An Efficient Synthesis of Thyrotropin Releasing Hormone (TRH)

Abstract

Sz~rnmary.A convenient and simple synthesis of L-pyroglutamyl-L-histidyl-L-prolinamide is descril~cd. Two pathways using the solid phase tcchnique have led to the pure tripeptide.Thyrotropin releasing hormone (TRH) was isolated from porcine hypothalamus by Schally et al. [l] and from ovine hypothalamus by Guillemin et al. [ Z ] , and proved to be identical with r*-pyroglutamyl-L-histidyl-L-prolinaniide [3]. A classical synthesis of this tripeptide was reported recently j4] ; the solid phase technique 1 5 1 used here gives better yields, is simpler and quicker.The following reaction scheme was used. t-Butyloxycarbonyl (Boc) is introduced in proline, glutamic acid or glutamine as the amine protecting group j6] using Bocazide at constant alcaline pH. The purity of the Boc-aminoacids was checked by measuring the rotation and by thin-layer chromatography. The histidine imidazole group was protected with o,p-dinitrophenyl before condensation, as selective removal of this group can easily be performed by thiolysis at pH 8 [7].Protected histidine was reacted with proline linked on the chloromethylresin in the presence of dicyclohexyl-carbodiimide (Dcc). The glutamyl residue was introduced in two ways: (A) Boc-pyroglutamic acid was condensed in the presence of Dcc; (B) Boc-glutam.ine dinitrophenylester j 81 was reacted with the dipeptide linked to the resin in dimeth:ylformaniide. The tripeptide was split from the resin by aminolysis 191, the imidazole -protecting group removed, and the glutamine converted into pyroglutamic acid by boiling acetic acid [lo].The tripept.ide was purified by Sepliadex G10 filtration followed by silica-gel chromatography. The pure material did not react with ninhydrin, as could be anticipated due to the presence of prolinamide and pyroglutamic acid. After silica-gel thin layer chromatography in several solvant systems [ll], the product gave a single spot when detected with Pauly's reagent. The optical rotation was of the order of the one published 141. After acid hydrolysis, the three initial aminoacids could be detected by spraying the thin layer chromatoplaque with ninhydrin.

Response of Normal, Hypothyroid and Hypothalamo-Pituitary Insufficient Children to Synthetic Thyrotrophin-Releasing Hormone

“…Thyrotrophin-releasing hormone has been shown to be L-pyroglutamyl-L-histidyl-L-proline-amide) (Burgus, Dunn, Desiderio & Guillemin, 1969; Schally, Redding, Bowers & Barrett, 1969;Gillessen, Felix, Lergier & Studer, 1970) using the classical technique to prepare the synthetic tripeptide. We used the solid phase technique to obtain a product which could be easily purified and which was highly active biologically (Milhaud et al 1970;Rivalile & Milhaud, 1971). …”

“…The hormone used in this study was made in the Isotope Laboratory by the solid phase technique. The synthesis of L-pyroglutamyl-L-histidyl-L-prolinamide is described elsewhere (Milhaud, Rivalile, Moukhtar, Job & Binet, 1970;Rivalile & Milhaud, 1971). The compound was chromatographically pure in several solvent systems ; its potency was assessed by rat bioassay.…”

Solid Phase Synthesis of Porcine Lh‐releasing Hormone With Hydrogen Chloride in Formic Acid as the Deblocking Reagent*

“…(1972) employed HCl in AcOH containing 1% ME with concomitant replacement of air in the reaction vessel with nitrogen. Other sulfhydryl reagents such as 1,2ethanedithiol (Rivaille et al, 1971;Rivier er al., 1972) and thioglycolic acid (Voelter & Zech, 1972) were also employed in the synthesis of LH-RH. All procedures described principally used sulfhydryl reagent as a scavenger for protecting tryptophan from oxidation.…”