Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues – Cyclic peptides
Reexamination Certificate
2001-09-06
2004-04-06
Russel, Jeffrey E. (Department: 1654)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
Cyclic peptides
C435S071300, C530S323000, C530S344000
Reexamination Certificate
active
06716962
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to methods of extractively purifying and/or isolating antibiotics and/or antimicrobial agents. More particularly, the present invention relates to an inexpensive and efficient extractive method for purifying or isolating lipopeptide antibiotics.
BACKGROUND OF THE INVENTION
An important class of antibiotics that inhibit gram-positive bacteria are the acidic lipopeptide antibiotics. Generally, acidic lipopeptide antibiotics consist of either a cyclic peptide core or a cyclic depsipeptide core acylated with a lipophilic fragment and have an isoelectric point of less than about pH 7.0. The lipophilic fragment, typically an unsaturated fatty acid, may be of varying length. Frequently, the antibiotic activity of lipopeptide antibiotics is related to the length of the lipophilic fragment.
Examples of acidic lipopeptide antibiotics include, but are not limited to, laspartomycin (Umezawa et al., U.S. Pat. No. 3,639,582; Naganawa et al., 1968,
J Antibiot.,
21, 55; Naganawa et al., 1970,
J Antibiot.,
23, 423), zaomycin (Kuroya, 1960,
Antibiotics Ann.,
194; Kuroya, JP 8150), crystallomycin (Gauze et al, 1957,
Antibiotiki,
2, 9), aspartocin (Shay et al., 1960,
Antibiotics Annual,
194; Hausman et al., 1964,
Antimicrob. Ag. Chemother.,
352; Hausman et al., 1969,
J Antibiot.,
22, 207; Martin et al., 1960,
J Am. Chem. Soc.,
2079), amphomycin (Bodanszky et. al., 1973,
J Am. Chem. Soc.,
95, 2352), glumamycin (Fujino et al., 1965,
Bull. Chem. Soc. Jap.,
38, 515), brevistin (Shoji et al., 1976,
J Antibiotics,
29, 380), cerexin A (Shoji et al., 1976,
J Antibiotics,
29, 1268), cerexin B (Shoji et al., 1976,
J Antibiotics,
29, 1275) Antibiotic A-30912 (Hoehn et al., U.S. Pat. No. 5,039,789), Antibiotic A-1437 (Hammann et al., EP 0 629 636 B1; Lattrell et al., U.S. Pat. No. 5,629,288), Antibiotic A-54145 (Fukada et al., U.S. Pat. No. 5,039,789; Boeck et al., 1990,
J Antibiotics,
43, 587), Antibiotic A-21978C (Debono et al., 1988,
J. Antibiotics,
41, 1093) and tsushimycin (Shoji et. al., 1968,
J Antibiot.,
21, 439). See also Berdy, “CRC Handbook of Antibiotic Compounds,” Volume IV, Part 1, pages 313-327, CRC Press, Boca Raton, Fla., (1980); Korzybinski et al., “Antibiotics-Origin Nature and Properties,” Vol. 1, Pergamon Press, pp. 397-401 and 404-408, New York, N.Y. (1967).
The acidic lipopeptide antibiotics are typically active against Gram-positive microbes and constitute important therapeutics in the treatment of infections caused by these bacteria. However, conventional procedures used to isolate and purify acidic lipopeptide antibiotics from fermentation broths involve a number of extraction and chromatography steps, which are time consuming, labor intensive and expensive to carry out on a commercial scale. Thus, there is a need in the art for improved methods of isolating and/or purifying acidic lipopeptide antibiotics.
SUMMARY OF THE INVENTION
These and other needs are addressed by the present invention, which provides a rapid and inexpensive extractive method for purifying large quantities of lipopeptide antibiotics in high yield. Quite surprisingly, it has been discovered that acidic lipopeptide antibiotics such as laspartomycin, amphomycin and aspartocin, which have cyclic peptide nuclei and Antibiotic A-21978C, which has a cyclic depsipeptide nucleus, may under conditions of pH that are above the isoelectric point of the lipopeptide antibiotic and in the presence of divalent metal cations such as Ca
+2
, be directly extracted into water-immiscible organic solvents such as 1-butanol.
While not intending to be bound by any particular theory of operation, it is believed that acidic lipopeptide antibiotics form chelates with divalent metal cations such as, for example, Ca
+2
that are stable under basic conditions and that are soluble in water-immiscible organic solvents such as 1-butanol. Under acidic conditions, the chelates are disrupted and acidic lipopeptide antibiotics may be extracted into aqueous solution at basic or near neutral pH. Thus, according to one embodiment of the invention, the method comprises contacting an aqueous composition comprising a lipopeptide antibiotic and a divalent metal cation and having a pH above the isoelectric point of the lipopeptide antibiotic with a water immiscible organic solvent, thereby extracting the lipopeptide antibiotic into organic solvent. Preferably, the pH of the aqueous composition is neutral or basic.
The lipopeptide antibiotic may then be extracted into aqueous solution from organic solvent by acidifying the organic solvent at a pH below the isoelectric point of the lipopeptide antibiotic followed by contacting the acidified organic solvent with an aqueous solution, which is at neutral or basic pH. The lipopeptide antibiotic, which now behaves like a conventional carboxylic acid, may be extracted back into organic solvent by acidifying the aqueous solution and extracting the aqueous solution with organic solvent. At this point, if necessary, the lipopeptide antibiotic may be further purified using extractive or chromatographic purification.
The extractive isolation methods of the current invention may be used to isolate and/or purify acidic lipopeptide antibiotics directly from fermentation or culture broths, either before or after removal of cells and/or cell debris and/or insoluble matter. Alternatively, the extractive isolation methods of the invention may be used in combination with conventional isolation and purification techniques. For example, an acidic lipopeptide antibiotic may be first precipitated from fermentation or culture medium and the antibiotic isolated and/or purified from the precipitate according to the extractive isolation methods of the invention. The methods of the current invention may be used to advantageously isolate and/or purify synthetic acidic lipopeptide antibiotics and/or derivatives such as the synthetic lipopeptide derivatives described, for example, in Debono et al., 1988,
J Antibiotics,
41, 1093 and Lattrell et al., U.S. Pat. No. 5,629,288.
Thus, when used either alone or in combination with standard extraction and chromatographic techniques, the extractive methods of the invention allow for the isolation of acidic lipopeptide antibiotics in high yield and high purity with fewer steps than are required by conventional methods.
DETAILED DESCRIPTION OF THE INVENTION
Reference will now be made in detail to preferred embodiments of the invention. While the invention will be described in conjunction with preferred embodiments, it should be understood that it is not intended to limit the invention to these preferred embodiments. To the contrary, it is intended to cover alternatives, modifications, and equivalents as may be included within the spirit and scope of the invention as defined by the appended claims.
The extractive purification methods of the invention may be used to rapidly and inexpensively isolate and/or purify virtually any acidic lipopeptide antibiotic. As used herein, “acidic lipopeptide antibiotic” refers to those antibiotics that have a cyclic peptidic nucleus with a lipophilic fragment such as a fatty acid chain attached thereto and an isoelectric point of less than about pH 7.0. The cyclic peptidic nucleus may be a cyclic peptide or a cyclic depsipeptide. The lipophilic fragment may be attached directly to the nucleus or through a linker, which is typically peptidic.
Acidic lipopeptide antibiotics may be natural products, synthetic or semisynthetic. Acidic lipopeptide antibiotics may also be derivatives of natural or synthetic acidic lipopeptide antibiotics, provided that the derivatives include carboxyl groups which permit extractive isolation according to the principles of the current invention.
Exemplary acidic lipopeptide antibiotics which can be advantageously isolated and/or purified according to the extractive methods of the invention include, but are not limited to, laspartomycin (Umezawa et al., U.S. Pat. No. 3,639,582; Naganawa et al., 1968,
J Antibiot.,
21, 55; Naganawa et al, 1970,
Borders Donald B
Fantini Amedeo A
Francis Noreen D
Micrologix Biotech Inc.
Russel Jeffrey E.
Seed Intellectual Property Law Group PLLC
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