Page - (000313) - in Biomedical Chemistry: Current Trends and Developments

two primary amide functions of the side chains of the glutamine in the C-terminal of SP1–7 resulted in a considerable decrease in affinity (7 and 8). The C-terminal phenylalanine was absolutely crucial for strong affinity in both SP1–7 and EM-2. Replacement of the C-terminal phenylalanine in SP1–7 gave analog 9, which was devoid of affinity. Making the same substitution in EM-2 resulted in compound 13 with a Ki value of 1460 nM. The finding that the C-terminal phenylalanine plays such an important role in binding affinity is in line with the peptide scan discussed above, where the C-terminal fragment SP1–6 possessed very weak binding affinity to the SP1–7 binding site (Botros, 2006; Igwe, 1990). Moreover, the potency of SP1–7 showed a five-fold increase upon amidation of the terminal carboxyl group (15). Similarly, the binding affinity of EM-2 was reduced by a factor of four upon removal of the amide function (16). Since SP1–7 is a proteolytic product of SP resulting in a C-terminal carboxylic acid, it was surprising that the amidated analog 15 led to improved binding affinity. As deduced from the two Ala scans, the N-terminal parts of SP1–7 and EM-2 does not seem to be engaged in molecular recognition or binding to the target protein, a fact that prompted synthesis of the truncated analogs 17–24 and 25–27. For SP1–7, removal of the N-terminal arginine rendered the hexapeptide 17 with a 20-fold reduction in affinity compared to SP1–7 and a little more than 2-times lower affinity than the alanine derivative 3. The affinity could, however, be recovered by amidation of 17, resulting in peptide 18, which was 10 times more potent. Further truncation down to the tripeptide level was possible without loss of affinity, and C-terminal amidation of all the truncated SP1–7 analogs improved the affinity 5–10-fold. Hence, the tripeptide H- Gln-Gln-Phe-NH2 (24) exhibited a Ki of 1.9 nM. For EM-2, simultaneous removal of the two N-terminal amino acids tyrosine and proline improved the binding affinity six times, resulting in the notable discovery of the dipeptide H-Phe-Phe-NH2 (26) with a Ki value similar to that of SP1–7 itself. It should be emphasized that the Tyr- Pro sequence is the critical fragment for binding to the µ-receptor, whereas the two C-terminal phenylalanines are not, facts that highlight