Radioactive analogs of neurotensin. II. Preparation of tritiated neurotensin from a synthetic multi-unsaturated derivative

In this second paper on the synthesis of neurotensin analogues as precursors for radiolabelling, solid phase synthesis of two polyunsaturated peptides, Dah6, delta Pro7,10]-neurotensin and acetyl-delta Pro10]-neurotensin-(8-13), are described. The first one contains one triple bond and two double bonds susceptible to tritiation in the same molecule, the second one contains one double bond in the shortest sequence having neurotensin activity. The C-terminal residue, Boc-Leu, was esterified on the chloromethyl-resin by its cesium salt. For the other amino acids a double coupling was carried out, the first one with dicyclohexylcarbodimide and the second one with the amino acid hydroxybenzotriazole ester. Acylation of the second amino acid, on the resin, presented some difficulties to achieve completeness and several acetylations and benzoylations had to be performed in order to block the last 4 per cent of free amines. It seems that these difficulties are related to some batches of chloromethyl-resin. Incorporation of both acetylenic lysine, N alpha-Boc-N epsilon-Z-L-2,6-diamino-4-hexynoic acid, whose synthesis is described, and N alpha-Boc-L-3,4-dehydroproline was without problems in this synthesis. After cleavage by hydrofluoric acid the crude peptides were purified by gel filtration on Bio-Gel P2 and ion exchange chromatography on carboxymethylcellulose (CM 52). Dah6, delta Pro7,10]-neurotensin so obtained (51 per cent compared to starting Boc-Leu-resin) was in homogeneous form as characterized by amino acid analysis, thin layer chromatography in different systems and high performance liquid chromatography. The hydrogenation or tritiation product was identical with native neurotensin. Unsaturated derivative and neurotensin obtained after catalytic hydrogenation were as active as native neurotensin in inhibition of 125I-Trp11]-neurotensin binding to rat brain synaptic membranes and in guinea pig ileum contractility test. Substitution of proline and lysine by their dehydro-derivatives did not affect the biological properties of neurotensin. The tritiated neurotensin (160-180 Ci/mmol) should be a good agent for biological characterization of neurotensin receptors and for investigation of the peptide metabolism.