WO2003062266A2 - Hybrid synthetic method for antimicrobial peptides - Google Patents

Hybrid synthetic method for antimicrobial peptides

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Publication number
WO2003062266A2
WO2003062266A2 PCT/US2003/001998 US0301998W WO2003062266A2 WO 2003062266 A2 WO2003062266 A2 WO 2003062266A2 US 0301998 W US0301998 W US 0301998W WO 2003062266 A2 WO2003062266 A2 WO 2003062266A2
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WO
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Patent type
Prior art keywords
peptide
solution
solvent
amino
fmoc
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Application number
PCT/US2003/001998
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French (fr)
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WO2003062266A3 (en )
Inventor
Gene Merutka
Zoulin Zhu
Sheri A. Almeda
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Intrabiotics Pharmaceuticals, Inc.
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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K7/00Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04Linear peptides containing only normal peptide links
    • C07K7/08Linear peptides containing only normal peptide links having 12 to 20 amino acids

Abstract

The present invention relates to methods for the synthesis and isolation of the protegrin IB 367; as well as analogs and derivatives thereof. The present method utilizes both solid phase and liquid phase peptide synthesis procedures to synthesize and combine specific peptide fragment intermediates. This invention relates to processes comprising solid phase peptide synthesis procedures in which glycine is the amino acid attached to the solid support, and both solid and liquid phase synthesis methods that use side-chain-unprotected arginine residues.

Description

HYBRID SYNTHETIC METHOD FOR ANTIMICROBIAL PEPTIDES

1. FIELD OF THE INVENTION

5

The present invention relates to methods for peptide synthesis. In particular, the present invention relates to the synthesis of the protegrin IB 367, as well as analogs and derivatives thereof. The present method utilizes both solid phase peptide synthesis procedures and liquid phase peptide synthesis procedures to synthesize and combine 10 specific peptide fragment intermediates. Even more particularly the present invention relates to processes comprising solid phase peptide synthesis procedures in which glycine is the amino acid attached to the solid support, and both solid and liquid phase synthesis methods that use side-chain-unprotected arginine residues.

15 2. BACKGROUND OF THE INVENTION

A large number of biologically-active peptides, particularly those with antimicrobial activity have been identified that may be useful for the prevention and treatment of disease, and, more particularly, infectious disease. Antimicrobial peptides include, but are not

20 limited to, cationic amphipathic peptides such as dermaseptins or derivatives or analogues thereof (Moτ et al, 1991, Biochemistry 30: 8824; Mor et al, 1991, Biochemistry 33: 6642; Mor et al, 1994, Eur. J. Biochem. 219: 145); magainin peptides or derivatives or analogues thereof (Zasloff, 1987, Proc. Natl. Acad. Sci. U.S.A. 84: 5449); PGLa or XPF peptides or derivatives or analogues thereof (Hoffman et al, 1983, EMBO J. 2:711; Andreu et al, "'

25 1985, Biochem. J. 149: 531; Gibson et al, 1986, J. Biol. Chem. 261: 5341; Giovannini et al, 1987, Biochem. J. 243: 113); CPF peptides (Richter et al, 1986, J. Biol. Chem. 261: ** 3676; U.S. Pat. No. 5,073,542); adrenoregulins or derivatives or analogues thereof (Donly et al, 1992, Proc. Natl. Acad. Sci. U.S.A. 89: 10960; Amiche et al, 1993, Biochem. Biophys. Res. Commun. 191: 983); performs or derivatives or analogues thereof (Henkart

30 et al, 1984, J. Exp. Med. 160: 75-93; Podack et al, 1984, J. Exp. Med. 160: 695-710); caerulein or derivatives or analogues thereof (Richter et al, 1988, J. Biol. Chem. 261: 3676-3680; and Gibson et al, 1986, J. Biol. Chem. 261: 5341-5349); Bacterial/Permeability-Increasing Protein (BPI) or peptide derivatives or analogues thereof (Ooi et al, 1987, J. Biol. Chem. 262: 14891-14898; Qi et al, 1994, Biochem. J.

35 298: 771-718; Gray and Haseman, 1994, Infection and Immunity 62:2732-2739; Little et al, 1994, J. Biol. Chem. 269: 1865-1872; and U.S. Pat. No. 5,348,942); insect defensins (also called sapecins) or analogues or derivatives thereof (Alvarez-Bravo et al, 1994, Biochem. J. 302: 535-538; Yamada and Natori, 1994, Biochem. J. 298: 623-628; Kum et al, 1994, FEBS Letters 342: 189-192; Shimoda et al, 1994, FEBS Letters 339: 59-62; Yamada and Natori, 1993, Biochem. J. 291: 275-279; Homma et al, 1992, Biochem. J. 288: 281-284; Hanzawa et al, 1990, FEBS Letters 269: 413-420; Kuzuliara et al, 1990, Biochem. J. 107: 514-518; Matsuyama and Natori, 1990, Biochem. J. 108: 128-132; U.S. Pat. No. 5,107,486; European Patent No. 303,859; European Patent No. 280,859; U.S. Pat. No. 5,008,371; U.S. Pat. No. 5,106,735; and U.S. Pat. No. 5,118,789); rabbit or human FALL-39/CAP-18 (Cationic Antimicrobial Protein) or analogues or derivatives thereof (PCT Application WO 94/02589 and references cited therein; Agerberth et al, 1995, Proc. Natl. Acad. Sci. U.S.A. 92: 195-199; Larrick et al, 1991, Biochem. Biophys. Res. Commun. 179: 170-175; Hirata et al, 1990, Endotoxin: Advances in Experimental Medicine and Biology (Herman Friedman, T. W. Klein, Masayasu Nakano, and Alois Nowotny, eds.); Tossi et al, 1994, FEBS Letters 339: 108-112; Larrick et al, 1994, J. Immunol. 152: 231-240; Hirata et al, 1994, Infection and Immunity 62:1421-1426; and Larrick et al, 1993, Antimicrobial Agents and Chemotherapy 37: 2534-2539; PMAP (Porcine Myeloid Antibacterial Peptide) or analogues or derivatives thereof (Zanetti et al, 1994, J. Biol. Chem. 269: 7855-7858; Storici et al, 1994, FEBS Letters 37: 303-307; and Tossi et al, 1995, Eur. J. Biochem. 228: 941-948); aibellin or analogues or derivatives thereof (Hino et al, 1994, J. Dairy Sci. 77: 3426-3461; Kumazawa et al, 1994, J. Antibiot. 47: 1136-1144; and Hino et al, 1993, J. Dairy Sci. 76: 2213-2221); caerin or analogues or derivatives thereof (Stone et al, 1992, J. Chem. Soc. Perkin Trans. 1: 3173-3178; and PCT WO 92/13881, published Aug. 20, 1992); bombinin or analogues or derivatives thereof (Simmaco et al, 1991, Eur. J. Biochem. 199: 217-222 and Gibson et al, 1991, J. Biol. Chem. 266: 23103-23111); brevenin or analogues or derivatives thereof (Morikawa et al. , 1992, Biochem. Biophys. Res. Commun. 189: 184-190; and Japanese Patent Application No. 6,080,695 A); esculetin or analogues or derivatives thereof (Simmaco et al, 1993, FEBS letters 324: 159-161; and Simmaco etal, 1994, J. Biol. Chem. 269: 11956-11961); lactoferrin or analogues or derivatives thereof (U.S. Pat. No. 5,317,084; U.S. Pat. No. 5,304,633; European Patent Application No. 519,726 A2; European Patent Application No. 503,939 Al; PCT Application WO 93/22348, published Nov. 11, 1993; PCT Application WO 90/13642; and To ita et al, hi: Lactoferrin Structure and Function, Hutchens, T. W., et al, Eds., Plenum Press, NY, 1994, pp. 209-218); CEMA peptides or analogues or derivatives thereof (PCT Application WO 94/04688, published Mar. 3, 1994); tachyplesins and analogues of tachyplesins such as polyphemusins (Nakamura et al, 1988, J. Biol. Chem. 263: 16709-16713; Miyata et al, 1989, J. Biochem. 106: 663-668), defensins (Lehrer et al, 1991, Cell 64: 229-230; Lehrer et al, 1993, Ann. Rev. Immunol. U: 105-128; U.S. Pat. No. 4,705,777; U.S. Pat. No. 4,659,692; U.S. Pat. No. 4,543,252), β-defensins (Selsted et al, 1993, J. Biol. Chem. 288: 6641-6648; Diamond et al, 1991,

5 Proc. Natl. Acad. Sci. U.S.A. 88: 3952-3958), histatins and analogues or derivatives thereof (Oppenheim et al. 1988, J. Biol. Chem. 263:7472-77; Xu et al. 1990, J. Dent. Res. 69(11): 1717-23; U.S. Patent No. 5,912,230); and protegrins (Kokryakov et α ., 1993, FEBS 337: 231-236; Zhao et al, 1994, FEBS Letters 346: 285-288; Migorodskaya et al, 1993, FEBS 330: 339-342; Storici et al, 1993, Biochem. Biophys. Res. Commun.

10 196: 1363-1367; Zhao et al, 1994, FEBS Lett. 346: 285-288; Manzoni et al, 1996, FEBS Lett. 383: 93-98; U.S. Pat. No. 5,464,823).

Also included within the scope of biologically-active peptides are the protegrin peptides, which are a recognized class of naturally occurring peptides that exhibit broad-spectrum antimicrobial activity. Protegrin peptides, as well as derivatives and

15 analogues thereof are described, inter alia, in U.S. Pat. Nos. 5,464,823, 5,464,823,

5,804,558, 5,994,306, and 6,025,326, in patent publication WO 95/03325, and U.S. Ser. Nos. 08/451,832, 08/562,346, 08/649,811 and 08/960,921, each of which is incorporated herein by reference in its entirety.

An example of such an antimicrobial peptide with therapeutic activity is the 0 protegrin peptide designated IB-367, which has been shown to be active both in vitro and in vivo against microflora associated with oral mucositis. This condition is believed to arise, for example, as a result of the toxic effects of radiation and chemotherapy on rapidly dividing epithelial cells, particularly those cells of the oropharyngeal mucosa, and to be exacerbated by endogenous microflora (Mosca et al. 2000, Antimicrobial Agents and 5 Chemotherapy, 44 (7): 1803-1808).

Widespread use of such biologically active peptides, particularly for the prevention and treatment of infectious diseases such as oral mucositis, creates a demand for the development of efficient, large-scale, commercially viable processes for their production. Such processes will involve the effective integration of methods that are rapid, reproducible,

30 robust, and that provide high-purity peptides at good yields, and that will meet regulatory standards for the manufacture of human therapeutics. Development of such processes is critical for the clinical and commercial success of biologically active peptides, including, but not limited to protegrins and their derivatives and analogs.

There are, currently, a number of techniques available for the synthesis of peptides,

35 as disclosed in the following: U.S. Patent No. 6,015,881; Mergler et al, 1988, Tetrahedron Letters 29: 4005-4008; Mergler et al, 1988, Tetrahedron Letters 29: 4009-4012; Kamber et al. (eds), Peptides, Chemistry and Biology, ESCOM, Leiden, 1992, 525-526; Riniker et al, 1993, Tetrahedron Letters 49: 9307-9320; Lloyd- Williams et al. 1993, Tetrahedron Letters 49: 11065-11133; and Andersson et al, 2000, Biopolymers 55: 227-250. However, although synthetic methods are known in the art, there is a need for large scale, commercially viable processes for the production of, inter alia, a therapeutic peptide such as IB-367 or a derivative or analog thereof.

3. SUMMARY OF THE INVENTION

The present invention relates to processes for the synthesis of peptides, and, more specifically, to processes for the production of IB-367

NH2-RGGLCYCRGRFCVCVGR-C(O)NH2 (SEQ ID NO: 1, FIG. 1) and derivatives and analogues thereof. The processes of the present invention are "hybrid processes" that comprise both solid phase peptide synthesis methods as well as liquid phase peptide synthesis methods for the synthesis and joining of peptide fragment intermediates to provide the peptide of interest. Generally, the processes of the invention comprise solid phase synthesis of specific protected peptide fragment intermediates, wherein some, but not all of the reactive side-chain moieties are protected. The peptide fragment intermediates, which comprise one or more protecting groups, are cleaved from the solid support, coupled in solution, and deprotected to provide the final peptide product, e.g., IB-367 or an analogue or derivative thereof. A specific embodiment of the invention is directed toward the synthesis of IB-367 peptide depicted in FIG. 1.

The present invention also relates to specific peptide fragments that are intermediates in the synthesis of IB-367 or to analogues and derivatives thereof. Such fragments include, but are not limited to those having the amino acid sequences depicted in Table 1:

TABLE 1

Corresponding

Numbered Amino

Amino Acid Sequence

Peptide Acid Sequence of No. IB-367

1 RGGLCYCRG (SEQ ID NO. : 2) 1-9

2 RFCVCVG (SEQ ID NO. : 3) 10-16

3 RFCVCVGX (X - Arg amide) (SEQ ID NO. : : 4) 10-17

The amino acid notations used herein are conventional and are as follows:

Common Amino Acid Abbreviations

Amino Acid One-Letter Symbol Common Abbreviation

Alanine A Ala

Arginine R Arg

Asparagine N Asn

Aspartic Acid D Asp

Cysteine C Cys

Glutamine Q Gin

Glutamic Acid E Glu

Glycine G Gly

Histidine H His

Isoleucine I He

Leucine L Leu

Lysine K Lys Methionine M Met

Phenylalanine F Phe

Proline P Pro

Serine s Ser

Threonine T Thr Tryptophan W Tip

Tyrosine Y Tyr

Valine V Val

5

In another embodiment of the present invention, specific peptide fragments other than those of Table 1, are synthesized and used to assemble IB-367 according the methods disclosed herein; for example the following, specific peptides could be used, RGG, LCY,

CRG, RFCV, and CVGR. That is, one of ordinary skill would appreciate that peptides

, disclosed herein can be assembled according to the hybrid processes disclosed herein using various combinations of intermediate peptides. Moreover, as one of ordinary skill in the art would appreciate, the assembly of a peptide may also comprise synthesis of a plurality of peptide fragments on separate solid supports, cleavage of a first peptide from a first solid support, and condensation of that first peptide to a second peptide, which is attached to a

^ _- second solid support.

Generally, solid phase peptide synthesis is carried out in the carboxy-terminal to the amino-terminal direction, in which the C-terminal amino acid residue of a peptide fragments intermediate is attached to a solid support and the remaining amino acids of the peptide fragment intermediate of interest are added sequentially thereafter. The present

2 invention further relates to processes for large scale peptide synthesis comprising solid phase synthesis methods wherein the amino acid attached to the solid support, either directly or through a cleavable linking group, is glycine. In this manner, racemization of the amino acid as it is attached to the solid support is obviated. Moreover, solid supports to which glycine has already been attached, are commercially available (Bachem Biosciences, Inc., Torrance CA; Senn Chemicals, Dielsdorf, Switzerland), or may be synthesized using techniques and reagents well-know to those of ordinary skill in the art, thereby providing reagent cost savings, and decreasing the number of synthetic cycles required for solid phase synthesis of each peptide fragment intermediate. In addition, the use of the same resin having a glycine residue attached, also simplifies the synthetic process by providing a A uniform step that is common to the solid phase synthesis of each peptide fragment intermediate designed to have a glycine residue at the carboxy-terminal end. Consequently, the present invention relates to solid phase peptide synthesis methods comprising the deliberate design and selection of peptide fragment intermediates having a carboxy-terminal glycine residue. This design, in which glycine is the C-terminal residue of fragments to be o_- joined, also obviates the commonly-recognized problem of racemization which can occur when the peptide bond is formed between the fragments. Accordingly, the present invention is particularly directed to processes for the production of peptides comprising at least one glycine residue. An example of such a design is depicted in FIG. 2, which presents one, non-limiting approach to the synthesis of IB-367. Table 2 provides other, non-limiting examples of the amino acid sequence of exemplary biologically-active peptides

5 and representative peptide fragment intermediates to be used according to the present invention for large-scale synthesis of the following biologically-active peptides: magainin (SEQ LD NO: 6), and peptide intermediates therefor (SEQ ID NOS.: 7-9); defensin (SEQ ID NO: 10), and peptide intermediates therefor (SEQ LD NOS.: 11-14); bombinin (SEQ ID NO: 15), and peptide inteπnediates thereof (SEQ ID NOS.: 16-18); apidaecin (SEQ ID NO:

10 19), and peptide intermediates thereof (SEQ ID NOS.: 20-21); and histatins including but not limited to the histatin of SEQ LD NO: 22, and peptide intermediates thereof (SEQ ID NOS: 23-24).

TABLE 2

15

Magainin Amino Acid Sequence and Peptide Intermediates therefor:

GIGKFLKS AGKFGKAFVGSrMNS (SEQ ID NO. : 6)

GIGKFLKS AG (SEQ LD NO. : 7)

KFGKAFVG (SEQ ID NO. : 8) 0 SLMNS (SEQ ID NO.: 9)

Defensin Amino Acid Sequence and Peptide Intermediates therefor: ACYCRYPACIAGERRYGTClYQGALNAYCC (SEQ ID NO. : 10)

ACYCRYPACIAG (SEQ ID NO. : 11) 5 ERRYG (SEQ ID NO.: 12)

TCIYQG (SEQ ID NO. : 13)

ALNAYCC (SEQ ID NO.: 14)

Bombinin Amino Acid Sequence and Peptide Intermediates therefor: 0 GIGSAILSAGKSALKGLAKGLAEHF (SEQ ID NO.: 15)

GIGS AJLS AG (SEQ ID NO. : 16)

KS ALKGLAKG (SEQ ID NO. : 17)

AKGLAEHF (SEQ ID NO.: 18) 5 Apidaecin Amino Acid Sequence and Peptide Intermediates therefor: REPEAEPGNNRPVYΓPQPRPPHPRLR (SEQ ID NO. : 19)

REPEAEPG (SEQ ID NO. : 20)

NNRPVYΓPQPRPPHPRLR (SEQ ID NO.: 21)

Histatin Amino Acid Sequence and Peptide Intermediates therefor:

AKRHHGYKRKFH (SEQ IDNO.: 22) AKRHHG (SEQ ID NO.: 23)

YKRKFH (SEQ ID NO.: 24)

The present invention is further directed to processes comprising solid phase peptide synthesis methods as well as solution phase synthesis methods utilizing arginine residues having unprotected side chains. The side chain guanidine group of arginine is typically protected during peptide synthesis reactions because this group is very nucleophilic and can be acylated during the coupling process. The side chain of arginine is usually protected by Mtr (4-methoxy-2,3,6-trimethylbenzenesulfonyl) or Pmc

(2,2,5,7,8-pentamethylchromane-6-sulfonyl) (Pmc) groups (Ramage et al. 1991, Tet. Lett. 47: 6353-70) or Pbf (2,3,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl) groups (Carpino et al 1993, Tet. Lett. 34: 7829-332). In addition, removal of side chain arginine protecting groups can also cause side reactions with the side chains of tryptophan, tyrosine, serine, and threonine (Fields et al. 1993, Tet. Lett. 34: 6661-64). Furthermore, it is sometimes problematic to achieve complete removal of all of the arginine protecting groups of peptides containing multiple arginines (Fischer et al. 1992 Int. J. Pept. Protein Res. 40: 19-24).

In this embodiment, arginine residues are coupled to an amino acid or to a peptide fragment intermediate, either in solution or attached to a solid support, in the presence of, inter alia, an additive such as pyridine»HBr (Rubina et al., 2000, Russian Journal of Bioorganic Chemistry, 26: 235-244) or other pyridinium halide such as, but not limited to pyridmerHCl, or pyridinium tosylate, butyl ammonium bromide, butyl ammonium chloride, or any other alkyl ammonium halide where the alkyl group comprises at least two carbon atoms. It has been found that such use of side-chain-nonprotected arginine reagents is particularly effective where the arginine residue to be added is at or near the amino-terminus of the peptide fragment intermediate being synthesized in a carboxy-terminal to amino-terminal direction. In certain embodiments, a peptide fragment intermediate can be cleaved from a solid support and a side-chain-nonprotected arginine residue coupled thereto without removal of the α-amino protecting group (e.g. Fmoc) from the peptide fragment intermediate. In this manner a side-chain-nonprotected arginine residue is added to the carboxy-terminus of the peptide fragment intermediate synthesized on a solid support. Since side-chain-nonprotected arginine reagents are less expensive than fully-protected arginine reagents, this aspect of the invention provides substantial cost savings for large scale peptide synthesis processes. The present invention still further relates to the coupling of modified, side-chain-nonprotected arginine derivatives to a peptide intermediate, using either solid phase or solution phase peptide synthesis methods. It has also been discovered that, for example, an amidated nonprotected arginine residue can be readily coupled to a Fmoc-protected peptide intermediate in solution, as depicted in FIG. 3. The following abbreviations are used herein: Nα-9-fluorenylmethyloxycarbonyl

(Fmoc); Nα-t-butoxycarbonyl (Boc); N,N'-Dicyclohexylcarbodiimide (DCC); N,N-diisopropylcarbodiimide (DJJPCDI); 1-hydroxybenzotriazole (HOBt); N-methylpyrrolidone (NMP); dichloromethane (DCM); 2-(H-benzotriazol- 1 -yl)- 1 , 1 ,3 ,3 ,-tetramethyluronium tetrafluoroborate (TBTU); O-benzotriazol-1 -yl-N, N, N', N',-tetramethyluroniumhexafluo hosphate (HBTU); N,N-diisopropylethylamine (DL A); acetamidomethyl (Acm);

4-methoxy-2,3,6-trimethylbenzenesulfonyl (Mtr); 2,4,6-trimethylbenzenesulfonyl (Mts); 2,2,5,7,8-pentamethylchromane-6-sulfonyl (Pmc); and 2,3,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf). hi one embodiment, the present invention is directed toward a method for the synthesis of a peptide having the formula RGGLCYCRGRFCVCVGX, where X = Arg-amide, (SEQ ID NO.: 1), in which a side-chain-protected peptide of the formula: Fmoc-ZGGLCYCRG-COOH (Z = side-chain-nonprotected Arg) (SEQ ID NO.: 2), is reacted with a side-chain-protected peptide of the formula: NH2-ZFCVCVGZ-C(O)NH2, where Z = side-chain-nonprotected Arg, (SEQ ID NO.: 4) to yield a side-chain-protected peptide of the formula:

Fmoc-ZGGLCYCRGZFCVCVGZ-C(O)NH2, where Z = side-chain-nonprotected Arg, (SEQ JD NO.: 5). The amino terminus of the terminus of the side-chain-protected peptide produced in this manner is deprotected, then the side chains of the side-chain-protected peptide are deprotected to provide a peptide of the following formula: RGGLCYCRGRFCVCVGX (X = Arg-amide) (SEQ ID NO.: 1).

In one aspect of this embodiment, the side-chain protected peptide of formula NH2-ZFCVCVGZ-C(O)NH2, where Z = side-chain-nonprotected Arg, (SEQ ID NO.: 4) is formed by a method comprising reacting a side-chain-protected peptide of the formula: Fmoc-ZFCVCVG-COOH Arg (SEQ ID NO.: 3) with arginine-amide to yield a side-chain-protected peptide of the formula Fmoc-ZFCVCVGZ-C(O)NH2, (SEQ TD NO.: 4), and then deprotecting the amino terminus of the side-chain-protected peptide produced in this manner, to yield a peptide of the formula NH2-ZFCVCVGZ-C(O)NH2, where Z = side-chain-nonprotected Arg, (SEQ ID NO.: 4).

In another aspect of this embodiment, Fmoc-ZGGLCYCRG-COOH , where Z represents a side-chain-nonprotected Arg (SEQ ID NO.: 2) is synthesized by solid phase peptide synthesis.

In a further aspect of this embodiment of the invention, the side-chain-protected peptide of the formula Fmoc-ZFCVCVG-COOH, where Z represents a side-chain-nonprotected Arg (SEQ LD NO.: 3), is synthesized by solid phase peptide synthesis.

In another embodiment, the present invention is directed toward a method for the precipitation of a peptide having the formula RGGLCYCRGRFCVCVGR (SEQ ID NO.: 1). The method comprises providing a first volume of solution comprising the peptide RGGLCYCRGRFCVCVGR (SEQ ID NO.: 1) dissolved in a solvent comprising ethanol and water; and adding a second volume of a co-solvent to the peptide solution to provide a mixture, wherein the co-solvent is selected from the group consisting of tefrahydrofuran, methyl acetate, and combinations thereof, and wherein the ratio of the second volume to said first volume is greater than one. In certain aspects of this embodiment, the peptide solution comprises about ImM HCl.

In a further embodiment, the present invention is directed toward a method for precipitation of a peptide having the formula RGGLCYCRGRFCVCVGR (SEQ ID NO.: 1), comprising providing a first volume of solution comprising this peptide dissolved in a solvent comprising methanol and water; and adding a second volume of a co-solvent to said solution to provide a mixture. In this embodiment, the co-solvent is selected from the group consisting of tefrahydrofuran, ethyl acetate, methyl acetate, methyl tert-butyl ether, and combinations thereof, and the ratio of the second volume to the first volume is greater than one. In certain aspects of this embodiment, the peptide solution comprises about ImM HCl. Another embodiment of the present invention is directed toward a method for precipitation of a peptide having the formula RGGLCYCRGRFCVCVGR (SEQ ID NO.: 1), comprising providing a first volume of solution comprising this peptide dissolved in a solvent comprising acetonitrile and water, and adding a second volume of tefrahydrofuran to the solution to provide a mixture in which the ratio of the second volume to the first volume is greater than two. In certain aspects of this embodiment, the peptide solution comprises about ImM HCl.

4. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Depicts the structure of LB-367.

FIG. 2 Depicts a hybrid peptide synthesis method for the production of IB-367. FIG. 3 Depicts the synthesis of Fragment lb from Fragment 1, comprising coupling of arginine amide to Fmoc-Fragment 1, followed by removal of the Fmoc protecting group.

FIG. 4 Depicts the synthesis of Fragment lb from Fragment 1, comprising removal of the Fmoc protecting group of Fragment 1, followed by coupling of arginine amide to Fragment 1.

5. DETAILED DESCRIPTION OF THE INVENTION

5.1 Synthesis of Full-length Peptides

The present invention relates to processes for the synthesis of full-length, biologically-active peptides, including but not limited to IB-367 and analogues and derivatives thereof, where UB-367 is a 17-amino acid residue peptide having the following sequence, reading from amino-terminus to carboxy-terminus:

NH2-RGGLCYCRGRFCVCVGR-C(O)NH2 (SEQ ID NO.: 1) It will be understood that the processes of the present invention, which comprise the methods and techniques disclosed herein can be applied generally to the synthesis of peptides, particularly biologically-active peptides, and more particularly to peptides having antimicrobial activity. The processes of the present invention are particularly directed toward the synthesis of such peptides wherein the peptide comprises at least one internal or carboxy-terminal glycine residue and/or at least one arginine residue. Such processes of the present invention include, but are not limited to, methods for: (1) the design of peptide fragment intermediates in such a manner that such fragments comprise either or both of: (a) a carboxy-terminal glycine residue and (b) an amino-terminal arginine residue; (2) the coupling of such an amino-terminal arginine residue to the peptide fragment intermediate using side-chain-nonprotected arginine reagents in the presence of an additive such as pyridine'HBr or other pyridinium halide such as, but not limited to pyridine»HCl, or pyridinium tosylate, butyl ammonium bromide, butyl ammonium chloride, or any other alkyl ammonium halide where the alkyl group comprises at least two carbon atoms, using either solid phase or solution phase peptide synthesis methods; and (3) methods for coupling modified and unmodified side-chain-nonprotected arginine residues, to either or both of the carboxy or amino termini of partially-protected peptide fragment intermediates in solution, in the presence of an additive such as pyridine»HBr or other pyridinium halide such as, but not limited to pyridineΗCl, or pyridinium tosylate, butyl ammonium bromide, butyl ammonium chloride, or any other alkyl ammonium halide where the alkyl group comprises at least two carbon atoms.

In addition to the specific peptides disclosed herein, the present methods are also used for the synthesis of peptides comprising either or both a modified amino-terminus and a modified carboxy-terminus, where the modification consists of, but is not limited to attachment of a hydrophobic group selected from the group consisting of carbobenzoxyl, dansyl, and t-butyloxycarbonyl; an acetyl group; a 9-fluoroenyl-methoxy-carbonyl (FMOC) group; or a macromolecular carrier group selected from the group consisting of lipid-fatty acid conjugates, polyethylene glycol, and carbohydrates; a t-butyloxycarbonyl group; and a para-nitrobenzyl ester group. Techniques for addition of such groups are well known to those of skill in the art. h certain embodiments of the present invention, peptide fragment intermediates can be purified using any silica or non-silica based column packing including but not limited to zirconium-based packings, poly-styrene, poly-acrylic or other polymer based packings. Columns packed with such material can be run in low, medium or high pressure chromatography. However, in preferred embodiments, the peptide fragment intermediates are used without chromatographic purification. For example, in the process for the synthesis of IB-367, as depicted in FIGS. 2 and 3, the Fmoc-protected peptide fragment intermediate, designated Fmoc-Fragment 1, Fmoc-R-F-C(Acm)-V-C(Acm)V-G (SEQ ID NO.: 3) is cleaved from the solid support, isolated and then, without chromatographic purification, coupled to side-chain-nonprotected arginine-amide. The product of this reaction is deprotected directly, without chromatographic purification, e.g., by addition of piperidine to the coupling reaction mixture to a final concentration of about 20% (v/v).

5.2 Peptide Fragment Intermediates

The processes of the present invention are also used for the synthesis of peptide fragment intermediates including but not limited to those having the specific amino acid sequences as those depicted in Table 1 and Table 2. That is, as one of ordinary skill would appreciate, for example, analogues and derivatives of IB-367 having a different amino acid sequence and/or length are synthesized with different peptide fragments intermediates. Moreover, although not indicated in the sequences provided in Table 1 and Table 2, as would be appreciated by one of ordinary skill, one or more of the side chains of those amino acids can be protected with standard protecting groups, including but not limited to, t-butyl (t-Bu), trityl (trt), t-butyloxycarbonyl (Boc), acetamidomethyl (Acm),

4-methoxy-2,3,6-trimethylbenzenesulfonyl (Mtr); 2,4,6-trimethylbenzenesulfonyl (Mts); 2,2,5,7,8-pentamethylchromane-6-sulfonyl (Pmc); and

2,3,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf). The t-Bu group is a preferred side-chain protecting group for amino acid residues Tyr (Y), Thr (T), Ser (S) and Asp (D); the trt group is a preferred side-chain protecting group for amino acid residues His (H), Gln(Q) and Asn (N); the Boc group is a preferred side-chain protecting group for amino acid residues Lys (K) and Trp (W); in certain embodiments, the Acm group is used as a side-chain-protecting group for Cys (C); and, where appropriate, the Pbf group is a preferred side-chain-protecting group for Arg (R). Peptides of the invention may alternatively be synthesized such that one or more of the bonds which link the amino acid residues of the peptides are non-peptide bonds. These alternative non-peptide bonds may be formed by utilizing reactions well known to those in the art, and may include, but are not limited to imino, ester, hydrazide, semicarbazide, and azo bonds, to name but a few. h yet another embodiment of the invention, a biologically active peptide, particularly IB-367 or an analogue or derivative thereof, comprising, but not limited to, the sequences described above in Table 1 and Table 2, may be synthesized with additional chemical groups present at their amino and/or carboxy termini, such that, for example, the stability, reactivity and/or solubility of the peptides is enhanced. For example groups such as carbobenzoxyl, dansyl, acetyl or t-butyloxycarbonyl groups, may be added to the peptides' amino termini. Additionally, groups such as t-butyloxycarbonyl, or an amido group may be added to the peptides' carboxy termini, such as the amido group of IB-367 (SEQ ID NO.: 1). Similarly, a para-nitrobenzyl ester group may be placed at the peptides' carboxy termini. Techniques for introducing such modifications are well known to those of skill in the art.

Moreover, in another embodiment of the present invention, peptides maybe synthesized such that their steric configuration is altered. For example, the D-isomer of one or more of the amino acid residues of the peptide may be used, rather than the usual L-isomer. In still another embodiment of the present invention, at least one of the amino acid residues of the peptides of the invention may be substituted by one of the well known non-naturally occurring amino acid residues. Alterations such as these may serve to increase the stability, reactivity and/or solubility of the peptides of the invention. Examples of such amino acids include but are not limited to β-alanine (β-Ala) and other omega-amino acids such as 3-aminopropionic acid, 2,3-diaminopropionic acid; (Dpr), 4-aminobutyric acid and so forth; α-aminoisobutyric acid (Aib); ε-aminohexanoic acid (Aha); Δ-aminovaleric acid (Ava); N-methylglycine or sarcosine (MeGly); ornithine (Orn); citrulline (Cit); t-butylalanine (t-BuA); t-butylglycine (t-BuG); N-methylisoleucine (Melle); phenylglycine (Phg); cyclohexylalanine (Cha); norleucine (Nle); 1-naphthylalanine (1-Nal); 2-naphthylalanine (2-Nal); 4-chlorophenylalanine (Phe(4-Cl)); 2-fluorophenylalanine (Phe(2-F)); 3-fluorophenylalanine (Phe(3-F)); 4-fluorophenylalanine (Phe(4-F)); penicillamine (Pen); l,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic); β-2-thienylalanine (Thi); methionine sulfoxide (MSO); homoarginine (Har); N-acetyl lysine (AcLys); 2,3-diaminobutyric acid (Dab); 2,4-diaminobutyric acid (Dbu); p-aminophenylalanine (Phe(pNH2)); N-methyl valine (MeNal); homocysteine (hCys) and homoserine (hSer).

In other embodiments, peptides synthesized according to the processes described herein are synthesized to comprise an additional macromolecular carrier group covalently attached to the amino and/or carboxy termini of the peptide. Such macromolecular carrier groups may include, for example, lipid-fatty acid conjugates, polyethylene glycol, carbohydrates or additional peptides. In other embodiments, peptides synthesized according to the processes of the present invention are coupled, either directly or via a suitable linking group, in non-limiting examples, to a therapeutic agent, a reporter molecule or diagnostic agent, or an antibody molecule.

5.3 Peptide Synthesis individual peptide fragment intermediates of the invention are assembled using a combination of solid phase and solution phase synthesis techniques, where the synthetic process culminates in the production of a biologically active peptide, particularly IB-367 or an analogue or derivative thereof. hi certain embodiments, peptide fragment intermediates of the present invention are synthesized by solid phase peptide synthesis (SPPS) techniques using standard FMOC protocols. See, e.g., Carpino et al, 1970, J. Am. Chem. Soc. 92(19): 5748-5749; Carpino et al, 1972, J. Org. Chem. 37(22): 3404-3409, "Fmoc Solid Phase Peptide Synthesis" Weng C. Chan and Peter D. White Eds. (2000) Oxford University Press Oxford Eng.. In certain embodiments, the solid phase syntheses of the peptide fragment intermediates of the present invention are carried out on an acid sensitive solid support, such as "Wang" resin, which comprises a copolymer of styrene and divinylbenzene with

4-hydroxymethylphenyloxymethyl anchoring groups (Wang, S.S. 1973, J. Am. Chem. Soc, 95: 1328-33, and Lu et al, (1981) J. Org. Chem. 46, 3433-3436, which is hereby incorporated by reference in its entirety), 2-chlorotrityl chloride resin (see, e.g., Barlos et al, 1989, Tetrahedron Letters 30(30): 3943-3946) and

4-hydroxymethyl-3-methoxyphenoxybutyric acid resin (see, e.g., Richter et al, 199 , Tetrahedron Letters 35(27): 4705-4706). The Wang, 2-chlorotrityl chloride, and 4-hydroxymethyl-3-methoxyphenoxy butyric acid resins may be purchased from Calbiochem-Novabiochem Corp., San Diego, CA.

General procedures for production and loading of resins which can be utilized in solid phase peptide synthesis are described in "Principles and Practice of Solid Phase Peptide Synthesis" (Edited by Gregory A. Grant, 1992, W. H. Freeman and Company) and references therein and are well-known to those of ordinary skill in the art. Specific procedures for loading of Wang resins are described in Sieber, 1987, Tet. Lett. 28: 6147-50; and Granadas et al. 1989, hit. J. Pept. Protein Res. 33: 386-90. Resin loading can be performed, for example, using the following techniques. The resin, preferably a "Wang" resin, as described above, is charged to the reaction chamber and washed with a solvent such as DCM or NMP. The resin is drained and a solution of about three equivalents of an amino acid, about 3 equivalents of hydroxybenzotriazole (HOBt) dissolved in a minimal volume of NMP is added. The amino-terminus of the amino acid should be protected, preferably with Fmoc, (although, in other embodiments of the present invention, alternative protecting groups such as Boc are used), and the side chain of the amino acid may be protected where necessary or appropriate. Diisopropylcarbodiimide (DIG) (about 1 equivalent) is added followed by about 0.1 equivalent of p-dimethylaminopyridine (DMAP). The mixture is agitated with nitrogen bubbling for 2 to 3 hours at room temperature.

After agitation, the bed is drained and washed with about ten volumes of NMP. About two equivalents of acetic anhydride are added and mixed for 30 minutes at room temperature to end-cap any unreacted hydroxyl groups on the resin.

The bed is drained, washed four times with about ten volumes of NMP and dried with a nitrogen purge, to provide the loaded resin. As noted above, Fmoc is a protecting group used in certain embodiments for protection of the α-amino moiety of an amino acid. Depending on which amino acid is being loaded, and at what point in the peptide fragment intermediate it is to be attached, the side chain of the amino acid may or may not be protected. For example, when Tip is loaded, its side chain should be protected with Boc, the side chain of Tyr is protected with tBu, and the side chain of Cys with Acm. Similarly, the side-chain of Gin may be protected with trt. However, in certain embodiments of the present invention, where the amino acid to be coupled is arginine and, when attached, it will be the amino-terminal residue of the peptide fragment intermediate, the coupling is carried out in the presence of an additive such as pyridine»HBr or other pyridinium halide such as, but not limited to pyridineΗCl, or pyridinium tosylate, butyl ammonium bromide, butyl ammonium chloride, or any other alkyl ammonium halide where the alkyl group comprises at least two carbon atoms, and the side arginine side chain guanidino moiety is not modified with a side-chain-protecting group. In other embodiments of the present invention, where the amino acid to be coupled is arginine and, when attached, it will not be the amino-terminal residue of the peptide intermediate, the side chain is often protected with a Pbf protecting group.

The Fmoc-protected amino acids, either with or without side-chain protecting groups as required, that are used in loading the resin and in peptide synthesis are available commercially from, ter alia, Genzyme Pharmaceuticals Inc., Cambridge, MA, Bachem Biosciences Inc. Torrance, CA, Senn Chemicals, Dielsdorf, Switzerland, and Orpegen Pharma, Heidelberg, Germany, or are readily synthesized using materials and methods well known in the art. As an alternative to the above procedure, the resin may be purchased, e.g. pre-loaded with the appropriate Fmoc-α-N-protected amino acid, (Bachem Biosciences Inc. Torrance, CA, Senn Chemicals, Dielsdorf, Switzerland), which, in certain preferred embodiments of the present invention is glycine.

Solid phase peptide synthesis techniques can be performed, for example, according to the following techniques. The loaded resin is added to the reaction chamber and washed with about ten volumes of a solvent, such as NMP. The resin is then agitated with nitrogen bubbling, preferably in ten volumes methylene chloride (DCM) for about 15 minutes to swell the resin beads. After the resin is swelled, solvent is drained from the reaction chamber and the resin is again washed with about ten volumes of NMP.

Fmoc (9-fluorenylmethyloxycarbonyl) removal from the terminal amine is accomplished using about 10 volumes (about 100 mL) of a 20% solution of piperidine in NMP for about 5 minutes. The deprotected resin is then washed once with about 10 volumes (about 100 mL) of NMP The resin is treated a second time with a 20% solution of piperidine in NMP ( about 10 volumes) for about 15 minutes. The resin is then washed about 5-7 times with about 10 volumes of NMP to remove Fmoc by-products (i.e., dibenzofulvene and its piperidine adduct) and residual piperidine. A chloranil test may be used to determine if the removal of Fmoc by-products and residual pyridine is complete. The chloranil test solution is prepared by adding a drop of a saturated solution of chloranil in toluene to about 1 mL of acetone. The NMP washings may be tested by adding a drop of the washing to the chloranil test solution. A blue or violet color is a positive indication for the presence of secondary amine, indicating that Fmoc by-products and/or residual piperidine are still present. The NMP washing is repeated until the blue or violet color is no longer observed in the chloranil test.

The amino acid or fragment to be coupled is activated for reaction at its carboxy terminus and is intended to react with either an amino acid in solution or another peptide fragment intermediate either in solution or attached to a solid support. The amino-terminus of each amino acid or fragment should be protected with, as a non-limiting example, Fmoc, while, as noted above, the side chain may or may not be protected, h certain embodiments, the side-chains of Tyr (Y), Thr (T), Ser (S) and Asp (D) are protected with t-Bu, the side-chains of His (H), Gin (Q) and Asn (N) are protected with trt, the side-chains of Lys (K) and Trp (W) are protected with Boc, and the side chain of Cys (C) with Acm. As discussed above, the side-chain of Arg may or may not be protected with the Pbf protecting group.

Activation of the amino acid residue to be coupled to the resin bound amino acid is carried out as follows. The Fmoc-protected amino acid (about 3 eq), 1 -hydroxybenzotriazole hydrate (HOBT) (about 3 eq), and about 3 equivalents of

2-(H-benzotriazol-l-yl)-l,l,3,3,-tetramethyluronium tetrafluoroborate (TBTU) are dissolved in about ten volumes of NMP at room temperature. This solution is added to the reaction vessel and agitated via N2 bubbling for about one minute. About 4.5 eq of N,N-diisopropylethylamine (DIEA) in NMP is added to the vessel and the coupling is allowed to proceed for about one hour with agitation via N2 bubbling. Where an arginine residue to be coupled to the resin-bound peptide as a side-chain-nonprotected reagent, the coupling mixture further comprises about one equivalent of an additive such as, but not limited to, pyridineΗBr or other pyridinium halide such as, but not limited to pyridine»HCl, or pyridinium tosylate, butyl ammonium bromide, butyl ammonium chloride, or any other alkyl ammonium halide where the alkyl group comprises at least two carbon atoms.

Coupling completion may be monitored with a qualitative ninhydrin test as described below.

To check for completion of the reaction using the qualitative ninhydrin test, a 2-20 mg sample of resin is withdrawn and washed with methanol. Three drops of a 76% solution of phenol in ethanol, four or five drops of a 0.2 mM KCN solution in pyridine, and three drops of a 0.28 M solution of ninhydrin in ethanol are added to the sample and placed in a heat block at about 100°C for about 5 minutes. The sample is removed and immediately diluted with approximately 5 mL ethanol/water solution (95:5). A blue or violet color is a positive indication of the presence of free amines, indicating that the reaction is not yet complete. If a positive ninhydrin test is observed after one hour of reaction time, the coupling reaction is continued for an additional hour. If the positive ninhydrin test is observed after 2 hours of reaction time, the resin is drained, washed three times in approximately 10 volumes of NMP, and the coupling reaction is repeated using about one equivalent of activated amino acid. After the coupling is complete, the resin is drained and washed with about ten volumes of NMP, completing a coupling cycle. The coupling cycle is repeated for each of the subsequent amino acid residues of the peptide fragment intermediate. Following the final coupling cycle, the resin is washed with about ten volumes of NMP, and then about ten volumes of DCM. The resin-bound peptide fragment intermediate may be dried with a nitrogen purge.

Peptide fragment intermediates synthesized via solid phase synthesis techniques can be cleaved and isolated, for example, according to the following techniques: the peptide is cleaved from the resin using techniques well known to those skilled in the art, which include addition of a solution of TFA, water, and triisopropylsilane (90:5:5) and reacting for 2 to 3 hours.

After cleavage the solution is subjected to standard work-up procedures to isolate the peptide. Typically, the peptide in the cleavage mixture is precipitated by the addition of cold ether, and the precipitate collected by vacuum filtration.

In one embodiment of the present invention, the Fmoc-protected peptide fragment intermediate assembled as described above on a solid support, is cleaved from the solid support, isolated, and coupled with side-chain-unprotected arginine amide to provide an Fmoc-protected peptide intermediate fragment (Fmoc-Fragment No. lb),

Fmoc-R-F-C(Acm)-N-C(Acm)-N-G-R-C(O)NH2 (SEQ LD NO.: 4), comprising an Fmoc-protected amino terminus, and two side-chain-protected cysteine residues.

The peptide intermediates listed in Table 1, above, can be coupled together to produce IB-367 full-length linear peptide. Representative examples of such synthesis of full-length IB-367 from peptide fragment intermediates are presented in Examples 6 and 7, below, and are depicted schematically in FIGS. 2 and 3.

In one embodiment of the present invention, a process for the synthesis of the antimicrobial peptide IB-367 is designed that exemplifies the invention disclosed herein. In this embodiment of the present invention, three peptide fragment intermediates (wherein one of the peptide fragment intermediates is a modified amino acid monomer, i.e. amidated, side-chain-nonprotected arginine) are first assembled and then joined to form the desired peptide product, IB-367, as depicted in FIGS. 2-4.

As shown in FIG. 2, a nine-amino-acid-residue peptide fragment intermediate, designated Fragment 2, Fmoc-R-G-G-L-C(Acm)-Y(tBu)-C(Acm)-R(Pbf)-G-OH (SEQ ID NO.: 2), is synthesized using solid phase peptide synthesis methods. Fragment 2 comprises three glycine, two cysteine, and two arginine residues. Fragment 2 is designed so that the glycine is positioned at the carboxy-terminus of the peptide intermediate fragment, and which, therefore is attached to the solid support, and one of the arginine residues is positioned at the amino-terminal residue of Fragment 2, allowing that arginine residue to be coupled as a side-chain-nonprotected arginine residue. As indicated above, the two reactive side chains of cysteine are protected by the Acm side-chain protecting group.

The reactive guanidino side chain moiety of the second arginine residue of Fragment 2, i.e. the arginine adjacent to the carboxy-terminal glycine, must be stable to seven cycles of coupling and α-amino protecting group removal, to obviate the generation of unwanted byproducts, diminished yield and lower purity. The present inventors have deteπnined that, in the overall context of a large-scale process for the synthesis of IB-367, the increased cost associated with the use of a side-chain-protected arginine in this instance is more than balanced by the observed increases in yield and purity. Accordingly, the second arginine residue of Fragment 2 is preferably coupled to the carboxy-terminal glycine using a side-chain-protected arginine reagent, Pbf.

Therefore, in one embodiment of the invention, Fragment 2 is assembled by coupling the following Fmoc-α-amino-protected amino acids, in order, to the resin-bound carboxy terminal glycine residue: Pbf-protected arginine, Acm-protected cysteine, t-Bu-protected tyrosine, Acm-protected cysteine, leucine, glycine, glycine, and side-chain-nonprotected arginine. The resulting peptide fragment intermediate

Fmoc-R-G-G-L-C(Acm)-Y(tBu)-C(Acm)-R(Pbf)-G-OH (SEQ ID NO.: 2), is cleaved from the solid support with TFA, and precipitated, thereby providing Fmoc-α-amino-protected Fragment 2.

Similarly, Fragment 1 R-F-C(Acm)-N-C(Acm)-V-G (SEQ ID NO.: 3) is also synthesized using solid phase peptide synthesis methods as described in Section 5.3 above, and Example 6, below. As designed, Fragment 1 comprises a carboxy-terminal glycine residue and a single arginine residue positioned at the amino-terminus of this peptide fragment intermediate. Fragment 1 further comprises two cysteine residues with reactive side chains that are protected using an Acm protecting group. Accordingly, in this embodiment, Fragment 1 is assembled by coupling the following Fmoc-α-amino-protected amino acids, in order, to the resin-bound carboxy terminal glycine residue: valine, Acm-protected cysteine, valine, Acm-protected cysteine, phenylalanine, and side-chain-nonprotected arginine. The resulting peptide fragment intermediate Fmoc-R-F-C(Acm)-N-C(Acm)-N-G (SEQ ID NO.: 3) is cleaved from the solid support with TFA, and precipitated, thereby providing Fmoc-α-amino-protected Fragment 1.

Fragment 1 is coupled with amidated, side-chain-nonprotected arginine to provide Fragment lb, either Fmoc-α-amino-protected Fragment lb

(Fmoc-R-F-C(Acm)-N-C(Acm)-V-G-R ), e.g. according to the synthetic scheme depicted in FIG. 3, or Fragment lb lacking the Fmoc-α-amino protecting moiety (R-F-C(Acm)-N-C(Acm)-N-G-R ), e.g. according to the synthetic scheme depicted in FIG. 4.

In the synthetic scheme depicted in Figure 2, Fmoc-α-amino-protected Fragment 1 is cleaved from the solid phase support, precipitated and resuspended, generally using reagents and methods well known to those in the art and as disclosed above. The resuspended Fmoc-α-amino-protected Fragment 1 is coupled directly with amidated-arginine, which lacks a side-chain protecting group, according to solution phase peptide synthetic procedures well known to those in the art, but in the presence of an additive such as, but not limited to pyridineΗBr or other pyridinium halide such as, but not limited to pyridineΗCl, or pyridinium tosylate, butyl ammonium bromide, butyl ammonium chloride, or any other alkyl ammonium halide where the alkyl group comprises at least two carbon atoms, and/or reacting at low temperature (< 10°C) (see Section 6.3, below). Use of these conditions eliminates the reactions between TBTU and the side chain guanidino group. Previously it has been observed that HBTU or TBTU can form a Schiff base with free amines (Gausepohl H., Pieles, U., Frank, R.W., Smith, J.A., and Rivier, J.E., Eds., "Peptides: Chemistry and Biology," Twelfth American Peptide Symposium, Cambridge, MA, USA, June 16-21, 1991, LNm, 989 P, Escom Science Publishers B.V., Leiden, Netherlands, 1992). It has been observed that this same reaction can, unexpectedly, also occur with free guanidino groups. Upon completion of the coupling reaction, piperidine is added directly to the reaction to remove the Fmoc-α-amino-protecting group, yielding Fragment lb. The peptide product is precipitated from the solution by addition of acetonitrile at room temperature, solids are collected by vacuum filtration or centrifugation, washed with acetonitrile and dried to provide crude Fragment Fib, R-F-C(Acm)N-C(Acm)-V-G-R-C(O)NH2 (SEQ ID NO.: 4). In an alternative embodiment of the present invention, represented by the synthetic scheme depicted in FIG. 4, Fmoc-α-amino-protected Fragment 1 is cleaved from the solid phase support, and the Fmoc-α-amino-protecting group is removed from the peptide fragment intermediate prior to coupling with amidated-arginine, which lacks a side-chain protecting group, according to solution phase peptide synthetic procedures well know to those in the art, but in the presence of an additive such as, but not limited to pyridine»HBr or other pyridinium halide such as, but not limited to pyridineΗCl, or pyridinium tosylate, butyl ammonium bromide, butyl ammonium chloride, or any other alkyl ammonium halide where the alkyl group comprises at least two carbon atoms.

Peptide fragment intermediates Fragment lb NH2-R-F-C(Acm)-V-C(Acm)-V-G-R-C(O)NH2 (SEQ ID NO.: 4), and Fragment 2 Fmoc-R-G-G-L-C(Acm)-Y(tBu)-C(Acm)-R(Pbf)-G-OH (SEQ ID NO.: 2) are coupled using solution phase peptide synthesis materials and procedures well known to those in the art; deprotection of that the product formed provides the linear IB-367 peptide depicted in FIG. 1 and FIG. 2, which has the amino acid sequence NH2-RGGLCYCRGRFCVCVGR-C(O)NH2 (SEQ ID NO.: 1).

In a preferred embodiment, removal of the Acm groups protecting the cysteines and formation of the two, intrachain disulfide bonds of IB-367, which are depicted in FIGS, land 2, is conducted concurrently using a solution of iodine dissolved in methanol and acetic acid (see Kamber, B. et al, Helv Chim Acta (1980) 63:899-915). Alternatively, orthogonal protecting groups, which are removed under different conditions, may be used to protect pairs of cysteine residues to insure formation of those disulfide linkages depicted in FIGS. 1 and 2. Various methods for the formation of disulfide linkages are known in the art and include but are not limited to those described by Tarn, J. P. et al, Synthesis (1979) 955-957; Stewart, J. M. et al, Solid Phase Peptide Synthesis, 2d Ed. Pierce Chemical Company Rockford, II. (1984); Ahmed A. K. et al, J. Biol. Chem. (1975) 250: 8477-8482 and Pennington M. W. et al, Peptides 1990, E. Giralt et al, ESCOM Leiden, The Netherlands (1991) 164-166. An additional method is described by Kamber, B. et al, Helv Chim Acta (1980) 63:899-915. A method conducted on solid supports is described by Albericio t. J. Pept. Protein Res (1985) 26: 92-97.

Accordingly, the hybrid assembly of IB-367 disclosed herein is an improvement upon purely solid phase peptide synthesis routes, which the present inventors have found to be less efficient and more costly, in which: (1) the carboxy-terminal arginine residue of IB-367 would be the residue attached to the solid support; (2) the remaining amino acid residues added in sequential manner to the carboxy-terminal arginine; (3) the arginine side chains are protected; and (4) the carboxy-terminal arginine would be amidated when the chain was cleaved from the solid phase support. Moreover, the hybrid assembly IB-367 as disclosed herein is also an improvement upon purely solution phase peptide synthesis routes which require significantly more time and may involve multiple, time-consuming chromatographic separations of intermediates and the final peptide product.

In certain embodiments of the present invention, solid-phase synthesis of a peptide or peptide intermediate is designed in such a manner that the N-terminal residue of that peptide is arginine, permitting that residue to be added as side-chain-unprotected arginine. Attention to this aspect of the invention is important, particularly in a commercial process for peptide synthesis, since (a) it obviates the use of expensive, side-chain-protected arginine reagents; (b) it obviates acylation of the unprotected side-chain guanidine moiety of arginine in subsequent condensations where further amino acid residues are added to a growing chain, i.e., where arginine is not the last residue added; and (c) it obviates the need for any additional washing step intended to protonate the unprotected side-chain guanidine moiety of arginine before a subsequent coupling reaction, thereby saving time, and further reagent expense, which, again, are fundamental considerations in the design of commercially viable, large-scale peptide synthetic schemes for the production of antimicrobial peptides.

5.4 Precipitation of Peptides

The present invention is also directed to methods for precipitating peptides, such as but not limited to LB-367, from solutions that are typical of those obtained by pooling chromatographic fractions containing the peptide of interest, particularly those pools generated by pooling fractions collected in preparative HPLC. The method of the present invention comprises addition of one or more co-solvents to a peptide solution to precipitate the peptide. h one aspect of this embodiment, the concentration of peptide in the solvent, prior to the addition of a co-solvent or co-solvent mixture is at least about 5 mg/n L, at least about 10 mg/mL, at least about 15 mg/mL, at least about 20 mg/mL.

In certain aspects of this embodiment of the present invention, the precipitation is carried out at a temperature within the range of about 0°C to about 30 °C, of about 0°C to about 20 °C, of about 0°C to about 10°C. In a certain aspect of this embodiment, the precipitation is carried out at a temperature of about 5 °C.

In one embodiment, the present invention is directed toward a method for precipitating a peptide having the formula RGGLCYCRGRFCVCVGR (SEQ ID NO.: 1) which is dissolved in a solvent comprising ethanol and water. In certain aspects of this embodiment, the solvent comprises about 30% ethanol to about 70% ethanol, about 40% ethanol to about 60% ethanol. In another aspect of this embodiment the solvent comprises about 50%) ethanol. In still another aspect of this embodiment, the peptide solution further comprises 1 mM hydrochloric acid. Precipitation of the peptide is achieved by adding to a first volume of a solution comprising a peptide having the formula RGGLCYCRGRFCVCVGR (SEQ ID NO.: 1) dissolved in a solvent comprising ethanol and water, a second volume of a co-solvent provide a mixture, where the co-solvent is selected from the group consisting of tefrahydrofuran, methyl acetate, and combinations thereof. In certain aspects of this embodiment, the ratio of the second volume to the first volume is greater than one, is in the range of about two to about four. In a certain aspect of this embodiment, the ratio is about three. Ln one aspect of this embodiment, the co-solvent is tefrahydrofuran, and at least two volumes of co-solvent are added to peptide solution comprising a peptide having the formula RGGLCYCRGRFCVCVGR (SEQ ID NO.: 1) dissolved in a solvent comprising ethanol and water. In certain embodiments, at least three volumes of the co-solvent tetrahychofiiran are added to one volume of the peptide solution. In certain embodiments, at least four volumes of the co-solvent tetrahydrofuran are added to one volume of the peptide solution. In a certain embodiment, the co-solvent is tetrahydrofuran and about four volumes of co-solvent are added to the peptide solution comprising a peptide having the formula RGGLCYCRGRFCVCVGR (SEQ ID NO.: 1) dissolved in a solvent comprising ethanol and water. hi one aspect of this embodiment, the co-solvent is ethyl acetate, and at least one volume of co-solvent is added to peptide solution comprising a peptide having the formula RGGLCYCRGRFCVCVGR (SEQ ID NO.: 1) dissolved in a solvent comprising ethanol and water. In certain embodiments, at least two volumes of the co-solvent ethyl acetate are added to one volume of the peptide solution. In certain embodiments, at least three volumes of the co-solvent ethyl acetate are added to one volume of the peptide solution. In a certain embodiment, the co-solvent is ethyl acetate and about three volumes of co-solvent are added to the peptide solution comprising a peptide having the formula

RGGLCYCRGRFCVCVGR (SEQ ID NO.: 1) dissolved in a solvent comprising ethanol and water. in one aspect of this embodiment, the co-solvent is a 1 : 1 mixture of ethyl acetate and tetrahydrofuran, and at least two volumes of the co-solvent mixture are added to peptide solution comprising a peptide having the formula RGGLCYCRGRFCVCVGR (SEQ LD NO.: 1) dissolved in a solvent comprising ethanol and water. In certain embodiments, at least three volumes of the co-solvent mixture are added to one volume of the peptide solution. In certain embodiments, at least four volumes of the co-solvent mixture are added to one volume of the peptide solution. In a certain embodiment, the co-solvent is a 1:1 mixture of ethyl acetate and tetrahydrofuran, and about four volumes of the co-solvent mixture are added to the peptide solution comprising a peptide having the formula RGGLCYCRGRFCVCVGR (SEQ ID NO.: 1) dissolved in a solvent comprising ethanol

5 and water.

In one embodiment, the present invention is directed toward a method for precipitating a peptide having the formula RGGLCYCRGRFCVCVGR (SEQ ID NO.: 1) which is dissolved in a solvent comprising methanol and water. In certain aspects of this embodiment, the solvent comprises about 40% methanol to about 90% methanol, about

10 50%) methanol to about 90% methanol. In another aspect of this embodiment the solvent comprises about 65% methanol. In still another aspect of this embodiment, the peptide solution further comprises 1 mM hydrochloric acid. Precipitation of the peptide is achieved by adding to a first volume of a solution comprising a peptide having the formula RGGLCYCRGRFCVCVGR (SEQ ID NO.: 1) dissolved in a solvent comprising methanol

15 and water, a second volume of a co-solvent provide a mixture, where the co-solvent is selected from the group consisting of tetrahydrofuran, ethyl acetate, methyl acetate, methyl tert-butyl ether, and combinations thereof. In certain aspects of this embodiment, the ratio of the second volume to the first volume is greater than one, is in the range of about two to about four. In a certain aspect of this embodiment, the ratio is about three.

20 In one aspect of this embodiment, the co-solvent is tetrahydrofuran, and at least two volumes of co-solvent are added to peptide solution comprising a peptide having the formula RGGLCYCRGRFCVCVGR (SEQ ID NO.: 1) dissolved in a solvent comprising methanol and water, hi certain embodiments, at least three volumes of the co-solvent tetrahydrofuran are added to one volume of the peptide solution. In a certain embodiment,

25 the co-solvent is tetrahydrofuran and about three volumes of co-solvent are added to the peptide solution comprising a peptide having the formula RGGLCYCRGRFCVCVGR (SEQ ED NO.: 1) dissolved in a solvent comprising methanol and water. one aspect of this embodiment, the co-solvent is ethyl acetate, and at least one volume of co-solvent is added to peptide solution comprising a peptide having the formula

30 RGGLCYCRGRFCVCVGR (SEQ ID NO.: 1) dissolved in a solvent comprising methanol and water. In certain embodiments, at least two volumes of the co-solvent ethyl acetate are added to one volume of the peptide solution, hi certain embodiments, at least three volumes of the co-solvent ethyl acetate are added to one volume of the peptide solution. In a certain embodiment, the co-solvent is ethyl acetate and about three volumes of co-solvent are added

35 to the peptide solution comprising a peptide having the formula RGGLCYCRGRFCVCVGR (SEQ ID NO.: 1) dissolved in a solvent comprising methanol and water.

In another aspect of this embodiment, the co-solvent is methyl acetate, and at least one volume of co-solvent is added to peptide solution comprising a peptide having the formula RGGLCYCRGRFCVCVGR (SEQ ID NO.: 1) dissolved in a solvent comprising methanol and water. In certain embodiments, at least two volumes of the co-solvent methyl acetate are added to one volume of the peptide solution. In certain embodiments, at least three volumes of the co-solvent methyl acetate are added to one volume of the peptide solution. In a certain embodiment, the co-solvent is methyl acetate and about three volumes of co-solvent are added to the peptide solution comprising a peptide having the formula RGGLCYCRGRFCVCVGR (SEQ ID NO.: 1) dissolved in a solvent comprising methanol and water.

In still another aspect of this embodiment, the co-solvent is methyl tert-butyl ether, and at least one volume of co-solvent is added to peptide solution comprising a peptide having the foπnula RGGLCYCRGRFCVCVGR (SEQ ID NO.: 1) dissolved in a solvent comprising methanol and water. In certain embodiments, at least two volumes of the co-solvent methyl tert-butyl ether are added to one volume of the peptide solution, i certain embodiments, at least three volumes of the co-solvent methyl tert-butyl ether are added to one volume of the peptide solution. In a certain embodiment, the co-solvent is methyl tert-butyl ether and about three volumes of co-solvent are added to the peptide solution comprising a peptide having the formula RGGLCYCRGRFCVCVGR (SEQ ID NO.: 1) dissolved in a solvent comprising methanol and water.

In a further embodiment, the present invention is directed toward a method for precipitating a peptide having the formula RGGLCYCRGRFCVCVGR (SEQ ID NO.: 1) which is dissolved in a solvent comprising acetonitrile and water. In certain aspects of this embodiment, the solvent comprises about 20% acetonitrile to about 60% acetonitrile, about 30% acetonitrile to about 50% acetonitrile. In another aspect of this embodiment the solvent comprises about 40% acetonitrile. In still another aspect of this embodiment, the peptide solution further comprises 1 mM hydrochloric acid. Precipitation of the peptide is achieved by adding to a first volume of a solution comprising a peptide having the formula RGGLCYCRGRFCVCVGR (SEQ ID NO.: 1) dissolved in a solvent comprising acetonitrile and water, a second volume of a co-solvent to provide a mixture, where the co-solvent is tetrahydrofuran. In certain aspects of this embodiment, the ratio of the second volume to the first volume is greater than one, is in the range of about two to about four, ha a certain aspect of this embodiment, the ratio is about four. In one aspect of this embodiment, the co-solvent is tetrahydrofuran, and at least two volumes of co-solvent are added to peptide solution comprising a peptide having the formula RGGLCYCRGRFCVCVGR (SEQ ID NO.: 1) dissolved in a solvent comprising methanol and water. In certain embodiments, at least three volumes of the co-solvent tetrahydrofuran are added to one volume of the peptide solution. In certain embodiments, at least four volumes of the co-solvent tetrahydrofuran are added to one volume of the peptide solution. In a certain embodiment, the co-solvent is tetrahydrofuran and about four volumes of co-solvent are added to the peptide solution comprising a peptide having the formula RGGLCYCRGRFCVCVGR (SEQ LD NO.: 1) dissolved in a solvent comprising acetonitrile and water.

As is apparent to one of ordinary skill in the art, a peptide may be precipitated from solutions comprising ethanol, methanol, or acetonitrile, by the addition of the appropriate co-solvents disclosed herein. Moreover, it would also be apparent to one of ordinary skill in the art, that a peptide, particularly but not limited to IB-367, may be precipitated from solutions comprising mixtures of ethanol, methanol, and/or acetonitrile, by the addition of mixtures of co-solvents disclosed herein as being useful in the methods of the present invention for precipitating peptides.

5.5 Isolation of Precipitated Peptides by Filtration The present invention is also directed to methods for concentrating peptides from solution, which comprises collecting precipitated peptides, such as but not limited to EB-367. In certain aspects of this embodiment of the present invention, peptide precipitates are generated by adding at least one co-solvent to a solution comprising the peptide of interest, where that solution is typical of those obtained by pooling chromatographic fractions, particularly those pools generated by pooling fractions collected in preparative HPLC, and collecting the precipitated material comprising the peptide of interest. In certain aspects of this embodiment, a peptide such as, but not limited to IB-367, can be precipitated according to the methods disclosed in section 5.3, supra, and the precipitate can be collected and recovered as disclosed herein. hi one embodiment, the present invention is directed toward a method for concentrating a peptide having the formula RGGLCYCRGRFCVCVGR (SEQ ID NO.: 1), comprising providing a first volume of solution comprising said peptide dissolved in a solvent comprising methanol and water, precipitating the peptide by adding a second volume of a co-solvent to the solution to provide a mixture comprising a precipitate which comprises the peptide of interest. In certain embodiments, the co-solvent is selected from the group consisting of tetrahydrofuran, methyl acetate, and combinations thereof, and the ratio of the second volume to said first volume is greater than one. The precipitate generated in this manner is then collected. In one aspect of this embodiment, the precipitation is carried out at a temperature within the range of 0°C to about 30 °C, about 0°C to about 20°C, about 0°C to about 10°C, and, in one aspect, at about 5°C. In a certain aspect of this embodiment, the co-solvent is ethyl acetate and the ratio of co-solvent to peptide solution is about three. hi another embodiment, the present invention is directed toward a method for concentrating a peptide having the formula RGGLCYCRGRFCVCVGR (SEQ ID NO.: 1), comprising providing a first volume of solution comprising said peptide dissolved in a solvent comprising ethanol and water, precipitating the peptide by adding a second volume of a co-solvent to the solution to provide a mixture comprising a precipitate which comprises the peptide of interest. In certain embodiments, the co-solvent is selected from the group consisting of tetrahydrofuran, methyl acetate, and combinations thereof, and the ratio of the second volume to said first volume is greater than one. The precipitate generated in this manner is then collected. In one aspect of this embodiment, the precipitation is carried out a temperature within the range of 0°C to about 30°C, about 0°C to about 20 °C, about 0°C to about 10°C, and, in one aspect, at about 5°C. In a certain aspect of this embodiment, the co-solvent is methyl acetate and the ratio of co-solvent to peptide solution is about three.

In a further embodiment, the present invention is directed toward a method for concentrating a peptide having the formula RGGLCYCRGRFCVCVGR (SEQ ID NO.: 1), comprising providing a first volume of solution comprising said peptide dissolved in a solvent comprising ethanol and water, precipitating the peptide by adding a second volume of the co-solvent tetrahydrofuran to the solution to provide a mixture comprising a precipitate which comprises the peptide of interest, h certain embodiments, the ratio of the second volume to said first volume is greater than one. The precipitate generated in this manner is then collected, hi one aspect of this embodiment, the precipitation is carried out a temperature within the range of 0°C to about 30°C, about 0°C to about 20°C, about 0°C to about 10°C, and, in one aspect, at about 5°C. In a certain aspect of this embodiment, the ratio of co-solvent to peptide solution is about three. h another embodiment, the present invention is directed toward a method for concentrating a peptide having the formula RGGLCYCRGRFCVCVGR (SEQ ID NO.: 1), comprising providing a first volume of solution comprising said peptide dissolved in a solvent comprising ethanol and water, precipitating the peptide by adding a second volume of a co-solvent, which is a 1:1 mixture of methyl acetate and tetrahydrofuran, to the solution to provide a mixture comprising a precipitate which comprises the peptide of interest. In certain embodiments, the ratio of the second volume to said first volume is greater than one. The precipitate generated in this manner is then collected. In one aspect of this embodiment, the precipitation is carried out a temperature within the range of 0 ° C to about 30°C, about 0°C to about 20°C, about 0°C to about 10°C, and, in one aspect, at about 5°C. h a certain aspect of this embodiment, the ratio of co-solvent to peptide solution is about four.

Precipitated peptide can be collected by various methods well known in the art including, but not limited to filtration and centrifugation. In certain embodiments, precipitated peptide is collected by vacuum filtration through glass-fiber filters.

As is apparent to one of ordinary skill in the art, a peptide, including but not limited to IB-367, may be precipitated from solutions comprising ethanol, methanol, or acetonitrile, by the addition of the appropriate co-solvents disclosed herein, and that the precipitate can be recovered in high yield according to the methods and using the reagents disclosed herein. Moreover, it would also be apparent to one of ordinary skill in the art, that a peptide, including but not limited to IB-367, may be precipitated from solutions comprising mixtures of ethanol, methanol, and/or acetonitrile, by the addition of mixtures of co-solvents disclosed herein as being useful in the methods of the present invention for precipitating peptides and that the precipitate can be recovered in high yield according to the methods and using the reagents disclosed herein.

6. EXAMPLE: SOLID PHASE SYNTHESIS OF PEPTIDE FRAGMENT

INTERMEDIATES Presented below, in Sections 6.1 - 6.4 are examples of solid phase synthesis of peptide intermediates listed in Table 1.

6.1 Preparation of Fragment 2:

Fmoc-R-G-G-L-(Acm)C-(tBu)Y-(Acm)C-(Pbf)R-G (SEQ ID NO.: 2) Solid phase synthesis procedure:

A peptide reaction chamber is charged with 10.0 g

Fmoc-Gly-styrene:divinylbenzene copolymer resin comprising

4-hydroxymethylphenyloxymethyl anchoring groups (Peptides International, Louisville,

KY). The resin is washed with about 10 volumes (about 100 mL) NMP, and then conditioned in about 10 volumes (about 100 mL) of DCM with nitrogen agitation for about 15 minutes to swell the resin beads. The DCM is then drained from the chamber and the resin washed with about 10 volumes (about 100 mL) of NMP.

Fmoc (9-fluorenylmethyloxycarbonyl) removal from the terminal amine is accomplished using about 10 volumes (about 100 mL) of a 20%) solution of piperidine in NMP for about 5 minutes. The deprotected resin is then washed once with about 10 volumes (about 100 mL) of NMP. The resin is treated a second time with a 20% solution of piperidine in NMP (about 10 volumes) for about 15 minutes. This reaction can be extended for up to 60 minutes without negative consequences. The deprotected resin is then washed with about 10 volumes (about 100 mL) of NMP. The resin is then washed about 5 to 7 times with about 10 volumes of NMP to remove Fmoc by-products (i.e. dibenzofulvene and its piperidine adduct) and residual piperidine.

A chloranil test maybe used to determine if the removal of Fmoc by-products and residual piperidine is complete. The chloranil test solution is prepared by adding a drop of a saturated solution of chrloanil in toluene to about lmL of acetone. The NMP washings may be tested by adding a drop of the washing to the chloranil test solution. A blue or violet color is a positive indication for the presence of secondary amine, indicating that Fmoc by-products and/or residual piperidine are still present. The NMP washing is repeated until the blue or violet color is no longer observed.

Meanwhile, Fmoc- Arg (Pbf) the subsequent amino acid in the sequence, is activated for reaction at the carboxyl terminus, i.e., the resin-attached carboxy-terminal glycine residue. The Fmoc-protected amino acid (about 3eq), HOBT (about 3eq), and TBTU (about 3 eq) are dissolved in about 7 to 8 volumes (about 75 mL) of NMP at room temperature. The solution of activated amino acid is charged to the drained resin, and about 4.5 eq of DIEA in about 25 mL of NMP is added to the resin. The reaction is agitated with N2 bubbling for 1 hr. Coupling completion is monitored with the qualitative ninhydrin test. In the qualitative ninhydrin test, a 2-20 mg sample of the resin is withdrawn and washed with NMP and subsequently DCM or methanol. Three drops of a 76% solution of phenol in ethanol, six drops of a 0.2 mM KCN solution in pyridine, and three drops of a 0.28 M solution of ninhydrin in ethanol are added to the sample and the sample placed in a heating block at about 100°C for about 5 minutes. The sample is removed and immediately diluted with an ethanol/water solution (9:1). A blue or violet color is a positive indication of the presence of free amines, indicating that the reaction is not yet complete. If a positive ninhydrin test is observed after one hour reaction, the coupling reaction is continued for an additional hour. If a positive ninhydrin test occurs after 2 hours of coupling reaction, the resin is drained, washed tliree times in approximately 10 volumes of NMP, and the coupling reaction is repeated using about one equivalent of activated amino acid. After the coupling is complete, the resin is drained and washed with about ten volumes of NMP, completing a coupling cycle.

The cycle is repeated for subsequent amino acid residues of the peptide fragment using about 3 equivalents each of Fmoc-protected amino acids Cys (Acm), Tyr (tBu), Cys (Acm), Leu, and Gly. The last amino acid added is Arg. The Fmoc- side-chain-unprotected Arg (about 3 eq), HOBT (about 3 eq), and TBTU (about 2.9 eq) are dissolved in about 7 to 8 volumes (about 75 mL) of NMP at room temperature. To this is added about three equivalents of pyridinerHBr. The solution of activated amino acid is charged to the drained resin, and about 4.5 eq of DLEA is added to the resin. The reaction is agitated with N2 bubbling for 1 hour. The reaction is monitored by the qualitative ninhydrin procedure described above. Following the final coupling reaction, the resin is washed five times with about 10 volumes (about 100 mL) of NMP followed by four washes of about 10 volumes of a mixture of methanol/dichlormethane mixture (1 : 1 v/v). The resin is dried with a nitrogen purge.

The peptide is cleaved from the resin using about 100 mL of 90% TFA, 5% water and 5% triisopropylsilane (TIS) for about 2 hours. The solid resin is filtered out and washed twice with a minimal volume of TFA. The solutions are combined and the peptide product is precipitated with the addition of about 300 mL of cold ether. Solids are collected by vacuum filtration and washed with about 300 mL of cold ether. The product is dried under vacuum to provide Fmoc-R-G-G-L-C(Acm)-Y(tBu)-C(Acm)-R(Pbf)-G-OH of greater than 90%) purity as measured by the following HPLC, analytical procedure. Analytical Procedure: Column: YMC Propack column (5 μm, 4 mm x 25 cm); C8, reverse phase Flow rate: 0.8 mL/min Injection Volume: 10 μL Detection: UV at 214 run

Two Mobile phases: A = 0.1% TFA in water, B = 0.1% TFA, 90% Acetonitrile in water Time (minutes): 0 19 29 34 44 o/o A. 97 0 0 55 97 97

% B: 3 100 100 45 3 3

6.2 Preparation of Fragment 1 :

Fmoc-R-F-C(Acm)-V-C(Acm)-V-G-OH (SEQ ID NO.: 3) Solid phase synthesis procedure: A peptide reaction chamber was charged with 10.0 g Fmoc-Gly-styrene:divinylbenzene copolymer resin comprising 4-hydroxymethylphenyloxymethyl anchoring groups (Peptides International, Louisville,

5 KY). The resin is washed with about 10 volumes (about 100 mL) NMP, and then conditioned in about 10 volumes (about 100 mL) of DCM with nitrogen agitation for about 15 to 30 minutes to swell the resin beads. The DCM is then drained from the chamber and the resin washed with about 10 volumes (about 100 mL) of NMP.

Fmoc (9-fluorenylmethyloxycarbonyl) removal from the terminal amine is

10 accomplished using about 10 volumes (about 100 mL) of a 20%> solution of piperidine in NMP for about 5 minutes. The deprotected resin is then washed once with about 10 volumes (about 100 mL) of NMP. The resin is treated a second time with a 20%> solution of piperidine in NMP (about 10 volumes) for about 15 minutes. This reaction can be extended for up to 60 minutes without negative consequences. The deprotected resin is then washed

15 with about 10 volumes (about 100 mL) of NMP. The resin is then washed about 5 to 7 times with about 10 volumes of NMP to remove Fmoc by-products (i.e. dibenzofulvene and its piperidine adduct) and residual piperidine.

A chloranil test maybe used to determine if the removal of Fmoc by-products and residual piperidine is complete. The chloranil test solution is prepared by adding a drop of a

20 saturated solution of chrloranil in toluene to about lmL of acetone. The NMP washings may be tested by adding a drop of the washing to the chloranil test solution. A blue or violet color is a positive indication for the presence of secondary amine, indicating that Fmoc by-products and/or residual piperidine are still present. The NMP washing is repeated until the blue or violet color is no longer observed.

25 Meanwhile, Fmoc-valine the subsequent amino acid in the sequence is activated for reaction at the carboxyl terminus, i.e., the resin-attached carboxy-tenninal glycine residue. The Fmoc-protected amino acid (about 3eq), HOBT (about 3eq), and TBTU (about 2.9 eq) are dissolved in about 7 to 8 volumes (about 75 mL) of NMP at room temperature. The solution of activated acid is charged to the drained resin, and about 4.5 eq of DLEA in about

30 25 mL of NMP is added to the resin. The reaction is agitated with N2 bubbling for 1 hr. Coupling completion is monitored with the qualitative ninl ydrin test.

In the qualitative ninliydrin test, a 2-20 mg sample of the resin is withdrawn and washed with NMP and subsequently DCM or methanol. Tliree drops of a 76%> solution of phenol in ethanol, six drops of a 0.2 mM KCN solution in pyridine, and tliree drops of a

35 0.28 M solution of ninhydrin in ethanol are added to the sample and the sample placed in a heating block at about 100°C for about 5 minutes. The sample is removed and immediately diluted with an ethanol/water solution (95:5). A blue or violet color is a positive indication of the presence of free amines, indicating that the reaction is not yet complete. If a positive ninhydrin test is observed after one hour reaction, the coupling reaction is continued for an

5 additional hour. If a positive ninhydrin test occurs after 2 hours of coupling reaction, the resin is drained, washed three times with approximately 10 volumes of NMP, and the coupling reaction is repeated using about one equivalent of activated amino acid. After the coupling reaction is complete, the resin is drained and washed five times with about 3 volumes (about 30 mL) of NMP.

10 The cycle is repeated for subsequent amino acid residues of the peptide fragment using about 3 equivalents each of Fmoc-protected amino acids Cys (Acm), Val, Cys (Acm), Phe. The cycle is repeated for subsequent amino acid residues of the peptide fragment using about 3 equivalents each of Fmoc-protected amino acids Cys (Acm), Tyr (tBu), Cys (Acm), Leu, and Gly. The last amino acid added is Arg. The Fmoc- side-chain-unprotected Arg

15 (about 3 eq), HOBT (about 3 eq), and TBTU (about 2.9 eq) are dissolved in about 7 to 8 volumes (about 75 mL) of NMP at room temperature. To this is added about three equivalents of pyridine»HBr. The solution of activated amino acid is charged to the drained resin, and about 4.5 eq of DLEA is added to the resin. The reaction is agitated with N2 bubbling for 1 hour. The reaction is monitored by the qualitative ninhydrin procedure

20 described above. Following the final coupling reaction, the resin is washed five times with about 10 volumes (about 100 mL) of NMP followed by four washes of about 10 volumes of a mixture of methanol/dichloromethane mixture (1 : 1 v/v). The resin is dried with a nitrogen purge.

The peptide is cleaved from the resin using about 100 mL of 90% TFA, 5% water

25 and 5%> triisopropylsilane (TIS) for about 2 hours. The solid resin is filtered out and washed twice with a minimal volume of TFA. The solutions are combined and the product is precipitated with the addition of about 300 mL of cold ether. Solids are collected by vacuum filtration or centrifugation and washed with about 300 mL of cold ether. The product is dried under vacuum to provide Fmoc-R-F-C(Acm)-V-C(Acm)-V-G-OH of greater than

30 90 ) purity as measured by the following HPLC, analytical procedure.

Analytical Procedure: Column: YMC Propack C18 column (5 μm, 4.mm x 25 cm); C8, reverse phase Flow rate: 0.8 mL/min Injection Volume: 10 μL

35 Detection: UV at 214 nm Two Mobile phases: A = 0.1% TFA in water; B = 0.1% TFA, 90% Acetonitrile in water

Time (minutes): 0 25 30

% A: 97 0 97 % B: 03 100 03

6.3 Hybrid Synthetic Method for the Preparation of Fmoc-Fragment lb:

Fmoc-R-F-C(Acm)-V-C(Acm)-V-G-R-C(O)NH2 (SEQ ID NO.: 4) Solid phase synthesis procedure:

A peptide reaction chamber is charged with 10.0 g Fmoc-Gly-styrene:divinylbenzene copolymer resin comprising

4-hydroxymethylphenyloxymethyl anchoring groups. The resin is washed with about 10 volumes (about 100 mL) NMP, and then conditioned in about 10 volumes (about 100 L) of DCM with nitrogen agitation for about 15 minutes to swell the resin beads. The DCM is then drained from the chamber and the resin washed with about 10 volumes (about 100 mL) ofNMP.

Fmoc (9-fluorenylmethyloxycarbonyl) removal from the terminal amine is accomplished using about 10 volumes (about 100 mL) of a 20% solution of piperidine in NMP for about 5 minutes. The deprotected resin is then washed once with about 10 volumes (about 100 mL) of NMP. The resin is treated a second time with a 20% solution of piperidine in NMP (about 10 volumes) for about 15 minutes. This reaction can be extended for up to 60 minutes without negative consequences. The deprotected resin is then washed with about 10 volumes (about 100 mL) of NMP. The resin is then washed about 5 to 7 times with about 10 volumes of NMP to remove Fmoc by-products (i.e. dibenzofulvene and its piperidine adduct) and residual piperidine.

A chloranil test maybe used to determine if the removal of Fmoc by-products and residual piperidine is complete. The chloranil test solution is prepared by adding a drop of a saturated solution of chloranil in toluene to about 1 mL of acetone. The NMP washings may be tested by adding a drop of the washing to the chloranil test solution. A blue or violet color is a positive indication for the presence of secondary amine, indicating that Fmoc by-products and/or residual piperidine are still present. The NMP washing is repeated until the blue or violet color is no longer observed.

Meanwhile, Fmoc-valine, the subsequent amino acid in the sequence, is activated for reaction at the carboxyl terminus, i.e., the resin-attached carboxy-terminal glycine residue. The Fmoc-protected amino acid (about 3eq), HOBT (about 3eq), and TBTU (about 3 eq) are dissolved in about 7 to 8 volumes (about 75 mL) of NMP at room temperature. The solution of activated amino acid is charged to the drained resin, and about 4.5 eq of DIEA in about 25 mL of NMP is added to the resin. The reaction is agitated with N2 bubbling for 1 hr. Coupling completion is monitored with the qualitative ninhydrin test.

In the qualitative ninhydrin test, a 2-20 mg sample of the resin is withdrawn and washed with NMP and subsequently with DCM or methanol. Tliree drops of a 76%> solution of phenol in ethanol, six drops of a 0.2 mM KCN solution in pyridine, and three drops of a 0.28 M solution of ninhydrin in ethanol are added to the sample and the sample placed in a heating block at about 100°C for about 5 minutes. The sample is removed and immediately diluted with an ethanol/water solution (95:5). A blue or violet color is a positive indication of the presence of free amines, indicating that the reaction is not yet complete. If a positive ninhydrin test is observed after one hour reaction, the coupling reaction is continued for an additional hour. If the positive ninhydrin test occurs after 2 hours of the coupling reaction, the resin is drained, washed three times in approximately 10 volumes of NMP, and the coupling reaction is repeated using about one equivalent of activated amino acid. After the coupling reaction is complete, the resin is drained and washed five times with about 3 volumes (about 30 mL) of NMP.

The cycle is repeated for subsequent amino acid residues of the peptide fragment using about 3 equivalents each of Fmoc-protected amino acids Cys (Acm), Val, Cys (Acm), and Phe. The last amino acid added is Arg. The Fmoc- side-chain-unprotected Arg (about 3 eq), HOBT (about 3 eq), and TBTU (about 2.9 eq) are dissolved in about 7 to 8 volumes (about 75 mL) of NMP at room temperature. To this is added one equivalent of pyridine»HBr. The solution of activated amino acid is charged to the drained resin, and about 4.5 eq of DIEA is added to the resin. The reaction is agitated with N2 bubbling for 1 hour. The reaction is monitored by the qualitative ninhydrin procedure described above. Following the final coupling reaction, the resin is washed five times with about 10 volumes (about 100 mL) of NMP followed by four washes of about 10 volumes of a mixture of methanol/dichloromethane mixture (1 : 1 v/v). The resin is dried with a nitrogen purge. The peptide is cleaved from the resin using about 100 mL of 90%> TFA, 5% water and 5% triisopropylsilane (TIS) for about 2 hours. Product is precipitated with the addition of about 100 mL of cold ether, and the resultant slurry stirred at room temperature for about 30 minutes. Solids are collected by vacuum filtration and washed with about 100 mL of cold ether. The product is dried under vacuum to provide Fmoc-R-F-C(Acm)-V-C(Acm)-V-G-OH. Solution phase synthesis procedure:

Dried product, Fmoc-R-F-C(Acm)-V-C(Acm)-V-G-OH (Fragment 1), (0.1 lmmol), is suspended in a 5mL NMP solution of side-chain-nonprotected arginine-amide (about 2 eq), TBTU (about 1 eq), and pyridineΗBr (about 2 eq). Once the solids are dissolved to provide a clear solution, DLEA (about 5.5 eq; about 4 eq to neutralize HCl from arginine amide) is added in one portion. The solution coupling is monitored by electrospray mass spectroscopy using an ion trap, according to methods well known in the art. The solution coupling of arginine amide to yield Fmoc-R-F-C(Acm)-V-C(Acm)-V-G-R-(O)NH2 (Fmoc-Fragment lb) is complete within 10 minutes, at 0-10°C. Lower temperature, or the use of pyridineΗBr, greatly reduces possible side reactions, which include: reaction between fragment Fmoc-Fl carboxy reacting (acylating) with an unprotected sidechain moiety, R; the formation of a Schiff base between TBTU Schiff and an unprotected side chain, R, of fragment FmocFl ; formation of a Schiff base between TBTU and the N-terminus of R-NH2; formation of Schiff base between TBTU and an unprotected side chain amino moiety, R-NH2; and formation of a Schiff base between TBTU and a reactive sidechain, R, of fragment Fmoclb). The temperature dependence of the formation of side products, which primarily comprise Fmoc-Fragment 1 -Schiff base adducts, was examined and the following results were obtained:

Reaction % Desired Product % Side Product Temperature ( Fmoc-Fragment IB) ( Fmoc-Fragment lB-Schiff base)

Room 60 % 35%

Temperature

0°C to l0°C > 95 % Trace

Cleavage of the Fmoc α- amino protecting group is accomplished by adding piperidine directly to the coupling reaction to a final concentration of about 20% (v/v), providing Fragment lb of greater than about 85% purity as measured by direct-infusion mass spectroscopy and by the analytical HPLC procedure described in Sections 6.1 and 6.2, above.

6.4 Hybrid Synthetic Method for the Preparation of Fragment lb:

R-F-C(Acm)-V-C(Acm)-V-G-R-C(O)NH2 (SEQ ID NO.: 4)

Solid phase synthesis procedure:

A peptide reaction chamber is charged with 10.0 g Fmoc-Gly-styrene:divinylbenzene coplymer resin comprising

4-hydroxymethylphenyloxymethyl anchoring groups. The resin is washed with about 10 volumes (about 100 mL) NMP, and then conditioned in about 10 volumes (about 100 L) of DCM with nitrogen agitation for about 15 minutes to swell the resin beads. The DCM is then drained from the chamber and the resin is washed with about 10 volumes (about 100 mL) of NMP. Fmoc (9-fluorenylmethyloxycarbonyl) removal from the terminal amine is accomplished using about 10 volumes (about 100 mL) of a 20%> solution of piperidine in NMP for about 5 minutes. The deprotected resin is then washed once with about 10 volumes (about 100 mL) of NMP. The resin is treated a second time with a 20% solution of piperidine in NMP (about 10 volumes) for about 15 minutes. This reaction can be extended for up to 60 minutes without negative consequences. The deprotected resin is then washed with about 10 volumes (about 100 mL) of NMP. The resin is then washed about 5 to 7 times with about 10 volumes of NMP to remove Fmoc by-products (i.e. dibenzofulvene and its piperidine adduct) and residual piperidine.

A chloranil test may be used to determine if the removal of Fmoc by-products and residual piperidine is complete. The chloranil test solution is prepared by adding a drop of a saturated solution of chrloanil in toluene to about 1 mL of acetone. The NMP washings may be tested by adding a drop of the washing to the chloranil test solution. A blue or violet color is a positive indication for the presence of secondary amine, indicating that Fmoc by-products and/or residual piperidine are still present. The NMP washing is repeated until the blue or violet color is no longer observed.

Meanwhile, Fmoc-valine the subsequent amino acid in the sequence is activated for reaction at the carboxyl terminus, i.e., the resin-attached carboxy-terminal glycine residue. The Fmoc-protected amino acid (about 3eq), HOBT (about 3eq), and TBTU (about 3 eq) are dissolved in about 7 to 8 volumes (about 75 mL) of NMP at room temperature. The solution of activated acid is charged to the drained resin, and about 4.5 eq of DIEA in about 25 mL of NMP is added to the resin. The reaction is agitated with N2 bubbling for 1 hr. Coupling completion is monitored with the qualitative ninhydrin test.

In the qualitative ninhydrin test, a 2-20 mg sample of the resin is withdrawn and washed with NMP and subsequently DCM or methanol. Three drops of a 76%> solution of phenol in ethanol, six drops of a 0.2 mM KCN solution in pyridine, and three drops of a 0.28 M solution of ninhydrin in ethanol are added to the sample and the sample is placed in a heating block at about 100°C for about 5 minutes. The sample is removed and immediately diluted with an ethanol/water solution (95:5). A blue or violet color is a positive indication of the presence of free amines, indicating that the reaction is not yet complete. If a positive ninhydrin test is observed after one hour of reaction time, the coupling reaction is continued for an additional hour. If the positive ninhydrin test occurs after 2 hours of coupling reaction, the resin is drained, washed three times in approximately 10 volumes of NMP, and the coupling reaction is repeated using about one equivalent of activated amino acid. After the coupling reaction is complete, the resin is drained and washed five times with about 3 volumes (about 30 mL) of NMP.

The cycle is repeated for subsequent amino acid residues of the peptide fragment using about 3 equivalents each of Fmoc-protected amino acids Cys (Amc), Val, Cys (Amc), and Phe. The last amino acid added is Arg. The Fmoc- side-chain-unprotected Arg (about 3 eq), HOBT (about 3 eq), and TBTU (about 2.9 eq) are dissolved in about 7 to 8 volumes (about 75 mL) of NMP at room temperature. To this is added about three equivalents of pyridineΗBr. The solution of activated amino acid is charged to the drained resin, and about 4.5 eq of DIEA is added to the resin. The reaction is agitated with N2 bubbling for 1 hour. The reaction is monitored by the qualitative ninhydrin procedure described above. Following the final coupling reaction, the N-terminal Fmoc group is removed. Fmoc (9-fluorenylmethyloxycarbonyl) removal from the terminal amine is accomplished using about 10 volumes (about 100 mL) of a 20% solution of piperidine in NMP for about 5 minutes. The deprotected resin is then washed once with about 10 volumes (about 100 mL) of NMP. The resin is treated a second time with a 20% solution of piperidine in NMP (about 10 volumes) for about 15 minutes. The deprotected resin is then washed with about 10 volumes (about 100 mL) of NMP. The resin is then washed about 5-7 times with about 10 volumes of NMP, to remove Fmoc by-products (i.e., dibenzofulvene and its piperidine adduct) and residual piperidine, followed by four washes of about 10 volumes of a mixture of methanol/dichlormethane mixture (1:1 v/v). The resin is dried with a nitrogen purge. The peptide is cleaved from the resin using about 100 mL of 90%> TFA, 5%> water and 5% triisopropylsilane (TIS) for about 2 hours. The solid resin is washed twice with a minimal volume of TFA. The solutions are combined and the product is precipitated with the addition of about 300 mL of cold ether. Solids are collected by vacuum filtration and washed with about 300 mL of cold ether. The product is dried under vacuum to provide NH2-R-F-C(Acm)-V-C(Acm)-V-G-OH.

Solution phase synthesis procedure:

Dried product, R-F-C(Acm)-V-C(Acm)-V-G-OH (Fragment 1), (1 eq), is suspended in NMP comprising side-chain-nonprotected arginine-amide (about 1.1 eq), TBTU (about 1 eq), and HOBt (about 1 eq). Once the solids are dissolved to provide a clear solution, the solution is cooled to 0-10 °C and DIEA (about 5.5 eq; about 4 eq to neutralize HCl from arginine amide), is added. The reaction is kept at 0-10°C. The solution coupling is monitored by electrospray mass spectroscopy using an ion trap. Solution coupling of arginine amide to yield R-F-C(Acm)-V-C(Acm)-V-G-R-(O)NH2 (Fragment lb) was complete within 10 minutes, yielding Fragment lb of greater than 85% purity as measured by electrospray mass spectroscopy and by the analytical HPLC procedure described in Sections 6.1 and 6.2, above.

7. Example: Preparation of IB-367 (NH2-RGGLCYCRGRFCVCVGR-C(O)NH2) (SEQ ID NO.: 1) by Solution-Phase Coupling of Fragment 2

(Fmoc-R-G-G-L-C(Acm)-Y(tBu)-C(Acm)-R(Pbf)-G-OH) (SEQ ID NO.: 2) and Fragment lb (NH2-R-F-C(Acm)-V-C(Acm)-V-G-R-C(O)NH2) (SEQ ID NO.: 4)

Solution phase peptide synthesis procedure:

A reaction vessel is charged with peptide fragment intermediate designated Fragment 2 (about 1.1 eq) (as synthesized in Section 6.1, above), and peptide fragment intermediate designated Fragment lb (about 1 eq) (as synthesized in Section 6.3, above) and HOBT (about 1.1 eq). The solids are dissolved in DMSO (about 10 ml/g of Fragment 2). After the solids are dissolved, NMP is added so that the mixture is only 10% DMSO. The solution is cooled to 0-10°C. DLEA is added (about 6.5 eq) followed by TBTU (about 1.1 eq). The reaction mixture is stirred at 0-5 °C for 15 minutes, after which cooling is removed, and then stirred an additional 60 minutes. The peptide product is precipitated from the solution by addition of acetonitrile, solids are collected by vacuum filtration or by centrifugation, washed with acetonitrile and dried to provide crude, Fmoc-linear IB-367. The N-terminal Fmoc group on the linear IB-367 peptide is removed by dissolving the isolated Fmoc-linear IB-367 in a 30% solution of piperidine in NMP and reacting for about 60 minutes. Alternatively, the Fmoc group on the linear IB-367 can be removed before isolation by adding enough piperidine to the reaction mixture until piperidine is 30%> of the total volume and reacting for 60 minutes. Either cold acetonitrile or cold ether is added to precipitate the deprotected linear IB-367. Solids are collected by vacuum filtration or centrifugation, washed with cold acetonitrile or cold ether and dried to provide crude, linear IB-367.

The reaction is monitored either by electrospray mass spectroscopy or by the analytical HPLC method below. Analytical Procedure: Column: Zorbax stable bond column (5 μm, 4.5mm x 25 cm); C8, reverse phase Flow rate: 0.8 mL/min Injection Volume: 10 μL Detection: UV at 214 nm Two Mobile phases: A = 0.15% TFA in water; B = 0.12% TFA, 70% Acetonitrile in water

Time (minutes): 0 2 3 38 33 38

%A: 80 80 70 55 80 80 %B: 20 20 30 45 20 20

The Acm groups are removed from Linear IB-367 while simultaneously forming the correct disulfide bonds to give crude IB-367 (FIGS. 1 and 2). Linear IB-367 (0.5 g) added to an acetic acid and water mixture (2:1 v/v) to which 2.5 eq of HCl added. The solution is stirred for about an hour to dissolve. Separately I2 (6.25 eq) is dissolved in about 3 mL methanol. After dissolution an equal volume of acetic acid is added to the iodine solution. The iodine solution is added to the peptide solution and is stirred at room temperature. The reaction is momtored by mass spectroscopy or HPLC and is completed in about 2-5 hours. When the reaction is complete, a solution of about 0.6 M ascorbic acid is added until the solution becomes light yellow or colorless. Cold ether is added to precipitate the peptide and the precipitate is washed twice with cold ether. The precipitate is isolated by centrifugation or filtration and dried under vacuum.

In certain embodiments of the present invention, peptide fragment intermediates can be purified by counter current distribution or chromatography methods using any silica or non-silica based column packing including but not limited to poly-styrene, poly-acrylic or other polymer based packings. Columns packed with such material can be run in low, medium or high pressure chromatography. Additionally various solvents may be used to purify the peptide. This may include, but is not limited to, water mixtures of methanol, ethanol, isopropanol, or acetonitrile (or their mixtures). These solvents may also include modifiers such as ammonium acetate, trifluoroacetic acid, or hydrochloric acid or any other modifiers known to those skilled in the art.

Preparative HPLC fractions are monitored by analytical HPLC for purity, and suitable fractions are pooled together. Peptides may be concentrated after purification using methods well known in the art, including, but not limited lyophilization, spray drying, and precipitation. Although conditions for lyophilization and spray drying are well known to those skilled in the art, for most peptides, it is difficult to find conditions in which to precipitate the final product. Nevertheless, precipitation has the advantage in that it is a rapid, reproducible procedure that does not involve the costly equipment and maintenance required for lyophilization and spray drying methods. Accordingly, in preferred embodiments, suitable procedures are used in which the peptide is precipitated directly from the pool of collected fractions, either through addition of another solvent and/or change in temperature. Moreover, in such preferred embodiments, co-solvents used are acceptable for phaπnaceutical use, and include, but not limited to, methanol, ethanol, methyl acetate, ethyl acetate, tetrahydrofuran, methyl t-butyl ether, acetone and mixtures thereof, h addition, the precipitate must be readily filterable.

Alternatively, the pool of collected fractions can be concentrated either in peptide content or solvent content (by methods such as ultrafiltration, evaporation, distillation etc.) before precipitation. The precipitate is isolated by filtration and dried under vacuum.

8. Example: Precipitation of IB-367

LB-367 synthesized according to the methods disclosed above, can be precipitated from pools generated by combining peptide-containing fractions from, e.g., preparative HPLC, by the addition of co-solvents according to the methods and using the materials provided below.

8.1 Example: Precipitation of IB-367 from Ethanol solution

Co-solvents were screened to identify those capable of precipitating IB-367 from solutions typical of preparative chromatographic pools. One mL aliquots of each co-solvent were added to 1 mL of a 20 mg/mL (dry weight) solution of IB-367 (50%> ethanol containing 1 mM HCl) until the solution was 1 part peptide solution and 4 parts co-solvents. Co-solvents explored were: acetone, methyl acetate, ethyl acetate, tefrahydrofuran, methyl tetrahydrofuran, methyl tert-butyl ether. If a precipitate formed, a portion was removed and centrifuged and the supernatant was assayed by HPLC, according to the method described above in Example 7, to determine the residual IB-367 and the percentage of IB-367 precipitated was calculated. The results of this study are summarized in Table 3:

TABLE 3 lmL 20 mg/ IB-367 mL in 50% Ethanol-lmM HCl

Co-solvent 1:1 2:1 3:1 4:1

The mixtures that gave the best recovery (>95%ι) were: (a) 1 part peptide/ethanol solution: 4 parts THF; and (b) 1 part peptide/ethanol solution:3 parts methyl acetate.

8.2 Example: Precipitation of IB-367 from Methanol solution Co-solvents were screened to identify those capable of precipitating IB-367 from solutions typical of preparative chromatographic pools. One mL aliquots of each co-solvent were added to 1 mL of a 20 mg/mL solution of JJB-367 (65 % methanol containing 1 mM HCl) until the solution was 1 part peptide solution and 4 parts co-solvent(s). Co-solvents explored were: acetone, methyl acetate, ethyl acetate, tetrahydrofuran, methyl tetrahydrofuran, methyl tert-butyl ether. If a precipitate formed, a portion of the sample was removed and centrifuged, and the supernatant assayed by HPLC according to the method described above in Example 7, to determine the residual IB-367 and the percentage of IB-367 precipitated was calculated. The results of this study are summarized in Table 4:

10

15

20

25

The mixtures that gave the best recovery (>95%) were: (a) 1 part peptide/methanol solution: 3 parts THF; (b) 1 part peptide/methanol solution:3 parts ethyl acetate; (c) 1 part peptide/methanol solution:3 parts methyl acetate; and (d) 1 part peptide/methanol solution: ~n 3 parts methyl tert-butyl ether.

8.3 Example: Precipitation of IB-367 from acetonitrile solution

Co-solvents were screened to identify those capable of precipitating IB-367 from solutions typical of preparative chromatographic pools. One mL aliquots of each co-solvent ~<- were added to 1 mL of a 20 mg/mL solution of LB-367 (40% acetonitrile containing 1 mM HCl) until the solution was 1 part peptide solution and 4 parts co-solvent(s). Co-solvents that were explored were: acetone, methyl acetate, ethyl acetate, and methyl tert-butyl ether. If a precipitate formed, a portion of the sample was removed and centrifuged and the supernatant assayed by HPLC according to the method described above in Example 7, to determine the residual IB-367 and the percentage of IB-367 precipitated was calculated. The results of this study are summarized in Table 5:

The mixture that gave the best recovery (>90%) was: 1 part peptide/acetonitrile solution: 4 parts THF. 5 9. Example: Isolation of Precipitated IB-367 by Filtration

Precipitated IB-367 can be collected in good yield using the materials and methods disclosed below.

9.1 Example: Room Temperature Filtration of IB-367 Precipitated from 70% Methanol -ImM HCl with Ethyl Acetate (3 Volumes)

Co-solvent was added dropwise to the peptide solution with stirring at ambient temperature. Stirring was maintained for approximately 10 minutes. The solution was then allowed to stand, without stirring, at ambient temperature for approximately 20 minutes or longer. The precipitate was then separated using vacuum filtration onto glass fiber filters (Whatman GF/D, coarse, 2.7 μ) (Whatman Inc., Clifton, NJ). The collected precipitate was washed with lmL or 2 mL of the appropriate, corresponding precipitant solvent/co-solvent or solvent/co-solvent mixture. The recovered precipitate and the filter upon which it was collected were extracted with 1 mL of 20% acetonitrile- 1 mM HCl. The extract was clarified and recovered peptide assayed by HPLC according to the method described above in Example 7.

9.2 Example: Low Temperature Filtration of IB-367 Precipitated from 70% Methanol -ImM HCl with Ethyl Acetate (3 Volumes)

Co-solvent was added dropwise to the peptide solution with stirring on ice. Stirring was maintained for approximately 10 minutes. The solution was then allowed to stand, without stirring, on ice, for approximately 20 minutes or longer. The precipitate was then separated using vacuum filtration onto glass fiber filters (Whatman GF/D, coarse, 2.7 μ) (Whatman Inc., Clifton, NJ). The collected precipitate was washed with 1 mL or 2 mL of the appropriate, corresponding precipitant solvent/co-solvent or solvent/co-solvent mixture. The recovered precipitate and the filter upon which it was collected were extracted with 1 mL of 20%) acetonitrile- 1 mM HCl. The extract was clarified and recovered peptide assayed by HPLC according to the method described above in Example 7.

0

5

9.3 Example: Low Temperature Filtration of IB-367 Precipitated Q from 50% Methanol with Methyl Acetate (3 Volumes)

Co-solvent was added dropwise to the peptide solution with stirring on ice. Stirring was maintained for approximately 10 minutes. The solution was then allowed to stand, without stirring, on ice, for approximately 20 minutes or longer. The precipitate was then separated using vacuum filtration onto glass fiber filters (Whatman GF/D, coarse, 2.7 μ) (Whatman Inc., Clifton, NJ). The collected precipitate was washed with 1 mL or 2 mL of the appropriate, corresponding precipitant solvent/co-solvent or solvent/co-solvent mixture. The recovered precipitate and the filter upon which it was collected were extracted with 1 mL of 20%> acetonitrile- 1 mM HCl. The extract was clarified and recovered peptide assayed by HPLC according to the method described above in Example 7. 0

TABLE 8

Recovery by Filtration of IB-367 Precipitated on ice from 50% Ethanol-lmM HCl by 3 volumes of Methyl acetate

Initial Concentration of D3-367 (mg/mL) and Fraction Tested % Recovery 5

9.4 Example: Low Temperature Filtration of IB-367 Precipitated from 50% Ethanol with 4 Volumes of Tetrahydrofuran Co-solvent was added dropwise to the peptide solution with stirring either on ice.

Stirring was maintained for approximately 10 minutes. The solution was then allowed to stand, without stirring, on ice, for approximately 20 minutes or longer. The precipitate was then separated using vacuum filtration onto glass fiber filters (Whatman GF/D, coarse, 2.7 μ) (Whatman Inc., Clifton, NJ). The collected precipitate was washed with 1 mL or 2 mL of the appropriate, corresponding precipitant solvent/co-solvent or solvent/co-solvent mixture. The recovered precipitate and the filter upon which it was collected were extracted with 1 mL of 20% acetonitrile- 1 mM HCl. The extract was clarified and recovered peptide assayed by HPLC according to the method described above in Example 7.

9.5 Example: Low Temperature Filtration of IB-367 Precipitated from 50% Ethanol with 4 Volumes of a mixture (1:1) of Methyl Acetate and Tetrahydrofuran

The co-solvent mixture was added dropwise to the peptide solution with stirring on ice. Stirring was maintained for approximately 10 minutes. The solution was then allowed to stand, without stirring, on ice, for approximately 20 minutes or longer. The precipitate was then separated using vacuum filtration onto glass fiber filters (Whatman GF/D, coarse, 2.7 μ) (Whatman hie, Clifton, NJ). The collected precipitate was washed with 1 mL or 2 mL of the appropriate, corresponding precipitant solvent/co-solvent mixture. The recovered precipitate and the filter upon which it was collected were extracted with 1 mL of 20% acetonitrile- 1 mM HCl. The extract was clarified and recovered peptide assayed by HPLC according to the method described above in Example 7.

The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

Various publications are cited herein, the disclosures of which are incorporated herein in their entireties.

Claims

What is claimed is:
1. A method for the synthesis of a peptide having the formula RGGLCYCRGRFCVCVGX (X = Arg-amide) (SEQ LD NO.: 1) comprising:
(a) reacting a side-chain-protected peptide of the formula: Fmoc-ZGGLCYCRG-COOH (Z = side-chain-nonprotected Arg) (SEQ ID NO.: 2) with a side-chain-protected peptide of the formula:
NH2-ZFCVCVGZ-C(O)NH2 (Z = side-chain-nonprotected Arg) (SEQ ID NO.: 4) to yield a side-chain-protected peptide of the formula:
Fmoc-ZGGLCYCRGZFCVCVGZ-C(O)NH2 (Z = side-chain-nonprotected Arg) (SEQ ID NO.: 5);
(b) deprotecting the amino terminus of the side-chain-protected peptide produced in (a); and (c) deprotecting the side chains of the side-chain-protected peptide of (b) to yield a peptide of the formula:
RGGLCYCRGRFCVCVGX (X = Arg-amide) (SEQ ID NO.: 1).
2. The method of claim 1 , wherein the side-chain protected peptide of formula NH2-ZFCVCVGZ-C(O)NH2 (Z - side-chain-nonprotected Arg) (SEQ LD NO.: 4) is formed by a method comprising:
(a) reacting a side-chain-protected peptide of the formula: Fmoc-ZFCVCVG-COOH (Z = side-chain-nonprotected Arg) (SEQ ID NO.: 3) with arginine-amide to yield a side-chain-protected peptide of the formula: Fmoc-ZFCVCVGZ-C(O)NH2 (Z - side-chain-nonprotected Arg ) (SEQ ID NO.:
4); and
(b) deprotecting the amino terminus of the side-chain-protected peptide produced in (a), to yield a peptide of the formula
NH2-ZFCVCVGZ-C(O)NH2 (Z = side-chain-nonprotected Arg) (SEQ ID NO.: 4).
3. The method of claim 1 , wherein the side-chain-protected peptide of the formula:
Fmoc-ZGGLCYCRG-COOH (Z = side-chain-nonprotected Arg) (SEQ JD NO.: 2) is synthesized by solid phase peptide synthesis.
4. The method of claim 2, wherein the side-chain-protected peptide of the formula:
Fmoc-ZFCVCVG-COOH (Z = side-chain-nonprotected Arg) (SEQ ID NO.: 3) is synthesized by solid phase peptide synthesis.
5. A method for precipitation of a peptide having the formula RGGLCYCRGRFCVCVGR (SEQ LD NO.: 1), comprising:
(a) providing a first volume of solution comprising said peptide dissolved in a solvent comprising ethanol and water; and (b) adding a second volume of a co-solvent to said solution to provide a mixture, wherein the co-solvent is selected from the group consisting of tefrahydrofuran, methyl acetate, and combinations thereof, and wherein the ratio of said second volume to said first volume is greater than one.
6. The method of claim 5, wherein the solvent comprises about 30 % ethanol to about 70% ethanol.
7. The method of claim 5, wherein the solvent comprises about 40 % ethanol to about 60%) ethanol.
8. The method of claim 5, wherein the solvent comprises about 50 % ethanol.
9. The method of claim 5, wherein the ratio is in the range of about 2 to about 4.
10. The method of claim 5, wherein the ratio is about three.
11. The method of claim 5, wherein the concentration of the peptide in the solution is at least about 5 mg/mL.
12. The method of claim 5 , wherein the concentration of the peptide in the solution is at least about 10 mg/mL.
13. The method of claim 5, wherein the concentration of the peptide in the solution is at least about 15 mg/mL.
- so -
14. The method of claim 5, wherein the concentration of the peptide in the solution is at least about 20 mg/mL.
15. The method of claim 5, wherein the precipitating is carried out at a temperature within the range of about 0°C to about 30°C.
16. The method of claim 5, wherein the precipitating is carried out at a temperature within the range of about 0°C to about 20 °C.
17. The method of claim 5, wherein the precipitating is carried out at a temperature within the range of about 0°C to about 10 °C.
18. The method of claim 5, wherein the precipitating is carried out at a temperature of about 5°C.
19. The method of claim 5, wherein the co-solvent is tetrahydrofuran, and wherein the ratio is at least about three.
20. The method of claim 5, wherein the co-solvent is tetrahydrofuran, and wherein the ratio is at least about four.
21. The method of claim 5, wherein the co-solvent is methyl acetate, and wherein the ratio is at least about two.
22. The method of claim 5, wherein the co-solvent is methyl acetate, and wherein the ratio is at least about tliree.
23. The method of claim 5, wherein the solution comprises about 1 mM hydrochloric acid.
24. A method for precipitation of a peptide having the formula RGGLCYCRGRFCVCVGR (SEQ ID NO.: 1), comprising:
(a) providing a first volume of solution comprising said peptide dissolved in a solvent comprising methanol and water; and (b) adding a second volume of a co-solvent to said solution to provide a mixture, wherein the co-solvent is selected from the group consisting of tetrahydrofuran, ethyl acetate, methyl acetate, methyl tert-butyl ether, and combinations thereof, and wherein the ratio of said second volume to said first volume is greater than one.
25. The method of claim 24, wherein the solvent comprises about 40 % ethanol to about 90%) methanol.
26. The method of claim 24, wherein the solvent comprises about 50 % methanol to about 80% methanol.
27. The method of claim 24, wherein the solvent comprises about 65 % methanol.
28. The method of claim 24, wherein the ratio is in the range of about 2 to about 3.
29. The method of claim 24, wherein the ratio is about two.
29. The method of claim 24, wherein the ratio is about three.
31. The method of claim 24, wherein the concenfration of the peptide in the solution is at least about 5 mg/mL.
32. The method of claim 24, wherein the concentration of the peptide in the solution is at least about 10 mg/mL.
33. The method of claim 24, wherein the concentration of the peptide in the solution is at least about 15 mg/mL.
34. The method of claim 24, wherein the concentration of the peptide in the solution is at least about 20 mg/mL.
35. The method of claim 24, wherein the precipitating is carried out at a temperature within the range of about 0°C to about 30°C.
36. The method of claim 24, wherein the precipitating is carried out at a temperature within the range of about 0 ° C to about 20 ° C .
37. The method of claim 24, wherein the precipitating is carried out at a temperature within the range of about 0°C to about 10°C.
38. The method of claim 24, wherein the precipitating is carried out at a temperature of about 5°C.
39. The method of claim 24, wherein the co-solvent is tetrahydrofuran, and wherein the ratio is at least about two.
40. The method of claim 24, wherein the co-solvent is tetrahydrofuran, and wherein the ratio is at least about tliree.
41. The method of claim 24, wherein the co-solvent is ethyl acetate, and wherein the ratio is at least about two.
42. The method of claim 24, wherein the co-solvent is ethyl acetate, and wherein the ratio is at least about tliree.
43. The method of claim 24, wherein the co-solvent is methyl acetate, and wherein the ratio is at least about two.
44. The method of claim 24, wherein the co-solvent is methyl acetate, and wherein the ratio is at least about three.
45. The method of claim 24, wherein the co-solvent is methyl tert-butyl ether, and wherein the ratio is at least about two.
46. The method of claim 24, wherein the co-solvent is methyl tert-butyl ether, and wherein the ratio is at least about three.
47. The method of claim 24, wherein the solution comprises about ImM hydrochloric acid.
48. A method for precipitation of a peptide having the formula RGGLCYCRGRFCVCVGR (SEQ ID NO.: 1), comprising:
(a) providing a first volume of solution comprising said peptide dissolved in a solvent comprising acetonitrile and water; and
(b) adding a second volume of tetrahydrofuran to said solution to provide a mixture, wherein the ratio of said second volume to said first volume is greater than two .
49. The method of claim 48, wherein the solvent comprises about 20 % acetonitrile to about 60% acetonitrile.
50. The method of claim 48, wherein the solvent comprises about 30 %> acetonitrile to about 50% acetonitrile.
51. The method of claim 48, wherein the solvent comprises about 40 %> acetonitrile.
52. The method of claim 48, wherein the ratio is in the range of about 3 to about 4.
53. The method of claim 48, wherein the ratio is about four.
54. The method of claim 48, wherein the concentration of the peptide in the solution is at least about 5 mg/mL.
55. The method of claim 48, wherein the concentration of the peptide in the solution is at least about 10 mg/mL.
56. The method of claim 48, wherein the concentration of the peptide in the solution is at least about 15 mg/mL.
57. The method of claim 48, wherein the concentration of the peptide in the solution is at least about 20 mg/mL.
58. The method of claim 48, wherein the precipitating is carried out at a temperature within the range of about 0°C to about 30°C.
59. The method of claim 48, wherein the precipitating is carried out at a temperature within the range of about 0°C to about 20°C.
60. The method of claim 48, wherein the precipitating is carried out at a temperature within the range of about 0°C to about 10°C.
61. The method of claim 48, wherein the precipitating is carried out at a temperature of about 5°C.
62. The method of claim 48, wherein the solution comprises about 1 mM hydrochloric acid.
63. A method for concentrating a peptide having the formula RGGLCYCRGRFCVCVGR (SEQ ID NO.: 1), comprising:
(a) providing a first volume of solution comprising said peptide dissolved in a solvent comprising methanol and water;
(b) precipitating said peptide by adding a second volume of a co-solvent to said solution to provide a mixture comprising a precipitate, wherein the precipitate comprises said peptide, wherein the co-solvent is selected from the group consisting of tefrahydrofuran, methyl acetate, and combinations thereof, and wherein the ratio of said second volume to said first volume is greater than one;
(c) collecting the precipitate.
64. The method of claim 63, wherein the precipitating is carried out at a temperature within the range of from about 0°C to about 30°C.
65. The method of claim 63, wherein the precipitating is carried out at a temperature within the range of from about 0°C to about 20°C.
66. The method of claim 63, wherein the precipitating is carried out at a temperature within the range of from about 0°C to about 10°C.
67. The method of claim 63, wherein the precipitating is carried out at a temperature of about 5 ° C.
68. The method of claim 63, wherein the co-solvent is ethyl acetate and said ratio is about three.
69. A method for concentrating a peptide having the formula
RGGLCYCRGRFCVCVGR (SEQ ID NO.: 1), comprising:
(a) providing a first volume of solution comprising said peptide dissolved in a solvent comprising ethanol and water;
(b) precipitating said peptide by adding a second volume of a co-solvent to said solution to provide a mixture comprising a precipitate, wherein the precipitate comprises said peptide, wherein the co-solvent is selected from the group consisting of tefrahydrofuran, methyl acetate, and combinations thereof, and wherein the ratio of said second volume to said first volume is greater than one; (c) collecting the precipitate.
70. The method of claim 69, wherein the precipitating is carried out at a temperature within the range of from about 0°C to about 30 °C.
71. The method of claim 69, wherein the precipitating is carried out at a temperature within the range of from about 0°C to about 20°C.
72. The method of claim 69, wherein the precipitating is carried out at a temperature within the range of from about 0°C to about 10°C.
73. The method of claim 69, wherein the precipitating is carried out at a temperature of about 5 °C.
74. The method of claim 69, wherein the co-solvent is methyl acetate and said ratio is about 3.
75. The method of claim 69, wherein the co-solvent is tetrahydrofuran and said ratio is about 3.
76. The method of claim 69, wherein the co-solvent is a 1 :1 mixture of methyl acetate and tetrahydrofuran and said ratio is about 4.
77. The method of claim 63 or 69, wherein said collecting comprises a method selected from the group consisting of filtration and centrifugation.
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US20100222268A1 (en) * 2007-07-23 2010-09-02 Amp-Therapeutics Gmbh & Co. Kg Antibiotic peptides
CN102336813A (en) * 2011-07-01 2012-02-01 上海苏豪逸明制药有限公司 Preparation method for synthesizing proteidin with solid phase polypeptide
US20140080999A1 (en) * 2011-05-31 2014-03-20 Ajinomoto Co., Inc. Method for producing peptide
US8686113B2 (en) 2009-01-29 2014-04-01 Amp-Therapeutics Gmbh Antibiotic peptides
WO2016127902A1 (en) * 2015-02-13 2016-08-18 普生股份有限公司 Use of composition containing p-113 peptide in preparing cosmetics having moisturizing function

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Cited By (11)

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US7696311B2 (en) 2003-12-31 2010-04-13 Roche Colorado Corporation Process and systems for recovery of peptides
US20100222268A1 (en) * 2007-07-23 2010-09-02 Amp-Therapeutics Gmbh & Co. Kg Antibiotic peptides
US9060513B2 (en) * 2007-07-23 2015-06-23 Amp-Therapeutics Gmbh Antibiotic peptides
US20150315241A1 (en) * 2007-07-23 2015-11-05 Amp-Therapeutics Gmbh Antibiotic Peptides
US8686113B2 (en) 2009-01-29 2014-04-01 Amp-Therapeutics Gmbh Antibiotic peptides
US9353148B2 (en) * 2011-05-31 2016-05-31 Ajinomoto Co., Inc. Method for producing peptide
US20140080999A1 (en) * 2011-05-31 2014-03-20 Ajinomoto Co., Inc. Method for producing peptide
CN102336813B (en) * 2011-07-01 2016-01-06 上海苏豪逸明制药有限公司 One kind of solid phase peptide synthesis method for the preparation of protein lysozyme
CN102336813A (en) * 2011-07-01 2012-02-01 上海苏豪逸明制药有限公司 Preparation method for synthesizing proteidin with solid phase polypeptide
WO2016127902A1 (en) * 2015-02-13 2016-08-18 普生股份有限公司 Use of composition containing p-113 peptide in preparing cosmetics having moisturizing function
CN105982825A (en) * 2015-02-13 2016-10-05 普生股份有限公司 Use of composition in production of cosmetics with moisturizing function

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