Tris(2,4,6-tribromophenoxy)dichlorophosphorane as a condensing agent: a new type of activation of phosphodiester components in the phosphotriester method

The title compound (BDCP) was found to be effective for the rapid internucleotidic bond formation in the presence of 3-nitro-1,2,4-triazole (NT). Mechanism of the condensation reaction was elucidated by means of 31P-NMR. It was proved that there was no active species but the desired nucleotide derivatives, phosphorochloridate and phosphorylnitrotriazole, in the reaction mixture. Thus, the side-reaction of excess condensing agent in the conventional phosphotriester method was overcome.     

Synthesis of oligonucleotides with sequences identical with or analogous to the 3''-end of 16S ribosomal RNA of Escherichia coli: preparation of A-C-C-U-C-C via the modified phosphotriester method.

A combination of two different methods for the synthesis of oligoribonucleotides, i.e. the two-step phosphotriester method with 2-chlorophenyl phosphate as bifunctional phosphate source and the modified triester method with 2,2,2-trichloroethyl 2-chlorophenyl phosphorochloridate as monofunctional phosphate source, is applied for the synthesis of the fully-protected hexaribonucleotide A-C-C-U-C-C. The two-step method is used for the synthesis of the required dinucleotide monophosphates 9, 10 and 11. Application of the modified triester method for the coupling of the oligonucleotide blocks results in the formation of the fully-protected hexamer 15. Furthermore, attention is paid to 2,4,6-triisopropylbenzenesulphonyl 4-nitroimidazolide as a new condensing agent for the coupling of larger oligonucleotide blocks.     

O-alkyl O-nucleoside 3'-phosphonates as novel starting materials for oligonucleotide synthesis

It was found that the title compounds were activated rapidly into the corresponding highly reactive phosphorochloridites by use of tris(2,4,6-tribromophenoxy)dichloro-phosphorane (BDCP) as a chlorinating reagent. The reaction was applied to the synthesis of oligodeoxyribonucleotides.     

Study on reactivity and protection of the alpha-hydroxyphosphonate moiety in 5'-nucleotide analogues: formation of the 3'-O-P-C(OH)-C4' internucleotide linkage

The recently described epimeric nucleosidyl-5'-C-phosphonates (alpha-hydroxyphosphonates) represent novel nucleotide analogues that can be incorporated into chimeric oligonucleotides by the phosphotriester condensation method. In order to prepare suitable protected monomer(s) we have studied condensation reaction between protected 2'-deoxythymidine and 2'-deoxythymidinyl-5'-C-phosphonate, both as model compounds, in dependence on the nature of the 5'-hydroxyl protecting group. We have found that the O-acetyl group is unstable in the presence of TPSCl or MSNT used as condensing agents for activation of the phosphorus moiety. This instability negatively influences the scope of the condensation process. On the other hand, introduction of the O-methoxycarbonyl group gave excellent results. The O-methoxycarbonyl group does not participate in the condensation process, and its quantitative introduction into the nucleotide analo gues is accomplished using a novel acylating agent, methoxycarbonyl tetrazole.     

The use of barium salts of protected deoxyribonucleoside-3'' p-chlorophenyl phosphates for construction of oligonucleotides by the phosphotriester method: high-yield synthesis of dinucleotide blocks.

A new experimental approach to the synthesis of polydeoxyribonucleotides via the phosphotriester method involves construction of oligonucleotide blocks by direct use of the easily prepared barium salts of O5',N-protected deoxyribonucleoside-3' p-chlorophenyl phosphates as the key monomers in condensation reactions. The procedure has been demonstrated by the rapid synthesis in high yield and purity of all sixteen fuly protected dinucleotides (Formula: see text) (where dN' = dT, dbzC, dbzA, or dibG; (Formula: see text) This set of molecules constitutes a "syllabary" for the preparation of defined sequence oligonucleotides.     

Study on Reactivity and Protection of the α-Hydroxyphosphonate Moiety in 5′-Nucleotide Analogues: Formation of the 3′-O-P-C(OH)-C4′ Internucleotide Linkage

Abstract

The recently described epimeric nucleosidyl-5′-C-phosphonates (α-hydroxyphosphonates) represent novel nucleotide analogues that can be incorporated into chimeric oligonucleotides by the phosphotriester condensation method. In order to prepare suitable protected monomer(s) we have studied condensation reaction between protected 2′-deoxythymidine and 2′-deoxythymidinyl-5′-C-phosphonate, both as model compounds, in dependence on the nature of the 5′-hydroxyl protecting group. We have found that the O-acetyl group is unstable in the presence of TPSCl or MSNT used as condensing agents for activation of the phosphorus moiety. This instability negatively influences the scope of the condensation process. On the other hand, introduction of the O-methoxycarbonyl group gave excellent results. The O-methoxycarbonyl group does not participate in the condensation process, and its quantitative introduction into the nucleotide analogues is accomplished using a novel acylating agent, methoxycarbonyl tetrazole.     

Solid support synthesis of oligothymidylates using phosphorochloridates and 1-alkylimidazoles.

A study of the synthesis of oligothymidylates via phosphotriester intermediates on a polystyrene support is described. The sequence involves condensation of a phenyl nucleoside-3' -phosphorochloridate with the 5'-hydroxyl group of the carrier bound oligonucleotide derivative in the presence of 1-methylimidazole. Conditions for preparation of the phenyl nucleo-side phosphorochloridate as well as for the condensation on the support are discussed. d-TpTpTpT was obtained in 31% overall yield from carrier bound thymidine in one series of experiments, and d-TpTpTpTpT was obtained in 9% yield in another. The cycle for addition of one nucleotide unit can be completed in about six hours.     

Prebiotic condensation reactions in an aqueous medium: A review of condensing agents

Biopolymers are formed by dehydration-type condensation reactions. In aqueous solutions dehydration reactions are very unlikely to happen spontaneously. However, coupling of dehydration-condensation to the hydrolysis of condensing agents could facilitate the synthesis of biopolymers in an aqueous solution. The literature shows that the peptides, nucleosides, nucleotides and oligonucleotides can be formed in this way. A careful study of the literature pertaining to prebiotic condensing agents was conducted in order to determine the most plausible prebiotic synthesis of biopolymers. The condensing agents taken into consideration are cyanamide, carbodiimide, dicyanamide, dicyandiamide, hydrogec-cyanide-tetramer, cyanogen and the linear- and cyclic polyphosphates. From both a chemical as well as biological point of view the polyphosphates appear to be the most plausible general prebiotic condensing agent.     

Highly efficient oligodeoxyribonucleotide synthesis using fully base protected phosphodiester building blocks carrying 2-(1-methylimidazol-2-yl) phenyl protection of the phosphate.

Four fully base protected phosphodiester building blocks have been synthesised and fully characterised. The phosphate protecting group used was the 2-(1-methylimidazol-2-yl)phenyl group, enabling intramolecular catalysis of the condensation step in oligodeoxyribonucleotide synthesis by the solid phase phosphotriester method. Cycle times of about 12 min could thus be achieved. Moreover, the used of extra protecting groups on deoxythymidine and 2'-deoxyguanosine resulted in much cleaner oligodeoxyribonucleotides as evidenced by ion-exchange and reversed phase h.p.l.c.     

Further improvements on the phosphotriester synthesis of deoxyribooligonucleotides and the oligonucleotide directed site-specific mutagenesis of E. coli lipoprotein gene.

Two improvements that greatly enhance the rate of phosphotriester oligonucleotide synthesis are described: 1) use of hindered primary amines, e.g. t-butyl amine for decyanoethylation of oligonucleotide triester intermediates, and 2) a simplified isolation procedure that eliminates the tedious bicarbonate extraction after each condensing reaction. Using the improved procedures, oligonucleotide fragments can be synthesized as rapidly as using solid phase chemistry. The final products are purer than those obtained by solid phase chemistry since each intermediate block is purified by chromatography. The technique has been used to synthesize five oligonucleotide fragments (size 15 to 20) for the purpose of performing guided site-specific mutagenesis on a cloned E. coli lipoprotein gene.     

New effective method for the synthesis of oligonucleotides via phosphotriester intermediates.

A rapid and convenient method for the synthesis of deoxyribooligonucleotides has been developed using the phosphotriester approach. The advantage of this methodology for work in solution was successfully demonstrated in synthesis of a number of DNA fragments up to 32-long. Adaptation of the presented method to solid-phase synthesis allows a pentadecamer to be assembled in 4-5 hours using dinucleotides as coupling units.     

Simplications in the synthesis of short oligonucleotide blocks.

A rapid and convenient procedure has been developed for the synthesis of fully protected mono, di and trideoxyribonucleotides utilizing an aryl phosphoroditriazolide. It affords advantages over coupling strategies employing condensing reagents, such as 2,4,6-triisopropylbenzenesulfonyl tetrazolide in preparing small oligonucleotides and is relatively free of the drawbacks inherent in other approaches using bifunctional phosphorylating reagents. In particular, the synthesis of trinucleotide blocks without purification at the dimer stage is described.     

Improved rapid phosphotriester synthesis of oligodeoxyribonucleotides using oxygen-nucleophilic catalysts.

The use of different condensing and phosphorylating agents in conjunction with oxygen-nucleophilic catalysts, such as 4-substituted derivatives of pyridine N-oxide and quinoline N-oxide, leads to a dramatic increase of the rate of the phosphotriester bond formation and minimizes the amount of by-products caused by the modification of heterocyclic bases. The application of these catalysts to the solid-phase oligonucleotide synthesis allows to reduce the time needed for the performance of one elongation cycle on a polymer support to 10 min.     

Synthesis of the human insulin gene. Part III. Chemical synthesis of 5'-phosphomonoester group containing deoxyribooligonucleotides by the modified phosphotriester method. Its application in the synthesis of seventeen fragments constituting human insulin C-chain DNA.

A method for phosphorylating a protected deoxyribooligonucleotide containing phosphotriester linkages is described. The modified phosphotriester method of chemical synthesis is further refined in terms of (i) better final deblocking conditions and (ii) new chromatography solvent systems containing acetone-water-ethyl acetate to yield pure oligomers. The effectiveness of these improvements has been demonstrated in the rapid and efficient synthesis of seventeen fragments constituting the sequence of human insulin C-chain DNA.     

Silica gel: an improved support for the solid-phase phosphotriester synthesis of oligonucleotides.

The phosphotriester method for the stepwise synthesis of deoxyoligonucleotides has been employed using HPLC-grade silica gel (Porasil B) as the solid support. The procedure results in a convenient flow-through system for the synthesis of oligomers where all the reaction steps including the zinc bromide method of detritylation are compatible with the selected support. Deoxyoligonucleotides of 25-30 nucleotides in length can be synthesized in high yields utilising stable phosphotriester intermediates. Ease of handling of the solid support allows convenient synthesis of mixed oligonucleotide sequences.     

Improved method for the synthesis of phosphatidylcholines

An improved method for the synthesis of phosphatidylcholines from phosphatidic acid and choline is described. The technique utilizes the tetraphenylborate salt of choline together with the condensing agent 2,4,6-triisopropylbenzenesulfonyl chloride. The yields in the reaction are consistently in the range 70-75%.     

A practical synthesis of beta-D-GlcA-(1-->3)-beta-D-Gal-(1-->3)-beta-D-Gal-(1-->4)-D-Xyl,a part of the common linkage region of a glycosaminoglycan

A practical synthesis of beta-D-GlcA-(1-->3)-beta-D-Gal-(1-->3)-beta-D-Gal-(1-->4)-beta-D-Xyl-(1-->OMe) was achieved by coupling of methyl 2,3,4-tri-O-acetyl-alpha-D-glucopyranosyluronate trichloroacetimidate with a trisaccharide acceptor. The trisaccharide acceptor was obtained by condensation of 3-O-allyl-2,4,6-tri-O-benzoyl-beta-D-galactopyranosyl-(1-->3)-2,4,6-tri-O-benzoyl-alpha-D-galactopyranosyl trichloroacetimidate with methyl 2,3-di-O-benzoyl-beta-D-xylopyranoside, followed by deallylation. The beta-(1-->3)-linked disaccharide was prepared readily with p-methoxyphenyl 3-O-allyl-2,4,6-tri-O-benzoyl-beta-D-galactopyranoside as the key synthon. The alpha-(1-->3)-linkage was formed in considerable amount with galactose mono- and disaccharide trichloroacetimidate donors with C-2 neighboring group participation.     

A new condensing reagent, 1-(2,4,6-triisopropylbenzenesulfonyl)-5-(pyridin-2-yl)tetrazolide and its use in the synthesis of lambda cro binding heptadecanucleotide on a polymer support.

A new condensing reagent 1-(2,4,6,-triisopropylbenzenesulfonyl)-5-(pyridin-2-yl)tetrazolide (TPSPy) was found to give one diastereoisomer of dinucleoside monophosphate aryl esters. Several oligodeoxynucleotide blocks were prepared using this reagent. A heptadecanucleotide, dTATCCCTTGCGGTGATA, which had the same sequence as the lambda cro binding DNA sequence was synthesized by condensing mono-, tri- and dodecanucleotide blocks using this reagent on a polystyrene support.     

Synthesis and selected properties of oligonucleotidyl-(Pm----O)-amino acids

N-Cbz-Ser(OMe)-(Pm----O)-d(TpT) was synthesized as a diastereomeric mixture of approx. (1:1) by reacting d(TpT) with 2,4,6-triisopropylbenzenesulfonyl chloride and N-Cbz-Ser-OMe. The phosphoesteric bond of N-Cbz-Ser (OMe)-(Pm----O)-d(TpT) was found to be stable in acid (1 N HCl, 1 h, 37 degrees C), but labile in alkaline solution (0.1-1 N NaOH, 1 h, 37 degrees C). The products of alkaline hydrolysis were determined to be d(TpT) and the amino acid derivatives. Furthermore, the phosphotriester N-Cbz-Ser(OMe)-(Pm----O)-d(TpT) was more labile than the diesteric analogue N-Cbz-Ser (OMe)-pdT. The internucleotide phosphotriester linkage of N-Cbz-Ser (OMe)-(Pm----O)-d(TpT) was also found to be resistant to enzymatic digestion with spleen and snake venom phosphodiesterases.     

Propionyl-CoA condensing enzyme from Ascaris muscle mitochondria. I. Isolation and characterization of multiple forms

The condensation of two propionyl-CoA units or a propionyl-CoA with acetyl-CoA is required for the synthesis of 2-methylvalerate or 2-methylbutyrate, respectively, two of the major fermentation products of Ascaris anaerobic muscle metabolism. An enzyme that preferentially catalyzes the condensation of propionyl-CoA rather than acetyl-CoA has been purified from the mitochondria of the parasitic intestinal nematode Ascaris lumbricoides var. suum. The purified enzyme is over 10 times more active with propionyl-CoA than with acetyl-CoA as substrate. It also catalyzes the coenzyme A-dependent hydrolysis of acetoacetyl-CoA at a rate four times higher than the propionyl-CoA condensation reaction. The purified Ascaris condensing enzyme preferentially forms the 2-methyl-branched-chain keto acids rather than the corresponding straight chain compounds. The native molecular weight of the purified enzyme was estimated to be 160,000 by gel filtration chromatography and 158,000 by high pressure liquid chromatography. The enzyme migrated as a single protein band with Mr 40,000 during sodium dodecyl sulfate-polyacrylamide electrophoresis, indicating that the enzyme is composed of four subunits of the same molecular weight. Chromatography on CM-sephadex resulted in the isolation of two separate peaks of activity, designated as A and B. Both A and B had the same molecular weight and subunit composition. However, they differed in their specific activities and isoelectric points. The pIs of condensing enzymes A and B were 7.6 and 8.4, respectively. Propionyl-CoA was the best substrate for the condensation reaction with both enzymes. However, the specific activity of enzyme B for both propionyl-CoA condensation (3.4 mumol/min/mg protein) and acetoacetyl-CoA thiolysis (13.8 mumol/min/mg protein) was 2.4 times higher than that obtained with enzyme A. Similarly, chromatography on phosphocellulose resolved the Ascaris condensing enzyme activity into one minor and two major peaks. All of these components had the same molecular weight and subunit composition, but differed in their specific activities. The two major phosphocellulose peaks cross-reacted immunologically when examined by the Ouchterlony double immunodiffusion technique. In addition, antiserum against the phosphocellulose most active form cross-reacted with forms A and B isolated by chromatography of the enzyme on CM-Sephadex, indicating that all forms were immunochemically related.