Chemoenzymatic Assembly of Isotopically Labeled Folates

Abstract

Pterin-containing natural products have diverse functions in life, but an efficient and easy scheme for their in vitro synthesis is not available. Here we report a chemoenzymatic 14-step, one-pot synthesis that can be used to generate C- andN-labeled dihydrofolates (HF) from glucose, guanine, and p-aminobenzoyl-l-glutamic acid. This synthesis stands out from previous approaches to produce HF in that the average yield of each step is >91% and it requires only a single purification step. The use of a one-pot reaction allowed us to overcome potential problems with individual steps during the synthesis. The availability of labeled dihydrofolates allowed the measurement of heavy-atom isotope effects for the reaction catalyzed by the drug target dihydrofolate reductase and established that protonation at N5 of HF and hydride transfer to C6 occur in a stepwise mechanism. This chemoenzymatic pterin synthesis can be applied to the efficient production of other folates and a range of other natural compounds with applications in nutritional, medical, and cell-biological research.

Loss of Hyperconjugative Effects Drives Hydride Transfer during Dihydrofolate Reductase Catalysis

“…Subsequently, 6-FP was reduced to 6-hydroxymethylpterin (6-HMP) by dimethylaminoborane (DMAB). Further reduction of 6-HMP by sodium dithionite affords 6-hydroxymethyl-7,8-dihydropterin (6-HMDP), which was enzymatically transformed to H 2 F by the combined actions of 6-hydroxymethyl 7,8-dihydropterin pyrophosphokinase (HPPK) and dihydropteroate synthase (DHPS) . (b) Reduction of deuterated 6-FP (6-FP- d ) with either ( S )- or ( R )-alpine borane offers an alternative route to stereoselectively introduce a deuterium in 6-HMP .…”

“…(b) Reduction of deuterated 6-FP (6-FP- d ) with either ( S )- or ( R )-alpine borane offers an alternative route to stereoselectively introduce a deuterium in 6-HMP . Cofactor recycling was operated by myokinase (MK) and pyruvate kinase (PK). , Details can be found in the Supporting Information.…”

“…6-HMDP is a metabolite of the folate de novo biosynthetic pathway and thus can be transformed in vitro to H 2 F (Figure a) with 6-hydroxymethyl 7,8-dihydropterin pyrophosphokinase (HPPK) and dihydropteroate synthase (DHPS) . In the first step, 6-HMDP was added with pyrophosphate by HPPK.…”

“…Primary KIEs arise when atoms directly involved in the chemical transformation are replaced by their heavy counterparts. ,,− Primary KIE measurements for NADPH­(D) and heavy-atom ( 15 N, 13 C) isotope labeling of the primary reacting centers have generated evidence in support of a stepwise mechanism for DHFR from E. coli ( Ec DHFR). ,,, Secondary α-deuterium KIEs (α-KIEs) arising from the rehybridization of C4′ of NADPH provide atomistic insights into local environmental changes during the chemical transformation, as isotopic substitution influences the rehybridization process of the primary atoms, which is reflected as a change in reaction rate. ,− However, despite Ec DHFR being one of the most studied enzymes, the role of C6 rehybridization in H 2 F has never been investigated in detail. Because the four hydrogens on C7 and C9 of H 2 F are located in positions β to C6, secondary β-deuterium isotope effects (β-KIEs) can be measured to explore the extent of C6 rehybridization.…”

“…6,19,21−23 Primary KIE measurements for NADPH(D) and heavy-atom ( 15 N, 13 C) isotope labeling of the primary reacting centers have generated evidence in support of a stepwise mechanism for DHFR from E. coli (EcDHFR). 21,22,24,25 Secondary α-deuterium KIEs (α-KIEs) arising from the rehybridization of C4′ of NADPH provide atomistic insights into local environmental changes during the chemical transformation, as isotopic substitution influences the rehybridization process of the primary atoms, which is reflected as a change in reaction rate. 23,26−28 2), 29 and this has been attributed to hyperconjugation, a quantum mechanical effect where σ C β −H(D) bonds partially donate electrons to the neighboring electron-deficient π-bond.…”