PubMed · 1309193
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R P Singhal, S S DeSilva. 1992. Boronate affinity chromatography.. https://pubmed.ncbi.nlm.nih.gov/1309193/
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Direct PCR sequencing with boronated nucleotides provides an alternative to current PCR sequencing methods. The positions of boranophosphate-modified nucleotides incorporated randomly into DNA during PCR can be revealed directly by exonuclease digestion to give sequencing ladders. Cytosine nucleotides, however, are especially sensitive to exonuclease digestion and provide suboptimal sequencing ladders. Therefore, a series of 5-substituted analogs of 2'-deoxycytidine 5'-(alpha-P-borano)triphosphates (dCTPalphaB) were synthesized with the hope of increasing the nuclease resistance of deoxycytosine residues and thereby enhancing the deoxycytosine band intensities. These dCTP analogs contain a boranophosphate modification at the alpha-phosphate group in 2'-deoxycytidine 5'-triphosphate (dCTP) as well as a 5-methyl, 5-ethyl, 5-bromo or 5-iodo substitution for the 5-hydrogen of cytosine. The two diastereomers of each new dCTP derivative were separated by reverse phase HPLC. The first eluted diastereomer (putatively Rp) of each dCTP analog was a substrate for T7 DNA polymerase (Sequenase) and had an incorporation efficiency similar to normal dCTP and dCTPalphaB, with the 5-iodo-dCTPalphaB analog being the least efficient. Substitution at the C-5 position of cytosine by alkyl groups (ethyl and methyl) markedly enhanced the dCTPalphaB resistance towards exonuclease III (5-Et-dCTPalphaB >5-Me-dCTPalphaB >dCTPalphaB approximately 5-Br-dCTPalphaB >5-I-dCTPalphaB), thereby generating DNA sequences that better define the deoxycytosine positions. The introduction of modified dCTPalphaB should increase the utility of direct DNA sequencing with boronated nucleoside 5'-triphosphates.
The title compound, C7H20BNOSi, features an N,N-disubstituted six-membered morpholine ring in a chair conformation, with the trimethylsilyl group in the equatorial position and the borane group in the axial position. The least-squares plane formed by the four C atoms of the morpholine ring has a mean deviation of 0.013 (2) A. The O and N atoms are 0.672 (2) and 0.650 (2) A above and below the plane, respectively.
The location of a series of lipophilic and lipid-attached BODIPY (4, 4-difluoro-4-bora-3a,4a-diaza-s-indacene) membrane probes was analyzed by the quenching of BODIPY fluorescence by a series of nitroxide-labeled lipids in which the depth of the nitroxide group is varied. When attached to the polar headgroup of PE the BODIPY remained near the polar headgroup in depth. However, when attached at the end of free or phospholipid-attached fatty acyl chains, or when attached to two hydrocarbon chains, we observed two probe populations. One, usually dominant, population of BODIPY groups 'looped back' towards the surface, but a second population remained deeply embedded within the bilayer. When attached to a fatty acid or fatty acyl chain, the deep population appeared to locate at a depth related to its point of attachment to the acyl chain. In BODIPY linked to free fatty acids, the location of the deep population responded to the ionization of the carboxyl group. Because, unlike NBD (7-nitro-2,1,3-benzoxadiazol-4-yl) and most dansyl groups, acyl chain linked BODIPY groups can exist in a deeply buried form we conclude that BODIPY linked acyl chains are superior to NBD or dansyl linked acyl chains as membrane probes.