title: alpha-fluorinated phosphonates as substrate mimics for glucose 6-phosphate dehydrogenase: the CHF stereochemistry matters.
abstract: Reported is a systematic study of the "fitness" (in terms of kcat/Km) of a series of phosphonate mimics of glucose 6-phosphate (G6P) as unnatural substrates for G6P dehydrogenase from Leuconostoc mesenteroides. The four G6P analogues (9, 10, 15a, and 15b) differ only in the degree of fluorination at the "bridging" phosphonate carbon. All have been synthesized from benzyl 6-O-trifluoromethanesulfonyl-2,3,4-tri-O-benzyl beta-D-glucopyranoside (6). The phosphonates with bridging CH2 (9) and CF2 (10) groups are cleanly obtained by direct displacements with the appropriate LiX2CP(O)(OEt)2 reagents (X = H, F) in 15 min at -78 degrees C. For the (alpha-monofluoro)alkylphosphonates (15a/b), homologation of 6 is achieved via lithiodithiane-mediated triflate displacement, followed by aldehyde unmasking [CaCO3, Hg(ClO4)2, H2O]. Addition of diethyl phosphite anion produces diastereomeric, (alpha-hydroxy)phosphonates 13a/b (1.4:1 ratio) which may be readily separated by chromatography. The stereochemistry of the minor diastereomer was established as 7(S) via X-ray crystallographic structure determination of its p-bromobenzoate derivative, 16b. Treatment of the major 7(R) diastereomer with DAST produces alpha-fluorinated phosphonate 14a, in modest yield, with inversion of configuration, as established, again, by X-ray crystallography. To our knowledge, this is first example of DAST-mediated fluorination of a (nonbenzylic, nonpropargylic) secondary (alpha-hydroxy)phosphonate and thus establishes the stereochemical course of this transformation. alpha-Deprotonation/kinetic quenching of 14a provides access to the 7(R)-epimer (14b). For all four protected phosphonates (7, 8, 14a, and 14b), diethyl phosphonate ester deprotection was carried out with TMSBr, followed by global hydrogenolytic debenzylation to produce the free phosphonates, as alpha/beta anomeric mixtures. Titrations of G6P itself and the free phosphonic acids provides second pKa values of 6.5 (1, bridging-O), 5.4 (10, bridging-CF2), 6.2 (14a, bridging-CHF), and 7.6 (9, bridging-CH2). Leuconostoc mesenteroides G6PDH-mediated oxidation and Lineweaver-Burk analysis yields normalized kcat/Km values of 0.043 (14b, bridging-7(R)-CHF), 0.11 (10, bridging-CF2), 0.23 (14b, bridging-CH2), and 0.46 (14a, bridging-7(S)-CHF) relative to G6P itself, largely reflecting differences in Km. The fact that kcat/Km increases by more than an order of magnitude in going from the 7(R)-alpha-monofluoroalkyl phosphonate (worst substrate) to the 7(S)-diastereomer (best substrate) is especially notable and is discussed in the context of the known phosphate binding pocket of this enzyme as revealed by X-ray crystallography (Adams, M. J. et al. Structure 1994, 2, 1073-1087).
authors.affiliation: Department of Chemistry, University of Nebraska, Lincoln, 68588-0304, USA. dbb@unlserve.unl.edu
authors.forename: D B
authors.initials: DB
authors.lastname: Berkowitz
authors.forename: M
authors.initials: M
authors.lastname: Bose
authors.forename: T J
authors.initials: TJ
authors.lastname: Pfannenstiel
authors.forename: T
authors.initials: T
authors.lastname: Doukov
mesh_terms: D018360:Crystallography, X-Ray; D005461:Fluorine / Q000737:chemistry / Q000378:metabolism*; D019298:Glucose-6-Phosphate / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D005954:Glucosephosphate Dehydrogenase / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D007934:Leuconostoc / Q000201:enzymology*; D008958:Models, Molecular; D008968:Molecular Conformation; D063065:Organophosphonates / Q000737:chemistry / Q000378:metabolism*; D013237:Stereoisomerism; D013379:Substrate Specificity
chemical_list: D063065:Organophosphonates; D005461:Fluorine; D019298:Glucose-6-Phosphate; D005954:Glucosephosphate Dehydrogenase