Improvements to solid phase phosphotriester synthesis of deoxyoligonucleotides.

A solution of benzenesulphonic acid (3%, w/v) in a dimethylformamide and dichloromethane mixture (9:1, v/v) is shown to be a very effective reagent for the detritylation of deoxyoligonucleotides attached to a solid support. The levels of depurination with this reagent were lower than those observed with other reagents such as trichloroacetic acid. Coupling reactions are described using above ambient temperatures with no detectable increase in side products. Both procedures have been successfully incorporated into an automated system, which can compete with the rate of synthesis by the phosphite approach.     

Solid phase phosphotriester synthesis of large oligodeoxyribonucleotides on a polyamide support.

Phosphotriester solid phase methodology on a polyamide support [(1980) Nucleic Acids Research, 8, 1081-1096] has been extended for the rapid synthesis of the tetradecanucleotide, d(AGTTGTTTGTAGTT), the octadecanucleotide, d(GTGGGTTTGGGGCAGGTC), and the heneicosanucleotide, d(GTGCTCTTATCCTCTTGGCTC). Thus, oligodeoxyribonucleotides comparable in size to those obtained by solution synthesis are readily accessible using solid phase techniques. An approach to the purification of the synthetic octadecanucleotide without recourse to high performance liquid chromatography is described.     

Syhthesis of peptides by fragment condensation on a solid support. I. Application in preparation of bradykinin.

Two tripeptides and one dipeptide, protected except at the carboxyl ends, have been prepared in solution and used as intermediates in a new synthesis of bradykinin on a solid support. Condensation of the two tripeptides to the resin was effected in satisfactroy yield with dicyclohexylcarbodiimide plus N-hydroxysuccinimide. The partial epimerization that might occur by such an approach was explicitly verified to be without practical importance in this case. The crude product contained only traces of impurities and yielded, after purification, bradykinin of high purity. Both the crude and purified bradykinin exhibited full biological activity.     

The possible role of solid surface area in condensation reactions during chemical evolution: Reevaluation

Summary Published data on adsorption and condensation of amino acids, purine and pyrimidine bases, sugars, nucleosides, and nucleotides are analyzed in connection with Bernal's hypothesis that clays and other minerals may have provided the most likely surface for adsorption and condensation of these molecules in prebiotic times. Using surface concentration and reaction rate as the main criteria for the feasibility of condensation reactions, four types of prebiotic environments were analyzed: (1) an ocean-sediment system, (2) a dehydrated lagoon bed produced by evaporation, (3) the surface of a frozen sediment, and (4) a fluctuating system where hydration (rainstorms, tidal variations, flooding) and dehydration (evaporation) take place in a cyclic manner. With the possible exception of nucleotides, low adsorption of organomonomers on sediment surfaces of a prebiotic ocean (pH 8) is expected, and significant condensation is considered unlikely. In dehydrated and frozen systems, high surface concentrations are probable and condensation is more likely. In fluctuating environments, condensation rates will be enhanced and the size distribution of the oligomers formed during dehydration may be influenced by a redistribution mechanism in which adsorbed oligomers and monomers are desorbed and redistributed on the solid surface during the next hydration-dehydration cycle.On leave from the Faculty of Agriculture, Rehovot, The Hebrew University of Jerusalem, Israel.     

Modeling the kinetics of enzymic reactions in mainly solid reaction mixtures

There is currently considerable interest in using mainly solid reaction mixtures for enzymic catalysis. In these reactions starting materials dissolve into, and product materials crystalize out of, a small amount of liquid phase in which the catalytic reaction occurs. An initial mathematical model for mass transfer effects in such systems is constructed using some physically reasonable approximations. The model equations are solved numerically to determine how the reactant concentrations vary with time and position. To evaluate the extent to which mass transfer limits the overall rate of product formation, an effectiveness factor is defined as the ratio of the observed total reaction rate to the total reaction rate in the reaction limited limit. As expected, the value of the effectiveness factor in steady state is strongly dependent on the Thiele modulus. However, it is also observed that the effectiveness factor can vary widely as a result of changes in the other dimensionless groups characterizing the system. For example, there are situations with Thiele modulus equal to unity in which the value of the effectiveness factor varies between approximately 0.1 and 0.8 as the other parameters are varied in physically reasonable ranges. Analytical asymptotic solutions that provide good approximations to the numerically calculated results in various physically important limiting cases are also presented.     

Catalytically active azoaldolase. Preparation on solid support.

A procedure for the coupling at pH 7.2 of p-carboxy benzene diazonium chloride with rabbit muscle aldolase supported on phosphocellulose is described and some of the spectroscopic, structural and catalytic features of the material obtained are reported. The tetrameric azoenzyme is homogeneous in disc gel electrophoresis even in the presence of 8 M urea. Twelve molecules of the reactant are bound to the protein. Eight azocysteins are identified by both spectroscopic studies and amino acid analysis. The presence of one azohistidine is suggested by the spectroscopic data along with the presence of other, as yet unknown, chromophores. The azoaldolase shows unchanged catalytic properties using both D-fructose 1,6-bisphosphate and D-fructose 1-phosphate as substrates, as compared with the native enzyme. The pH profile of the enzyme activity is broadened towards the alkaline region but no changes occur in the physiological range of pH.     

Synthesis of peptides by fragment condensation on a solid support. II. A scheme for preparation of 4,8-disubstituted vasopressins evaluated on 8-arginine-vasopressin.

A convenient scheme for the synthesis of 4,8-disubstituted vasopressins has been designed and its usefulness evaluated in the preparation of one of the parent hormones, 8-arginine-vasopressin. The main feature of the scheme involves preparation of two protected tripeptide fragments, Z-Cys(Bzl)-Tyr(Bzl)-Phe and Boc-Asn(Mbh)-Cys(Bzl)-Pro which are incorporated in a synthesis on a solid support. Both tripeptides were prepared conventionally with the carboxyl groups protected as benzyl esters. The benzyl-ester groups were removed by transesterification with 2-dimethylaminoethanol and subsequent hydrolysis. To avoid racemization in the coupling step with the fragment containing a C-terminal phenylalanine, N-hydroxysuccinimide was added. After removal of the peptide from the resin, deprotection, oxidation and desalting, final purification was effected by ion-exchange chromatography. Apart from the main product, which exhibited full pressor activity, only small amounts of impurities could be isolated.     

Rapid synthesis of oligodeoxyribonucleotides. IV. Improved solid phase synthesis of oligodeoxyribonucleotides through phosphotriester intermediates.

A phosphotriester solid phase method on a polyamide support has been used to prepare oligodeoxyribonucleotides up to 12 units long. Compared to solid phase phosphodiester synthesis the new methodology is quicker, more flexible and gives 10-60-fold better overall yields.     

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.     

Influence of the microorganism support on the kinetics of anaerobic fermentation of condensation water from thermally concentrated olive mill wastewater

Two materials of different structure, sepiolite and bentonite, evaluated as supports for the microorganisms effecting anaerobic fermentation, behaved differently towards condensation water from thermally concentrated olive mill wastewater from a kinetic point of view. Assuming the overall anaerobic digestion process to conform to first-order kinetics, the apparent kinetic constant for the digester including sepiolite as support was 1.12 day^-1, while that of the digester using the bentonite support was 0.73 day^-1. Thus, the apparent kinetic constant of the process was increased by 35% with the use of sepiolite. The yield coefficient, Y_p/s, was 0.344 and 0.318 litres CH₄ STP/g COD for the sepiolite and bentonite supports respectively.     

Solid phase synthesis of polynucleotides. VIII. Synthesis of mixed oligodeoxyribonucleotides by the phosphotriester solid phase method.

A solid phase method for the simultaneous synthesis of mixed oligonucleotides using a phosphotriester approach has been developed. For this synthesis, a mixture of mono or dimeric coupling units is used, and a slight difference in the reactivity of those units is found. However, this difference does not hamper the simultaneous, mixed oligonucleotide synthesis, and the sequence analysis of a product demonstrates the existence of all desired sequences in the final mixture.     

The enzymatic segment condensation of peptides on a solid phase in organic medium

The segment condensation of peptides on a solid phase (Aminosilochrom) in organic medium catalyzed by a subtilisin complex with sodium dodecylsulfate was studied. The dependence of the efficiency of the enzymatic coupling of tripeptides with the basic structure X-Ala-Ala-Y-OMe [where X = Z, Boc, or Dnp and Y = Leu or Glu(OMe)] on the spacer content on the support and on the structure of the acylating component was investigated. The tripeptide segments were successively coupled to Aminosilochrom containing the Met-Ala-Gly spacer, and the peptidylaminosilochroms Dnp-Ala-Ala-Leu-Ala-Ala-Leu-Ala-Ala-Glu(OMe)-Met-Ala-Gly-A and Dnp-Ala-Ala-Leu-Ala-Ala-Glu(OMe)-Ala-Ala-Leu-Met-Ala-Gly-A (A is the Aminosilochrom residue) were obtained in satisfactory yields. It was shown by these examples that the second and third segments are attached in yields higher than that for the first segment and the coupling efficiency does not depend on the amino acid composition of the acylating component.     

General scheme of the phosphotriester condensation in the oligodeoxyribonucleotide synthesis with arylsulfonyl chlorides and arylsulfonyl azolides

Phosphotriester condensation (RO)(R'O)PO-2 (PDE) + R"OH (RO)(R'O)(R"O)PO (PTE) in the presence of arylsulfonyl chloride (ArSO2Cl) as well as arylsulfonyl azolides proceeds in two steps as revealed by 31P NMR spectroscopy. Pyrophosphotetraester (PPTE) accumulates in over 80% yield in the first step and converts to PTE in the second one. Nucleophilic catalysts of pyridine type (Nu1) are necessary in the first step. The second step is catalyzed by Nu1 as well as by catalysts of the tetrazole type (Nu2H). Base catalysis operates in the latter case. With Nu1 catalysts (pyridine, 4-N,N-dimethylaminopyridine, N-methylimidazole) the general scheme may be presented as follows: ArSO2Cl + Nu1 in equilibrium ArSO2Nu+1 + Cl-; ArSO2Nu+1 + PDE----(RO)(R'O)P(O)OSO2Ar (I); I + Nu+1----(RO)(R'O)P(O)Nu+1 (II); II + PDE in equilibrium [(RO)(R'O)PO]20; II + R"OH----(RO)(R'O)(R"O)PO. Catalysts of Nu2H type don't accelerate PPTE formation. In the second step they participate most probably in the process PPTE + Nu2H in equilibrium (RO)(R'O)P(O)Nu2 (III) + PDE; III + R"OH----(RO)(R'O)(R"O)PO + H+. The latter step is subjected to strong base catalysis.     

Synthesis of ppTppp via phosphotriester intermediates.

The bifunctional and crystalline phosphorylating agent morpholino-0,0-bis[1-benzotriazolyl]phosphate has been used for the preparation of a 3',5'-bis-phosphotriester intermediate of thymidine. The latter has been converted into ppTppp by the following consecutive steps; removal of the benzotriazolyl group followed by the addition of phosphoric acid and removal of the 2-(4-nitrophenyl)-ethyl group followed by the addition of pyrophosphoric acid.