected acceptor in a transglycosylation reaction catalysed by the same enzyme to yield 6-O,N,N'-triacetylchitobiose (4).     

Editorial board page for “Preparative Biochemistry”, Volume 6, Number 6

This is a scanned image of the original Editorial Board page(s) for this issue.