A New Method for the Synthesis of Furanose Derivatives of Aldohexoses<sup>1</sup>

Kohn, Paul M.; Samaritano, Rita H.; Lerner, Léon M.

doi:10.1021/ja00951a041

Cited by 65 publications

(19 citation statements)

References 3 publications

(4 reference statements)

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“…These compounds have allowed us to reinvestigate the mechanism of inhibition of the E. coli RNR and to report for the first time on the details of the inhibition of the human RNR. The present communication shows that incubation of human and E. (25,26). The 2-deoxy-3,5-di-O-benzoyl-3,3-difluororibonolactone was reduced with [ 3 H]-disiamylborane made from NaB 3 H 4 and 2-methyl-2-butene.…”

mentioning

confidence: 58%

Enhanced subunit interactions with gemcitabine-5′-diphosphate inhibit ribonucleotide reductases

Wang

Lohman²,

Stubbe³

2007

Proc. Natl. Acad. Sci. U.S.A.

175

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Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides in all organisms. The class I RNRs are composed of two subunits, ␣ and ␤, with proposed quaternary structures of ␣2␤2, ␣6␤2, or ␣6␤6, depending on the organism. The ␣ subunits bind the nucleoside diphosphate substrates and the dNTP/ATP allosteric effectors that govern specificity and turnover. The ␤2 subunit houses the diferric Y • (1 radical per ␤2) cofactor that is required to initiate nucleotide reduction. 2 ,2 -Difluoro-2 -deoxycytidine (F2C) is presently used clinically in a variety of cancer treatments and the 5 -diphosphorylated F2C (F2CDP) is a potent inhibitor of RNRs. The studies with [1 -3 H]-F2CDP and [5-3 H]-F2CDP have established that F2CDP is a substoichiometric mechanism based inhibitor (0.5 eq F2CDP/␣) of both the Escherichia coli and the human RNRs in the presence of reductant. Inactivation is caused by covalent labeling of RNR by the sugar of F2CDP (0.5 eq/␣) and is accompanied by release of 0.5 eq cytosine/␣. Inactivation also results in loss of 40% of ␤2 activity. Studies using size exclusion chromatography reveal that in the E. coli RNR, an ␣2␤2 tight complex is generated subsequent to enzyme inactivation by F2CDP, whereas in the human RNR, an ␣6␤6 tight complex is generated. Isolation of these complexes establishes that the weak interactions of the subunits in the absence of nucleotides are substantially increased in the presence of F2CDP and ATP. This information and the proposed asymmetry between the interactions of ␣n␤n provide an explanation for complete inactivation of RNR with substoichiometric amounts of F2CDP.G emcitabine, or 2Ј,2Ј-difluoro-2Ј-deoxycytidine (F 2 C), is a drug that is used clinically in the treatment of advanced pancreatic cancer and non-small cell lung carcinomas (1-3). In humans, F 2 C enters the cell via CNT-type or ENT-type transporters (4-6) and must be phosphorylated to exhibit its cytotoxicity. The monophosphate of F 2 C (F 2 CMP) is generated by deoxycytidine kinase (7) and is rapidly phosphorylated to the di-and triphosphates (F 2 CDP and F 2 CTP) (8, 9). Diphosphorylated F 2 C (F 2 CDP) is an irreversible inhibitor of ribonucleotide reductase (RNR) (10-13), and F 2 CTP functions as a chain terminator in the DNA polymerase reaction (14, 15). Differentially phosphorylated states of gemzar can also interfere with other enzymes involved in nucleotide metabolism. The mechanisms of cytotoxicity of F 2 C depend on the phosphorylated state of the inhibitor and are likely to be cell specific and multifactoral. Our recent synthesis of [1Ј-3 H]-F 2 CDP has provided the required tool to investigate the mechanism by which this molecule inactivates RNRs. Studies reported herein provide a previously unrecognized approach for RNR inhibition, one in which the mechanism based inhibitor (F 2 CDP) enhances the interactions between the two subunits of RNR preventing nucleotide reduction despite substoichiometric labeling.RNRs catalyze the conversion on nucleoside di(tri)phosphates to de...

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mentioning

confidence: 58%

Enhanced subunit interactions with gemcitabine-5′-diphosphate inhibit ribonucleotide reductases

Wang

Lohman²,

Stubbe³

2007

Proc. Natl. Acad. Sci. U.S.A.

175

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“…The presence of the two fluorines required an increased ratio of disiamylborane to 6 relative to ribonolactone to obtain high yields. (36) The reduced 7 was then converted directly to the mesylate 8 with mesyl chloride and TEA without purification in 85% yield from the lactone 6 . The mesylate was coupled to bis -trimethylsilylcytosine 9 prepared by refluxing cytosine in 10:1 HMDS:TMSCl.…”

Section: Resultsmentioning

confidence: 99%

“…The concentration of BH 3 :THF was established by titration. (36, 37) The solution of BH 3 :THF was transferred to a ice/salt bath (−15°C) and 2-methyl-2-butene (1.03 mL, 9.6 mmol) was added slowly, dropwise via syringe over 1 h. The reaction mixture was stirred an additional 1 h at −15°C. The solution of disiamyl borane was transferred to an ice bath (0°C) and a solution of 6 (188 mg, 0.5 mmol) in 1 mL THF was added dropwise via syringe over 30 min.…”

Section: Methodsmentioning

confidence: 99%

Inactivation of Lactobacillus leichmannii Ribonucleotide Reductase by 2′,2′-Difluoro-2′-deoxycytidine 5′-Triphosphate: Covalent Modification

Lohman¹,

Stubbe²

2010

Biochemistry

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Ribonucleotide reductase (RNR) from Lactobacillus leichmannii, a 76 kDa monomer using adenosylcobalamin (AdoCbl) as a cofactor, catalyzes the conversion of nucleoside triphosphates to deoxynucleotides and is rapidly (<30 sec) inactivated by one equivalent (eq.) of 2′,2′-difluoro-2′-deoxycytidine 5′-triphosphate (F 2 CTP). [1′-3 H] and [5-3 H]-F 2 CTP were synthesized and used independently to inactivate RNR. Sephadex G-50 chromatography of the inactivation mixture revealed that 0.47 eq. of a sugar were covalently bound to RNR and that 0.71 eq. of cytosine were released. Alternatively, analysis of the inactivated RNR by SDS PAGE without boiling, resulted in 33% of RNR migrating as a 110 kDa protein. Inactivation of RNR with a mixture of [1′-3 H]-F 2 CTP/ [1′-2 H]-F 2 CTP followed by reduction with NaBH 4 , alklyation with iodoacetamide, trypsin digestion, and HPLC separation of the resulting peptides, allowed isolation and identification by MALDI-TOF mass spectrometry (MS) of a [ 3 H/ 2 H]-peptide containing C 731 and C 736 from the C-terminus of RNR accounting for 10% of the labeled protein. The MS analysis also revealed that the two cysteines were cross-linked to a furanone-species derived from the sugar of F 2 CTP. Incubation of [1′-3 H]-F 2 CTP with C119S-RNR resulted in 0.3 eq. of sugar covalently bound to the protein and incubation with NaBH 4 subsequent to inactivation resulted in trapping of 2′-fluoro-2′-deoxycytidine. These studies and the ones in the accompanying manuscript allow proposal of a mechanism of inactivation of RNR by F 2 CTP involving multiple reaction pathways. The proposed mechanisms share many common features with F 2 CDP inactivation of the class I RNRs.The nucleoside 2-deoxy-2′,2′-difluorocytidine (F 2 C, Gemzar ™ ) is a clinically used anti-cancer drug, which targets ribonucleotide reductase (RNR) and DNA polymerase in addition to several other proteins involved in nucleotide metabolism.(1-3) Studies have shown that F 2 C is therapeutically effective against a range of cancers(4-17) with tolerable levels of toxicity. The mechanism by which F 2 C inactivates RNRs has been the subject of a number of investigations (18-21), modeled on our detailed understanding of the mechanism by which 2′-substituted 2′-deoxynucleotides inhibit these enzymes (Scheme 1).(22) These studies revealed that gemcitabine 5′-diphosphate (F 2 CDP) in the case of the class I RNRs (E. coli and humans are both diphosphate reductases, RDPR) and gemcitabine-5-triphosphate (F 2 CTP) in the case of the class II RNR from Lactobacillus leichmannii (a nucleoside triphosphate reductase, RTPR) are potent, mechanism-based inhibitors. (20,21,(23)(24)(25) § Funding for this study was provided in part by NIH grant GM-29595.*To whom correspondence should be addressed. Tel: (617) 253-1814. Fax: (617) SUPPORTING INFORMATION AVAILABLE Outline of F 2 C synthesis (Scheme S1). Depiction of PSD peptide fragments (Scheme S2). MS/MS analysis of RTPR peptide of 2020 Da ( Figure S1). This material is available free of charge via th...

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“…This topic will thus not be covered here in detail; instead the focus will be on approaches to UDP‐Gal f and oligosaccharides containing Gal f residues. However, it should be appreciated that a number of approaches for the conversion of galactopyranose derivatives to the corresponding furanose forms have now been reported, including kinetically controlled Fisher glycosylation,102, 103 electrophile‐induced cyclization of dithioacetals or O , S ‐mixed acetals,101, 104–106 high‐temperature benzoylation of galactose in pyridine,107 anomeric alkylation,108 reduction of protected D ‐galactonolactones with borane reagents109 and reduction of furanoside derivatives of D ‐galacturonic acid with a mixture of sodium borohydride and iodine 100…”

Section: Chemical Synthesis Of Galf‐containing Glycansmentioning

confidence: 99%

Chemistry and Biology of Galactofuranose‐Containing Polysaccharides

2009

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The thermodynamically less stable form of galactose-galactofuranose (Galf)-is essential for the viability of several pathogenic species of bacteria and protozoa but absent in this form in mammals, so the biochemical pathways by which Galf-containing glycans are assembled and catabolysed are attractive sites for drug action. This potential has led to increasing interest in the synthesis of molecules containing Galf residues, their subsequent use in studies directed towards understanding the enzymes that process these residues and the identification of potential inhibitors of these pathways. Major achievements of the past several years have included an in-depth understanding of the mechanism of UDP-galactopyranose mutase (UGM), the enzyme that produces UDP-Galf, which is the donor species for galactofuranosyltransferases. A number of methods for the synthesis of galactofuranosides have also been developed, and practitioners in the field now have many options for the initiation of a synthesis of glycoconjugates containing either alpha- or beta-Galf residues. UDP-Galf has also been prepared by a number of approaches, and it appears that a chemoenzymatic approach is currently the most viable method for producing multi-milligram amounts of this important intermediate. Recent advances both in the understanding of the mechanism of UGM and in the synthesis of galactofuranose and its derivatives are highlighted in this review.

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A New Method for the Synthesis of Furanose Derivatives of Aldohexoses¹

Cited by 65 publications

References 3 publications

Enhanced subunit interactions with gemcitabine-5′-diphosphate inhibit ribonucleotide reductases

Enhanced subunit interactions with gemcitabine-5′-diphosphate inhibit ribonucleotide reductases

Inactivation of Lactobacillus leichmannii Ribonucleotide Reductase by 2′,2′-Difluoro-2′-deoxycytidine 5′-Triphosphate: Covalent Modification

Chemistry and Biology of Galactofuranose‐Containing Polysaccharides

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A New Method for the Synthesis of Furanose Derivatives of Aldohexoses1

Cited by 65 publications

References 3 publications

Enhanced subunit interactions with gemcitabine-5′-diphosphate inhibit ribonucleotide reductases

Enhanced subunit interactions with gemcitabine-5′-diphosphate inhibit ribonucleotide reductases

Inactivation of Lactobacillus leichmannii Ribonucleotide Reductase by 2′,2′-Difluoro-2′-deoxycytidine 5′-Triphosphate: Covalent Modification

Chemistry and Biology of Galactofuranose‐Containing Polysaccharides

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A New Method for the Synthesis of Furanose Derivatives of Aldohexoses¹