Thiophosphate Analogs of c-di-GMP: Impact on Polymorphism

Zhao, Jianwei; Veliath, Elizabeth; Kim, Se‐Ho; Gaffney, Barbara L.; Jones, R. A. C.

doi:10.1080/15257770903044523

Cited by 15 publications

(32 citation statements)

References 41 publications

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“…We identified this peak as c-(S p S p )-di-G ps because the molecule had an m/z value of 721 like the other two c-di-GMP diphosphorothioates and because the retention time was in between that of c-di-GMP and c-(R p S p )-di-G ps . This same order of elution was previously reported for the three c-di-GMP diphosphorothioate stereoisomers ( 55 ).…”

Section: Resultssupporting

confidence: 86%

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Enzymatic synthesis of cyclic dinucleotide analogs by a promiscuous cyclic-AMP-GMP synthetase and analysis of cyclic dinucleotide responsive riboswitches

Launer-Felty

Strobel

2018

Nucleic Acids Research

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Cyclic dinucleotides are second messenger molecules produced by both prokaryotes and eukaryotes in response to external stimuli. In bacteria, these molecules bind to RNA riboswitches and several protein receptors ultimately leading to phenotypic changes such as biofilm formation, ion transport and secretion of virulence factors. Some cyclic dinucleotide analogs bind differentially to biological receptors and can therefore be used to better understand cyclic dinucleotide mechanisms in vitro and in vivo. However, production of some of these analogs involves lengthy, multistep syntheses. Here, we describe a new, simple method for enzymatic synthesis of several 3′, 5′ linked cyclic dinucleotide analogs of c-di-GMP, c-di-AMP and c-AMP-GMP using the cyclic-AMP-GMP synthetase, DncV. The enzymatic reaction efficiently produced most cyclic dinucleotide analogs, such as 2′-amino sugar substitutions and phosphorothioate backbone modifications, for all three types of cyclic dinucleotides without the use of protecting groups or organic solvents. We used these novel analogs to explore differences in phosphate backbone and 2′-hydroxyl recognition between GEMM-I and GEMM-Ib riboswitches.

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Section: Resultssupporting

confidence: 86%

“…However, this method only allows synthesis of R p R p and R p S p phosphorothioate cyclic dinucleotides. A more complicated synthetic procedure, requiring additional purification steps, is necessary to prepare the S p S p phosphorothioate ( 55 ).…”

Section: Resultsmentioning

confidence: 99%

Enzymatic synthesis of cyclic dinucleotide analogs by a promiscuous cyclic-AMP-GMP synthetase and analysis of cyclic dinucleotide responsive riboswitches

Launer-Felty

Strobel

2018

Nucleic Acids Research

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“…Further, as a phosphate triester cyclization does not lend itself to the preparation of thiophosphates, prior to our recent work16 only the analog with a single thiophosphate had been reported 8,13. We have recently prepared and characterized all of the thiophosphate diastereomers of c-di-GMP containing one, two, or three sulfur atoms,16 but in such modest yields that opportunities for further study are limited, beyond confirming that they showed similar polymorphism to that we had found for c-di-GMP itself 17. Thiophosphate analogs of defined stereochemistry are of particular interest as probes of phosphate/metal interaction, and because of the resistance of thiophosphates to enzymatic hydrolysis.…”

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confidence: 99%

One-Flask Syntheses of c-di-GMP and the [R_p,R_p] and [R_p,S_p] Thiophosphate Analogues

et al. 2010

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An integrated set of reactions and conditions that allow an eight step one-flask synthesis of the protected derivatives of c-di-GMP and the [Rp,Rp] and [Rp,Sp] thiophosphate analogs is reported. Deprotection is also carried out as a one-flask procedure, with the final products isolated by crystallization from the reaction mixture. Chromatography is only used for separation of the thiophosphate diastereomers.

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“…Mono and dithiophosphate analogs were synthesized in solution as previously described 35 . The absolute stereochemistry of the thiophosphate c-di-GMP analogs was assigned as previously described based on the specificity of P1 and venom phosphodiesterases 36 . 3′- phosphate CPG beads, guanosine methylphosphonamidite precursors, and Poly-Pak II desalting columns were purchased from Glen Research.…”

Section: Methodsmentioning

confidence: 99%

Identification of c-di-GMP Derivatives Resistant to an EAL Domain Phosphodiesterase

et al. 2013

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The bacterial second messenger signaling molecule bis-(3′-5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) controls important biological processes such as biofilm formation, virulence response, and motility. This second messenger is sensed by macromolecular targets inside the cell, both protein and RNA, which induce specific phenotypic responses critical for bacterial survival. One class of enzymes responsible for regulating the intracellular concentration of c-di-GMP, and therefore the physiological behavior of the cell, is the EAL domain phosphodiesterases, which degrade the second messenger to its linear form, pGpG. Here, we investigate how base and backbone modifications to c-di-GMP affect the rate of cyclic dinucleotide degradation by an EAL domain protein (CC3396 from Caulobacter crescentus). The doubly substituted thiophosphate analog is highly resistant to hydrolysis by this metabolizing enzyme but can still bind c-di-GMP riboswitch targets. We used these findings to develop a novel ribosyl-phosphate modified derivative of c-di-GMP containing 2′-deoxy and methylphosphonate substitutions that is charge neutral and demonstrate that this analog is also resistant to EAL domain catalyzed degradation. This suggests a general strategy for designing c-di-GMP derivatives with increased enzymatic stability that also possess desirable properties for development as chemical probes of c-di-GMP signaling.

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Thiophosphate Analogs of c-di-GMP: Impact on Polymorphism

Cited by 15 publications

References 41 publications

Enzymatic synthesis of cyclic dinucleotide analogs by a promiscuous cyclic-AMP-GMP synthetase and analysis of cyclic dinucleotide responsive riboswitches

Enzymatic synthesis of cyclic dinucleotide analogs by a promiscuous cyclic-AMP-GMP synthetase and analysis of cyclic dinucleotide responsive riboswitches

One-Flask Syntheses of c-di-GMP and the [R_p,R_p] and [R_p,S_p] Thiophosphate Analogues

Identification of c-di-GMP Derivatives Resistant to an EAL Domain Phosphodiesterase

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Thiophosphate Analogs of c-di-GMP: Impact on Polymorphism

Cited by 15 publications

References 41 publications

Enzymatic synthesis of cyclic dinucleotide analogs by a promiscuous cyclic-AMP-GMP synthetase and analysis of cyclic dinucleotide responsive riboswitches

Enzymatic synthesis of cyclic dinucleotide analogs by a promiscuous cyclic-AMP-GMP synthetase and analysis of cyclic dinucleotide responsive riboswitches

One-Flask Syntheses of c-di-GMP and the [Rp,Rp] and [Rp,Sp] Thiophosphate Analogues

Identification of c-di-GMP Derivatives Resistant to an EAL Domain Phosphodiesterase

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One-Flask Syntheses of c-di-GMP and the [R_p,R_p] and [R_p,S_p] Thiophosphate Analogues