Use of isotopically chiral [4′‐ 13 C]penciclovir and 13 C NMR to determine the specificity and absolute configuration of penciclovir phosphate esters formed in HSV‐1‐ and HSV‐2‐infected cells and by HSV‐1‐encoded thymidine kinase

Penciclovir is a potent antiherpesvirus agent which is highly selective due to its phosphorylation only in virus infected cells. Phosphorylation of one of the hydroxymethyl groups of penciclovir (PCV) creates a chiral centre leading to the possible formation of ( R )‐ and ( S )‐enantiomers. The absolute configuration and stereospecificity of the PCV‐phosphates produced in cells infected with herpes simplex viruses types 1 and 2 (HSV‐1 and HSV‐2), as well as by HSV‐1‐encoded thymidine kinase, were determined using isotopically chiral [4′‐ 13 C]PCV precursors and 13 C NMR spectroscopy of the isolated metabolites. The absolute configuration of penciclovir‐triphosphate (PCV‐TP) produced in HSV‐1‐infected cells was shown to be S with an enantiomeric purity of greater than 95%. However, in contrast to HSV‐1‐infected cells in which none of the ( R ) enantiomer was detected, about 10% of ( R )‐PCV‐TP was produced in HSV‐2‐infected cells. Phosphorylation of PCV by HSV‐1‐encoded thymidine kinase was found to give 75% ( S )‐ and 25% ( R )‐PCV‐monophosphate. The proportion of the ( S )‐isomer appears to be amplified in the subsequent phosphorylations leading to the triphosphate. © 1993 Wiley‐Liss, Inc.