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US 6,013,764 AGranted
Liquid phase peptide synthesis of KL-4 pulmonary surfactant
AssigneeOrtho Pharmaceutical Corporation
InventorsAhmed F. Abdel-Magid, Urs Eggmann, Cynthia Anne Maryanoff, Adrian Thaler, Frank J. Villani
Life of this patent
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Abstract
The invention relates to improved liquid phase processes for the preparation of the 21 residue protein component, (Lys-Leu.sub.4).sub.4 -Lys, of the pulmonary surfactant KL-4. These process are amenable to large scale synthesis and one process employs a method of saponifying an ester which reduces the inherent racemization of the .alpha.-carbon.
Claims16
A process for producing the synthetic KL-4 pulmonary surfactant protein of the formula (Lys-Leu.sub.4).sub.4 Lys (SEQ. ID No. 1) comprising the steps of:
- a. reacting a 3-amino acid peptide residue of the formula H-Lys(Z)-Leu-Leu OH with a 5-amino acid peptide residue Boc-Leu-Leu-Lys(Z)-Leu-Leu-OH (SEQ. ID No. 2) to yield an 8-amino acid peptide residue, which is successively reacted with the 5-amino acid peptide to form an 18-amino acid peptide of the formula H-Leu-Leu(Lys(Z)-Leu.sub.4).sub.3 Lys(Z)-OBzl (SEQ. ID No. 3) wherein Z is benzyloxycarboxyl;
- b. reacting the 18 amino acid peptide residue with a 3-residue amino acid peptide of the formula:
- H-Leu-Leu-Lys(Z)-OBzl to form the protected 21-amino acid KL-4 protein;
- and
- c. removing the protecting group of the 21-amino acid KL-4 protein by reaction with a suitable acid to form the final KL-4-protein.
2 more elements
A process for producing (Lys-Leu.sub.4).sub.4 Lys (SEQ. ID No. 1) comprising the steps of:
- a. reacting H-Leu-Leu-Lys(Z)-OBzl, Boc-Leu-Leu-Lys(Z)-Leu-Leu-OH (SEQ. ID No. 2) wherein Z is benzyloxycarboxyl, and a peptide coupling agent in an inert organic solvent, to form:
- Boc-Leu-Leu-Lys(Z)-Leu-Leu-Leu-Leu-Lys(Z)-OBzl (SEQ. ID No. 6);
- b. reacting Boc-Leu-Leu-Lys(Z)-Leu-Leu-Leu-Leu-Lys(Z)-OBzl (SEQ. ID No. 6) with a suitable acid with or without an inert organic solvent to form:
- H-Leu-Leu-Lys(Z)-Leu-Leu-Leu-Leu-Lys(Z)-OBzl (SEQ. ID No. 6);
- c. reacting H-Leu-Leu-Lys(Z)-Leu-Leu-Leu-Leu-Lys(Z)-OBzl (SEQ. ID No. 6), Boc-Leu-Leu-Lys(Z)-Leu-Leu-OH (SEQ. ID No. 2) and a peptide coupling agent in an inert organic solvent to form:
- Boc-Leu-Leu-(Lys(Z)-Leu.sub.4).sub.2 -Lys(Z)-OBzl (SEQ. ID No. 5);
- d. reacting Boc-Leu-Leu-(Lys(Z)-Leu.sub.4).sub.2 -Lys(Z)-OBzl (SEQ. ID No. 5) with a suitable acid, with or without an inert organic solvent to form:
- H-Leu-Leu-(Lys(Z)-Leu.sub.4).sub.2 -Lys(Z)-OBzl (SEQ. ID No. 5);
- e. reacting, Boc-Leu-Leu-Lys(Z)-Leu-Leu-OH (SEQ. ID No. 2), a peptide coupling agent, a salt and H-Leu-Leu-(Lys(Z)-Leu.sub.4).sub.2 -Lys(Z)-OBzl (SEQ. ID No. 5) in an inert solvent to form:
- Boc-Leu-Leu-(Lys(Z)-Leu.sub.4).sub.3 -Lys(Z)-OBzl (SEQ. ID No. 3);
- f. reacting Boc-Leu-Leu-(Lys(Z)-Leu.sub.4).sub.3 -Lys(Z)-OBzl (SEQ. ID No. 3) with a suitable acid, with or without an inert organic solvent to form:
- H-Leu-Leu-(Lys(Z)-Leu.sub.4).sub.3 -Lys(Z)-OBzl (SEQ. ID No. 3);
- g. reacting Z-Lys(Z)-Leu-Leu-OH, water, a peptide coupling agent and H-Leu-Leu-(Lys(Z)-Leu.sub.4).sub.3 -Lys(Z)-OBzl (SEQ. ID No. 3) in an inert organic solvent to form:
- Z-(Lys(Z)-Leu.sub.4).sub.4 -Lys(Z)-OBzl (SEQ. ID No. 1);
- h. reacting, acetic acid, water, a catalyst and Z-(Lys(Z)-Leu.sub.4).sub.4 -Lys(Z)-OBzl (SEQ. ID No. 1) under an H.sub.2 atmosphere of 2 to 2.2 bar to form:
- (Lys-Leu.sub.4).sub.4 Lys (SEQ. ID No. 1).
13 more elements
A process for producing (Lys-Leu.sub.4).sub.4 Lys (SEQ. ID No. 1) comprising the steps of:
- a. reacting a compound of the formula Boc-Lys(Z)-Leu-Leu-OH wherein Z is benzyloxycarboxyl and a peptide
- coupling agent with a compound of the formula:
- H-Leu-Leu-Lys(Z)-Leu-Leu-OR (SEQ. ID No. 2), a suitable organic solvent to form a compound of the formula:
- Boc-Lys(Z)-Leu-Leu-Leu-Leu-Lys(Z)-Leu-Leu-OR (SEQ. ID No.4),
- wherein R is C.sub.1-8 lalkyl or phenylC.sub.1-8 alkyl;
- b. removing the carboxy protecting group by reacting a hydroxide, salt, water and Boc-Lys(Z)-Leu-Leu-Leu-Leu-Lys(Z)-Leu-Leu-OR (SEQ. ID No. 4) in an inert organic solvent, to form:
- Boc-Lys(Z)-Leu-Leu-Leu-Leu-Lys(Z)-Leu-Leu-OH (SEQ. ID No. 4);
- c. reacting Boc-Lys(Z)-Leu-Leu-Leu-Leu-Lys(Z)-Leu-Leu-OH (SEQ. ID No. 4), H-Leu-Leu-(Lys(Z)-Leu.sub.4).sub.2 -Lys(Z)-OBzl (SEQ. ID No. 5), a salt, water and a peptide coupling agent in an inert organic solvent, to form:
- Boc-(Lys(Z)-Leu.sub.4).sub.4 -Lys(Z)-OBzl (SEQ. ID No. 1);
- d. reacting trifluoroacetic acid, Boc-(Lys(Z)-Leu.sub.4).sub.4 -Lys(Z)-OBzl (SEQ. ID No. 1), acetic acid, water and a catalyst under a H.sub.2 atmosphere of 40 to 50 psi to form (Lys-Leu.sub.4).sub.4 Lys (SEQ. ID No. 1).
7 more elements
Intermediates useful in processes for the production of (Lys-Leu.sub.4).sub.4 Lys (SEQ. ID No. 1) selected from the group consisting of:
- Boc-Leu-Leu-Lys(Z)-Leu-Leu-OH, (SEQ. ID No. 2);
- Boc-Leu-Leu-Lys(Z)-Leu.sub.4 -Lys(Z)-OBzl (SEQ. ID No. 6);
- H-Leu-Leu-Lys(Z)-Leu.sub.4 -Lys(Z)-OBzl (SEQ. ID No. 6);
- Boc-Leu-Leu-(Lys(Z)-Leu.sub.4).sub.2 -Lys(Z)-OBzl (SEQ. ID No. 5);
- H-Leu-Leu-(Lys(Z)-Leu.sub.4).sub.2 -Lys(Z)-OBzl (SEQ. ID No. 5);
- Boc-Leu-Leu-(Lys(Z)-Leu.sub.4).sub.3 -Lys(Z)-OBzl (SEQ. ID No. 3);
- H-Leu-Leu-(Lys(Z)-Leu.sub.4).sub.3 -Lys(Z)-OBzl (SEQ. ID No. 3);
- and
- Boc-Lys(Z)-Leu.sub.4 -Lys(Z)-Leu-Leu-OH (SEQ. ID No. 4), wherein Z is benzyloxycarboxyl, and suitable acid salts thereof.
6 more elements
Dependencies are inferred from the claim text.