Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues – Synthesis of peptides
Reexamination Certificate
1998-11-27
2001-08-21
Low, Christopher S. F. (Department: 1654)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
Synthesis of peptides
C530S333000, C530S335000, C530S336000, C530S337000, C530S339000, C530S345000, C436S086000, C436S089000, C436S090000
Reexamination Certificate
active
06277958
ABSTRACT:
FIELD OF INVENTION
This invention relates to a method for producing a peptide thiol ester, and, more particularly, the method which uses a fluoren-9-ylmethoxycarbonyl amino acid (Fmoc-amino acid).
BACKGROUND OF THE INVENTION
Peptides have been synthesized by condensation of amino acids. The peptide synthesis methods fall into two general categories; liquid-phase and solid-phase. The liquid-phase method has disadvantages, resulting from use of a solution, of being highly time consuming and labor intensive before it is completed, because the intermediate must be extracted from the solution, refined and confirmed whenever amino acid is added to, and chain length elongation is conducted in the solution. The solid-phase method elongates the peptide chain by a simple procedure in which an activated amino acid derivative is condensed consecutively on a resin on which amino acid with the protected amino terminals is and removing the protective group for the amino terminals. It is a simple peptide-synthesis method, much more efficient than the liquid-phase method.
One of the solid-phase methods uses t-butoxycarbonyl amino acid (hereinafter “Boc-amino acid”). It immobilizes Boc-amino acid on a resin, and condenses activated Boc-amino acid, while removing the Boc groups as the protective group for the amino terminals. It needs a large quantity of a halogen-containing compound, such as trifluoroacetic acid and methylene chloride, to remove the Boc group, and also needs a strong acid, such as anhydrous hydrogen fluoride to take peptide out of the resin in the final stage. A peptide thiol ester can be easily synthesized by the aid of Boc-amino acid. Nevertheless, however, this method involves environmental and safety-related problems, resulting from use of a halogen-containing compound and strong acid.
Another solid-phase method uses fluoren-9-ylmethoxycarbonyl amino acid (hereinafter “Fmoc-amino acid”). It condenses Fmoc-amino acid, while removing the Fmoc groups as the protective group for the amino groups with the aid of an amine compound, such as piperidine. It has been widely used, because of its advantages of dispensing with a halogen-containing compound for elongating the peptide chains and a strong acid for taking peptide out of the resin. This method, although preferable in terms of environment and safety, has an inherent disadvantage, when applied to synthesis of peptide thiol ester: peptide thiol ester shows high reactivity with the amine, decomposing the thiol ester bond to prevent and vitiate peptide chain elongation.
It is an object of the present invention to provide a method for producing a peptide thiol ester relatively easily, while reducing problems related to environmental preservation and safety. It is highly desirable to provide a synthetic scheme which does not require halogen containing compounds such as methylene chloride and which does not require reagents which reduce yields by reacting with desired intermediates. Such a method is very useful for synthesis of peptides, such as a long-chain peptides and cyclopeptides, and, at the same time, can be widely used for a variety of medicines from peptides as the starting stocks.
SUMMARY OF THE INVENTION
In accordance with the invention, the Fmoc group as a protective group is bound to the &agr;-amino groups of amino acid(s), fixed on a resin via a thiol ester bond; the protective group can be removed by the aid of a specific reagent to remove the Fmoc group without causing decomposition of the thiol ester bond.
The present invention provides a method for producing a peptide thiol ester of formula (1):
H—A
1
—B—S—X (1)
(In which each of A
1
and B is an amino acid residue; and X is a component bound to a sulfur atom to constitute the thiol ester)
The method comprises stages (1) to (4):
In Stage (1) a fluoren-9-ylmethoxycarbonyl amino acid derivative (Fmoc-amino acid derivative) is reacted with a resin, to produce an Fmoc-amino acid thiol ester resin, shown by the formula (2):
Fmoc-B—S—X-Resin (2)
(Fmoc is fluoren-9-ylmethoxycarbonyl; B and X are the same as the above; and Resin is a synthetic resin).
In Stage (2) the Fmoc-amino acid thiol ester resin, produced by Stage (1), is reacted with a reagent capable of removing the Fmoc group, to produce an amino acid thiol ester resin, shown by the formula (3):
H—B—S—X-Resin (3)
(B,X and Resin are the same as the above).
In Stage (3) the amino acid thiol ester resin, produced by Stage (2), is reacted with Fmoc-amino acid to produce an Fmoc-peptide thiol ester resin shown by the formula (4):
Fmoc-A
1
—B—S—X-Resin (4)
(Fmoc, A
1
, B, X and Resin are the same as the above).
In Stage (4) the Fmoc-peptide thiol ester resin, produced by Stage (3), is reacted with a cleavage reagent, to produce an Fmoc-peptide thiol ester, shown by the formula (5):
Fmoc-A
1
—B—S—X (5)
(Fmoc, A
1
, B and X are the same as the above); and it is either preceded by or followed by the reaction of the Fmoc-peptide thiol ester with a reagent capable of removing the Fmoc group. Thus the Fmoc-peptide thiol ester resin, produced by Stage (3), is reacted with a reagent capable of removing the Fmoc group before or after it is treated with a cleavage reagent, to produce a peptide thiol ester shown by the formula (1):
H—A
1—B
—S—X (1)
(A
1
and B are each an amino acid residue; and X is a component bound to a sulfur atom to constitute the thiol ester).
In another embodiment the present invention provides a method for producing a peptide thiol ester shown by the formula (6):
H—A
n
—A
n−1
. . . —A
2
—A
1
—B—S—X (6)
(A
n
, A
n−1
. . . A
2
, A
1
and B are each an amino acid residue; X is a component bound to a sulfur atom to constitute the thiol ester; and n is a positive integer) by the following stages (1) to (4):
In Stage (1) a fluoren-9-ylmethoxycarbonyl amino acid derivative (Fmoc-amino acid derivative) is reacted with a resin, to produce an Fmoc-amino acid thiol ester resin, shown by the formula (2):
Fmoc-B—S—X-Resin (2)
(Fmoc is fluoren-9-ylmethoxycarbonyl; B and X are the same as the above; and Resin is a synthetic resin).
In Stage (2) the Fmoc-amino acid thiol ester resin, produced by Stage (1), is reacted with a reagent capable of removing the Fmoc group, to produce an amino acid thiol ester resin, shown by the formula (3):
H—B—S—X-Resin (3)
(B, X and Resin are the same as the above).
In Stage (3) the amino acid thiol ester resin, produced by Stage (2), is reacted with Fmoc-amino acid to produce an Fmoc-peptide thiol ester resin shown by the formula (4):
Fmoc-A
1
—B—S—X-Resin (4)
(Fmoc, A
1
, B, X and Resin are the same as the above). The Fmoc-peptide thiol ester resin is then subjected to procedures similar to Stages (2) and (3) above, to remove the Fmoc group, and then to add Fmoc-amino acid to the resultant peptide thiol ester resin repeatedly (n—1) times, to produce a Fmoc-peptide thiol ester resin shown by the formula (7):
Fmoc-A
n
—A
n−1
. . . —A
2
—A
1
—B—S—X-Resin (7)
(Fmoc, A
n
, A
n−1
. . . A
2
, A
1
, B, X and Resin are the same as the above; and n is a positive integer).
In Stage (4) the Fmoc-peptide thiol ester resin, produced by Stage (3), is reacted with a cleavage reagent, to produce an Fmoc-peptide thiol ester, shown by the formula (8):
Fmoc-A
n
—A
n−1
. . . —A
2
—A
1
—B—S—X (8)
(Fmoc, A
n
, A
n−1
. . . A
2
, A
1
, B, X and n are the same as the above), preceded by or followed by the reaction of the Fmoc-amino thiol ester with a reagent capable of removing the Fmoc group. The Fmoc-amino thiol ester resin, produced by Stage (3), is reacted with a reagent capable of removing the Fmoc group before or after it is treated with a cleavage reagent, to produce a peptide thiol ester shown by the formula (6):
H—A
n
—A
n−1
. . . —A
2
—A
1
—B—S—X (6)
(A
n
, A
n−1
. . . A
2
, A
1
and B are each an amino acid residue; X is a component bound to a sulfur atom to constitute the thiol ester; and n is a posit
Aimoto Saburho
Low Christopher S. F.
Mohamed Abdel A.
Schneller Marina V.
Venable
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