Preparation of Base-Deuterated 2?-Deoxyadenosine Nucleosides and Their Site-Specific Incorporation Into Dna

Chirakul, Panadda; Litzer, Justin R.; Sigurdsson, Snorri Th.

doi:10.1081/ncn-100108321

Cited by 10 publications

(8 citation statements)

References 11 publications

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“…The results demonstrated that the replacement of protonated methanol with its deuterated analog within the same concentration of 2 H 2 O in the growth media slightly decreased the growth characteristics (Table 1, (Table 1, experiment 1). In the intermediate experiments (2)(3)(4)(5)(6)(7)(8)(9)(10), these parameters varied proportionally to the 2 Н 2 О concentration ( Table 1). The observed effect consisted in the increase in the lag-phase period and cell generation time with a simultaneous decrease in microbial biomass outputs on media with increasing 2 Н 2 О-content.…”

Section: Resultsmentioning

confidence: 99%

Studying the Biosynthesis of 2H-labeled purine Ribonucleoside Inosine by a Chemoheterotrophic Bacterium Bacillus subtilis B-3157

2015

European Reviews of Chemical Research

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We studied the biosynthesis of 2 H-labeled purine ribonucleoside inosine excreted into liquid microbial culture (LC) by Gram-positive chemoheterotrophic bacterium Bacillus subtilis B-3157 while growing of this bacterium on heavy water (HW) medium with 2% (v/v) hydrolysate of deuterated biomass of the methylotrophic bacterium Brevibacterium methylicum B-5662 as a source of 2 H-labeled growth substrates. Isolation of 2 H-labeled inosine from the LC was performed by adsorption/desorption on activated carbon with following extraction by 0,3 M ammoniumformate buffer (pH = 8,9), crystallization in 80% (v/v) EtOH, and ion exchange chromatography (IEC) on a column with AG50WX 4 cation exchange resin equilibrated with 0,3 M ammoniumformate buffer and 0,045 M NH 4 Cl. The investigation of deuterium incorporation into the inosine molecule by FAB method demonstrated the incorporation of 5 deuterium atoms into the molecule (the total level of deuterium enrichment -65,5 atom% 2 H) with 3 deuterium atoms included into the ribose and 2 deuterium atoms -into the hypoxanthine residue of the molecule. Three nonexchangeable deuterium atoms were incorporated into the ribose residue owing to reactions of enzymatic izomerization of glucose in 2 H 2 O-medium. These non-exchangeable deuterium atoms in the ribose residue were originated from glycolysis, associated with the Embden-Meyerhof pathway, while two other deuterium atoms at C2,C8-positions in the hypoxanthine residue were synthesized from [ 2 H]amino acids that originated from the deuterated hydrolysate of the methylotrophic bacterium Brevibacterium methylicum B-5662. However, the effect of auxotrophy of this strain in tyrosine, histidine, adenine and uracilб presupposes the branched metabolic pathways, different those indicated above.

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Section: Resultsmentioning

confidence: 99%

Studying the Biosynthesis of 2H-labeled purine Ribonucleoside Inosine by a Chemoheterotrophic Bacterium Bacillus subtilis B-3157

2015

European Reviews of Chemical Research

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“…For this purpose, we used a stepwise increasing gradient of 2 H 2 O-concentration in M9 growth media (from 24,5; 49,0; 73,5 up to 98% (v/v) 2 Н 2 О) in the presence of 2% (v/v) methanol and its 2 H-labeled analog ([ 2 H]methanol), because we assumed that gradual cell adaptation to 2 Н 2 О would have a favorable effect on the growth parameters of the strain. (2)(3)(4)(5)(6)(7)(8)(9)(10), these parameters varied proportionally to the 2 Н 2 О concentration ( Table 1). The observed effect consisted in the increase in the lag-phase period and cell generation time with a simultaneous decrease in microbial biomass outputs on media with increasing 2 Н 2 О-content.…”

Section: Resultsmentioning

confidence: 99%

“…The recent advance in technical and computing capabilities of these analytical methods has allowed a considerable increase the efficiency of carrying out biological studies with 2 H-labeled molecules de novo, as well as to carry out the analysis of the structure and function of nucleosides and their analogs at the molecular level [7]. In particular, 2 H-labeled ribonucleosides and their analogs are used in template-directed syntheses of deuterated RNA macromolecules for studying their spatial structure and conformational changes [8]. Perdeuteration and selective deuteration techniquemay be useful approaches for simplification of NMR spectra and for other structural studies of large biomolecules.…”

Section: Introductionmentioning

confidence: 99%

Deuterated Methylotrophic Biomass as a Substrate for Microbiological Synthesis of 2H-Labeled Purine Ribonucleoside Inosine by Chemoheterotrophic Bacterium Bacillus Subtilis B-3157

Mosin¹,

Ignatov²,

State³

2015

European Reviews of Chemical Research

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We propose to use the hydrolyzed deuterated biomass of the facultative methylotrophic bacterium Brevibacterium methylicum B-5662 as a source of 2 H-labeled growth substrates for microbiological synthesis of 2 H-labeled purine ribonucleoside inosine, excreted into the liquid microbial culture (LC) by a Gram-positive chemoheterotrophic bacterium Bacillus subtilis B-3157. The bacterium was grown on heavy water (HW) medium with 2% (v/v) hydrolysate of deuterated biomass of the methylotrophic bacterium Brevibacterium methylicum B-5662 obtained on minimal salt media M9 supplemented with 2% (v/v) [ 2 H]methanol and increasing gradient of 2 Н 2 O concentration from 0; 24,5; 49,0; 73,5 up to 98% (v/v) 2 Н 2 O. Isolation of 2 H-labeled inosine from LC was performed by adsorption/desorption on activated carbon with following extraction by 0,3 M ammonium-formate buffer (pH = 8,9), crystallization in 80% (v/v) EtOH, and ion exchange chromatography (IEC) on a column with AG50WX 4 cation exchange resin equilibrated with 0,3 M ammonium-formate buffer and 0,045 M NH 4 Cl. The investigation of deuterium incorporation into the inosine molecule by FAB method demonstrated incorporation of 5 deuterium atoms into the molecule (the total level of deuterium enrichment -65,5 atom% 2 H) with 3 deuterium atoms being included into the ribose and 2 deuterium atoms -into the hypoxanthine residue of the molecule.

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Section: Preparation Of Deutero-biomass Of B Methylicum Vkpm B-3157mentioning

confidence: 99%

Fluorescent probing of biological particles suspensions

Varekhov¹

2016

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In this paper was demonstrated the possibility of the use of FAB mass spectrometry on an impulse mass spectrometer VG-70 SEQ ("Fisons VG Analytical", USA) for the study of biosynthetic pathways of 2 H-labeled purine ribonucleoside inosine secreted into the liquid culture (LC) by Gram-positive chemoheterotrophic bacterium Bacillus subtilis VKPM B-3157 when grown on heavy water (HW) medium with 2 % hydrolyzate of deuterated biomass of methylotrophic bacterium Brevibacterium methylicum VKPM B-5662 as a source of growth 2 H-labeled substrates. Isolation of 2 H-labeled inosine from the LC was performed by adsorption/desorption on activated carbon with following extraction by 0.3 M ammonium-formate buffer (pH = 8.9), crystallization in 80 % ethanol and ion exchange chromatography (IEC) on a column with AG50WX 4 cation exchange resin equilibrated with 0.3 M ammonium-formate buffer and 0.045 M NH 4 Cl. The investigation of deuterium incorporation into the molecule of biosynthetic [ 2 H]inosine by FAB mass spectrometry demonstrated the incorporation of 5 deuterium atoms into the molecule (the total level of deuterium enrichment-62.5 atom. % 2 H) with incorporation of 3 deuterium atoms into the ribose and 2 deuterium atoms-into the hypoxanthine residue of the molecule. Three non-exchangeable deuterium atoms were incorporated into the ribose residue owing to reactions of enzymatic izomerization of glucose in 2 H 2 O-medium due to reactions of glycolysis, associated with the Embden-Meyerhof pathway with participation of reactions of isotope (1 Н-2 Н) exchange, while two other deuterium atoms at C2, C8-positions in the hypoxanthine residue were synthesized from [ 2 H]amino acids that originated from the deuterated hydrolysate of the methylotrophic bacterium Brevibacterium methylicum VKPM B-5662.

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Preparation of Base-Deuterated 2?-Deoxyadenosine Nucleosides and Their Site-Specific Incorporation Into Dna

Cited by 10 publications

References 11 publications

Studying the Biosynthesis of 2H-labeled purine Ribonucleoside Inosine by a Chemoheterotrophic Bacterium Bacillus subtilis B-3157

Studying the Biosynthesis of 2H-labeled purine Ribonucleoside Inosine by a Chemoheterotrophic Bacterium Bacillus subtilis B-3157

Deuterated Methylotrophic Biomass as a Substrate for Microbiological Synthesis of 2H-Labeled Purine Ribonucleoside Inosine by Chemoheterotrophic Bacterium Bacillus Subtilis B-3157

Fluorescent probing of biological particles suspensions

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