Influence of Diastereomeric Ratios of Deoxyribonucleoside Phosphoramidites on the Synthesis of Phosphorothioate Oligonucleotides

Abstract

Extensive investigations on the influence of diastereomeric ratios of deoxyribonucleoside phosphoramidites on stereo-reproducibility of solid phase synthesis of phosphorothioate oligodeoxyribonucleotides via the phosphoramidite approach indicate that the process is stereoreproducible and under inherent process control.     

Substituent Influence on the Reaction Rate of Deoxyribonucleoside Phosphoramidites with Deoxyribonucleosides Catalysed by Tetrazole

Abstract

The rates of formation of some dinucleoside phosphites 3 from different deoxyribonuclroside phosphoramidites 1 and a common nucleoside 2a (Scheme) have been compared.     

Solution Stability and Degradation Pathway of Deoxyribonucleoside Phosphoramidites in Acetonitrile

The impuritiy profiles of acetonitrile solutions of the four standard O‐cyanoethyl‐N,N‐diisopropyl‐phosphoramidites of 5′‐O‐dimethoxytrityl (DMT) protected deoxyribonucleosides (dG^ib, dA^bz, dC^bz, T) were analyzed by HPLC‐MS. The solution stability of the phosphoramidites decreases in the order T, dC>dA>dG. After five weeks storage under inert gas atmosphere the amidite purity was reduced by 2% (T, dC), 6% (dA), and 39% (dG), respectively. The main degradation pathways involve hydrolysis, elimination of acrylonitrile and autocatalytic acrylonitrile‐induced formation of cyanoethyl phosphonoamidates. Consequently, the rate of degradation is reduced by reducing the water concentration in solution with molecular sieves and by lowering the amidite concentration. Acid‐catalyzed hydrolysis could also be reduced by addition of small amounts of base.     

Synthesis of Deoxyribonucleoside Phosphorodithioate Dimers by a Triester Method

Abstract

Modified oligodeoxynucleotides have recently received much attention due to their therapeutic applications. Among the more promising are phosphorodithioates where both nonbridging oxygen atoms in the phosphate diesters are replced by sulfur. Deoxynucleoside phosphorodithioate dimers have been prepared in several ways, using H-phosphonate, phosphordiamidite, phosphoramidite, and thiophosphoramidite methods. Reports have also appeared on the synthesis of oligonucleotides with alternating phosphodiester and dithiophosphodiester linkages, as well as one on ribonucleoside dimers. Of the above methods, the thiophosphoramidite method has been applied successfully for the preparation of mixed base oligonucleotides containing contiguous phosphorodithioate linkages. However, this method gives products which contain varying amounts of phosphorothioate linkages (2 ? 10%) due to factors associated with the involvement of trivalent thiophosphorus compounds. In addition, the thiophosphoramidite synthons are difficult to purify on silica gel column, and have a tendency to dismutate in presence of acidic catalysts such as tetrazole. The thiophosphite intermediate which is formed is also unstable to tetrazole. Similarly in the thio- and dithio-H-phosphonate method, the primary coupling products are unstable to catalysts, pivaloyl chloride and iodine. Recently, Dahl et al reported^1–2 synthesis of dimers and oligomers upto octamer which also leads to formation of small amounts of phosphorothioate linkages. In additon, about 1.2% per phosphorodithioate linkage of the oligomer is cleaved during     

Efficient Synthesis of Simvastatin by Use of Whole-Cell Biocatalysis

Simvastatin is a semisynthetic derivative of the fungal polyketide lovastatin and is an important drug for lowering cholesterol levels in adults. We have developed a one-step, whole-cell biocatalytic process for the synthesis of simvastatin from monacolin J. By using an Escherichia coli strain overexpressing the previously discovered acyltransferase LovD (X. Xie, K. Watanabe, W. A. Wojcicki, C. C. Wang, and Y. Tang, Chem. Biol. 13:1161-1169, 2006), we were able to achieve >99% conversion of monacolin J to simvastatin without the use of any chemical protection steps. The key finding was a membrane-permeable substrate, α-dimethylbutyryl-S-methyl-mercaptopropionate, that was efficiently utilized by LovD as the acyl donor. The process was scaled up for gram-scale synthesis of simvastatin. We also demonstrated that simvastatin synthesized via this method can be readily purified from the fermentation broth with >90% recovery and >98% purity as determined by high-performance liquid chromatography. Bioconversion using high-cell-density, fed-batch fermentation was also examined. The whole-cell biocatalysis can therefore be an attractive alternative to currently used multistep semisynthetic transformations.     

Synthesis of 5′-C-Branched Thymidines and Conversion to Phosphoramidites

Abstract

Thymidine was converted to its 5′-epoxy derivative, which was reacted with nucleophiles to give 5′-C-aminomethyl-, 5′-C-bromomethyl-, 5′-C-cyanomethyl, and 5′-C-methoxymethylthymidine derivatives with defined stereochemistry. 5′-C-ally-, 5′-C-hydroxymethyl-, 5′-C-hydroxypropyl-, and 5′-C-(imidazole-4-acetamido)methyl-thymidine derivatives were also prepared. The 5′-C-branched thymidines were converted to the corresponding phosphoramidites.     

Synthesis and Structural Analysis of Oxadiazole Carboxamide Deoxyribonucleoside Analogs

Two novel C-linked oxadiazole carboxamide nucleosides 5-(2′-deoxy-3′,5′-β-D-erythro-pentofuranosyl)-1,2,4-oxadiazole-5-carboxamide (1) and 5-(2′-deoxy-3′,5′-β-D-erythro-pentofuranosyl)-1,2,4-oxadiazole-3-carboxamide (2) were successfully synthesized and characterized by X-ray crystallography. The crystallographic analysis shows that both unnatural nucleoside analogs 1 and 2 adapt the C2′-endo (“south”) conformation. The orientation of the oxadiazole carboxamide nucleobase moiety was determined as anti (conformer A) and high anti (conformer B) in the case of the nucleoside analog 1 whereas the syn conformation is adapted by the unnatural nucleoside 2. Furthermore, nucleoside analogs 1 and 2 were converted with high efficiency to corresponding nucleoside triphosphates through the combination chemo-enzymatic approach. Oxadiazole carboxamide deoxyribonucleoside analogs represent valuable tools to study DNA polymerase recognition, fidelity of nucleotide incorporation, and extension.

     

Improved Synthesis of Trinucleotide Phosphoramidites and Generation of Randomized Oligonucleotide Libraries

A new method to produce a set of 20 high quality trinucleotide phosphoramidites on a 5–10 g scale each was developed. The procedure starts with condensation reactions of P-components with N-acyl nucleosides, bearing the 3 ′-hydroxyl function protected with 2-azidomethylbenzoyl, to give fully protected dinucleoside phosphates 13. Upon cleavage of dimethoxytrityl group from 13, dinucleoside phosphates 16 are initially transformed into trinucleoside diphosphates 19 and then the 2-azidomethylbenzoyl is selectively removed under neutral conditions to generate trinucleoside diphosphates 5 in excellent yield. Subsequent 3 ′-phosphitylation affords target trinucleotide phosphoramidites 7. When mutagenic oligonucleotides are synthesized employing mixtures of building blocks 7 as well as following the new synthetic protocol, representative oligonucleotide libraries are generated in good yields.     

Synthesis and NMR Spectra of Some New Carbohydrate Modified Uridine Phosphoramidites

Abstract

The one step reaction of 2′- and 3′-keto derivatives of uridine with bromodifluoromethyl[tris(dimethylamino)]phosphonium bromide and zinc gives the corresponding 2′- and 3′-difluoromethylene nucleosides in good yield. Desilylation and phosphitylation of the resultant 2′- or 3′-hydroxyls provides the target 2′- and 3′-phosphoramidites 7 and 8 for use in oligonucleotide synthesis¹.     

An Efficient System for Production of Recombinant Urokinase-Type Plasminogen Activator

A system was developed to produce recombinant urokinase-type plasminogen activator inEscherichia coli.The urokinase-type plasminogen activator was produced with a 6× His-tag at the C-terminus which was shown to have the same activity, after refolding, as the wild-type protein. Purification of the recombinant protein to homogeneity (95%) was possible by one-step affinity chromatography under denaturing conditions. As a result, proteolysis by bacterial proteases during purification was avoided. A higher refolding efficiency (40%) and a higher total recovery yield (25%) of the recombinant protein were obtained by this method.     

Synthesis of Nucleosides with Additional Nucleobases

The syntheses of two nucleosides with additional nucleobases in the 2′-position are presented. The nucleosides have two- and one-carbon linkers to the additional nucleobase, respectively. The two nucleosides are synthesized from different strategies. The nucleoside with two carbons in the linker has been incorporated into oligonucleotides and showed stabilization of a tree-way junction.     

Phosphoramidites of [180] Chiral (Rp)- and (Sp)-Configurated Dimer-Blocks and their Use in Automated Oligonucleotide Synthesis

Abstract

The N,N-diisopropylphosphoramidites 1 and 2 of appropriately protected chiral diastereoisomers of d(T-[P-180]-A) (6a and 6o, resp.) have been synthesized. They were employed in solid-phase synthesis to yield the octamers d(GAGT-(Rp)-[P180]-ACTC) and d(GAGT-(Sp)-[P180]-ACTC).     

N-6-Dialkylformamidine-2′-deoxyadenosine Phosphoramidites in Oligodeoxynucleotide Synthesis Raped Deprotection of Oligodeoxynucleotides

Abstract

A series of dialkylformamidine protected deoxyadenosine phosphoramidites were prepared for automated, solid-support DNA synthesis. The set of A^bz, G^dmf, C^bz, T phosphoramidites gave high purity and high yield oligodeoxynucleotides, with complete deprotection at 65 °C in one hour. Different times and temperatures of exposure to concentrated ammonium hydroxide were examined to establish the optimum conditions for deprotection of oligodeoxynucleotides.     

Synthesis of Fully Protected Nucleoside- Folic Acid Conjugated Phosphoramidites and Their Incorporation into Antisense Oligonuleotides

Abstract

A novel synthesis of the nucleoside-folic acid conjugates has been accomplished. This approach allowed us to synthesize several analogs, which were converted to phosphoramidites and successfully incorporated into therapeutically active antisense oligonucleotides.     

A New Approach to the Synthesis of Trinucleotide Phosphoramidites - Synthons for the Generation of Oligonucleotide/Peptide Libraries

Abstract

Trinucleotide phosphoramidites that correspond to the codons of all 20 amino acids were synthesized in high yield in 5g scale. Precursors of those amidites - trinucleotide phosphotriesters - have been prepared using the phosphotriester approach without protection of the 3′-hydroxyl function. More than 10 oligonucleotides up to 90 bases long have been synthesized by a phosphite-triester approach using new synthons. The 67-mer (12 random codons) has been used to generate a display library of 2 × 10⁸ complexity.     

大肠杆菌高效合成L-苯甘氨酸的研究进展

自然界存在着多种氨基酸,除用于蛋白质合成的20种外,大量用于合成具有生物活性的物质,广泛应用于食品、医药等多个领域.其中,非天然芳香族氨基酸L-苯甘氨酸作为一种重要的组成单元广泛的应用于盘尼西林、维吉霉素S、原始霉素Ⅰ等β-内酰胺类抗生素的生物合成当中.目前L苯甘氨酸主要通过化学法合成,但该方法合成收率低、污染大,且不...     

Synthesis of Urokinase-Type Plasminogen Activator and of Type- 1 Plasminogen Activator Inhibitor in Neuronal Cultures of Human Fetal Brain: Stimulation by Phorbol Ester

Human neuronal brain cultures established from 12- and 14-week-old fetuses synthesize and secrete urokinase-type plasminogen activator (uPA) and limited amounts of tissue-type plasminogen activator (tPA). These cells also produce and secrete the endothelial cell-type PA inhibitor (PAI-1), which forms sodium dodecyl sulfate-stable tPA/PAI-1 complexes in the culture medium. Immunocytochemistry shows a predominant localization of uPA, tPA, and PAI-1 in neuronal cells, with only a very weak positivity detectable in the few glial cells present in these cultures. The protein kinase C (PKC) activator 12-O-tetradecanoylphorbol 13-acetate (TPA) stimulates the synthesis of both uPA and PAI-1, resulting in a final increase in the plasmin-generating capacity of neuronal cell cultures. No significant effect is observed, however, when cells are treated with the TPA analogue 4 alpha-phorbol 12,13-didecanoate, which is inactive as a PKC inducer, or with the neurotrophic polypeptide basic fibroblast growth factor. These data represent the first characterization of the plasmin-generating system in human fetal brain neurons and suggest a role for PKC in the modulation of uPA and PAI-1 synthesis.     

Synthesis of 6-Aza- & 6-Methyl-pyrimidine Ribonucleoside Phosphoramidites and Their Incorporation in Hammerhead Ribozymes

Abstract

The synthesis of phosphoramidites of 6-modified pyrimidine ribonucleosides and their incorporation into hammerhead ribozymes and influence on nuclease stability and catalytic activity is described.