Completing the β,γ-CXY-dNTP Stereochemical Probe Toolkit: Synthetic Access to the dCTP Diastereomers and 31 P and 19 F NMR Correlations with Absolute Configurations Pouya Haratipour, Corinne Minard, Maryam Nakhjiri, Amirsoheil Negahbani, Brian T. Chamberlain, Jorge Osuna, Thomas G. Upton, Michelle Zhao, Boris A. Kashemirov, and Charles E. McKenna* Cite This: J. Org. Chem. 2020, 85, 14592-14609 Read Online ACCESS Metrics & More Article Recommendations * sı Supporting Information ABSTRACT: Nucleoside 5′-triphosphate (dNTP) analogues in which the β,γ-oxygen is mimicked by a CXY group (β,γ-CXY- dNTPs) have provided information about DNA polymerase catalysis and fidelity. Definition of CXY stereochemistry is important to elucidate precise binding modes. We previously reported the (R)- and (S)-β,γ-CHX-dGTP diastereomers (X = F, Cl), prepared via P,C-dimorpholinamide CHCl (6a, 6b) and CHF (7a, 7b) bisphosphonates (BPs) equipped with an (R)-mandelic acid as a chiral auxiliary, with final deprotection using H 2 /Pd. This method also affords the β,γ-CHCl-dTTP (11a, 11b), β,γ-CHF (12a, 12b), and β,γ-CHCl (13a, 13b) dATP diastereomers as documented here, but the reductive deprotection step is not compatible with dCTP or the bromo substituent in β,γ-CHBr-dNTP analogues. To complete assembly of the toolkit, we describe an alternative synthetic strategy featuring ethylbenzylamine or phenylglycine-derived chiral BP synthons incorporating a photolabile protecting group. After acid-catalyzed removal of the (R)-(+)-α-ethylbenzylamine auxiliary, coupling with activated dCMP and photochemical deprotection, the individual diastereomers of β,γ-CHBr- (33a, 33b), β,γ-CHCl- (34a,34b), β,γ-CHF-dCTP (35a, 35b) were obtained. The β,γ-CH(CH 3 )-dATPs (44a, 44b) were obtained using a methyl (R)-(-)-phenylglycinate auxiliary. 31 P and 19 F NMR Δδ values are correlated with CXY stereochemistry and pK a2-4 values for 13 CXY-bisphosphonic acids and imidodiphosphonic acid are tabulated. ■ INTRODUCTION Nucleoside triphosphates play critical roles in innumerable aspects of biology and medicine. 1,2 High fidelity in DNA replication is essential to maintain the integrity of the genome and avoid mutations which may lead to human diseases such as cancer. 2-9 The molecular interactions and processes under- lying the catalytic efficiency and exquisite base-specific selectivity of DNA polymerases have therefore been a continuing focus of intense study. 10-19 Nucleotide analogues have proven to be essential tools in the effort to identify intermediate structures and individual steps in the mechanisms of different DNA polymerases. 20-25 We previously reported 26-30 deoxynucleoside 5′-triphos- phate bisphosphonate analogues in which the β,γ-bridging oxygen of the triphosphate moiety (dNP α -O-P β -O-P γ ) is replaced by a substituted methylene group (CXY) as probes of the ground state (GS) and the transition state (TS) in pol β- catalyzed DNA repair, including by the cancer-associated K289M mutant of pol β. 29,31 In addition to β,γ-CXY-dNTPs (N = G, T, and most recently 29 C and A), β,γ-imido-dNTP (β,γ-NH-dNTP, N = A and G) analogues have also been used to study the mechanism of DNA polymerization catalyzed by pol η, which is implicated in the prevention of skin cancer by copying past cyclobutene dimers in UV-damaged DNA. 32 This approach has been extended to unnatural nucleoside triphosphates (β,γ-CF 2 -dNaMTP and β,γ-CF 2 -dTPT3TP) as a strategy to avoid degradation by cellular and secreted phosphatases after cellular uptake. 33,34 These β,γ-modified dNTP analogues (Figure 1) mimic natural dNTP substrates of DNA polymerases in enabling template-dependent incorporation of dNMP into DNA primer, but their complexes with the enzymes can exhibit different ground-state dissociation constants (K d ) and turnover rate constants (k pol ) due to their modified triphosphate groups. The latter case will be expected for a rate-determining step corresponding to a transition state sensitive to triphosphate charge stabilization. This should be the case in a “chemical” rate-determining catalytic step in which the pyrophosphate (PPi) leaving group acquires a negative charge as a result of attack of the DNA primer strand terminal 3′-OH oxygen on P α Special Issue: The New Golden Age of Organo- phosphorus Chemistry Received: May 29, 2020 Published: October 30, 2020 Article pubs.acs.org/joc © 2020 American Chemical Society 14592 https://dx.doi.org/10.1021/acs.joc.0c01204 J. Org. Chem. 2020, 85, 14592-14609 Downloaded via CITY OF HOPE NATL MEDICAL CTR on March 22, 2021 at 23:36:56 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.