1 3.11.96) 2'-Deoxy-5-(isothiazol-5-yl)uridine (12) was synthesized starting from 2'-deoxy-5-iodouridine using a Pdcatalysed cross-coupling reaction with propiolaldehyde diethyl acetal followed by deprotection and ring closure using thiosulfate. 2'-Deoxyuridine 12 has a particular place among the 5-heteroaryl-substituted 2'-deoxyuridines in that it has a high affinity for herpes simplex virus type 1(HSV-1)-encoded thymidine kinase (TK) without antiviral activity. Biochemical studies revealed that 12 is a substrate for viral TK. We further investigated the interaction of 12 with the HSV-1 thymidine kinase. The conformation of 12 in solution was established by NMR spectroscopy. The most stable conformer 12A has the S-atom of the isothiazole ring placed in the neighbourhood of the C(4)=0 group of the pyrimidine moiety. The compound was docked in its most stable conformation in the active site of HSV-1 TK and subjected to energy minimization. This demonstrated that the isothiazole moiety binds in a cavity lined by the side chains ofTyr-132, Arg-163, Ala-167, and Ala-168 and that the C(3) atom of the isothiazole moiety is located in close proximity of the phenolic 0-atom of Tyr-132 and the aliphatic part of the Arg-163 side chain.