TY - JOUR
T1 - α-Fluorinated phosphonates as substrate mimics for glucose 6-phosphate dehydrogenase
T2 - The CHF stereochemistry matters
AU - Berkowitz, David B.
AU - Bose, Mohua
AU - Pfannenstiel, Travis J.
AU - Doukov, Tzanko
PY - 2000/7/28
Y1 - 2000/7/28
N2 - Reported is a systematic study of the 'fitness' (in terms of k(cat)/K(m)) 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-trifluoromethane-sulfonyl-2,3,4-tri-O-benzyl β-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 °C. For the (α-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, (α-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 α-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 (α-hydroxy)-phosphonate and thus establishes the stereochemical course of this transformation. α-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 α/β anomeric mixtures. Titrations of G6P itself and the free phosphonic acids provides 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) increases by more than an order of magnitude in going from the 7(R)-α-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).
AB - Reported is a systematic study of the 'fitness' (in terms of k(cat)/K(m)) 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-trifluoromethane-sulfonyl-2,3,4-tri-O-benzyl β-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 °C. For the (α-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, (α-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 α-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 (α-hydroxy)-phosphonate and thus establishes the stereochemical course of this transformation. α-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 α/β anomeric mixtures. Titrations of G6P itself and the free phosphonic acids provides 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) increases by more than an order of magnitude in going from the 7(R)-α-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).
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U2 - 10.1021/jo000220v
DO - 10.1021/jo000220v
M3 - Article
C2 - 10959850
AN - SCOPUS:0034725770
SN - 0022-3263
VL - 65
SP - 4498
EP - 4508
JO - Journal of Organic Chemistry
JF - Journal of Organic Chemistry
IS - 15
ER -