On the repetitive excess mixed anhydride method for the synthesis of peptides. Synthesis of the sequence 1‐10 of human growth hormone

Abstract

The synthesis of the fully protected N‐terminal decapeptide of human growth hormone, Boc‐Phe‐Pro‐Thr(Bzl)‐Ile‐Pro‐Leu‐Ser(Bzl)‐Arg(NO2)‐Leu‐Phe‐OMe (HGH 1‐10) by the repetitive excess mixed anhydride (REMA) method is described. Starting from Phe‐OMe this sequence was synthesized in a stepwise manner in an overall yield of 59%. In the coupling of Boc‐Ile to Pro‐Leu‐Ser(Bzl)‐Arg‐(NO2)‐Leu‐Phe‐OMe the isobutoxycarbonyl derivative of the amino‐component was isolated. This problem of reaction on the undesirable side of the mixed anhydride in the couplings to proline‐peptides was circumvented by the coupling of dipeptides, viz. Boc‐Ile‐Pro and Boc‐Phe‐Pro. Also a larger peptide fragment, ZPhe‐Pro‐Thr‐Ile‐Pro (HGH 1‐5), was conveniently coupled to the C‐terminal pentapeptide. Synthesis of Boc‐Leu‐Ser(Bzl)‐Arg(NO2)‐Leu‐Phe‐OMe (HGH 6‐10) was performed with and without intermediate purification. It was found that the yield was higher when no purifications were applied. HGH 6‐10 was compared with the corresponding peptide synthesized by the solid phase method. Both products were found to be identical with regard to melting point, optical rotation and chromatographic behaviour. The decapeptide, on deprotection, showed identical chromatographic behaviour with a product obtained by the solid phase method. The optical rotations showed a small difference; no significant racemization could be detected on digestion with L‐amino‐acid oxidase.

Synthesis of the Gastrointestinal Peptide Hormone Secretin by the Repetitive Excess Mixed Anhydride (REMA) Method

“…-By the method which one of us has called the repetitive excess mixed anhydride or REMA method [l] a noriapeptide was synthesized by Tilak [2]. Subsequently we effected by this method the complete synthesis of sequence 1-10 of human growth hormone (HGH 1-10) [3], the luteinising hormonereleasing hormone (LH-RH, or Gn-RH) [4], also a decapeptide, substance P [5], an undecapeptide, and sequences of human calcitonin of approximately the same size [6].…”

“…The difference in basicity of the various species on elimination of protecting groups and, in consequence, the purification by ion-exchange chromatography, will be discussed below. However, anomalous coupling to a considerable extent in a particular step, as established after deblocking, has to be avoided, just as in the case of coupling to prolyl peptides [3]. I n order to ascertain the necessary stepwise yields in a sequential (seq.)…”

The Minimal Amino Acid Sequence of the Insulin-potentiating Fragments of Human Growth Hormone: Its Mechanism of Action

“…22 This preliminary evidence was later substantiated with the demonstration of the in vivo and in vitro insulin-potentiating action of synthetic hGH 1-20, 1-15, 1-13, 3-13, and 6-13. 23 Unfortunately, hGH 1-10 synthesized by Beyerman 24 and hGH 1-35 synthesized by Niall and Tregear 25 were not investigated for this biologic activity, and hGH 1-24 and 25-51 were shown devoid only of growth-promoting activity by Chillemi and Pecile. 26 The present paper reports the definitive identification of an active core of hGH capable of insulin-potentiating action and demonstrates the mechanism of action of this core using intravenous insulin tolerance tests, radioreceptor binding studies, and the modulation of insulin-dependent enzymes.…”

Repetitive Excess Mixed Anhydride (REMA) Synthesis of Peptides. The protected C‐terminal hexadecapeptide of secretin

“…Tilak et al succeeded in producing a nonapeptide in this way [6]. I n our Laboratory the sequence 1-10 of human growth hormone (HGH 1-10) was synthesized by the REMA method [l] [7] ; the product, on deprotection, was found to be identical with the corresponding peptide synthesized by the solid-phase method [S]. The problem of reaction on the undesirable side of the mixed anhydride, which results in carbamates, e.g.…”

“…One of us has discussed a comparison of the highly satisfactory REMA method [7] with Merrifield's solid-phase peptide synthesis [9]. A salient point still to be investigated was the extension limit with respect to the chain length.…”