Generally Applicable, Convenient Solid-Phase Synthesis and Receptor Affinities of Octreotide Analogs

octreotide was assembled by Fmoc solid-phase synthesis and the intramolecular disulfide bond formed by treatment of the resin-bound peptide with thallium trifluoroacetate [Tl(Tfa)J Sidechain protection of Trp by the Boc group was found to preserve Trp integrity during Tl(Tfa)s treatment. The protected peptide was cleaved from the resin by aminolysis with threoninol and purified by semipreparative RP-HPLC. Isolated [~-Trp(Boc)~,Lys(Boc)~,Thr(tBu)~loctreotide had the correct molecular mass ([M + HI+ = 1275 Da) and sequence and was obtained in 14% yield at ’98% purity. [~-Trp(Boc)~,Lys(Boc)~,Thr(tBu)~loctreotide was utilized for the solutionphase synthesis of CPTA-D-Phel-octreotide, where CPTA is 4-[(1,4,8,1 l-tetraazacyclotetradec1-y1)methyllbenzoic acid. Cyclic dianhydride of diethylenetriaminepentaacetic acid (DTPA) was coupled to a portion of the protected peptide-resin following disulfide bond formation. The DTPA-conjugated, side-chain-protected peptide was cleaved from the resin by aminolysis with threoninol, side-chain deprotected with trifluoroacetic acid, and purified by semipreparative RP-HPLC. The isolated DTPA-D-Phel-octreotide had the correct molecular mass ([M + HI+ = 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) handles and the resin andor resin particle size. The somatostatin receptor binding affinities of synthetic DTPA-D-Phel-octreotide and CPTA-D-Phel-octreotide to AtT-20 mouse pituitary carcinoma cell membranes were examined by labeling with lllIn and 64Cu, respectively, and performing Scatchard analyses. The dissociation constant (&) for our synthetic [lllInlDTPA-~-Pheloctreotide was 4.31 nM, which is comparable to a & = 5.57 nM obtained with commercially available DTPA-D-Phel-octreotide. The & for [64C~lCPTA-~-Phe1-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 synthetic 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 for the preparation of octreotide.