Synthesis of 5-fluorouridine 5′-diphosphate galactose from 5-fluorouridine by chemical phosphorylation and microbial uridylyl transfer

Fujita, Ken‐ichi; Matsukawa, Akihiro; Shibata, Kozo; Tanaka, Toshio; Taniguchi, Makoto; Oi, Susumu

doi:10.1016/0008-6215(94)00231-2

Cited by 5 publications

(4 citation statements)

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“…5-Fluorouridine 5′-monophosphate was prepared as its triethylammonium salt from 5-fluorouridine using modifications of a literature procedure . Phosphorus(V) oxychloride (0.13 mL, 1.4 mmol) and 5-fluorouridine (0.20 g, 0.76 mmol) were dissolved in dry trimethyl phosphate (2 mL) at 0 °C.…”

Section: Methodsmentioning

confidence: 99%

“…5-Fluorouridine 5′-monophosphate was prepared as its triethylammonium salt from 5fluorouridine using modifications of a literature procedure. 28 Phosphorus(V) oxychloride (0.13 mL, 1.4 mmol) and 5-fluorouridine (0.20 g, 0.76 mmol) were dissolved in dry trimethyl phosphate (2 mL) at 0 °C. After 16 h the mixture was poured into cold water (100 mL), the solution was adjusted to pH 7 using 1 M NaOH, and water was added to give a total volume of 180 mL.…”

Section: ■ Introductionmentioning

confidence: 99%

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Proton Transfer from C-6 of Uridine 5′-Monophosphate Catalyzed by Orotidine 5′-Monophosphate Decarboxylase: Formation and Stability of a Vinyl Carbanion Intermediate and the Effect of a 5-Fluoro Substituent

et al. 2012

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The exchange for deuterium of the C-6 protons of uridine 5′-monophosphate (UMP) and 5-fluorouridine 5′-monophosphate (F-UMP) catalyzed by yeast orotidine 5′-monophosphate decarboxylase (ScOMPDC) at pD 6.5 – 9.3 and 25 °C was monitored by 1H NMR spectroscopy. Deuterium exchange proceeds by proton transfer from C-6 of the bound nucleotide to the deprotonated side chain of Lys-93 to give the enzyme-bound vinyl carbanion. The pD-rate profiles for kcat give turnover numbers for deuterium exchange into enzyme-bound UMP and F-UMP of 1.2 × 10−5 and 0.041 s−1, respectively, so that the 5-fluoro substituent results in a 3400-fold increase in the first-order rate constant for deuterium exchange. The binding of UMP and F-UMP to ScOMPDC results in 0.5 and 1.4 unit decreases, respectively, in the pKa of the side chain of the catalytic base Lys-93, showing that these nucleotides bind preferentially to the deprotonated enzyme. We also report the first carbon acid pKas for proton transfer from C-6 of uridine (pKCH = 28.8) and 5-fluorouridine (pKCH = 25.1) in aqueous solution. The stabilizing effects of the 5-fluoro substituent on C-6 carbanion formation in solution (5 kcal/mol) and at ScOMPDC (6 kcal/mol) are similar. The binding of UMP and F-UMP to ScOMPDC results in a greater than 5 × 109-fold increase in the equilibrium constant for proton transfer from C-6 so that ScOMPDC stabilizes the bound vinyl carbanions, relative to the bound nucleotides, by at least 13 kcal/mol. The pD-rate profile for kcat/Km for deuterium exchange into F-UMP gives the intrinsic second-order rate constant for exchange catalyzed by the deprotonated enzyme as 2300 M−1 s−1. This was used to calculate a total rate acceleration for ScOMPDC-catalyzed deuterium exchange of 3 × 1010 M−1, which corresponds to a transition state stabilization for deuterium exchange of 14 kcal/mol. We conclude that a large portion of the total transition state stabilization for the decarboxylation of orotidine 5′-monophosphate can be accounted for by stabilization of the enzyme-bound vinyl carbanion intermediate of the stepwise reaction.

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Section: Methodsmentioning

confidence: 99%

Section: ■ Introductionmentioning

confidence: 99%

Proton Transfer from C-6 of Uridine 5′-Monophosphate Catalyzed by Orotidine 5′-Monophosphate Decarboxylase: Formation and Stability of a Vinyl Carbanion Intermediate and the Effect of a 5-Fluoro Substituent

et al. 2012

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“…Synthesis of FUMP. The procedure of Fujita et al was followed with modifications (26). Phosphorus oxychloride (330 μL) was added under dry nitrogen to a stirred mixture of 5-fluorouridine (1.9 mmol) and 5 mL of anhydrous trimethyl phosphate.…”

Section: Methodsmentioning

confidence: 99%

Mechanism of the Orotidine 5′-Monophosphate Decarboxylase-Catalyzed Reaction: Evidence for Substrate Destabilization^,

et al. 2009

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The reaction catalyzed by orotidine 5'-monophosphate decarboxylase (OMPDC) involves a stabilized anionic intermediate, although the structural basis for the rate acceleration (k cat /k non , 7.1 × 10 16 ) and proficiency [(k cat /K M )/k non , 4.8 × 10 22 M −1 ] is uncertain. That the OMPDCs from Methanothermobacter thermautotrophicus (MtOMPDC) and Saccharomyces cerevisiae (ScOMPDC) catalyze the exchange of H6 of the UMP product with solvent deuterium allows an estimate of a lower limit on the rate acceleration associated with stabilization of the intermediate and . We investigated that hypothesis by structural and functional characterization of the D70N and D70G mutants of MtOMPDC and the D91N mutant of ScOMPDC. The substitutions for Asp 70 in MtOMPDC significantly decrease the value of k cat for decarboxylation of FOMP (a more reactive substrate analog) but have little effect on the value of k ex for exchange of H6 of FUMP with solvent deuterium; the structures of wild type MtOMPDC and its mutants are superimposable when complexed with 6-azaUMP. In contrast, the D91N mutant of ScOMPDC does not catalyze exchange of H6 of FUMP; the structures of wild type ScOMPDC and its D91N mutant are not superimposable when complexed with 6-azaUMP, with differences in both the conformation of the active site loop and the orientation of the ligand vis á vis the active site residues. We propose that the differential effects of substitutions for Asp 70 of MtOMPDC on decarboxylation and exchange provide additional evidence for a carbanionic intermediate as well as the involvement of Asp 70 in substrate destabilization. † This research was supported by NIH GM039754 (to J.P.R.) and GM065155 (to J.A.G.). Molecular graphics images were produced using the UCSF Chimera package from the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco (supported by NIH P41 RR-01081). The X-ray coordinates and structure factors for the following structures have been deposited in the Protein Data Bank with the indicated accession codes: wild type MtOMPDC in the absence of ligands (PDB accession code 3G18), wild type MtOMPDC complexed with 6-azaUMP (3G1A), wild type MtOMPDC complexed with UMP (3G1D), wild type MtOMPDC complexed with H 2 OMP (3D1F), wild type MtOMPDC complexed with H 2 UMP (3D1H), the D70N mutant of MtOMPDC in the absence of ligands (3G1Y), the D70N mutant of MtOMPDC complexed with 6-azaUMP (3G24), the D70N mutant of MtOMPDC complexed with UMP (3G22), the D70G mutant of MtOMPDC in the absence of ligands (3G1S), the D70G mutant of MtOMPDC complexed with UMP (3G1X), the D70G mutant of MtOMPDC complexed with FOMP (3G1V), wild type ScOMPDC in the absence of ligands (3GDK), wild type ScOMPDC complexed with 6-azaUMP (3GDL), the D91N mutant of ScOMPDC in the absence of ligands (3GDR), and the D91N mutant of ScOMPDC complexed with 6-azaUMP (3GDT).

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“…The 5-FdUMP (39) was synthesized from 5-FdUrd (51) (Abraham et al, 1996). 5-FUMP (42) was obtained from 5-FUrd (50) (Fujita et al, 1994). The synthetic route…”

Section: Synthesis Of 5-fu (4) Metabolitesmentioning

confidence: 99%

The importance of drug metabolites synthesis: the case-study of cardiotoxic anticancer drugs

Hrynchak

Sousa

Pinto

et al. 2017

Drug Metabolism Reviews

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Anticancer drugs are presently guarantying more survivors as a result of more powerful drugs or combinations of drugs used in therapy. Thus, it has become more crucial to study and overcome the side effects of these therapies. Cardiotoxicity is one of the most relevant side effects on the long-term cancer survivors, because of its high social and economic impact. Drug metabolism can result in active metabolites or toxic metabolites that can lead to important side effects. The metabolites of anticancer drugs are possible culprits of cardiotoxicity; however, the cardiotoxicity of many of the metabolites in several drug classes was not yet suitably studied so far. On the other hand, the use of prodrugs that are bioactivated through metabolism can be a good alternative to obtain more cardio safe drugs. In this review, the methods to obtain and study metabolites are summarized and their application to the study of a group of anticancer drugs with acknowledged cardiotoxicity is highlighted. In this group of drugs, doxorubicin (DOX, 1), mitoxantrone (MTX, 2), cyclophosphamide (CTX, 3) and 5-fluorouracil (5-FU, 4) are included, as well as the tyrosine kinase inhibitors, such as imatinib (5), sunitinib (6) and sorafenib (7). Only with the synthesis and purification of considerable amounts of the metabolites can reliable studies be performed, either in vitro or in vivo that allow accurate conclusions regarding the cardiotoxicity of anticancer drug metabolites and then pharmacological prevention or treatment of the cardiac side effects can be done.

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Synthesis of 5-fluorouridine 5′-diphosphate galactose from 5-fluorouridine by chemical phosphorylation and microbial uridylyl transfer

Cited by 5 publications

References 9 publications

Proton Transfer from C-6 of Uridine 5′-Monophosphate Catalyzed by Orotidine 5′-Monophosphate Decarboxylase: Formation and Stability of a Vinyl Carbanion Intermediate and the Effect of a 5-Fluoro Substituent

Proton Transfer from C-6 of Uridine 5′-Monophosphate Catalyzed by Orotidine 5′-Monophosphate Decarboxylase: Formation and Stability of a Vinyl Carbanion Intermediate and the Effect of a 5-Fluoro Substituent

Mechanism of the Orotidine 5′-Monophosphate Decarboxylase-Catalyzed Reaction: Evidence for Substrate Destabilization^,

The importance of drug metabolites synthesis: the case-study of cardiotoxic anticancer drugs

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Synthesis of 5-fluorouridine 5′-diphosphate galactose from 5-fluorouridine by chemical phosphorylation and microbial uridylyl transfer

Cited by 5 publications

References 9 publications

Proton Transfer from C-6 of Uridine 5′-Monophosphate Catalyzed by Orotidine 5′-Monophosphate Decarboxylase: Formation and Stability of a Vinyl Carbanion Intermediate and the Effect of a 5-Fluoro Substituent

Proton Transfer from C-6 of Uridine 5′-Monophosphate Catalyzed by Orotidine 5′-Monophosphate Decarboxylase: Formation and Stability of a Vinyl Carbanion Intermediate and the Effect of a 5-Fluoro Substituent

Mechanism of the Orotidine 5′-Monophosphate Decarboxylase-Catalyzed Reaction: Evidence for Substrate Destabilization,

The importance of drug metabolites synthesis: the case-study of cardiotoxic anticancer drugs

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Mechanism of the Orotidine 5′-Monophosphate Decarboxylase-Catalyzed Reaction: Evidence for Substrate Destabilization^,