Chemical peptide ligation with three or more components
View Patent ↗The invention provides a method of assembling oligopeptide intermediates in a native chemical ligation reaction that eliminates self-ligation of bi-functional intermediates. An important aspect of the invention is a bi-functional intermediate with an N-terminal heterocyclic protecting group which effectively prevents self-ligation in the chemical assembly process. The present invention is useful in methods for convergent synthesis of polypeptides and proteins and improves the efficiency of native chemical ligation reactions, particularly where three or more peptide fragments are used to assemble a polypeptide or protein product.
1. A composition of matter comprising
an N-terminal heterocyclic-protected oligopeptide thioester defined by the following formula:
wherein:
each of Xaa 1 to Xaa n , is independently a protected or unprotected amino acid;
n is an integer from 2 to 120;
R 1 is hydrogen;
R 2 is hydrogen, unsubstituted alkyl having from 1 to 3 carbon atoms or an electron withdrawing-substituted alkyl having from 1 to 3 carbon atoms, said electron withdrawing substituent-selected from the group consisting of: halo, cyano, nitro-substituted alkyl having from 1 to 3 carbon atoms, carbonyl, carboxy, carboxyester, carboxyamide, amidocarboxy, amidocarbonyl, sulfoxy, sulfone and quartenary ammonium salts,
wherein R 2 may optionally be attached to a solid support;
R 3 is hydrogen or an electron donating group having from 1 to 12 carbon atoms and from 0 to 4 heteroatoms selected from the group consisting of nitrogen, oxygen, sulfur, and phosphorus;
R 4 is hydrogen, alkyl having from 1 to 3 carbon atoms, aryl or electron donating-substituted aryl having 6-10 carbon atoms with 0-2 heteroatoms, or electron donating-substituted alkyl having from 1 to 3 carbon atoms;
R 6 is alkyl having from 1 to 6 carbon atoms, alkylaryl having from 6 to 8 carbon atoms, —CH 2 —CONH 2 , —CH 2 CH 2 CONH 2 , or —(CH 2 ) k —CO—Xaa, wherein k is an integer equal to 1 or 2 and Xaa is an amino acid; and
R 9 is a side chain of an amino acid selected from the group consisting of alanine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, lysine, leucine, methionine, asparagine, glutamine, arginine, serine, tryptophan, and tyrosine.
2. The composition of claim 1 wherein:
R 3 is hydrogen, or a group having from 1 to 8 carbon atoms and from 0 to 2 heteroatoms selected from the group consisting of nitrogen, oxygen, and sulfur;
R 4 is hydrogen; and
R 9 is a side chain of an amino acid selected from the group consisting of glycine, alanine, and histidine.
3. The composition of matter according to claim 1 , wherein n is an integer from 2 to 70.
4. The composition of claim 3 wherein:
R 3 is hydrogen, phenyl, substituted phenyl, 2-picolyl, 4-picolyl, substituted 2-picolyl, or substituted 4-picolyl;
R 4 is hydrogen; and
R 9 is a side chain of an amino acid selected from the group consisting of glycine, alanine, and histidine.
5. The composition of claim 4 wherein R 3 taken alone is hydrogen or methoxy-substituted phenyl.
6. The composition of claim 3
wherein
R 2 is electron withdrawing-substituted alkyl having from 1 to 3 carbon atoms.
7. The composition of claim 3
wherein
R 2 is hydrogen,
R 3 is 4-methoxyphenyl, and
R 4 is hydrogen.