GENERALLY APPLICABLE, CONVENIENT SOLID-PHASE SYNTHESIS AND RECEPTOR AFFINITIES OF OCTREOTIDE ANALOGS

Octreotide, an analogue of the hormone somatostatin, has applications as a therapeutic and imaging agent for somatostatin-positive tumors. W e have developed a generally applicable, convenient stepwise solid-pha se synthetic protocol for octreotide (D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys- threoninol). D-Trp(Boc)(4),Lys(Boc)(5),Thr(tBu)(6),Cys(Acm)(7), des(th reoninol)]- octreotide was assembled by Fmoc solid-phase synthesis and the intramolecular disulfide bond formed by treatment of the resin-bo und peptide with thallium trifluoroacetate [Tl(Tfa)(3)]. Sidechain pro tection of Trp by the Boc group was found to preserve Trp integrity du ring Tl(Tfa)(3) treatment. The protected peptide was cleaved from the resin by aminolysis with threoninol and purified by semipreparative RP -HPLC. Isolated [D-Trp (Boc)(4),Lys(Boc)(5),Thr(tBu)(6)]octreotide had the correct molecular mass ([M + H](+) = 1275 Da) and sequence and wa s obtained in 14% yield at >98% purity. [D-Trp(Boc)(4),Lys(Boc)(5),Thr (tBu)(6)]octreotide was utilized for the solution-phase synthesis of C PTA-D-Phe(1)-octreotide, where CPTA is 1,4,8,11-tetraazacyclotetradec- 1-yl)methyl]benzoic acid. Cyclic dianhydride of diethylenetriaminepent aacetic acid (DTPA) was coupled to a portion of the protected peptide- resin following disulfide bond formation. The DTPA-conjugated, side-ch ain-protected peptide was cleaved from the resin by aminolysis with th reoninol, side-chain deprotected with trifluoroacetic acid, and purifi ed by semipreparative RP-HPLC. The isolated DTPA-D-Phe(1)-octreotide h ad the correct molecular mass ([M + H](+) = 1395 Da) and was obtained in 5% yield at >90% purity. The efficiency of aminolysis was partially dependent upon the linkage between 4-(hydroxymethyl)phenoxy (HMP) han dles and the resin and/or resin particle size. The somatostatin recept or binding affinities of synthetic DTPA-D-Phe(1)-octreotide and CPTA-D -Phe(1)-octrebtide to AtT-20 mouse pituitary carcinoma cell membranes were examined by labeling with In-111 and Cu-64, respectively, and per forming Scatchard analyses. The dissociation constant (K-d)for our syn thetic [In-111]DTPA-D-Phe(1)-octreotide was 4.31 nM, which is comparab le to a K-d = 5.57 nM obtained with commercially available DTPA-D-Phe( 1)-octreotide. The K-d for [Cu-64]CPTA-D-Phe(1)-octreotide was 78.5 pM . On the basis of the criteria of molecular mass, RP-HPLC elution time , sequence analysis, and somatostatin receptor binding affinity, our s ynthetic octreotide is identical to commercially available octreotide. The aminolysis protocol used here;has distinct advantages Over either reductive cleavage or preformed linker methods described previously f or the preparation of octreotide.