Synthesis of Hydrophobically Modified Nucleotide Analogs and their Incorporation into Oligonucleotides for Enhanced Chromatographic Separations
Creators: name: Shukla, Brahmi
Publisher: name: Maryland Shared Open Access Repository
Year: 2013

description: Lipophilic nucleosides and oligonucleotides have been found to have an extensive range of applications. They have shown a wide variety of antiviral and antitumour activities and represent a new class of drug carriers for diagnostic and therapeutic applications. Lipophilic nucleosides and oligonucleotides are involved in the formation of monolayer films, micelles and/or vesicles, organogels or multilamellar layers and have a wide variety of biotechnological (eg. chip technology) and chromatographic applications. The objective of this project was to synthesize hydrophobically modified nucleotide analogs and incorporate them into oligonucleotides with the objective of achieving enhanced chromatographic separations. The synthesis of these analogs was achieved by introducing lipophilic functionalities at the 5'- position of deoxythymidine, a pyrimidine deoxynucleoside, commonly called thymidine. The scheme that evolved over the course of synthesis involved  TBS-protection of commercially available Thymidine at both the 3' and 5' hydroxyl groups, selective 5'-deprotection, 5'-tosylation, substitution of tosyloxy group by primary amines (CH3(CH2)nNH2, where n = 3,7,11,17),  acylation of the resulting 5'-amino 5'-deoxythymidine analogs with palmitoyl chloride and conversion of each of these analogs into their corresponding 3'-phosphoramidite derivatives. This was followed by incorporating these modified phosphoramidite analogs into an oligomeric sequence at the 5'-end by carrying out solid phase oligonucleotide synthesis. The oligomeric sequence chosen for incorporation of the modified thymidine phosphoramidite analogs was the human telomere nucleotide sequence, namely the telomeric repeat: 5'-TTAGGG-3' resulting in a heptameric sequence of T'TTAGGG where T' stands for the hydrophobically modified deoxythymidine analogs. Thus, the synthesis of four different lipophilically modified heptameric oligonucleotides was achieved and their characterization established using mass spectrometry. The lipophilic oligomers, which have structural similarity to an amphiphile, are proposed to have a reversible non-covalent interaction with the reverse phase surface of the C18-modified sorbent system by insertion of their lipophilic tails into the hydrophobic surface of the sorbent. Both solution and solid phase hybridization/binding studies were carried out. Solution phase binding/hybridization studies showed an interaction/binding between the modified oligonucleotides and their normal complementary strands by recording a decrease in absorbance at 260 nm(A260) with decrease in temperature. Retention/Adsorption properties of the modified as well as normal oligonucleotides on reverse phase sorbent systems were studied. ENVI-C18 discs were used and subsequently abandoned as the sorbent system of choice because they retained both the modified and normal oligonucleotides in equal measure and showed no selectivity. Also the glass microfiber discs lost their physical integrity when exposed to the analyte solution. Reverse Phase ZipTips were used as an alternative sorbent phase and it was found that the percent retention/adsorption of the lipophilic (modified) oligonucleotide under a given condition of temperature and/or ionic strength is appreciably greater than the percent retention of the normal unmodified complementary strand. Also, it was found that the increase in percent retention/adsorption of the modified oligomers with increase in temperature and/or ionic strength was greater than the increase observed with the normal unmodified complementary strands. It was observed that the RP sorbent phase preloaded with the modified oligomer showed a greater percent retention of the normal complementary strand under given conditions of temperature and/or ionic strength relative to the percent complementary strand retained when the RP sorbent phase is preloaded with the unmodified control oligomer . It was also observed that the modified oligomer analog with the longest hydrophobic chain length showed greatest percent retention on the reverse phase sorbent system, namely the RP ZipTips.