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

Abstract

Octreotide, an analogue of the hormone somatostatin, has applications as a therapeutic and imaging agent for somatostatin-positive tumors. We have developed a generally applicable, convenient stepwise solid-phase synthetic protocol for octreotide (D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-threoninol). [Cys(Acm)2,D-Trp(Boc)4,Lys(Boc)5,Thr(fBu)6,Cys(Acm)7,des(threoninol)]octreotide was assembled by Fmoc solid-phase synthesis and the intramolecular disulfide bond formed by treatment of the resin-bound peptide with thallium trifluoroacetate [TKTfajg]. Sidechain protection of Trp by the Boc group was found to preserve Trp integrity during Tl(Tfa)a 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(fBu)6]octreotide had the correct molecular mass ([ + H]+ = 1275 Da) and sequence and was obtained in 14% yield at >98% purity. [D-Trp(Boc)4,Lys(Boc)5,Thr(fBu)6]octreotide was utilized for the solutionphase synthesis of CPTA-D-Phe^octreotide, where CPTAis 4-[(l,4,8,ll-tetraazacyclotetradecl-yl)methyl]benzoic 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-o-Phe1-octreotide had 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) handles and the resin and/or resin particle size. The somatostatin receptor binding affinities of synthetic DTPA-D-PheLoctreotide and CPTA-D-Phe^octreotide to AtT-20 mouse pituitary carcinoma cell membranes were examined by labeling with mIn and 64Cu, respectively, and performing Scatchard analyses. The dissociation constant ( &) for our synthetic [111In]DTPA-D-Phe1octreotide was 4.31 nM, which is comparable to a K¿ = 5.57 nM obtained with commercially available DTPA-D-Phe1-octreotide. The K¿ for [64Cu]CPTA-D-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.

Development of inductively coupled plasma–mass spectrometry-based protease assays

“…The biotin tag was incorporated into the peptide sequence during the solid phase synthesis using the commercially available Fmoc-lys(biotin)-OH building block [31, 32]. Lanthanides are known to form thermodynamically and kinetically stable complexes with diethylenetriaminepentaacetic acid (DTPA)-based ligands and this chelate was coupled to the N-terminus of the peptide substrate [33, 34]. The sequences of the synthesized α-chymotrypsin substrates are shown in Table 1.…”

Preparation and Biological Evaluation of Copper-64–Labeled Tyr3-Octreotate Using a Cross-Bridged Macrocyclic Chelator

“…The AR42J (32,33) using an Advanced Chem Tech peptide synthesizer. To facilitate on-board cyclization, acetamidomethyl thiol-protected cysteine was used to introduce the cysteine amino acids (34). After peptide assembly, intramolecular disulfide cyclization was performed on solid support with thallium trifluoroacetate (23 mg, 42 mol in 2 mL DMF) for 90 minutes, and the resin was thoroughly washed with DMF.…”

Exploring new near-infrared fluorescent disulfide-based cyclic RGD peptide analogs for potential integrin-targeted optical imaging

“…The protected RGD peptide Fmoc-C(Acm)-R(Pbf)-G-D(OBut)-C(Acm) was first assembled on Rink amide MBHA resin (1 equiv) using conventional Fmoc chemistry (Scheme 1). The disulfide-based cyclization was realized by swirling the resin-bound peptide with a solution of Tl(F 3 CCOO) 3 in DMF for 2 h, yielding the disulfide-containing cyclic peptide on a resin, Fmoc- c [CR(Pbf)GD(OBut)C]-Resin 39, 40. After the Fmoc protecting group was removed with piperidine/DMF, the free amino group at the N-terminus was conjugated with cypate in the presence of DIC and HOBT.…”