US 3,978,035 A · 13-Norleucine-14-desamido motilin, a method for preparing it and an agent containing it

Abstract

The specification describes the total synthesis of L-norleucine-13-motilin ased on the replacement of the L-methionine radical located in the 13-position by an L-norleucine radical. In accordance with the present day development of peptide synthesis the central arginyl-methionyl-bond (amino acids 12 and 13 of motilin) can in practice hardly be synthesised, while on the other hand the norleucine-13-docosapeptide corresponding to motilin can be more readily produced synthetically, and this provides for a comparatively rapid determination of structure and can give important information as regards the biological effect specificity of methionine.

A method for the production of 13-norleucine-14-desamido-motilin comprising:

a. forming fragment I, which consists of arginyl (hydrobromide)-asparagine-N.epsilon.-tert.butyloxycarbonyl-lysyl-glycyl-gl utamine-tert.butyl ester (amino acids 18 to 22), by esterifying N.alpha.-benzyloxycarbonyl-glutamine with acetic acid tert.butyl ester using a sulfuric acid catalysis, hydrogenolytic removing the N-protective group to form glutamine tert.butyl ester (amino acid 22), reacting the glutamine tert.butyl ester with N.alpha.-benzyloxycarbonyl-glycine-N-hydroxy-succinimide ester (amino acid 21) and hydrogenolytically removing the N-protected group from the dipeptide ester, reacting the glycyl-glutamine-tert.butyl ester (amino acids 21 to 22) with N.alpha.-benzyloxycarbonyl-asparaginyl-N.epsilon.-tert.butyloxycarbonyl-ly sine (amino acids 19 to 20) by means of the carbodiimide-N-hydroxysuccinimide method to form benzyloxy-carbonyl-tetra-peptide ester, removing the N-protective group from the benzyloxy-carbonyl-tetra-peptide ester by means of catalytic hydrogenation, reacting the resultant asparaginyl-N.epsilon.-tert.butyloxycarbonyl-lysyl-glycyl-glutamine-tert.b utyl ester (amino acids 19 to 22) with N.alpha.-benzyloxycarbonyl-N.delta.,.omega.-dibenzyloxycarbonyl-arginine-4 -nitrophenyl ester (amino acid 18) to form N.alpha.-benzyloxycarbonyl-N.delta.,.omega.-di-benzyloxycarbonyl-arginyl-a sparaginyl-N.epsilon.-tert.butyloxycarbonyl-lysyl-glycyl-glutamine-tert.but yl ester, and removing the three benzylocarbonyl protective groups from the resultant ester by means of catalytic hydrogenolysis with the addition of 2 equivalents of hydrogen bromide whereby fragment I is formed;
b. forming fragment II, which consists of N.alpha.-benzyloxycarbonyl-glutamyl- (.gamma.-tert.butyl ester)-glutamyl-(.gamma.-tert.butyl ester)-N.epsilon.-tert.butyloxycarbonyl-lysyl-glutamic acid-.gamma.-tert.butyl ester (amino acids 14 to 17), by reacting N.alpha.-benzyloxycarbonyl-N.epsilon.-tert.butyloxy-carbonyl-lysine-N-hydr oxysuccinimide ester (amino acid 16) and glutaminic acid -.gamma.-tert.butyl-.alpha.-methyl ester (amino acid 17), converting the resultant compound by alkaline ester saponification and subsequent catalytic deacylating to N.epsilon.-tert.butyloxycarbonyl-lysyl-glutamic acid-.gamma.-tert.butyl ester (amino acids 16 and 17), and twice, sequentially, reacting said ester with N.alpha.-benzyloxycarbonyl-glutamic acid (.gamma.-tert.butyl ester)-N-hydroxysuccinimide ester (amino acids 15 and 14, respectively), such respectively being a head component;
c. forming fragment III which consists of N.alpha.-benzyloxy-carbonyl-N.delta., N.omega.-di-benzyloxycarbonyl-arginyl-norleucine (amino acids 12 and 13), by aminoacylating norleucin (amino acid 13) with N.alpha.-benzyloxycarbonyl N.delta., N.omega.-dibenzyloxy-carbonyl-arginine-N-hydroxysuccinimide ester (amino acid 12);
d. forming fragment IV which consists of N.alpha.-benzylocarbonyl-glutamyl-(.gamma.-tert.butyl ester)-leucyl-glutamine (amino acids 9 to 11), by reacting leucyl-glutamine (amino acids 10 and 11) with N.alpha.-benzyloxy-carbonyl-glutaminic acid-(.gamma.-tert.butyl ester) N-hydroxysuccinimide ester;
e. forming fragment V which consists of 0-tert.butyl-threonyl-0-tert.butyl-tyrosyl-glycine (amino acids 6 to 8), by reacting 0-tert.-butyl-tyrosyl-glycine (amino acids 7 and 8) with N.alpha.-benzyloxycarbonyl-0-tert.-butyl-threonine-N-hydroxysuccinimide ester (amino acid 6) and converting the compound into fragment V by catalytic hydrogenation of the N-protective group;
f. forming fragment VI which consists of N.alpha.-tert.butyloxy-carbonyl-phenylalanyl-valyl-prolyl-isoleucyl-phenyl alanine (amino acids 1 to 5), by reacting N.alpha.-benzyloxy-carbonyl-isoleucine (amino acid 4) and phenylalanine-tert.-butyl ester (amino acid 5) using the carbodiimide method, catalytic deacylating of the resultant compound, reacting the resultant isoleucyl-phenylalanin-tert.-butyl ester (amino acids 4 and 5) with N.alpha.-tert.-butyloxycarbonyl-valyl-proline using the carbodiimide method, whereby N.alpha.-tert.butyloxycarbonyl valyl-prolyl-isoleucyl-phenylalanine-tert.-butyl ester is formed, converting the resultant ester by treatment with trifluoro acetic acid to valyl-prolyl-isoleucylphenylalanine (amino acids 2 to 5), and reacting the amino function with N.alpha.-tert.butyl-oxycarbonyl-phenylalanin-N-hydroxysuccinimide ester (amino acid 1), whereby fragment VI is formed,
wherein, in fragments I to VI, all of the side chain functions of the polyfunctional amino acids have tert.butyl alcohol derived protective groups, which can acidolytically readily be removed, and the complex function of the arginine is masked either by N.delta., .omega.-diacylation or by protonisation by salt formation with hydrogen bromide, said fragments I to VI being separately formed, in any desired sequence or concurrently;
g. connecting fragments I to VI together by means of the N,N'-dicyclohexylcarbodiimide-N-hydroxysuccinimide method or a modification thereof;
h. removing all of the protective groups from the so-obtained polypeptide, masked on all sides and having the overall sequence of the amino acids 1 to 22, by means of tri-fluoro-acetic acid;
i. removing. the trifluoroacetate and bromide ions by means of an anion exchange resin;
and
j. isolating and purifying the resultant 13-norleucine-14-desamido-motilin.

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13-Norleucine-14-desamido motilin, a method for preparing it and an agent containing it

Life of this patent

13-Norleucine-14-desamido motilin, a method for preparing it and an agent containing it

Life of this patent

Abstract

Claims7