Biomedical subjects
Publications and source records attributed to R L Letsinger.
Phosphorothioate oligonucleotide analogs conjugated to cholesteryl by a neutral, 6 atom linker are more effective inhibitors of HIV-1 in cell culture than the corresponding analogs conjugated via a phosphorothioate group. The antiviral activity correlates with the hydrophobic character of the oligonucleotide. Some new synthetic methodology is also discussed.
A family of oligonucleotides and phosphorothioate oligonucleotide analogues was synthesized with a cholesteryl group tethered at the 3'-terminal internucleoside link. This modification, introduced to enhance interaction of the polyanions with cell membranes, significantly increases the antiviral activity of the oligomers, as judged by inhibition of syncytia formation and expression of viral proteins p17, p24, and reverse transcriptase for human immunodeficiency virus 1 in Molt-3 cells. In the most favorable case, with a 20-mer cholesteryl-phosphorothioate derivative, complete inhibition by all assays was obtained with an oligomer concentration of 0.2 microM. Even decamers were active, and some antiviral activity was observed for a heptanucleotide cholesteryl-phosphorothioate derivative, which binds very poorly to complementary oligonucleotides. These facts, and the finding that the activity of the phosphorothioate decamers does not correlate with a specific sequence, suggests that a mechanism other than "antisense inhibition" may be operative in these systems.
The interaction of several synthetic analogues of d-ApA with Poly U and Poly dT was examined to explore the effects of substituents at phosphorus on binding properties of oligonucleotides. These analogues contained a bulky, lipophilic group (2,2,2-trichloroethoxy or 2,2,2-trichloro-1,1-dimethylethoxy) a small, uncharged hydrogen-bonding group (amido), or a cationic phosphoramidate (2-aminoethylamido, protonated in neutral aqueous media) in place of the anionic oxygen of the internucleotide phosphate. As determined by "melting curves" each formed a complex with Poly U more stable than the Poly U.d-ApA complex. Binding to Poly dT was comparable or in some cases stronger. Checks on composition (mixing curves) revealed the expected stoichiometry of ldA:2U (or 2dT). Stereochemistry at phosphorus influenced stability of the complexes, but the effect was not a major one. These results suggest that oligonucleotides containing large, lipophilic groups, as well as small non-ionic groups (e.g., the methyl phosphonates) or polar groups, could be useful as probes in hybridization experiments.
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.
A simple procedure is described for synthesis of oligonucleotides by phosphate chemistry. Chains can be constructed rapidly with minimal equipment (a syringe and reagent bottles). The method is illustrated by synthesis of d-TGCAGGTT. Pertinent supporting data on the effect of variations in the detritylation, condensation, oxidation, capping and cleavage steps in the synthetic approach and in isolation procedures are also presented.
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A method for peptide synthesis is proposed based on a template-directed scheme that parallels that of the native ribosomal mechanism. In this procedure, peptide bond formation is facilitated by the juxtaposition of aminoacyl and peptidyl oligonucleotide carriers bound adjacent to one another on an oligonucleotide template. The general strategy of the synthesis and relevant model studies are described. The scheme provides an intrinsic mechanism by which oligonucleotides can direct the synthesis of polypeptides in the absence of protein or ribosomal machinery and, as such, suggests a model for the origin of prebiotic protein synthesis.
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Duplex circular phiX174 DNA (RF I) containing some phosphoramidate links in the backbone chain of the (-) strand was synthesized by reaction of 5'-amino-5'-deoxythymidine 5'-triphosphate, dCTP, dGTP, and 3H-dATP with DNA polymerase I and DNA ligase (T4) on a (+) strand phiX174 amber 3 DNA template. The yield of duplex DNA was higher when dTTP was included along with the amino analog in the initial reaction system or was added late in the synthesis. RF I DNA was observed as a rapidly sedimenting species in an alkaline sucrose gradient, and the presence of phosphoramidate linkages was demonstrated by the unusual lability of the duplex DNA in a weakly acidic solution.
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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.
The stepwise synthesis of oligomers derived from 5'-amino-5'-deoxythymidine 3'-phosphate and 5'-amino-5'-deoxy-3'-O-mono-p-methoxytrityl-thymidine is described. The internucleotide phosphoramidate links were formed by condensation of nucleoside 3'-phosphates with aminonucleosides by means of triphenylphosphine and dipridyl disulfide.
Procedures are described for phosphorylating protected nucleotides, oligonucleotides and phosphoramidate oligonucleotide derivatives at the 3'-hydroxyl group. The conditions (phosphorylation with phosphorus oxychloride and pyridine in dioxane followed by hydrolysis with aqueous pyridine) are sufficiently mild that base labile (trifluoroacetylamino; beta-cyanoethyl phosphotriester) and acid labile (O-monomethoxytrityl; phosphoramidate) functions are retained intact. Application of the technique is illustrated by the synthesis of dpT, dTp, d(CF(3)CONH)Tp, dTp(N)Tp, and dTp(N)Tp(N)Tp. In addition, the utilization of phosphorus oxychloride in joining thymidine derivatives and dinucleoside phosphotriester blocks via phosphodiester links is described.
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