Five 2-substituted 2'-deoxyinosine triphosphates (dRITP) were synthesized and tested as substrates in enzymatic synthesis of minor-groove base-modified DNA. Only 2-methyl and 2-vinyl derivatives proved to be good substrates for Therminator DNA polymerase, whilst all other dRITPs and other tested DNA polymerases did not give full length products in primer extension. The DNA containing 2-vinylhypoxanthine was then further modified through thiol-ene reactions with thiols. Cross-linking reaction between cysteine-containing minor-groove binding dodecapeptide and DNA proceeded thanks to the proximity effect between thiol and vinyl groups inside the minor groove. 2-Substituted dIRTPs and also previously prepared 2-substituted 2'-deoxyadenosine triphosphates (dRATP) were then used for enzymatic synthesis of minor-groove modified DNA to study the effect of minor-groove modifications on cleavage of DNA by type II restriction endonucleases (REs). Although the REs should recognize the sequence through H-bonds in the major groove, some minor-groove modifications also had an inhibiting effect on the cleavage.