Synthesis of novel 3′-azido-3′-deoxy-α-L-<i>ribo</i> configured nucleosides: A comparative study between chemical and chemo-enzymatic methodologies

<p></p> <p>Syntheses of novel 3′-azido-3′-deoxy-2′-<i>O</i>,4′-<i>C</i>-methylene-<i>α-</i>L-<i>ribo</i>furanosyl nucleosides have been carried out from 3′-azido-3′-deoxy-4′-<i>C</i>-hydroxymethyl-β-D-<i>xylo</i>furanosyl nucleosides following both chemical and chemo-enzymatic methodologies. The precursor nucleoside in turn was synthesized from a common glycosyl donor 4-<i>C</i>-acetoxymethyl-1,2,5-tri-<i>O</i>-acetyl-3-azido-3-deoxy-<i>α,β</i>-D-<i>xylo</i>furanose, which was obtained by the acetolysis of 4-<i>C</i>-acetoxymethyl-5-<i>O</i>-acetyl-3-azido-3-deoxy-1,2-<i>O</i>-isopropylidene-α-D-<i>xylo</i>furanose in 96% yield. It has been observed that a chemo-enzymatic pathway for the synthesis of targeted nucleosides is much more efficient than a chemical pathway, leading to the improvement in yield for the synthesis of 3′-azido-3′-deoxy-<i>α-</i>L-<i>ribo</i>furanosyl thymine and uracil from 49 to 89% and 55 to 93%, respectively.</p

Time course of luciferase expression under control of the <i>AOX1</i> promoter of <i>P. pastoris</i>.

<p>Cultures of recombinant strain <i>GS115</i> harboring the <i>Fluc</i>-containing expression vector pPIC3.5-Fluc and the control vector pPIC3.5 were grown in suitable medium up to six days. Luciferase assay was performed in triplicate and activity was plotted against indicated time points.</p

Constitutive expression of the Firefly luciferase (<i>Fluc</i>) under lsd90 promoter.

<p>Cultures of strain <i>SPJ25</i> harboring the vector constructs pJH6c-Fluc and pJH6c were grown in selective media (PMA ura⁻) at 30°C and 200 rpm. Aliquots were taken at the indicated time points and subjected to protein extraction. (A) Luciferase activity was determined using the Luciferase Assay System (Promega, USA). Assays were done in triplicate and the average luminescence values were plotted against the indicated time points. (B) Graph showing growth kinetics of cultures up to early stationary phase. (C) The histogram shows the cell density in Cells/ml at the indicated time points of culture.</p

Relative-qPCR analysis of Luciferase mRNA.

<p>RNA isolated form cells harvested at time points showing maximum level of Fluc RNA under the control of different vectors was subjected to real time RTPCR analysis. Samples were analyzed in triplicate and displayed as histogram. The relative Fluc/<i>act1</i> RNA levels expressed by different vectors are displayed after normalization with respect to the vector pJH6c-Fluc. The time points were: pJH6c-Fluc: 32 hrs; pART1-Fluc: 48 hrs; pREP3X-Fluc: 16 hrs, pPIC3.5-Fluc: 96 hrs.</p

Time course of <i>Fluc</i>-expression under control of the <i>adh1</i> promoter of <i>S. pombe</i>.

<p>Cultures of strain <i>SPJ25</i> harboring the vector constructs pART1-Fluc and pART1 were grown in selective media (PMA leu⁻) at 30°C and 200 rpm. (A) Luciferase activity measured in RLU and (B) growth kinetics of cultures.</p

Schematic diagram of the Fluc-expression vectors used in this study.

<p>Restriction map of Fluc-expression vectors; (A) pJH6c-Fluc, (B) pART1-Fluc, (C) pREP3X-Fluc and (D) pPIC3.5-Fluc. Approximately 1.0 kb upstream region of gene <i>SPAC1F8.02C/lsd90</i> of <i>S. pombe</i> was PCR amplified and inserted into the plasmid pJH5 at SphI/NdeI sites as promoter <i>Plsd90</i> respectively. The resulting vectors were designated as pJH6c (A). The strategy of <i>Fluc</i> cloning is described in the methods section.</p

<b>P</b>rimers used in this study.

<p><b>P</b>rimers used in this study.</p

Comparison of levels of expression of luciferase using different expression systems.

<p>Comparison of levels of expression of luciferase using different expression systems.</p

Time course of Fluc-expression under control of the <i>nmt1</i> promoter of <i>S. pombe</i>.

<p>Cultures of strain <i>SPJ25</i> harboring the vector constructs pREP3X-Fluc and pREP3X were grown in selective media (PMA leu⁻) at 30°C and 200 rpm. Initially the cultures were grown in medium containing thiamine and then sub-cultured in medium lacking thiamine for the indicated time points. (A) Luciferase activity measured in RLU and (B) growth kinetics of cultures.</p