HPLC evidence is reported demonstrating the occurrence in some human urine samples of a novel catecholic metabolite, (3R,7S)-3, 7-dicarboxy-10,11-dihydroxy-2,3,4,5,6,7,8,9-octahydropyrido[ 4,3-g][1, 4]benzothiazepine (2). The compound was shown to arise by a double Pictet-Spengler condensation of the urinary melanogen 5-S-cysteinyldopa (1) with formaldehyde, in which regioselective formation of the six-membered ring ortho to the activating hydroxyl group lends assistance to the subsequent closure of the seven-membered 1,4-thiazepine moiety. Under physiologically relevant conditions, i.e., in 0.1 M phosphate buffer pH 7.4 and at 37 degrees C, the 7,8-tetrahydroisoquinoline 5 was the sole detectable intermediate in the formation of 2. N-Acetylcysteinyldopa (4) reacted likewise with formaldehyde to give the 7, 8-dihydroxytetrahydroisoquinoline 6. The anomalous regiochemistry underlying formation of 5 and 6 was rationalized with the aid of AM1/PM3 calculations on the model alkylthiocatechol 10, predicting a higher HOMO-controlled reactivity on the position ortho rather than para to the activating hydroxyl group. The potential of the reported chemistry as a convenient synthetic access to the 2,3,4, 5-tetrahydro[1,4]benzothiazepine ring system is suggested by the efficient conversion of the cysteinylcatechol 3 to 8 in the presence of formaldehyde.