The synthesis and uses of modified oligodeoxynucleotides
The synthesis and uses of modified oligodeoxynucleotides
A brief review of oligodeoxynucleotide synthesis is presented with special reference to the synthesis of modified oligodeoxynucleotides. The uses of 5'-phosphorylated, 5'-thiol containing and phosphorothioate containing oligodeoxynucleotides are discussed. One method for the synthesis of thiol containing oligodeoxynucleotides utilises S-trityl-O-cyanoethyl-N,N-diisopropylphosphityl-3-mercaptopropan-1-ol. Occasionally the trityl group is labile during DNA synthesis producing previously unidentified products. Additionally it has been reported that disulphide formation is the result of iodine detritylation of trityl protected thiols. However, detritylation of trityl protected mercaptopropanol containing oligodeoxynucleotides with iodine results in the formation of several additional products. The major product of iodine detritylation has been found to be the sulphonic acid. A second product produced in high yield is dimeric possibly a thiolsulphonate or thiolsulphinate. The phosphite described above is a viscous oil. Significant advantages would be gained if a solid reagent was available. Described here are attempts to synthesise such a reagent by varying the protecting groups employed for the thiol moiety in attempt to induce crystallinity or make the molecule sufficiently polar to be precipitated from pentane. The syntheses of S-dimethoxytrityl- and S-(9-phenylxanthenyl)- O-cynaoethyl-N,N-diisopropylphosphityl-2-mercaptoethanol are described. These reagents can be used for the 5'-phosphorylation of synthetic oligodeoxynucleotides with one slight modification to the deprotection procedures namely the addition of DTT prior to ammonia deprotection. The protecting group is labile during iodine treatment and therefore compatible with any type of exocyclic amino group protection. The synthesis and resolution of the diastereoisomers of d(GACunderline GATsATCGTC) is described. This oligodeoxynucleotide contains the EcoRV restriction site (underlined) with a phosphorothioate group replacing the scissile phosphodiester bond. The Rp diastereoisomer is a substrate for the enzyme and performing the reaction in H218O results in the formation of a chiral product. In this way the stereochemical course of the EcoRV catalysed hydrolysis of DNA has been monitored and determined to proceed with inversion of configuration at phosphorus. This result implies that the reaction proceeds via direct in-line attack by H2O without the intermediacy of a covalent enzyme intermediate.
University of Southampton
1992
Grasby, Jane Alison
(1992)
The synthesis and uses of modified oligodeoxynucleotides.
University of Southampton, Doctoral Thesis.
Record type:
Thesis
(Doctoral)
Abstract
A brief review of oligodeoxynucleotide synthesis is presented with special reference to the synthesis of modified oligodeoxynucleotides. The uses of 5'-phosphorylated, 5'-thiol containing and phosphorothioate containing oligodeoxynucleotides are discussed. One method for the synthesis of thiol containing oligodeoxynucleotides utilises S-trityl-O-cyanoethyl-N,N-diisopropylphosphityl-3-mercaptopropan-1-ol. Occasionally the trityl group is labile during DNA synthesis producing previously unidentified products. Additionally it has been reported that disulphide formation is the result of iodine detritylation of trityl protected thiols. However, detritylation of trityl protected mercaptopropanol containing oligodeoxynucleotides with iodine results in the formation of several additional products. The major product of iodine detritylation has been found to be the sulphonic acid. A second product produced in high yield is dimeric possibly a thiolsulphonate or thiolsulphinate. The phosphite described above is a viscous oil. Significant advantages would be gained if a solid reagent was available. Described here are attempts to synthesise such a reagent by varying the protecting groups employed for the thiol moiety in attempt to induce crystallinity or make the molecule sufficiently polar to be precipitated from pentane. The syntheses of S-dimethoxytrityl- and S-(9-phenylxanthenyl)- O-cynaoethyl-N,N-diisopropylphosphityl-2-mercaptoethanol are described. These reagents can be used for the 5'-phosphorylation of synthetic oligodeoxynucleotides with one slight modification to the deprotection procedures namely the addition of DTT prior to ammonia deprotection. The protecting group is labile during iodine treatment and therefore compatible with any type of exocyclic amino group protection. The synthesis and resolution of the diastereoisomers of d(GACunderline GATsATCGTC) is described. This oligodeoxynucleotide contains the EcoRV restriction site (underlined) with a phosphorothioate group replacing the scissile phosphodiester bond. The Rp diastereoisomer is a substrate for the enzyme and performing the reaction in H218O results in the formation of a chiral product. In this way the stereochemical course of the EcoRV catalysed hydrolysis of DNA has been monitored and determined to proceed with inversion of configuration at phosphorus. This result implies that the reaction proceeds via direct in-line attack by H2O without the intermediacy of a covalent enzyme intermediate.
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Published date: 1992
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Local EPrints ID: 461268
URI: http://eprints.soton.ac.uk/id/eprint/461268
PURE UUID: 78225e8a-469c-4fcc-b842-0c32dc2f1379
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Date deposited: 04 Jul 2022 18:41
Last modified: 04 Jul 2022 18:41
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Author:
Jane Alison Grasby
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