Irradiation of DNA with UV-B light causes the formation of mutagenic DNA lesions such as cis-syn and trans-syn cyclobutane pyrimidine dimers. DNA photolyases are flavin-dependent repair enzymes which directly revert the mutagenic cis-syn pyrimidine dimers into the corresponding monomers by a light-facilitated repair reaction. To gain deeper insight into the repair process, we recently prepared flavin-containing model compounds which are able to mimic the repair reaction (Carell, T.; Epple, R.; Gramlich, V. Angew. Chem., Int. Ed. Engl. 1996, 35, 620-623). This publication now contains a detailed description of the synthesis of a series of related model compounds and a comprehensive investigation of their cleavage properties. The results obtained help to unravel the requirements necessary for an efficient, flavin-mediated cleavage of pyrimidine dimers and provide insight into the factors on which the enzymatic repair process depends. The investigation of the cleavage reaction with cis-syn, trans-syn, and hans-anti cyclobutane pyrimidine dimer model compounds reveal an enhanced vulnerability for the cis-syn isomer. The trans-syn dimer is 10 times more stable, These results are comparable to those observed in a recent study on the E. coli enzyme. The excellent solubility of some of the model compounds has allowed a medium-dependent investigation of the flavin-initiated cleavage reaction. increased cleavage efficiencies are observed in polar solvents such as water (phi = 0.06) and acetonitrile (phi = 0.05). The quantum yields decrease by a factor of 4 in solvents with very low polarity such as dioxane (phi = 0.01). These results are not in agreement with earlier solvent-dependent evaluations performed with non-flavin-containing model compounds (Hartzfeld, D. G.; Rose, S. D. J. Am. Chem. Soc. 1993, 115,850-854). The results, however, suggest that the unusually polar flavin-binding pocket, observed in the X-ray crystal structure of the E, coli. photolyase, might be required to increase the catalytic repair efficiency. Investigations of the cleavage reaction in the presence of acid and base in organic solvents emphasize the strict requirement for a deprotonated reduced riboflavin chromophore. The determined pH values for half-maximal (pH = 6.5) and maximal (7 less than or equal to pH less than or equal to 9) cleavage efficiencies are in agreement with the pK(a) value (pK(a) = 6.3) of the reduced riboflavin and reveal that physiological conditions are required to reach maximum catalytic cleavage efficiency.