A simple procedure for synthesis of diastereoisomers of thymidine cyclic 3',5'-phosphate derivatives

Diastereoisomeric thymidine cyclic (3',5')-methanephosphonates (3a), cyclic (3',5')-phosphoranilidates (3b) and cyclic (3',5')-phosphoranilidothioates (3c) were prepared by treatment of diastereoisomerically pure thymidine 3'-O-[O-(4-nitrophenyl)methanephosphonates] (2a), 3'-O-[O-(4-nitrophenyl)phosphoranilidates] (2b) or 3'-O-[O-(4-nitrophenyl)phosphoranilidothioates] (2c), respectively, with sodium hydroxide in dioxane-water solution.     

Attempted stereoselective synthesis of P-chiral analogues of oligodeoxyribonucleotides

Reaction of diastereomeric 5'MMT-nucleoside 3'-O-(4-nitrophenylmethanephosphate) or 3'-O-[O-(4-nitrophenyl)-S-(2-nitrobenzyl)phosphorothioate] with tBuMgCl-activated, 3'-protected nucleoside is stereospecific and leads, after appropriate work-up, to dinucleoside-3',5'-methanephosphonate or dinucleoside-3',5'-phosphorothioate, respectively. This procedure extended to 5'-activated nucleotides, allows to prepare short, stereoregular oligonucleotide analogues bearing at internucleotide positions methanephosphonate or phosphorothioate function of predetermined sense of chirality at P-centers.     

Synthesis and activity against HBV of novel Tetra-seconucleoside analogues of dyphlline having the acyclic chains of ACV and HBG

Selective alkylation of dyphylline (1) with (2-acetoxyethoxy)methyl bromide (2a) or 4-acetoxybutyl bromide (2b) afforded 3'-O-[(acetoxyethoxy)methyl]dyphylline (3a) and 3'-O-(4-acetoxybutyl)-dyphylline (3b), respectively. A trans esterification process rather than alkylation of the dihydroxy-propyl side chain in 1 had taken place during the reaction with 2-p-toluoyloxy)ethyl chloride (5) to afford the respective 3'-toluoyloxy derivative 7 and not the anticipated 3'-O-[(p-toluoyloxy)ethyl]-dyphylline (6). Deacylation of 3a,b and 7 afforded 4a,b and 1, respectively. Viral screening of selected compounds against HBV has been investigated.     

Preparation and characterization of NADP derivatives alkylated at 2'-phosphate and 6-amino groups

Reaction of NADP with 3-propiolactone at pH 6 gave new NADP derivatives carboxyethylated at the 2'-phosphate or 6-amino group, or both: 2'-O-(2-carboxyethyl)phosphono-NAD (I), N6-(2-carboxyethyl)-NADP (II), and 2'-O-(2-carboxyethyl)phosphono-N6-(2-carboxyethyl)-NAD (III). Their structures were assigned on the basis of ultraviolet, 1H-NMR and 31P-NMR spectra, and also treatment with nucleotide pyrophosphatase or alkaline phosphatase. Carbodiimide-promoted reaction of derivative I with 1,2-diaminoethane gave 2'-O-[N-(2-aminoethyl)carbamoylethyl]phosphono-NAD (IV); derivative III gave 2'-O-[N-(2-aminoethyl)carbamoylethyl]phosphono-N6-[N-(2-aminoethyl ) carbamoylethyl]-NAD (IV). The same reaction of derivative II, on the other hand, gave a mixture of N6-[N-(2-aminoethyl)carbamoylethyl]-NADP (Va) and its 3'-phosphate isomer (Vb). The mixture was converted to Va via the 2',3'-cyclic derivative (Vc). Their structures were assigned on the basis of ultraviolet and 1H-NMR spectra, and also treatment with alkaline phosphatase or 3'-nucleotidase. All the NADP derivatives obtained in this work could be reduced with yeast glucose-6-phosphate dehydrogenase.     

Synthesis of modified nucleotide building blocks containing electrophilic groups in the 2'-position

Chemical syntheses of 2'-O-(allyloxycarbonyl)methyladenosine, 2'-O-(methoxycarbonyl)methyladenosine and 2'-O-(2,3-dibenzoyloxy)propyluridine 3'-2-cyanoethyl-N,N-diisopropyl phosphoramidite building blocks are described. These monomers were used successfully to incorporate carboxylic acid, 1,2-diol and aldehyde functionalities into synthetic oligonucleotides.     

Galloyl, caffeoyl and hexahydroxydiphenoyl esters of dihydrochalcone glucosides from Balanophora tobiracola

Seven galloyl, caffeoyl and (S)-hexahydroxydiphenoyl (HHDP) esters of dihydrochalcone glucosides were isolated from Balanophora tobiracola; based on spectroscopic and chemical evidence, their structures were determined to be 6'-O-galloyl-, 3',4'-di-O-galloyl-, 4',6'-di-O-galloyl-, 4',6'-O-(S)-HHDP-, 3'-O-galloyl-4',6'-O-(S)-HHDP-, 3'-O-caffeoyl-4',6'-O-(S)-HHDP-3-hydroxyphloretin 4'-O-beta-D-glucosides and 3'-O-galloyl-4',6'-O-(S)-HHDP-phloretin 4'-O-beta-D-glucoside, respectively. By contrast, these compounds were not found in the taxonomically related B. japonica. The 3'-galloyl-4',6'-HHDP esters of the dihydrochalcone glucosides showed strong inhibitory activities against alpha-glucosidase. Four known compounds were also isolated namely, (+/-)-eriodictyol 7-O-beta-D-glucoside, 1-O-caffeoyl-3-O-galloyl-beta-D-glucose, phloretin 4'-O-beta-D-glucoside, and 3-hydroxyphloretin 4'-O-beta-D-glucoside.     

Di(azacrown) conjugates of 2'-O-methyl oligoribonucleotides as sequence-selective artificial ribonucleases

Functionalized 2'-O-methyl oligoribonucleotides bearing two 3-(3-hydroxypropyl)-1,5,9-triazacyclododecane ligands attached via a phosphodiester linkage to a single non-nucleosidic building block have been prepared on a solid-support by conventional phosphoramidite chemistry. The branching units employed for the purpose include 2,2-bis(3-hydroxypropylaminocarbonyl)propane-1,3-diol, 2-hydroxyethyl 3'-O-(2-hydroxyethyl)-beta-D-ribofuranoside, and 2-hydroxyethyl 2'-O-(2-hydroxyethyl)-beta-D-ribofuranoside. Each of these has been introduced as a phosphoramidite reagent either into the penultimate 3'-terminal site or in the middle of the oligonucleotide chain. The dinuclear Zn2+ complexes of these conjugates have been shown to exhibit enhanced catalytic activity over their monofunctionalized counterpart, the 3'-terminal conjugate derived from 2-hydroxyethyl 3'-O-(2-hydroxyethyl)-beta-D-ribofuranoside being the most efficient cleaving agent. This conjugate cleaves an oligoribonucleotide target at a single phosphodiester bond and shows turnover and 1000-fold cleaving activity compared to the free monomeric Zn2+ chelate of 1,5,9-triazacyclododecane.     

Separation of [Sp]-3'-O-(5'-dimethoxytritylthymidyl)-5'-O-(3'camphanoyl thymidyl)-O-methyl phosphite.

First isolation of diastereoisomerically enriched (d.p. 95%) [Sp]-3'-O-(5'-dimethoxytrityl tjymidyl)-5'-O-(3'-camphanoyl thymidyl)-O-methyl phosphite (1) allowed to assign, via sulfuration and deprotection, its absolute configuration as Sp. Its reactions with methyl iodide, dimethoxytrityl chloride, or water, respectively, allowed to confirm correctness of assignment of absolute configuration in diastereisomers of TPMeT, and to assign absolute configuration in corresponding diastereoisomers of TPDMTT and TPHT.     

Flavonoids and phenylpropanoid derivatives from Campanula barbata

Four new phenylpropanoid derivatives, barbatosides A-D, and a new catechin, barbatoflavan, were isolated from the whole plant of Campanula barbata L. (Campanulaceae) and identified as wahlenbergioside-3'-O-glucoside, wahlenbergioside-3'-O-(2'-(p-methoxycinnamoyl))-glucoside, wahlenbergioside-3'-O-(4'-(trans-p-coumaroyl))-glucoside, wahlenbergioside-3'-O-(4"'-(cis-p-coumaroyl))-glucoside and 3-acetyl-5-methoxy-7,3',4'-trihydroxy-8-O-glucoside-flavan-3-ol, respectively, by spectroscopic methods. In addition, four flavonols were isolated and identified as kaempferol-3-O-glucoside, kaempferol-3-O-rutinoside, quercetin-3-O-glucoside and quercetin-3-O-rutinoside. Barbatoflavan demonstrated scavenging properties towards the DPPH radical.     

An approach to the stereoselective synthesis of Sp-dinucleoside phosphorothioates using phosphotriester chemistry.

An approach to the stereoselective synthesis of Sp- dinucleoside phosphorothioates has been investigated which utilizes phosphotriester chemistry. The stereoselectivity of internucleotide bond formation between N4-benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxycytidine-3'-O-(S2-cyano-e thyl) phosphorothioate (3) and 3'-O-acetylthymidine has been studied using either mesitylenesulphonyl-5-(pyridin-2-yl)tetrazole (MSPy) or 1-mesitylenesulphonyl-3-nitro-1,2,4-triazole (MSNT) as the activating agent. The removal of the cyanoethyl group from the protected dinucleoside phosphorothioate has been studied, and conditions are reported which provide rapid deprotection without concomittant desulphurisation.     

Synthesis and properties of oligonucleotides bearing a pendant pyrene group

Two hexathymidine oligonucleotide derivatives bearing a pyrenylbutyl substituent at a designated internucleotide phosphorus were prepared by solid phase syntheses using 5'-O-(di-p-methoxytrityl)thymidyl-3'-4-(1-pyrenyl)butyl phosphorochloridite and 5'-O-(di-p-methoxytrityl)thymidyl-3'2,2,2-trichloro-1,1-dimethylet hyl phosphorochloridite as phosphitylating agents. Spectrophotometric studies showed that these oligomers bind to Poly A, but not to Poly C, Poly G, or Poly U, and that the complexes with Poly A are somewhat more stable than the duplex formed between hexathymidine pentaphosphate and Poly A.     

Activation of 5-lipoxygenase by guanosine 5'-O-(3-thiotriphosphate) and other nucleoside phosphorothioates: redox properties of thionucleotide analogs

The stable nucleotide analog guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) was found to be a very potent activator of 5-lipoxygenase in cell-free preparations from rat polymorphonuclear (PMN) leukocytes, causing a 10-fold stimulation of arachidonic acid oxidation at concentrations as low as 0.5-1 microM. The enhancement of enzyme activity was not directly related to G protein activation since the effect of GTP gamma S could not be abolished by GDP nor replaced by GTP or guanylyl-imidodiphosphate (up to 100 microM). Furthermore, other phosphorothioate analogs, such as guanosine 5'-O-(2-thiodiphosphate), adenosine 5'-O-(3-thiotriphosphate), adenosine 5'-O-(2-thiodiphosphate), and adenosine 5'-O-thiomonophosphate all stimulated 5-lipoxygenase activity at concentrations of 10 microM or lower. This effect could not be detected with any of the corresponding nucleoside phosphate derivatives. The stimulation of 5-lipoxygenase activity by nucleoside phosphorothioates was observed under conditions where the reaction is highly dependent on exogenous hydroperoxides, such as in the presence of beta-mercaptoethanol or using enzyme preparations pretreated with sodium borohydride or glutathione peroxidase. GTP gamma S stimulated arachidonic acid oxidation by 5-lipoxygenase to the same extent as the activating hydroperoxides but had no effect on the reaction measured in the presence of optimal concentrations of 13-hydroperoxyoctadecadienoic acid (1-5 microM). Finally, sodium thiophosphate, but not sodium phosphate, markedly stimulated 5-lipoxygenase activity with properties similar to those of GTP gamma S. These results indicate that GTP gamma S and other phosphorothioate derivatives have redox properties that can contribute to increase 5-lipoxygenase activity by replacing the effect of hydroperoxides.     

The use of nucleotide phosphorothioate diastereomers to define the structure of metal-nucleotide bound to GTP-AMP and ATP-AMP phosphotransferases from beef-heart mitochondria

The diastereomers of adenosine 5'-O-[1-thio]triphosphate (ATP[alpha S]) and adenosine 5'-O-[2-thio]triphosphate (ATP[beta S]) were utilized to seek unambiguous assignment of Mg2+ coordination to ATP when bound to ATP-AMP phosphotransferase from beef heart mitochondria (AK2). Similarly, the diastereomers of guanosine 5'-O-[thio]triphosphate (GTP[alpha S]) and guanosine 5'-O-[2-thio]triphosphate (GTP[beta S]) were utilized to seek unambiguous assignment of Mg2+ coordination to GTP when bound to GTP-AMP phosphotransferase from beef heart mitochondria (AK3). Furthermore the diastereomers of guanosine 5'-O-[1-thio]diphosphate (GDP-[alpha S]) have been used to assign Mg2+ coordination to GDP when bound to AK3. The ratios (V for isomer Sp)/(V for isomer Rp) obtained in the presence of Mg2+ and Cd2+ are compared to those already published for ATP-AMP phosphotransferases from pig muscle (AK1) [Kalbitzer et al. (1983) Eur. J. Biochem. 133, 221-227] and from baker's yeast (AKy) [Tomasselli and Noda (1983) Eur. J. Biochem. 132, 109-115]. In all cases, coordination of Mg2+ to the beta-phosphate via the pro-R oxygen is present, as shown by reversal of specificity for the diastereomers of ATP [beta S] or GTP [beta S] respectively on changing the metal ion. In contrast, there is no reversal of specificity for the diastereomers of ATP [alpha S] or GTP[alpha S], or for GDP[alpha S] in the case of AK3 for the reverse reaction, indicating that there is no interaction of the metal with the alpha-phosphate group. The observed stereospecificity for the alpha-thiophosphate is consistent with the assumption of an interaction of the pro-R oxygen of the alpha-phosphate group with the enzyme.     

Synthesis of RNA containing inosine: analysis of the sequence requirements for the 5'' splice site of the Tetrahymena group I intron.

Two protected derivatives of the ribonucleoside inosine have been prepared to serve as building blocks for phosphoramidite-based synthesis of RNA. Two different synthetic routes address the unusual solubility characteristics of inosine and its derivatives. The final products of the different synthetic pathways, 5'-O-(dimethoxytrityl)-2'-O-(t-butyldimethylsiyl) inosine 3'-O-(beta-cyanoethyldiisopropylamino) phosphoramidite 5a, and O6-p-nitrophenylethyl-5'-O-(dimethoxytrityl)-2'-O-(t-butyldimethylsilyl) inosine 3'-O-(methyldiisopropylamino) phosphoramidite 5b, were chemically incorporated into short oligoribonucleotides which also contained the four standard ribonucleoside bases. The oligomers were chosen to study base-specific interactions between an RNA substrate and an RNA enzyme derived from the Group I Tetrahymena self-splicing intron. The oligomers were shown to be biochemically competent using a trans cleavage assay with the modified Tetrahymena intron. The results confirm the dependence of the catalytic activity on a wobble base pair, rather than a Watson-Crick base pair, in the helix at the 5'-splice site. Furthermore, comparison of guanosine and inosine in a wobble base pair allows one to assess the importance of the guanine 2-amino group for biological activity. The preparation of the inosine phosphoramidites adds to the repertoire of base analogues available for the study of RNA catalysis and RNA-protein interactions.     

Formation of 3'-O-beta-galactosyl compounds of 5-bromouridine by Sporobolomyces singularis

Two new compounds of 5-bromouridine, 3'-O-(beta-D-galactopyranosyl)-5-bromouridine and 3'-O-[beta-D-galactopyranosyl-(1-->4)-beta-D-galactopyranosyl]-5-bromouridine, were found to be selectively formed in a high yield in a culture filtrate of Sporobolomyces singularis, when grown on a medium containing lactose and 5-bromouridine.     

Process development for the synthesis of 5'-O-(4,4'-dimethoxytrityl)-N2-isobutyryl-2'-O-(2-methoxyethyl)-guanosine--a potential precursor for the second generation antisense oligonucleotides: an improved process for the preparation of 2'-O-alkyl-2,6-diaminopurine riboside

An efficient four step process for the preparation of 5'-O-(4,4'-dimethoxytrityl)-N2-isobutyryl-2'-O-(2-methoxyethyl)-guanosine 1 was developed. Direct 2'-O-alkylation of 2,6-diaminopurine riboside 2 was accomplished via inexpensive and commercially available reagents such as KOH, DMSO and alkyl halides at room temperature in 4-6 hrs. Pure 2'-O-(2-methoxyethyl)-DAPR 3 was isolated by crystallization from methanol. Enzymatic deamination of 3 followed by selective N2-isobutyrylation and 5'-O-dimethoxytritylation furnished desired 1 in high yield and purity. Fully optimized four step synthetic process has been scaled up to the pilot plant level.     

Conformationally locked nucleosides. Synthesis of oligodeoxynucleotides containing 3'-amino-3'-deoxy-3'-N,5'(R)-C-ethylenethymidine

3'-Amino-3'-deoxy-5'-O-(4,4'-dimethoxytrityl)-3'-N,5'(R)-C-ethylenethymidine (6) was synthesized starting from 3'-azido-3'-deoxythymidine. Condensation of 6 with 5'O-(H-phosphonyl)thymidine and 5'-O-(p-nitrophenoxycarbonyl)thymidine derivatives gave dinucleotide and dinucleoside derivatives, respectively, which were incorporated into oligodeoxynucleotides (ODNs). Tm data of the modified ODNs are also presented.     

Chromatographic investigation on acyl migration in betacyanins and their decarboxylated derivatives

Chemopreventive and antioxidant action of betalain pigments can differ in dependence on their stereoselective properties, therefore, it is necessary to use relevant methods for monitoring of their possible stereoisomers. Chromatographic characterisation of a group of new isomers of various 6'-O-acylated betacyanins and decarboxylated betacyanins which were generated at low concentration by intramolecular pH-dependent acyl migration was studied in aqueous solutions by HPLC separation with diode-array and mass spectrometric detection. Under alkaline conditions (pH 10.5) the rate of migration was dramatically accelerated, however, always favouring the 6'-O-position and it was much less prominent at lower pH (under 7.0). The possible products of the partial rearrangement were tentatively identified as the 3'-O- and 4'-O-acylated forms and their relative retention times were provided. In malonylated betacyanins and 17-decarboxy-betacyanins the 4'-O-forms were characterised in RP-HPLC by higher retention than the 6'-O forms, whereas the 3'-O-forms were always the most polar. In contrast, the isomerisation of hylocerenin and 17-decarboxy-hylocerenin resulted in different chromatographic profiles of the migration products. In 2-decarboxy- and 2,17-bidecarboxy-betacyanins the 3'-O- and 4'-O-acylated forms eluted always before the 6'-O-acylated betacyanins. The investigations on acyl migration in isolated 4'-O-malonyl-betanin confirmed the strong tendency of reverse acyl migration (4'-->6') and also partial 4'-->3' rearrangement which were leading to the final monoester regioisomeric distribution (%) close to 87:7:6 (6'-O-, 4'-O-, 3'-O-).     

Synthesis and properties of 5'-triphosphoryl 2'-5' oligoadenylates (2-5A), and a general method for synthesis of 3', 5'-bisphosphorylated oligonucleotides.

2'-5'-Linked oligoadenylic acid 5'-triphosphates (2-5A) having chain lengths of 2-4 have been synthesized by polymerization of 3'-O-(o-nitrobenzyl)-N-benzoyladenosine 5'-phosphate followed by 5'-triphosphorylation via the imidazolidates. A large scale preparation of 5'-O-phosphoryladenylyl-(2'-5')-adenylyl-(2'-5')-adenosine was performed by the phosphotriester method using 5'-O-monomethoxytrityl-3'-O-(o-nitrobenzyl)-N-benzoyladenosine 2'-O-p-chlorophenylphosphate and 5'-O-phosphorodianilido-3'-O-(o-nitrobenzyl)-N-benzoyladenosine 2'-O-p-chlorophenylphosphate as intermediates. The trimer was also triphosphorylated by the imidazolide method. CD spectra for 5'-mono and triphosphorylated 2'-5' adenylates were measured as well as their UV hypochromicities. This triester method was also applied to the synthesis of 3',5'-bisphosphorylated protected oligoadenylic acids with natural 3'-5' linkages which could be used for further condensations to yield 5'-phosphorylated polynucleotides.     

Synthesis and characteristics of modified oligodeoxyribonucleotides containing 2'-O-(2,3-dihydroxypropyl)uridine and 2'-O-(2-exoethyl)uridine

A new uridine derivative, 2'-O-(2,3-dihydroxypropyl)uridine, and its 3'-phosphoramidite were obtained for direct introduction into oligonucleotides during automated chemical synthesis. Oligonucleotides 10 to 15 nt long harboring 2'-O-(2,3-dihydroxypropyl)uridine residues were synthesized; periodate oxidation of these oligomers gave oligonucleotides containing 2'-O-(2-oxoethyl)uridine residues. The presence of a reactive aldehyde group in 2' position of the carbohydrate moiety was confirmed by the interaction with p-nitrophenylhydrazine and methionine methyl ester. The thermostability of DNA duplexes containing modified units is practically indistinguishable from that of the natural analogues.