alpha-fluorinated phosphonates as substrate mimics for glucose 6-phosphatedehydrogenase: the CHF stereochemistry matters

Reported is a systematic study of the "fitness" tin terms of k(cat)/K-m) of a series of phosphonate mimics of glucose 6-phosphate (G6P) as unnatural s ubstrates for G6P dehydrogenase from Leuconostoc mesenteroides. The four G6 P analogues (9, 10, 15a, and 15b) differ only in the degree of fluorination at the "bridging" phosphonate carbon. All have been synthesized from benzy l 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 o btained by direct displacements with the appropriate LiX2CP(O)-(OEt)(2) rea gents (X = H, F) in 15 min at -78 degrees C. For the (alpha-monofluoro)alky lphosphonates (15a/b), homologation of 6 is achieved via lithiodithiane-med iated triflate displacement, followed by aldehyde unmasking [CaCO3, Hg(ClO4 )(2), H2O]. Addition of diethyl phosphite anion produces diastereomeric, (a lpha-hydroxy)phosphonates 13a/b (1.4:1 ratio) which may be readily separate d by chromatography. The stereochemistry of the minor diastereomer was esta blished as 7(S) via X-ray crystallographic structure determination of its p -bromobenzoate derivative, 16b. Treatment of the major 7!R) diastereomer wi th DAST produces alpha-fluorinated phosphonate 14a, in modest yield, with i nversion 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 th us establishes the stereochemical course of this transformation. alpha-Depr otonation/kinetic quenching of 14a provides access to the 7(R)-epimer (14b) . For all four protected phosphonates (7, 8, 14a, and 14b), diethyl phospho nate ester deprotection was carried out with TMSBr, followed by global hydr ogenolytic debenzylation to produce the free phosphonates, as alpha/beta no meric mixtures. Titrations of G6P itself and the free phosphonic acids prov ides second pK(a) 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 k( cat)/K-m 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 K-m. The fact that k(cat)/K-m in creases by more than an order of magnitude in going from the 7(R)-alpha-mon ofluoroalkyl phosphonate (worst substrate) to the 7(S)-diastereomer (best s ubstrate) is especially notable and is discussed in the context of the know n phosphate binding pocket of this enzyme as revealed by X-ray crystallogra phy.