Liquid phase peptide syntheses of KL-4 pulmonary surfactant

Details Liquid phase peptide syntheses of KL-4 pulmonary surfactant Liquid phase peptide syntheses of KL-4 pulmonary surfactant

1999-11-09

2002-12-10

Low, Christopher S. F. (Department: 1654)

Chemistry: natural resins or derivatives; peptides or proteins;

Peptides of 3 to 100 amino acid residues

4 to 5 amino acid residues in defined sequence

C530S333000, C530S335000, C530S345000

Reexamination Certificate

active

06492490

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to the field of peptide syntheses and intermediates useful therein. More particularly, the invention relates to the synthesis of the polypeptide component of KL-4, a synthetic pulmonary surfactant.
BACKGROUND
The epithelium of mammalian lungs are lined with an endogenous pulmonary surfactant (PS) which facilitates breathing by aiding the transport of oxygen across the lung air-liquid interface. A deficiency in this surfactant is the primary cause of neonatal respiratory distress syndrome (RDS) and is linked to RDS in adults as well. Native PS is a mixture of lipids and proteins, and although its exact composition is unknown, researchers have prepared a number of exogenous surfactants which are useful in the treatment of RDS in pre-term infants. KL-4 is an example of an exogenous surfactant which is useful in the treatment of RDS as disclosed in U.S. Pat. Nos. 5,164,369, 5,260,273 and 5,407,914, hereby incorporated by reference.
KL-4 is a mixture of a pharmaceutically acceptable phospholipid and a 21 residue polypeptide, (L-lysine-(L-leucine)
4
)
4
L-lysine. (SEQ. ID No. 1) As disclosed in the aforementioned patents, this peptide was prepared by solid phase synthesis and recombinant DNA techniques. The solid phase synthesis comprises sequential addition of one or more amino acid residues coupled with suitable protection of amino or carboxyl groups. Although this process is effective, it is not amenable to the large scale synthesis necessary to manufacture a drug substance.
The object of the present invention is the production of the peptide component of KL
4
, namely (Lys-Leu
4
)
4
Lys (SEQ. ID No. 1) by a liquid phase peptide synthesis (“LPPS”). Unlike the methods disclosed in the aforementioned patents, this process is amenable to large scale synthesis.
An additional embodiment of the invention concerns the deprotection of the carboxy terminus of a peptide which was protected as an ester. Most peptide syntheses require manipulation of carboxyl and amino protecting groups. Typically, terminal carboxyl groups are protected as their ester derivative. However deprotection methods may be accompanied by racemization of the &agr;-carbon; a problem that is compounded as the length of the peptide increases. In several steps of the synthesis of (Lys-Leu
4
)
4
Lys, an ester protected carboxl group is deprotected. As with most biomimetic products, the configuration of the peptide is crucial and the active configuration of the residues of (Lys-Leu
4
)
4
Lys is “L”. This invention discloses a method of deprotecting a peptide's ester protected carboxyl group which reduces the amount of racemized product. Although this method is applied in the synthesis of (Lys-Leu
4
)
4
Lys, it may be used in the synthesis of other peptides as illustrated hereinafter.
SUMMARY OF THE INVENTION
The invention relates to improved LPPS processes for the preparation of the 21 residue protein component of the pulmonary surfactant, KL-4, which is amenable to large scale synthesis. The first method uses three peptide fragments:
The process starts off with a 3-residue right hand fragment which is successively reacted with the 5-residue body fragment to build an 18-amino acid fragment of the formula H-Leu-Leu-(Lys(Z)-Leu
4
)
3
-Lys(Z)-OBzl (SEQ. ID No. 3). This 18-amino acid fragment is then reacted with the 3-residue right hand fragment to form the final 21 residue protein of the present invention.
In the second method, a convergent synthesis is employed in which an 8-residue protected polypeptide of the formula: Boc-Lys(Z)-Leu-Leu-Leu-Leu-Lys(Z)-Leu-Leu-OR (SEQ. ID No. 4) is prepared and saponified with tetraalkylammonium hydroxide. The saponified peptide is then reacted with a 13-residue peptide of the formula H-Leu-Leu-(Lys(Z)-Leu
4
)
2
-Lys-OBzl (SEQ. ID No. 5) to yield the protected 21-amino acid peptide. Removal of the protecting group by reaction with a suitable acid yields the final KL-4 polypeptide. This convergent method exhibits certain advantages in solubility and control over unwanted by-products, which makes the method particularly suitable to large scale synthesis.
In another aspect of the present invention is a novel method for the deprotection of the carboxy terminus of a peptide protected as an ester. This method comprises saponifying the protected peptide with tetraalkylammonium hydroxide reagent in a suitable solvent. This process provides a method of deprotecting the peptide which reduces the amount of racemization at the &agr;-carbon.
DETAILED DESCRIPTION OF THE INVENTION
Herein, the amino acid nomenclature corresponds to standard conventions where: L-leucine is “L” or “Leu”, L-lysine is “K” or “Lys”, L-alanine is “A” or “Ala”, benzyloxycarbonyl is “Z” or CBZ, D-(1-naphthyl)alanine is “D-Nal”, 4-chlorophenylalanine is “D-Cal”, L-serine is “L-Ser” and D-3-pyridylalanine is “D-Pal”. In addition all amino acid residue sequences are represented by formulae whose left to right orientation is in the conventional direction of amino-terminus to carboxy-terminus. A dash at the beginning or end of the sequence indicates a bond to a radical such as H, OH or OBzl; and a dash in the middle of the sequence indicates a conventional amide bond. Other abbreviations and symbols are as follows: DMF is N,N-dimethylformamide, MeOH is methanol, HOBT is 1-hydroxybenzotriazole, THF is tetrahydrofuran, DCC is 1,3 dicyclohexylcarbodiimide, EtOH is ethanol, iPrOH is isopropanol, HBTU is N,N,N′,N′-tetramethyl-O-(1H-benzotriazol-1-yl)uronium-hexafluororphosphate), DIPEA is N,N-diisopropylethylamine, NMP is 1-methyl-2-pyrrolidone, HOOBT is 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine, WSCD.I (water soluble carbodiimide) is 1-(dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and DIC is 1,3-diisopropylcarbodiimide. The term “alkyl” includes straight and branched groups; the term “hydroxide” includes Group I metal hydroxides (NaOH, LiOH and the like) as well as tetraalkylammonium hydroxides; the term “salts” includes tetraalkylammonium halides and tetrahalo borates and “Ac” is acetyl.
The synthesis of (Lys-Leu
4
)
4
Lys (SEQ. ID No. 1) in accordance with the present invention, follows two pathways. Procedure A uses three peptides fragments: a 3-residue left-hand (amino-end), a three residue right hand (carboxy terminus) and a five residue body fragment to construct the molecule.
As illustrated in Scheme A, H-Leu-Leu-Lys(Z)-OBzl and Boc-Leu-Leu-Lys(Z)-Leu-Leu-OH (SEQ. ID No. 2) are reacted together in the presence of a peptide coupling agent and an inert solvent at about 0° C. to room temperature to give Boc-Leu-Leu-Lys(Z)-Leu-Leu-Leu-Leu-Lys(Z)-OBzl. (SEQ. ID No. 6) Examples of suitable peptide coupling agents include: DCC, DIC, HBTU, WSCDI, HOBT, HOOBt, where the preferred agents are HOOBT and HTBU. Solvents are chosen for their compatibility with the chosen coupling agent. Suitable solvents include DMF, THF, NMP and acetonitrile, where a mixture of DMF and acetonitrile is preferred. If the acid salt of H-Leu-Leu-Lys(Z)-OBzl is used, this peptide is neutralized with an organic base. The Boc protecting group is cleaved by treating Boc-Leu-Leu-Lys(Z)-Leu-Leu--Leu-Leu-Lys(Z)-OBzl (SEQ. ID No. 6) with an acid at about −20 to 0° C. under an inert atmosphere. Although a solvent may be used with a gaseous acid, such as HCl, the preferred method uses neat trifluoroacetic acid at about 0° C.
H-Leu-Leu-Lys(Z)-Leu
4
-Lys(Z)-OBzl (SEQ. ID No. 6) is treated with a peptide coupling agent, an organic base and Boc-Leu-Leu-Lys(Z)-Leu-Leu-OH (SEQ. ID No. 2) in an inert solvent at about −4 to 10° C. to give Boc-Leu-Leu-(Lys(Z)-Leu
4
)
2
-Lys(Z)-OBzl. (SEQ. ID No. 5) The preferred peptide coupling agents are HOOBT and HBTU, the preferred solvent is DMF and the preferred organic base is DIPEA. The Boc protecting group is cleaved by treating Boc-Leu-Leu-(Lys(Z)-Leu
4
)
2
-Lys(Z)-OBzl (SEQ. ID No. 5) with an acid at about −20 to 0° C. under an inert atmosphere. The preferred acid was HCl and the inert solvent was ethyl acetate.
H-Leu-Leu-(Lys(Z)-Leu
4

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