Synthesis of nucleotide coenzymes via nucleoside 5'-phosphorothioate intermediates

Hata, Tsujiaki; Nakagawa, Iwao

doi:10.1021/ja00721a037

Cited by 8 publications

(5 citation statements)

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“…When diphenyl phosphorochloridate is used as an activating agent for FAD synthesis, the yield is 70-80% and the reaction is complete within a few hours [28] . However, when FAD and NAD analogues were synthesized by this method, the yield drops to 25-30% [13,14,29] Another method uses riboflavin phosphorothioate with silver salt of adenosine monophosphate, achieving 51% yield in 5 hours [30] . However, obtaining of the starting material is a limiting factor, as it is not commercially available.…”

Section: Resultsmentioning

confidence: 99%

Efficient chemical and enzymatic syntheses of FAD nucleobase analogues and their analysis as enzyme cofactors

Shah

Kumar

Rohan

et al. 2023

Preprint

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Flavin adenine dinucleotide (FAD), an essential cofactor in cellular metabolism, catalyses a wide range of redox reactions. The organic synthesis of FAD is typically conducted by coupling flavin mononucleotide (FMN) and adenosine monophosphate. The reported synthesis routes have certain limitations such as multiple reaction steps, low yields, and/or difficult-to-obtain starting materials. In this study, we report the synthesis of FAD nucleobase analogues using chemical and enzymatic methods with readily available starting materials achieved in 1-3 steps with moderate yields (10-51%). Further, we demonstrate that Escherichia coli glutathione reductase can use these analogues to catalyse the reduction of glutathione. Finally, we show that FAD nucleobase analogues can also be synthesized inside a cell from cellular substrates FMN and nucleoside triphosphates. This lays the foundation for their use in studying the molecular role of FAD in cellular metabolism and as biorthogonal reagents in biotechnology and synthetic biology applications.

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Section: Resultsmentioning

confidence: 99%

Efficient chemical and enzymatic syntheses of FAD nucleobase analogues and their analysis as enzyme cofactors

Shah

Kumar

Rohan

et al. 2023

Preprint

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“…The synthesis of unsymmetrical diesters of pyrophosphoric acid such as occur in sugar nucleotides can be accomplished by several methods [31]. They all depend on the condensation of an appropriately activated nucleotide with the corresponding glycosyl phosphate.…”

Section: Synthesis Of Nucleoside 5'-diphospho-sulfbguinovosesmentioning

confidence: 99%

“…We used the tris(tri-n-octylammonium) salt of 6-sulfo-a-~-quinovopyranosyl phosphate in which both sulfonate and phosphate groups were fully neutralized, although there is some variation in the literature regarding the use of mono or bis(tri-n-octylammonium) salts of glycosyl phosphates or even of the free acid form [24, 32, 331. Depending on the type of condensation reaction, the phosphate group of the nucleotide moiety is used in different forms of derivatisation to increase the electrophilicity of the phosphorus [31]. In most cases we used the nucleoside 5'-phosphomorpholidates [20, 241 ( Fig.…”

Section: Synthesis Of Nucleoside 5'-diphospho-sulfbguinovosesmentioning

confidence: 99%

Synthesis of different nucleoside 5′‐diphospho‐sulfoquinovoses and their use for studies on sulfolipid biosynthesis in chloroplasts

Heinz

Schmidt

Hoch

et al. 1989

European Journal of Biochemistry

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6-Su~fo-a-~-quinovopyranosyl phosphate was reacted with different nucleoside monophosphate morpholidates to form ADP-, CDP-, GDP-and UDP-sulfoquinovose. Analytical and preparative HPLC of these nucleotides was performed on reversed-phase columns using volatile buffer systems as eluant. The isolated compounds were characterized by NMR spectroscopy (except the CDP derivative) and used for an investigation of sulfolipid biosynthesis by chloroplasts. For this purpose intact spinach chloroplasts were biosynthetically preloaded with radioactive diacylglycerol to provide a sulfoquinovosyl acceptor. When sulfosugar nucleotides were added to such prelabelled intact organelles, the background levels of sulfolipid biosynthesis did not rise. On the other hand, after osmotic shock of prelabelled chloroplasts sulfolipid labelling was significantly increased by the addition of UDP-or GDP-sulfoquinovose. The same stimulation was observed with isolated envelope membranes, and UDP-sulfoquinovose proved to be twice as active as the GDP derivative. From these results it was concluded that the final step in sulfolipid biosynthesis is catalyzed by a UDP-sulfoquinovose: 1,2-diacylglycero13-0-a-D-sulfoquinovosyltransferase. This chloroplast enzyme cannot use exogenously supplied sulfosugar nucleotides, which as membrane-impermeable compounds are expected to be formed in viwo within chloroplasts.The plant sulfolipid 1 ',2'-di-0-acyl-3'-0-(6-deoxy-6-sulfoa-D-glucopyranosy1)-sn-glycerol [I] is a membrane lipid found in higher plants, cyanobacteria and some other photosynthetic procaryotes [2]. With regard to the sulfur budget of plants, the sulfolipid represents a major component which accounts for about half of the organic sulfur in leaves [3]. In higher plants this unique glycolipid is confined to plastids [4]. In these organelles it is located in the two surrounding envelopes as well as in the inner thylakoid membranes [5], where part of it may be firmly bound to specific protein complexes [6 -81.After application of radioactive sulfate to plants, the sulfolipid is rapidly labelled [I, 2, 91. Despite this specific and easy labelling, several steps in sulfolipid biosynthesis have not been resolved and at present two alternative routes are discussed. It has been suggested that a sulfoglycolytic sequencc [lo -121 produces 3-sulfo-~-lactaldehyde which instead of 3-phospho-~-glyceraldehyde can be used by aldolase [lo] to form 6-deoxy-6-sulfo-~-fructofuranosyl 1-phosphate. Subsequent hydrolysis and isomerisation to 6-sulfoquinovose (quinovose = 6-deoxy-~-glucose), rephosphorylation at C-I and activation by a corresponding pyrophosphorylase could result in a nucleoside diphospho-sulfoquinovose. Such a com-

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“…When diphenyl phosphorochloridate is used as an activating agent for FAD synthesis, the yield is 70–80 %, and the reaction is complete within a few hours [25] . However, when FAD and NAD analogues were synthesized by this method, the yield dropped to 25–30 % [17,18,26] Another method uses riboflavin phosphorothioate with silver salt of adenosine monophosphate, achieving 51 % yield in 5 h [27] . However, obtaining the starting material is a limiting factor, as it is not commercially available.…”

Section: Introductionmentioning

confidence: 99%

“…When diphenyl phosphorochloridate is used as an activating agent for FAD synthesis, the yield is 70-80 %, and the reaction is complete within a few hours. [25] However, when FAD and NAD analogues were synthesized by this method, the yield dropped to 25-30 % [17,18,26] Another method uses riboflavin phosphorothioate with silver salt of adenosine monophosphate, achieving 51 % yield in 5 h. [27] However, obtaining the starting material is a limiting factor, as it is not commercially available. Interestingly, synthesis of NAD + nucleobase analogues have been conducted by activation of the nicotinamide mononucleotide phosphate with the redox pair triphenylphosphine/2,2'-dipyridyl disulfide (Ph 3 P/(PyS) 2 ) using a nucleophilic catalyst 1-methylimidazole in DMSO/DMF solvent mixture, followed by the coupling with the other mononucleotide.…”

Section: Introductionmentioning

confidence: 99%

Efficient Chemical and Enzymatic Syntheses of FAD Nucleobase Analogues and Their Analysis as Enzyme Cofactors**

et al. 2023

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Flavin adenine dinucleotide (FAD) is an essential redox cofactor in cellular metabolism. The organic synthesis of FAD typically involves coupling flavin mononucleotide (FMN) with adenosine monophosphate, however, existing synthesis routes present limitations such as multiple steps, low yields, and/or difficult‐to‐obtain starting materials. In this study, we report the synthesis of FAD nucleobase analogues with guanine/cytosine/uracil in place of adenine and deoxyadenosine in place of adenosine using chemical and enzymatic approaches with readily available starting materials, achieved in 1–3 steps with moderate yields (10–57 %). We find that the enzymatic route using Methanocaldococcus jannaschii FMN adenylyltransferase (MjFMNAT) is versatile and can produce these FAD analogues in high yields. Further, we demonstrate that Escherichia coli glutathione reductase is capable of binding and using these analogues as cofactors. Finally, we show that FAD nucleobase analogues can be synthesized inside a cell from cellular substrates FMN and nucleoside triphosphates by the heterologous expression of MjFMNAT. This lays the foundation for their use in studying the molecular role of FAD in cellular metabolism and as biorthogonal reagents in biotechnology and synthetic biology.

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Synthesis of nucleotide coenzymes via nucleoside 5'-phosphorothioate intermediates

Cited by 8 publications

References 0 publications

Efficient chemical and enzymatic syntheses of FAD nucleobase analogues and their analysis as enzyme cofactors

Efficient chemical and enzymatic syntheses of FAD nucleobase analogues and their analysis as enzyme cofactors

Synthesis of different nucleoside 5′‐diphospho‐sulfoquinovoses and their use for studies on sulfolipid biosynthesis in chloroplasts

Efficient Chemical and Enzymatic Syntheses of FAD Nucleobase Analogues and Their Analysis as Enzyme Cofactors**

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