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  Liquid phase peptide syntheses of kl-4 pulmonary surfactantRelated Patent Categories: Chemistry: Natural Resins Or Derivatives; Peptides Or Proteins; Lignins Or Reaction Products Thereof, Peptides Of 3 To 100 Amino Acid Residues, 8 To 10 Amino Acid Residues In Defined SequenceLiquid phase peptide syntheses of kl-4 pulmonary surfactant description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20070149765, Liquid phase peptide syntheses of kl-4 pulmonary surfactant. Brief Patent Description - Full Patent Description - Patent Application Claims
FIELD OF THE INVENTION [0001] 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 [0002] 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. [0003] KL-4 is a mixture of a pharmaceutically acceptable phospholipid and a 21 residue polypeptide, (L-lysine-(L-leucine).sub.4).sub.4L-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. [0004] The object of the present invention is the production of the peptide component of KL.sub.4, namely (Lys-Leu.sub.4).sub.4Lys (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. [0005] 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 .alpha.-carbon; a problem that is compounded as the length of the peptide increases. In several steps of the synthesis of (Lys-Leu.sub.4).sub.4Lys, 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.sub.4).sub.4Lys 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.sub.4).sub.4Lys, it may be used in the synthesis of other peptides as illustrated hereinafter. SUMMARY OF THE INVENTION [0006] 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: TABLE-US-00001 H-Lys(Z)Leu-Leu-OH H-Leu-Leu-Lys(Z)OBzl left-hand right-hand BocLeu-Leu-Lys(Z)Leu-Leu-OH (SEQ. ID No. 2) body fragment [0007] 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.sub.4).sub.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. [0008] In the second method, a convergent synthesis is employed in which an 8-residue protected polypeptide of the formula: [0009] 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.sub.4).sub.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. [0010] 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 .alpha.-carbon. DETAILED DESCRIPTION OF THE INVENTION [0011] 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 convential 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-hexafluororphosphat- e), DIPEA is N,N-diisopropylethylamine, NMP is 1-methyl-2-pyrrolidone, HOOBT is 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine, WSCDI (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. [0012] The synthesis of (Lys-Leu.sub.4).sub.4Lys (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. TABLE-US-00002 H-Lys(Z)Leu-Leu-OH H-Leu-Leu-Lys(Z)OBzl left-hand right-hand BocLeu-Leu-Lys(Z)Leu-Leu-OH (SEQ. ID No. 2) body fragment [0013] 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.degree. 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.degree. 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.degree. C. [0014] H-Leu-Leu-Lys(Z)-Leu.sub.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.degree. C. to give Boc-Leu-Leu-(Lys(Z)-Leu.sub.4).sub.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.sub.4).sub.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. [0015] H-Leu-Leu-(Lys(Z)-Leu.sub.4).sub.2-Lys(Z)-OBzl (SEQ. ID No. 5) 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.degree. C. to give Boc-Leu-Leu-(Lys(Z)-Leu.sub.4).sub.3-Lys(Z)-OBzl. (SEQ. ID No. 3) The preferred peptide coupling agents are HOOBT and DIC, the preferred solvent is THF and the preferred organic base is DIPEA. The Boc protecting group is cleaved by treating Boc-Leu-Leu-(Lys(Z)-Leu.sub.4).sub.3-Lys(Z)-OBzl (SEQ. ID No. 3) with an acid at about -20 to 0.degree. C. under an inert atmosphere. [0016] H-Leu-Leu-(Lys(Z)-Leu.sub.4).sub.3-Lys(Z)-OBzl (SEQ. ID No. 3) was treated with a salt, an organic base, a peptide coupling agent, water and Z-Lys(Z)-Leu-Leu-OH in an inert solvent at about 0 to 20.degree. C. over 2 to 5 h to give Z-(Lys(Z)-Leu.sub.4).sub.4-Lys(Z)-OBzl. (SEQ. ID No. 1) The preferred coupling agents are DIC and HOOBT, the preferred solvent is THF and the preferred salt is LiBF.sub.4. The benzyl protecting groups are removed by hydrogenating Z-(Lys(Z)-Leu.sub.4).sub.4-Lys(Z)-OBzl (SEQ. ID No. 1) in the presence of a catalyst and an inert solvent under a positive H.sub.2 atmosphere. The preferred catalyst is Pd/C, the preferred solvent is acetic acid and the preferred H.sub.2 pressure is 2 to 2.2 bar. [0017] Procedure B follows another approach. This method employs a convergent pathway where two larger fragments are combined to produce the protected final product. In addition this method uses tetrabutylammonium hydroxide to saponify an ester protected carboxy group with less than 1% racemization of the .alpha.-carbon. [0018] As illustrated by Scheme B, H-Leu-Leu-Lys(Z)-Leu-Leu-OR (SEQ. ID No. 2) is treated with Boc-Lys(Z)-Leu-Leu-OH, a peptide coupling agent, an organic amine in an inert solvent at about -5 to -2.degree. C. for about 2 to 3 h to give Boc-Lys(Z)-Leu-Leu-Leu-Leu-Lys(Z)-Leu-Leu-OR. (SEQ. ID No. 4) The preferred coupling agents are HOOBT and HBTU, the preferred organic base is DIPEA, the preferred solvent is DMF and the preferred group for R is methyl. The terminal ester protecting group is saponified by treating Boc-Lys(Z)-Leu-Leu-Leu-Leu-Lys(Z)-Leu-Leu-OR (SEQ. ID No. 4) with a tetraalkylammonium hydroxide and water in an inert organic solvent at about -17 to 0.degree. C. over 10 to 160 min. Inert solvents include DMF and THF, tetraalkylammonium hydroxide reagents include tetrabutylammonium hydroxide, tetramethylammonium hydroxide, tetraethylammonium hydroxide and tetrabenzylammonium hydroxide. The preferred solvent is THF, the preferred tetraalkylammonium hydroxide is tetrabutylammonium hydroxide and the preferred temperature is -5 to 0.degree. C. The reaction should be monitored by analytical methods, particularly HPLC in order to determine when the starting ester is saponified, for racemization increases over time. [0019] Boc-(Lys(Z)-Leu.sub.4)Lys(Z)-Leu-Leu-OH (SEQ. ID No. 4) is treated with a salt, a peptide coupling agent, an organic base and H-Leu-Leu-(Lys(Z)Leu.sub.4).sub.2-Lys-OBzl (SEQ. ID No. 5) in an inert organic solvent at about 0 to 25.degree. C. over 1 to 30 h to give Boc-(Lys(Z)-Leu.sub.4).sub.4Lys(Z)-OBzl. (SEQ. ID No. 1) The preferred coupling agents are HOOBT and DIC, the preferred organic base is DIPEA, the preferred solvent is THF/water and the preferred salt is tetrabutylammonium chloride. [0020] The protecting groups of Boc-(Lys(Z)-Leu.sub.4).sub.4Lys(Z)-OBzl (SEQ. ID No. 1) are removed by treatment with trifluoroacetic acid followed by hydrogenation in the presence of a catalyst and an inert solvent under a positive H.sub.2 atmosphere. The preferred catalyst is Pd/C, the preferred solvent is acetic acid and the preferred H.sub.2 pressure is 2 to 2.2 bar. Continue reading about Liquid phase peptide syntheses of kl-4 pulmonary surfactant... Full patent description for Liquid phase peptide syntheses of kl-4 pulmonary surfactant Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Liquid phase peptide syntheses of kl-4 pulmonary surfactant patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. Each week you receive an email with patent applications related to your keywords. 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