Chemical Synthesis of (R_P)- and (S_P)-[¹⁶O,¹⁷O,¹⁸O]Phosphoenol Pyruvate

Abstract

Enzymes and chirality are intimately associated. For their mechanisms to be studied, chiral substrates are needed as probes. Here, we report a concise synthesis of (R)- and (S)-[O,O,O]phosphoenol pyruvate starting from enantiomerically pure (R)-2-chloro-1-phenylethanol, which was transformed into O-labeled 3-methyl-1-phenylbutane-1,3-diol. The diol was reacted with tris(dimethylamino)phosphane and consecutively with HO to yield a mixture of cyclic H-phosphonates labeled with O andO. They were silylated and subjected to a Perkow reaction with ethyl 3-chloropyruvate. Two protected-[O,O,O]phosphoenol pyruvates were formed and finally globally deprotected. Their configuration was reassessed by a known enzymatic test in combination with conversion of the formed d-glucose-6-phosphate into mixtures of labeled methyl d-glucose-4,6-phosphates, which were analyzed by P NMR spectroscopy. The enzymatic test supported the configuration assigned to labeled stereogenic phosphorus atoms on the basis of synthesis.

Monitoring of phosphatase and kinase activity using 31P NMR spectroscopy

“…All chemical shifts detected were similar to the reported data. 20,22,23 Notably, the 31 P chemical shift (Fig. 1a, δ = 0.10 ppm) of AMP is obviously different from that of cAMP (Fig.…”

Essential Functional Interplay of the Catalytic Groups in Acid Phosphatase

“…In accordance with the proposed catalytic mechanism of 18 O isotope exchange (Figure S9), variants of ecAGP having the catalytic nucleophile (His18) or the catalytic acid−base (Asp290) replaced by an incompetent alanine residue were 53 We prepared (R P )-and (S P )-[ 16 O, 17 O, 18 O]phosphoenolpyruvate (PEP) as two complementary substrates to unravel the stereochemical course of the enzymatic phosphoryl transfer. The synthetic route used was reported recently, 54 but we here improved substantially on its overall efficiency (Sections S1.20−S1.27). Although PEP was hydrolyzed by both wildtype enzyme (k cat = 0.8 s −1 ) and the H18D variant (k cat = 6 × 10 −6 s −1 ), it was not a usable phosphate donor substrate for transphosphorylation of glucose.…”

“…The phosphoryl transfer from PEP to mannose occurs in two enzymatic steps, each of which is known to proceed with inversion of configuration. 54,55 The Man1P thus obtained has the same (R P )-or (S P )configuration at the phosphorus atom as the P-chiral PEP substrate used. (R P )-or (S P )-Man1P (10 mM) was reacted with glucose (800 mM) in the presence of wild-type enzyme S7 and S8.…”

The Essential Functional Interplay of the Catalytic Groups in Acid Phosphatase

“…53 We prepared (RP)-and (SP)-[ 16 O, 17 O, 18 O]phosphoenolpyruvate (PEP) as two complementary substrates to unravel the stereochemical course of the enzymatic phosphoryl transfer. The synthetic route used was reported recently 54 , but we here improved substantially on its overall efficiency (Supporting Information S1.20-S1.27). Although PEP was hydrolyzed by both wildtype enzyme (kcat = 0.8 s -1 ) and the H18D variant (kcat = 6  10-6 s -1 ), it was not a usable phosphate donor substrate for transphosphorylation of glucose.…”

“…The phosphoryl transfer from PEP to mannose occurs in two enzymatic steps, each of which is known to proceed with inversion of configuration. 54,55 The Man1P thus obtained has the same (RP)-or (SP)configuration at the phosphorus atom as the P-chiral PEP substrate used. (RP)-or (SP)-Man1P (10 mM) was reacted with glucose (800 mM) in the presence of wildtype enzyme (0.07 µM; 60 min) or H18D variant (22 µM; 8 days).…”