Deoxynucleoside S'-triphosphate analogues in which the beta,gamma-bridging oxygen has been replaced with a CXY group are useful chemical probes to investigate DNA polymerase catalytic and base-selection mechanisms. A limitation of such probes has been that conventional synthetic methods generate a mixture of diastereomers when the bridging carbon substitution is nonequivalent (X not equal Y). We report here a general solution to this longstanding problem with four examples of /beta,gamma-CXY dNTP diastereomers: (S)- and (R)-beta,gamma-CHCI-dGTP (12a-1/12a-2) and (S)- and (R)-beta,gamma-CHF-cIGTP (12b-1/12b-2). Central to their preparation was conversion of the prochiral parent bisphosphonic acids to the P,Cdimorpholinamide derivatives 7 of their (R)-mandelic acid monoesters, which provided access to the individual diastereomers 7a-1, 7a-2, 7b-1, and 7b-2 by preparative HPLC. Selective acidic hydrolysis of the P N bond then afforded "portal" diastereomers, which were readily coupled to morpholine-activated dGMP. Removal of the chiral auxiliary by H-2 (Pd/C) gave the four individual diastereomeric nucleotides 12, which were characterized by P-31, H-1, and F-19 NMR spectroscopy and by mass spectrometry. After treatment with Chelex-100 to remove traces of paramagnetic ions, at pH similar to 10 the diastereomer pairs 12a,b exhibit discrete P-alpha and P-beta P-31 resonances. The more upheld P-alpha and more downfield P-beta resonances (and also the more upheld F-19 NMR resonance in 12b) are assigned to the R configuration at the P-beta-CHX-P-gamma, carbons on the basis of the absolute configurations of the individual diastereomers as determined from the X-ray crystallographic structures of their ternary complexes with DNA and polyrnerase beta.