EP0850316A1

EP0850316A1 - Cryptophycins from aberrant biosynthesis

Info

Publication number: EP0850316A1
Application number: EP96932217A
Authority: EP
Inventors: Richard E. Moore; Thomas K. Hemscheidt
Original assignee: University of Hawaii; Wayne State University
Current assignee: University of Hawaii; Wayne State University
Priority date: 1995-08-30
Filing date: 1996-08-30
Publication date: 1998-07-01
Also published as: EP0850057A1; JP2000501067A; BR9610214A; WO1997007798A1; AU7109096A; AU709828B2; EP0850316A4; CA2246117A1; WO1997008334A1; CA2230540A1; AU6904896A; JP2000507805A; EP0850057A4

Abstract

Methods for producing cryptophycin compounds by bacterial fermentation are disclosed, together with novel compositions produced thereby.

Description

Cryptophycins from Aberrant Biosynthesis

This invention was made in part with U.S. Government support under Grant

No. CHE-9024748 from the National Science Foundation. Accordingly, the U.S. Government may have certain rights in this invention.

Technical Field The present invention relates generally to the production of cryptophycin compounds, and, more particularly, to the production of such compounds by the use of bacterial fermentation.

Background of the Invention The cryptophycins are a group of structurally related compounds originally isolated from cyanobacteria (blue-green algae). This family of compounds has been found to exhibit a broad spectrum of antineoplastic activity similar to presently-used antineoplastic agents, such as vinblastine, taxol, and adriamycin.

Of the previously known cryptophycins, Cryptophycin 1 (also termed Cryptophycin A) is the major cytotoxin produced by certain Nostoc species of cyanobacteria. It has shown excellent activity against drug-sensitive and drug- resistant solid tumors. This cyclic depsipeptide consists of four units; two hydroxy acid units and two amino acid units. The stoichiometry of the two hydroxy acid units is as follows: (5S, 6S, 7R, SR)-7,8-epoxy-5-hydroxy-6-methyl-8-phenyl- 2-octenoic acid (Unit A) and (25)-2-hydroxy-4-methylvaleric acid (Unit D; leucic acid). The stoichiometry of the two amino acid units is as follows: (2/?)-3-(3-chloro-4-methoxyphenyl)alanine (Unit B) and (2 -3-amino- 2-methylpropionic acid (Unit C). The units are connected in an ABCD sequence. In addition to the cryptophycin isolates of Nostoc spp. , other members of the cryptophycin group have been produced by chemically modifying the isolates.

Moreover, additional cryptophycins have been produced by total synthesis. Many of these non-bacterial cryptophycins demonstrate activities and in vivo stability which could prove beneficial in therapeutic applications. However, fermentation is far more cost-effective for producing a compound in a commercial setting than the use of partial or total chemical synthesis. Unfortunately, the variety of cryptophycins obtained via native bacterial fermentation is limited.

Thus, it is considered desirable to provide a means for obtaining both known and novel cryptophycin compounds by bacterial fermentation.

It is also considered desirable to produce novel cryptophycin compounds via fermentation which demonstrate improved properties of stability and activity when compared to compounds obtained via native bacterial fermentation.

Disclosure of the Invention

The present invention provides a method for producing cryptophycin compounds as metabolites by the controlled use of metabolic substrates in bacterial fermentation. In one aspect, the present invention comprises culturing bacteria capable of producing cryptophycin compounds in the presence of a substimted amino acid under conditions sufficient to produce the desired cryptophycin compound.

Also provided in accordance with an aspect of the present invention are novel cryptophycin compounds produced by culturing bacteria capable of producing cryptophycins in the presence of a substimted amino acid under conditions sufficient to produce the desired cryptophycin compound. Typically, such cryptophycin compounds will have a stable macrolide and possess substituent groups which have been shown to provide beneficial activities.

Brief Description of the Drawings

Figure 1 provides a general structure of selected cryptophycin compounds of the present invention and the four different acid units, two hydroxy acid groups (A

& D) and two amino acid groups (B & C), of which cryptophycins are characteristically comprised. Figure 1 also provides a numbering system for the hydroxy acid units A and D and two amino acid units B and C in selected embodiments; and Figure 2 provides a schematic representation of the fragment ionic species obtained when selected cryptophycin compounds are subjected to electron impact mass spectrometric analysis. Detailed Description of the Invention

The present invention provides a method for producing cryptophycin compounds as metabolites by the controlled use of metabolic substrates in bacterial fermentation. In one aspect, the present invention comprises culturing bacteria capable of producing cryptophycins in the presence of a substimted amino acid under conditions sufficient to produce the desired cryptophycin compound.

The present invention thus provides a means for overcoming the problem of producing only a limited number of cryptophycins from native bacterial fermentation. By the use of the present invention one can obtain pre-determined cryptophycins produced by fermentation of a bacteria capable of producing a cryptophycin compound. Typically, such pre-determined cryptophycin compounds will have a stable macrolide and possess substituent groups which have been shown to provide beneficial activities.

Also provided in accordance with the present invention are novel cryptophycin compounds produced by culturing bacteria capable of producing cryptophycins in the presence of a substimted amino acid under conditions sufficient to produce the desired cryptophycin compound.

Selection of Desirable Cryptophycin Compounds It has been found that, in addition to Cryptophycin 1, over 20 different cryptophycin compounds can be isolated from one strain of cyanobacteria (Nostoc spp.). Moreover, numerous additional cryptophycin compounds have been produced by total chemical synthesis or semi-synthesis. Many of these compounds have been subjected to structure-activity relationship (SAR) studies in order to determine the strucmral feamres of the class of compounds which are most important in providing the beneficial therapeutic properties.

These SAR studies have indicated that in vivo activity is largely premised upon an intact macrolide. Also significant are the epoxide group, the chloro group in Unit B and me methyl group in Unit C. The SAR studies further showed that the lack of the methyl group in Unit C, as in Cryptophycin 21, enhances the lability of the ester bond between Units C and D (thereby decreasing the probability that the macrolide will remain intact in vivo prior to reaching the tumor site). This ester bond was in excess of 100 times more labile in Cryptophycin 21 compared to Cryptophycin 1.

Cryptophycin 21 differs from Cryptophycin 1 only in that it does not contain the methyl group in Unit C. Although Cryptophycin 1 and Cryptophycin 21 show essentially the same cytotoxicity in vitro, Cryptophycin 21 was found to be inactive in vivo. This suggested that the C-D ester bond of the macrolide was being broken prior to the compound reaching the tumor site.

Thus, based upon the results of such studies, desirable feamres of cryptophycin compounds can be engineered into the metabolites produced in bacterial fermentation by controlled selection of metabolic substrates.

Bacteria Capable of Producing Cryptophycin Compounds

The cryptophycins are a group of structurally related compounds originally isolated from cyanobacteria (blue-green algae). Cryptophycin 1 (also termed Cryptophycin A) is the major cytotoxin produced by certain Nostoc species of cyanobacteria. As disclosed above, a limited number of other cryptophycin compounds can be obtained by native bacterial fermentation.

The morphological characteristics of the Nostoc sp. of cyanobacteria, as described in U.S. Patent No. 4,946,835, are well known, and the basis for the identification of a Nostoc sp. is described in detail in J. Gen. Micro. , 111:1-61 (1979).

The present invention provides that a bacteria capable of producing a cryptophycin compound, for example a Nostoc sp., may be cultured under appropriate conditions and that novel cryptophycin metabolites, as well as previously disclosed cryptophycin metabolites, may be isolated from this culture. In an embodiment of the present invention, the Nostoc sp. strain designated GSV 224 is the strain which is cultivated and from which are isolated both previously known and novel cryptophycins.

In one aspect, the method of the present invention is directed to any strain of the Nostoc sp. and preferably to the Nostoc sp. GSV 224 strain to produce non- naturally occurring cryptophycin compounds. To that end, the GSV 224 strain of Nostoc sp. was deposited on October 7, 1993 pursuant to the Budapest Treaty on the International Deposit of Microorganisms for the Purposes of Patent Procedure with the Patent Culture Depository of the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852 U.S.A. under ATCC Accession No. 55483. Other strains of Nostoc sp., in particular strain MB 5357 previously deposited by Merck and Co. under ATCC Accession No. 53789, are strains contemplated to be utilized to practice the present invention.

As is the case with other organisms, the characteristics of Nostoc sp. are subject to variation. For example, recombinants, variants, or mutants of the specified strains may be obtained by treatment with various known physical and chemical mutagens, such as ultraviolet ray, X-rays, gamma rays, and N-memyl- N'-nitro-N-nitrosoguanidine. All natural and induced variants, mutants, and recombinants of the specified strains which retain the characteristic of producing a cryptophycin compound are intended to be within the scope of the claimed invention.

The cryptophycin compounds of the present invention can be prepared by culturing a strain of Nostoc sp. under submerged aerobic conditions in a suitable culture medium until substantial antibiotic activity is produced. Other culture techniques, such as surface growth on solidified media, can also be used to produce these compounds. The culture medium used to grow the specified strains can include any of one of many nitrogen and carbon sources and inorganic salts that are known to those of ordinary skill in the art. Economy in production, optimal yields, and ease of product isolation are factors to consider when choosing the carbon and nitrogen sources to be used. Among the nutrient inorganic salts which can be incorporated in the culture media are the customary soluble salts capable of yielding iron, potassium, sodium, magnesium, calcium, ammonium, chloride, carbonate, phosphate, sulfate, nitrate, and like ions. Essential trace elements which are necessary for the growth and development of the organisms should also be included in the culture medium. Such trace elements commonly occur as impurities in other constituents of the medium in amounts sufficient to meet the growth requirements of the organisms. It may be desirable to add small amounts (i.e. 0.2mL/L) of an antifoam agent such as polypropylene glycol (M.W. about 2000) to large scale cultivation media if foaming becomes a problem.

For production of substantial quantities of the cryptophycin compounds, submerged aerobic cultivation in tanks can be used. Small quantities may be obtained by shake-flask culmre. Because of the time lag in metabolite production commonly associated with inoculation of large tanks with the organisms, it is preferable to use a vegetative inoculum. The vegetative inoculum is prepared by inoculating a small volume of culture medium with fragments of the vegetative trichome or heterocyst-containing form of the organism to obtain a fresh, actively growing culmre of the organism. The vegetative inoculum is then transferred to a larger tank. The medium used for the vegetative inoculum can be the same as that used for larger cultivations or fermentation, but other media can also be used.

The bacterial organisms may be grown at temperatures between about 20 °C and 30 °C and at an incident illumination intensity of from about 100 to about 200 μmol photons m²Sec^"* (photosynthetically active radiation).

As is customary in aerobic submerged culture processes of this type, carbon dioxide gas is introduced into the culmre by addition to the sterile air stream bubbled through the culmre medium. For efficient production of the cryptophycin compounds, the proportion of carbon dioxide should be about 1% (at 24°C and one atmosphere of pressure).

The prior art, specifically U.S. Patent No. 4,946,835, the contents of which are hereby incorporated by reference, provides methods of cultivating Nostoc sp.

Cryptophycin compound production can be followed during the cultivation by testing samples of the broth against organisms known to be sensitive to these antibiotics. One useful assay organism is Candida albicans.

Following their production under fermentation culmre conditions, cryptophycin compounds of the invention can be recovered from the culmre and from the culmre media by methods known to those of ordinary skill in this art. Recovery is generally accomplished by initially filtering the culmre medium to separate the algal cells and then freeze-drying the separated cells. The freeze-dried alga can be extracted with a suitable solvent such as a mixmre of acetonitrile and dichloromethane. The cryptophycins can be separated by subjecting this extract, as well as the culmre media, to rapid chromatography on reversed-phase column. The cryptophycins can be purified by reversed-phase high-performance liquid chromatography (HPLC).

The novel cryptophycin compounds of the present invention and the previously disclosed cryptophycin compounds can be therapeutically employed as anti-neoplastic agents and thereby used in methods to treat neoplastic diseases. Five cryptophycin compounds, designated Cryptophycins 1, 3, 5, 13 and 15, were disclosed in U.S. Patent Nos. 4,946,835, 4,845,085, 4,845,086, and 4,868,208, such compounds either having been isolated from a strain of Nostoc sp. designated MB 5357 or having been synthesized from such an isolated compound. The present invention provides methods of producing these compounds via aberrant biosynthesis. Additional Cryptophycins, not to mention the disclosure of their use as anti¬ neoplastic agents, are disclosed in U.S. Application Serial No. 08/400,057 filed March 7, 1995, International Application Serial No. PCT\US94\ 14740 filed December 21, 1994, U.S. Application Serial No. 08/249,955 filed May 27, 1994 and U.S. Application Serial No. 08/172,632, filed December 21, 1993. These patent applications are incorporated herein by reference.

Selection of Metabolic Substrates In the biosynthesis of Cryptophycin 1, L-phenylalanine is the precursor of the phenyl and epoxide carbons of Unit A, a polyketide assembled from phenylacetate and three equivalents of acetate. L-Tyrosine is the precursor of Unit B with the O-methyl group arising from the methyl group of S-adenosyl-L-methionine. Not only is L-Tyrosine incorporated well into Unit B, L-O-methyltyrosine is also incorporated well into Unit B, indicating that O-methylation of tyrosine occurs first and this is followed by assimilation of O-methyltyrosine or the chlorinated O-methyltyrosine into the depsipeptide. In the biosynthesis of Cryptophycin 1, (25^*, 5R)-3-methylaspartic acid is the precursor of Unit C. (2R)-2-Methyl-β -alanine, however, is also incorporated well into Unit C, suggesting that (2S, 5R)-3-methylaspartic acid is decarboxylated into (2R)-2-methyl-β-alanine first and this is followed by assimilation of the (2i?)-2-methyl-β -alanine into the depsipeptide. In the biosynthesis of Cryptophycin 1, L-leucine is the precursor of Unit D and is incoφorated well into this unit. L-Leucic acid is not incorporated into Unit D, suggesting that L-leucine is taken up into the depsipeptide synthase (the multifunctional enzyme that carries out the assembly of the depsipeptide) and the nitrogen in L-leucine is lost and replaced by an oxygen during the assembly of the Unit A, B, C, and D precursors into the depsipeptide. In order to produce desirable cryptophycin compounds which have the properties noted previously, e.g., an intact macrolide, an epoxide group, a chloro group in Unit B and/or a methyl group in Unit C, selected substimted amino acids can be employed as metabolic substrates for fermentation cultures of bacteria capable of producing cryptophycin compounds. In the broadest sense, a substimted amino acid is considered to be any amino acid other than the protein amino acids which ordinarily form the basis for bacterial fermentation cultures. More usually, such substimted amino acids will include amino acids which provide the substiment groups in the appropriate positions to form a desirable cryptophycin compound as a metabolite of a bacterial fermentation culmre. Such amino acids include, for example, substimted α-amino acids, substituted phenylalanines, substimted tyrosines, substimted O-methyltyrosines and substimted β-alanines.

Typically substiment groups will be chosen so as to provide the desired strucmral feamres in the cryptophycin metabolites. For example, as halogens attached to aryl groups have been shown to be desirable feamres in Unit B of the cryptophycin compounds, amino acids substimted with at least one halogen moiety will prove to be of use. Commonly, alanine or phenylalanine will be the amino acids of choice in this regard, typically substimted with a halo-substituted aryl compound. Similarly, a methyl group in Unit C can be included as a feamre of an appropriately substimted β-alanine.

Thus in many cases, the amino acid of choice is substimted with at least one substiment selected from the group consisting of alkyl, alkoxy, alkaryl and alkoxyaryl compounds and halo-substituted derivatives thereof. Thus, the present invention provides methods of producing previously known cryptophycins and new cryptophycins through the culturing of a strain of the Nostoc sp. and introducing into the culmre one or more of the following pre-selected compounds: a substimted phenylalanine, a substimted β-alanine, substimted tyrosine or O-methyltyrosine and a substimted α-amino acid. Specifically, the present invention provides a method for producing previously disclosed Cryptophycin-52, Cryptophycin- 110 and Cryptophycin-115 by aberrant biosynthesis, as well as the novel cryptophycin compounds described hereafter. For example, Cryptophycin-52 is produced, along with other cryptophycins, when the cyanobacterium is grown in the presence of 2 , 2-dimethyl- β -alanine :

CRYPTOPHYCIN-52

Cryptophycin-110 and Cryptophycin-115 are produced when the cyanobacterium is grown in the presence of DL-p-fluorophenylalanine:

CRYPTOPHYCIN-110

CRYPTOPHYCIN-115

The present method can be used to produce novel cryptophycins that differ in the aryl group of Unit A of Figure 1 by growing the cyanobacterium in the presence of the appropriately substimted phenylalanine. Likewise, the present method can be used to produce novel cryptophycins that differ (1) in Unit B by growing the cyanobacterium in the presence of the appropriately substimted tyrosine or

O-methyltyrosine, (2) in Unit C by growing the cyanobacterium in the presence of the appropriate substimted β-alanine, and (3) in Unit D by growing the cyanobacterium in the presence of the appropriately substimted α-amino acid.

An example of a novel cryptophycin compound of the present invention is Cryptophycin- 189 (also called Cryptophycin B-8 which is produced by growing the cyanobacterium in the presence of DL-3-(3-methyl-4-methoxyphenyl)alanine:

CRYPTOPHYCIN-189

Two further examples of novel cryptophycin compounds of the present invention are Cryptophycin- 190 (also called Cryptophycin B-l) and Cryptophycin-210 which are produced by growing the cyanobacterium in the presence of DL-3-(3-fluoro-4-methoxyphenyl)alanine:

CRYPTOPHYCIN-190

CRYPTOPHYCIN-210

Another example of a novel cryptophycin compound of the present invention is Cryptophycin B-2 which would be produced by growing the cyanobacterium in the presence of (2R)-3-(3-bromo-4-methoxyphenyl) alanine or (2R)-3-(3-bromo- 4-hydroxyphenyl)alanine :

B - 2

Another example of a novel cryptophycin compound of the present invention is Cryptophycin B-3 which would be produced by growing the cyanobacterium in the presence of (2R)-3-(3,4-dimethoxyphenyl)alanine or (2R)-3-(3-hydroxy- 4-methoxyphenyl)alanine, or (2R)-3-(3,4-dihydroxyphenyl)alanine:

B - 3

Two further examples of novel cryptophycin compounds of the present invention are Cryptophycin-208 and Cryptophycin-209 (also called Cryptophycin B- 4) which are produced by growing the cyanobacterium in the presence of DL-3-(3,4-methylenedioxyphenyl)alanine:

CRYPTOPHYCIN-208

CRYPTOPHYCIN-209

Another example of a novel cryptophycin compound of the present invention is Cryptophycin-211 which is produced by growing the cyanobacterium in the presence of DL-3-(3-fluoro-4-hydroxyphenyl)alanine:

CRYPTOPHYCIN-211

Another example of a novel cryptophycin compound of the present invention is Cryptophycin B-5 which would be produced by growing the cyanobacterium in the presence of (2R)-3-(4-methoxy-2-pyridyl)alanine or (2R)-3-(4-hydroxy- 2-pyridyl)alanine:

B - 5

Another example of a novel cryptophycin compound of the present invention is Cryptophycin B-6 which would be produced by growing the cyanobacterium in the presence of (2R)-3-(4-methoxy-3-pyridyl)alanine:

B - 6

Another example of a novel cryptophycin compound of the present invention is Cryptophycin B-7 which would be produced by growing the cyanobacterium in the presence of (2R)-3-(4-ethoxyphenyl)alanine:

B - 7

Another example of a novel cryptophycin compound of the present invention is Cryptophycin-213 which is produced by growing the cyanobacterium in the presence of (35)-3-aminobutanoic acid:

CRYPTOPHYCIN-213

Another example of a novel cryptophycin compound of the present invention is Cryptophycin C-1 which would be produced by growing the cyanobacterium in the presence of (2R)-2-ethyl- β-alanine:

C - 1

Another example of a novel cryptophycin compound of the present invention is Cryptophycin C-2 which would be produced by growing the cyanobacterium in the presence of (2R)-2-isopropyl- β-alanine:

C - 2

Another example of a novel cryptophycin compound of the present invention is Cryptophycin C-3 which would be produced by growing the cyanobacterium in the presence of (2R)-2-t-butyl- β-alanine:

C - 3

Another example of a novel cryptophycin compound of the present invention is Cryptophycin C-4 which would be produced by growing the cyanobacterium in the presence of (2R)-2-dimethylamino- β-alanine:

C - 4

Another example of a novel cryptophycin compound of the present invention is Cryptophycin C-5 which would be produced by growing the cyanobacterium in the presence of (2R)-2-dimethylaminomethyl- β-alanine:

C - 5

Another example of a novel cryptophycin compound of the present invention is Cryptophycin C-6 which would be produced by growing the cyanobacterium in the presence of 2, 2-diethyl- β-alanine:

Another example of a novel cryptophycin compound of the present invention is Cryptophycin C-7 which would be produced by growing the cyanobacterium in the presence of 2, 2-di(methoxy Imethy 1)- β-alanine :

C - 7

Another example of a novel cryptophycin compound of the present invention is Cryptophycin C-8 which would be produced by growing the cyanobacterium in the presence of 1-aminomethylcyclopropane-l -carboxylic acid:

C - 8

Two further examples of novel cryptophycin compounds of the present invention are Cryptophycin-214 and Cryptophycin-215 which are produced by growing the cyanobacterium in the presence of (2<S)-2-aminobutyric acid:

CRYPTOPHYCIN-214

CRYPTOPHYCIN-215

Another example of a novel cryptophycin compound of the present invention is Cryptophycin D-1 which would be produced by growing the cyanobacterium in the presence of 3-cyclopropyl-α-alanine:

D - 1

Another example of a novel cryptophycin compound of the present invention is Cryptophycin D-2 which would be produced by growing the cyanobacterium in the presence of 3-t-butyl-α-alanine:

D - 2

Another example of a novel cryptophycin compound of the present invention is Cryptophycin D-3 which would be produced by growing the cyanobacterium in the presence of 3-vinyl-α-alanine:

D - 3

Experimental

In the experimental disclosure which follows, all weights are given in grams (g), milligrams (mg), micrograms (mg), nanograms (ng), picograms (pg) or moles (mol), all concentrations are given as percent by volume (%), molar (M), millimolar (mM), micromolar (μM), nanomolar (nM), or picomolar (pM), normal (N) and all volumes are given in liters (L), milliliters (mL) or microliters (μL), and measures in millimeters (mm), unless otherwise indicated.

Example 1 General Procedure for Feeding Substimted Amino Acids For the production of cryptophycin compounds by bacterial fermentation utilizing substimted amino acids, the selected amino acid is dissolved in 0.5N HCl to a concentration in the range of 15-25mg/mL. Typically, a 0.5mL portion of the solution is added to each of 2-4 carbouys of the bacterial culmre in two-day intervals beginning on day 7-10 after bacterial innoculation. after 7-9 additions of the amino acid solution, the culmres are allowed to grow for an additional 3-5 days, and then harvested.

Example 2 Identification of Cryptophycins bv El Mass Spectrometry

Mass spectra, including high resolution mass measurements are determined in the electron-impact mode on a VG-70SE Instrument (source ??). Cryptophycin-1, -2, -3, and -4 were found to give characteristic fragmentation patterns as shown in Figure 2. Ion c is useful for identifying analogs that differ in the aryl group found in Unit A. Comparison of ions a, b and d is useful for identifying analogs that differ in Unit B, Unit C and/or Unit D.

Example 3 Aberrant Biosynthesis of Cryptophycin 52

To each of nine carboys (20L) of Nostoc sp. GSV 224 growing under standard conditions was added 2, 2-dimethyl- β-alanine (400mg) in four equal portions of lOOmg each on days 6, 8, 14 and 22 after inoculation. The culmres were harvested 30 days after inoculation and lyophilized to yield 103.6g of dried alga. Extraction was carried out in 52g batches with 2L of 4: 1 acetonitrile/dichloromethane. The extract was evaporated in vacuo and the residue fractionated by C-18 flash chromatography. The crude cryptophycin fraction (300mg) eluted with 65:35 acetonitrile/water was subjected to HPLC on C-18 (Econosil, lOμ, 250 X 22mm column, 65:35 MeCN/H₂O, 6mL/min). The fraction eluting after 66.5 min was purified by normal phase HPLC on silica (Econosil, 5μ, 250 x 4.6mm column, 3:2 EtOAc/hexanes, 2.5mL/min). The fraction eluting after 21.4 min, which was a mixmre of Cryptophycin-52 and Cryptophycin-28, was purified further by HPLC on C-18 (Econosil, 10μ, 250 x 10mm, 85:15 methano/water, 2mL/min). Cryptophycin-52 (2mg), identical with authentic material by spectroscopic comparison, eluted after 18.4 min.

The spectral data for Cryptophycin 52 is as follows: [α]_D +19.9° (c 0.5, CHC1₃); EIMS m/z 668/670 (4/2, M⁺), 445 (35), 244 (12), 227 (22, ion c), 195/197 (66/27, ion d), 184 (45), 155/157 (38/10), 91 (100, benzyl ion = tropylium ion); HREIMS m/z 668.2873 (C₃₆H₄₅N₂O₈ ³⁵Cl, Δ -0.9 mmu), 445.2497 (C^H^NO_e, Δ -3.3 mmu); UV (MeOH) λ„ (e) 204 (35100), 218 (20900)nm; IR (NaCI) v^ 3415, 3270, 2960, 1748, 1721, 1650, 1536, 1504, 1260, 1192, 1150, 1066, 1013, 800, 698 cm ¹. Η NMR (CDC1₃) δ Unit A 7.33-7.38 (11-H/12-H/13-H; bm, W_1/2*25 Hz), 7.24 (10-H/14-H; m, W_I/2*15 Hz), 6.76 (3-H; ddd, 15.1/10.8/4.3), 5.71 (2-H; dd, 15.1/1.7), 5.20 (5-H; ddd, 11.0/5.0/1.8), 3.68 (8-H; d, 1.9), 2.92 (7-H; dd, 7.5/1.9), 2.57 (4-H_b; ddd, -14.6/1.8/1.7), 2.45 (4-H_a; ddd, -14.6/11.0/10.8), 1.78 (6-H; bm, W_1/2*15 Hz), 1.14 (6-Me; d, 6.9); Unit B 7.18 (5-H; d, 2.2), 7.04 (9-H; dd, 8.4/2.2), 6.83 (8-H; d, 8.4), 5.56 (NH; d, 7.9), 4.73 (2-H; ddd, 7.9/7.4/5.3), 3.87 (OMe; s), 3.09 (3-H_b; dd, -14.6/5.3), 3.05 (3-H_a; dd, -14.6/7.4); Unit C 7.20 (NH; dd, 8.6/3.2), 3.41 (3-H_b; dd, -13.4/8.6), 3.10 (3-H_a; dd, -13.4/3.2), 1.22 (2-Me'; s), 1.15 (2-Me'; s); Unit D 4.82 (2-H; dd, 10.2/3.5), 1.73 (3-H_b; bm, W_1/2=20 Hz), 1.66 (4-H; bm, W_IQ ^*=20 Hz), 1.31 (3-H_a; ddd, -13.8/9.1/3.5), 0.84* (4-Me; d, 6.6), 0.82* (5-H₃; d, 6.6); ^!3C NMR (CDC1₃) δ Unit A 164.9 (1), 141.8 (3), 136.7 (9), 128.7 (11/13), 128.3 (12), 125.6 (10/14), 124.7 (2), 75.9 (5), 63.0 (7), 59.0 (8), 40.7 (6), 36.9 (4), 13.5 (6-Me), Unit B 170.3 (1), 154.1 (7), 130.9 (5), 129.5 (4), 128.5 (9), 122.6 (6), 112.4 (8), 56.1 (7-OMe), 54.3 (2), 35.3 (3), Unit C 178.0 (1), 46.5 (3), 42.8 (2), 22.8 (2-Me), 22.8 (2-Me'), Unit D 170.5 (1), 71.2 (2), 39.3 (3), 24.6 (4), 22.7* (4-Me), 21.2* (5).

Example 4 Aberrant Biosynthesis of Crvptophvcin-110 and Cryptophycin- 115

To each of two carboys (20L) of Nostoc sp. GSV 224 growing under standard conditions was added DL-3-(4-fluorophenyl)alanine (150mg) as described in the general procedure. After harvesting, the freeze-dried alga (26g) was extracted with IL of 5:1 CH_JCN/CH_JCL_J for 24 hours, and the extract (lg) was concentrated in vacuo to give a dark green solid. The crude extract was applied to an ODS-coated silica column and subjected to flash chromatography with 25:75 CH₃CN/H₂O (250mL), 50:50 CH₃CN/H₂O (250mL), 65:35 CH₃CN/H₂O (250mL), 80:20 CH₃CN/H₂O (500mL), CH₃OH (600mL), and CH₂C1₂ (500mL). The fractions that were eluted with 65:35 CH₂CN/H₂O (98mg) and 80:20 CH₃CN/H₂O (45mg) were evaporated and further separated by reversed-phase HPLC (Econosil C-18, lOμ, 250 X 22mm column, 65:35 MeCN/H₂O, 6mL/min). The fraction containing Cryptophycin-115 eluted at 45min<t_R<59min and the fraction containing Cryptophycin-110 eluted at 75min<t_R<90min. Purification was achieved by normal-phase HPLC (Econosil silica, 5μ, 250 x 4.6mm column, 50:50

EtOAc/hexanes, 3mL/min) to give Cryptophycin- 110 (t_R = 14 min, O.lmg) and Cryptophycin-115 (18min<t_R<23min, 0.7mg) The 'H-NMR spectra of the isolated cryptophycins were identical to those of synthetic samples.

Cryptophycin-110: 'H NMR (CDC1₃) δ (carbon position, multiplicities, J in Hz) Unit A 7.29 (10-H/14-H; dd, 8.6, 5.6), 6.99 (11-H/13-H, dt, 8.6, 8.5), 6.68 (3-H; ddd, 15.3, 9.7, 5.6), 6.38 (8-H; d, 15.8), 5.83 (7-H; dd, 15.8, 8.8), 5.78 (2-H, d, 15.3), 5.00 (5-H, ddd, 10.8, 7.3, 1.3), 2.53 (4-H/6-H, m), 2.63 (4-H\ m), 1.13 (6-CH₃, d, 6.8); Unit B 7.21 (5-H; d, 1.8), 7.07 (9-H, dd, 8.4, 1.8), 6.84 (8-H; d, 8.4), 5.68 (NH, d, 8.5), 4.82 (2-H, m), 3.87 (OMe, s), 3.14 (3-H, dd, 5.6, -14.4), 3.04 (3-H\ dd, 7.2, -14.4); Unit C 6.95 (NH, bdd, 6.8, 5.9), 3.50

(3-H, td, 4.4, -13.5), 3.28 (3-H\ ddd, 6.8, 6.7, -13.5), 2.72 (2-H, m), 1.23 (2-Me, d, 7.2); Unit D 4.82 (2-H, m), 1.65 (3-H/4-H; m), 1.35 (3-H\ ddd, 4.5, 3.8, -10.9), 0.78 (5-H, d, 6.4), 0.74 (5'-H, d, 6.4).

Cryptophycin-115: EIMS m/z (relative intensity %) 672 (1.9, M⁺), 412 (5.8, ion a), 280 (10, ion b), 245 (17, ion c), 195 (52, ion d), 155 (31), 141 (23), 135 (15), 109 (100, -fluorobenzyl ion = fluorotropylium ion); high-resolution EIMS 668.2853 (calcd for C₃₅H₄₂ClFN₂O₈, Δ +3.4mmu, M⁺). Η NMR (CDCL) δ (carbon position, multiplicities, J in Hz) Unit A 7.22 (10-H/14-H; ddt, 8.7, 5.2, 2.0), 7.01 (11-H/13-H, ddt, 8.7, 8.5, 2.0), 6.68 (3-H, ddd, 15.2, 9.7, 5.2), 5.74 (2-H, dd, 15.2, 0.8), 5.15 (5-H, ddd, 11.2, 5.0, 1.8), 3.67 (8-H, d, 2.0), 2.88 (7-H, dd, 7.4, 2.0), 2.54 (4-H, dtd, 5.2, 1.8, -14.4), 2.44 (4-H\ ddd, 11.2, 9.7, -14.4), 1.79 (6-H, m), 1.13 (6-CH₃, d, 6.8); Unit B 7.21 (5-H; d, 2.0), 7.06 (9-H, dd, 8.3, 2.0), 6.83 (8-H; d, 8.3), 5.63 (NH, d, 8.4), 4.80 (2-H, ddd, 8.4, 7.2, 5.4), 3.87 (OMe, s), 3.14 (3-H, dd, 5.4, -14.4), 3.03 (3-H\ dd, 7.2, -14.4); Unit C 6.94 (NH, bdd, 6.7, 5.0), 3.48 (3-H, ddd, 5.0, 3.7, -13.4), 3.30 (3-H\ ddd, 6.8, 6.7, -13.4), 2.72 (2-H, m), 1.22 (2-Me, d, 7.4); Unit D 4.83 (2-H, dd, 9.9, 3.6), 1.70 (3-H/4-H; m), 1.35 (3-H\ m), 0.87 (5-H, d, 6.5), 0.85 (5'-H, d, 6.5).

Example 5 Aberrant Biosynthesis of Cryptophycin- 189

To each of two carboys (20L) of Nostoc sp. GSV 224 growing under standard conditions was added DL-3-(3-methyl-4-methoxyphenyl)alanine (120mg) as described in the general procedure. After harvesting, the freeze-dried alga (20g) was extracted with IL of 5:1 CH₃CN/CH₂CL, for 24 hours, and the extract (l.lg) was concentrated in vacuo to give a dark green solid. The crude extract was applied to an ODS-coated silica column and subjected to flash chromatography with 25:75 CH₃CN/H₂O (250mL), 50:50 CH₃CN/H₂O (250mL), 65:35 CH₃CN/H₂O (250mL), 80:20 CH₃CN/H₂O (250mL), CH₃OH (500mL), and CH₂C1₂ (500mL). The fractions that were eluted with 65:35 CH₂CN/H₂O (178mg) and 80:20 CH₃CN/H₂O (75mg) were evaporated and further separated by reversed-phase HPLC (Econosil C-18, 10μ, 250 X 22mm column, 65:35 MeCN/H₂O, 6mL/min). The fraction containing Cryptophycm- 189 eluted at 50min<t_R<64min. Normal phase HPLC (Econosil silica, lOμ, 250 x 10mm column, 60:40 EtOAc/hexane, 3mL/min) of this fraction (150mg) afforded 2.8mg of the pure compound (t_R = 28 min).

To facilitate the isolation and identification of Cryptophycin- 189, [O-methyl-Η₃]-DL-3-(3-methyl-4-methoxyphenyl)alanine was fed to the alga. The ²H₃-labeled Cryptophycin- 189 displayed the following spectral properties: Η NMR (500MHz, CDC1₃) δ (carbon position, multiplicity) for Unit B 3.74 (7-OCΗ₃, s); [αlD^2*5 = +27.8° (CHC1₃, c 1.9); IR (neat) λ„. 3412, 3272, 2959, 1749, 1725, 1667, 1503, 1263, 1176 cm¹; EIMS m/z (relative intensity %) 637 (13, M⁺), 395 (3, ion a), 178 (43, ion d), 91 (100); high resolution EIMS m/z (rel. intensity) 637.3477 (calcd for C₃₆H₄₃D₃N₂O₈, Δ-3.5mmu); Η NMR (CDC1₃) δ (carbon position, multiplicities, J in Hz) Unit A 5.70 (2, dd, 15.1, 1.4), 6.71 (3, ddd, 16.2, 8.4, 3.7), 2.45 (3, ddd, 14.5, 12.6, 3.7), 2.55 (3, br. dd, 12.1, 2.1), 5.20 (5, ddd, 11.2, 4.9, 1.7), 1.78 (6, m), 1.14 (6-Me, d, 6.9), 2.92 (7, dd, 7.5, 2.1), 3.68 (8, d, 1.7), 7.24 (10/14, m), 7.33-7.29 (11/12/13, m); Unit B 4.75 (2, dd, 13.1, 7.3), 3.09 (3, dd, 14.5, 5.4), 3.03 (3, dd, 14.4, 5.4), 7.07 (5, m), 2.16 (6-Me, s), 6.71 (8, d, 8.0), 6.95 (9, dd, 10.5, 2.3), 5.57 (NH, d, 7.8); Unit C 2.68 (2, m), 1.23 (2-Me, d, 7.2), 3.34 (3, dt, 13.7, 3.8), 3.45 (3, m), 6.71 (NH, br. m); Unit D 4.82 (2, dd, 10.0, 3.7), 1.70 (3, m), 1.32 (3, m), 1.70 (4, m), 0.85 (5, d, 6.5), 0.83 (5' , d, 6.5). "C NMR (CDC1₃) δ (carbon position) Unit A 165.1 (1), 125.1 (2), 141.2 (3), 36.7 (4), 76.0 (5), 40.6 (6), 13.5 (6-Me), 63.0 (7), 59.0 (8), 136.8 (9), 125.6 (10/14), 128.7 (11/13), 128.5 (12); Unit B 171.2 (1), 54.0 (2), 35.4 (3), 127.8 (4), 131.5 (5), 126.9 (6), 16.1 (6Me), 156.8 (7), 110.1 (8), 127.3 (9); Unit C 176.1 (1), 38.1 (2), 14.3 (2-Me), 40.7 (3); Unit D 170.6 (1), 71.3 (2), 39.4 (3), 24.5 (4), 22.9 (5), 21.3 (5').

Example 6 Aberrant Biosynthesis of Cryptophycin- 190 and Crvptophvcin-210

To each of three carboys (20L) of Nostoc sp. GSV 224 growing under standard conditions was added DL-3-(3-fluoro-4-methoxyphenyl)alanine (180mg) as described in the general procedure. After harvesting, the freeze-dried alga (32g) was extracted with 1.5L of 5:1 CH^N/OLCL, for 24 hours, and the extract (1.2g) was concentrated in vacuo to give a dark green solid. The crude extract was applied to an ODS-coated silica column and subjected to flash chromatography with 25:75 CH₃CN/H₂O (250mL), 50:50 CH₃CN/H₂O (250mL), 65:35 CH₃CN/H₂O (250mL), 80:20 CH₃CN/H₂O (500mL), CH₃OH (500mL), and CH₂C1₂ (500mL). The fractions that were eluted with 65:35 CH₂CN/H₂O and 80:20 CH₃CN/H₂O were evaporated and further separated by reversed-phase HPLC (Econosil C-18, 10 μ, 250 X 22mm column, 65:35 MeCN/H₂O, 6mL/min). The fraction containing Cryptophycin- 190 eluted at 44min<t_R <50min and the fraction containing Cryptophycin-210 eluted at 75min < t_R < 96min. Further purification of these two fractions by normal-phase HPLC (Econosil silica, 5μ, 250 x 4.6mm column, 2.5mL/min) afforded 4.3mg of Cryptophycin-190 (45:55 EtOAc/hexane, t_R = 22 min) and 1.5 mg of Cryptophycin-210 (50:50 EtOAc/hexane, t_R = 14.4min).

To facilitate the isolation and identification of Cryptophycin- 190 and Cryptophycin-210, [O-methyl-²H₃]-DL-3-(3-fluoro-4-methoxyphenyl)alanine was fed to the alga. The ²H₃-labeled Cryptophycin- 190 displayed the following spectral properties:

Η NMR (500MHZ, CDC1₃) amino or hydroxy acid δ (carbon position, multiplicity) Unit B 3.14 (7-OCΗ₃, S); ^,9F-NMR (CDC1₃) δ -135.6 (dd); [αJD²⁵ = +16.8° (CHC1₃, c 0.64); IR (neat) λ_^ 3416, 2959, 1748, 1651, 1517, 1276, 1244, 1177, 1126 cm ¹; EIMS m/z (relative intensity %) 641 (M⁺, 7), 399 (17, ion a), 267 (13, ion b), 227 (39, ion c), 182 (72, ion d), 91 (100); high resolution EIMS m/z 641.3198 (calcd for _sH^FN , Δ-0.6mmu);

Η NMR (CDCL) δ (carbon position, multiplicities, J in Hz) Unit A 5.73 (2, d, 15.8), 6.68 (3, ddd, 15.2, 9.8, 5.3), 2.45 (4, ddd, 14.0, 11.1, 3.6), 2.55 (4, dd, 14.1, 5.3), 5.16 (5, ddd, 11.2, 4.6, 1.7), 1.80 (6, m), 1.14 (6-Me, d, 6.8), 2.92 (7, dd, 7.5, 2.0), 3.69 (8, d, 2.0), 7.25 (10/14, m), 7.32-7.39 (11/12/13, m); Unit B 4.80 (2, m), 3.03 (3, dd, 14.4, 7.4), 6.84-6.97 (5/9, m), 3.85 (7-OMe, s), 7.32- 7.39 (8, m), 5.67 (NH, d, 8.6); Unit C 2.70 (2, m), 1.22 (2-Me, d, 6.8), 3.30 (3, dt, 13.6, 4.4), 3.46 (3, dt, 13.6, 4.4), 6.83-6.97 (NH, m); Unit D 4.82 (2, m), 1.66-1.74 (3, m), 1.66-1.74 (4, m), 0.86 (5, d, 6.6), 0.84 (5', d, 6.6). ¹³C NMR (125MHz, CDC1₃) δ (carbon position) Unit A 165.3 (1), 125.3 (2), 141.0 (3), 36.7 (4), 76.2 (5), 40.7 (6), 13.5 (6-Me), 63.0 (7), 59.0 (8), 136.8 (9), 125.6 (10/14), 128.7 (11/13), 128.5 (12); Unit B 170.9 (1), 53.6 (2), 35.2 (3), not determined (4), 117.5 (5), not determined (6), 141.1 (7), 59.0 (7-Me), 113.6 (8), 124.9 (9); Unit C 175.6 (1), 38.3 (2), 14.1 (2-Me), 41.1 (3); Unit D 170.7 (1), 71.3 (2), 39.4 (3), 24.5 (4), 22.9 (5), 21.3 (5').

Example 7 Aberrant Biosynthesis of Crvptophvcin-208 and Crvptophvcin-209

To each of two carboys (20L) of Nostoc sp. GSV 224 growing under standard conditions was added DL-3-(3,4-methylenedioxyphenyl)alanine (120mg) as described in the general procedure. After harvesting, the freeze-dried alga (25g) was extracted with IL of 5:1 CH₃CN/CH₂CL₂ for 24 hours, and the extract (510mg) was concentrated in vacuo to give a dark green solid. The crude extract was applied to an ODS-coated silica column and subjected to flash chromatography with 25:75 CH₃CN/H₂O (250mL), 50:50 CH₃CN/H₂O (250mL), 65:35 CH₃CN/H₂O (250mL), 80:20 CH₃CN/H₂O (250mL), CH₃OH (500mL), and CH₂C1₂ (500mL). The fractions that were eluted with 65:35 CH₂CN/H₂O (204mg) and 80:20 CH₃CN/H₂O (35mg) were evaporated and further separated by reversed-phase HPLC (Econosil C-18, lOμ, 250 X 22mm column, 65:35 MeCN/H₂O, 6mL/min). The fraction containing Cryptophycin-209 eluted at 42min<t_R <48.5min and the fraction containing Cryptophycin-208 eluted at 69min<t_R<88.5min. Normal phase HPLC (Econosil silica, 5μ, 250 x 4.6mm column, 45:55 EtOAc/hexane, 3mL/min) of the fraction containing Cryptophycin-209 afforded <0.1mg of the pure compound (t_R = 16.5 min). HPLC (Econosil silica, 5μ, 250 x 4.6mm column, 50:50 EtOAc/hexane, 2.5mL/min) of the fraction containing Cryptophycin-208 afforded <0.1mg of the pure compound (t_R = 12 min). To facilitate the isolation and identification of Cryptophycin-208 and

Cryptophycin-209, [methy lenedioxy-²HJ-DL-3-(3 ,4-methylenedioxyphenyl) alanine was fed to the alga. The ²H₂-labeled Cryptophycin-208 displayed the following spectral properties: Η NMR (500MHz, CHC1₃) δ (carbon position, multiplicity) Unit B 5.88 (6,7-OCΗ₂O, s); EIMS m/z (relative intensity, assignment) 620 (5, M⁺), 394 (68, ion a), 393 (49), 262 (12, ion b), 227 (45, ion c), 177 (100, ion d), 91 (79); high resolution EIMS m/z 620.3090 (calcd for _sH^NA, Δ-2.3mmu error). The ²H₂-labeled Cryptophycin-209 displayed the following spectral properties: Η NMR (500MHz, CHC1₃) δ (carbon position, multiplicity) Unit B 5.85 (6,7-OCΗ₂O, S); EIMS m/z (relative intensity, assignment) 636 (M⁺, 17), 394 (6, ion a), 262 (5, ion b), 193 (35), 177 (50, ion d), 91 (79); high resolution EIMS m/z 636.3031 (calcd for H^NA, Δ-1.5mmu). Example 8 Aberrant Biosynthesis of Crvptophvcin-211

To each of three carboys (20L) of Nostoc sp. GSV 224 growing under standard conditions was added DL-3-(3-fluoro-4-hydroxyphenyl)alanine (180mg) as described in the general procedure. After harvesting, the freeze-driεd alga (34g) was extracted with 1.5L of 5:1 CH-CN/CHjCLj for 24 hours, and the extract (l.lg) was concentrated in vacuo to give a dark green solid. The crude extract was applied to an ODS-coated silica column and subjected to flash chromatography with 25:75 CH₃CN/H₂O (250mL), 50:50 CH₃CN/H₂O (250mL), 65:35 CH₃CN/H₂O (250mL), 80:20 CH₃CN/H₂O (600mL), CH₃OH (600mL), and CH₂C1₂ (500mL). The fractions that were eluted with 65:35 CH₂CN/H₂O (302mg) and 80:20 CH₃CN/H₂O (49mg) were evaporated and further separated by reversed-phase HPLC (Econosil C-18, lOμ, 250 X 22mm column, 65:35 MeCN/H₂O, 6mL/min). The fraction containing Cryptophycin-211 eluted at t_R ^**•= 66 min. Further purification by normal-phase HPLC (Econosil silica, 5μ, 250 x 4.6mm, 50:50 EtOAc/hexane, 3mL/min) afforded 3.5mg of the pure compound (t_R = 5.5 min). Cryptophycin-211 :

¹⁹F-NMR (CDC1₃) δ -128.1 (d, 11); EIMS m/z (relative intensity) 674/672 (M⁺, 3/6), 432/430 (5/12, ion a), 300/298 (8/19, ion b), 227 (40, ion c), 215/213 (15/39, ion d), 91 (100); high resolution EIMS m/z 672.2580 (calcd for C₃₅H₄₂ClFN₂O₈, Δ + 3.4mmu error) ;

Η NMR (CDC1₃) δ (carbon position, multiplicities, J in Hz) Unit A 5.75 (2, dd, 16.4, 0.9), 6.66 (3, ddd, 15.3, 9.4, 5.6), 2.46 (4, dd, 10.3, 3.6), 2.56 (4, dd, 14.2, 5.5), 5.14 (5, ddd, 6.5, 4.8, 1.7), 1.81 (6, m), 1.14 (6-Me, d, 7.2), 2.93 (7, dd, 8.4, 2.0), 3.69 (8, d, 1.8), 7.25 (10/14, m), 7.30-7.40 (11/12/13, m); Unit B 4.81 (2, m), 3.13 (3, dd, 14.4, 5.4), 3.00 (4, dd, 14.5, 7.3), 7.01 (5, s), 3.92 (7-OMe, s), 6.90 (9, dd, 11.4, 2.0); Unit C 2.72 (2, m), 1.22 (2-Me, d, 7.2), 3.22 (3, m), 3.54 (3, m); Unit D 4.83 (2, m), 1.71 (3, m), 1.37 (3, m), 1.71 (4, m), 0.87 (5, d, 7.0), 0.85 (5', d, 7.2). ¹³C NMR (125MHz, CDC1₃) δ (carbon position) Unit A 165.5 (1), 125.4 (2), 141.0 (3), 36.7 (4), 76.3 (5), 40.6 (6), 13.5 (6-Me), 63.0 (7), 58.9 (8), 136.7 (9), 125.6 (10/14), 128.7 (11/13), 128.5 (12); Unit B 170.8 (1), 53.3 (2), 35.3 (3), 133.4 (4), 126.2 (5), 127.4 (6), 141.4 (7), 61.4 (7-OMe), 155.8 (8, d 248Hz), 116.4 (9, d, 28Hz); Unit C 175.3 (1), 38.3 (2), 14.0 (2-Me), 41.4 (3); UnU D 170.7 (1), 71.3 (2), 39.4 (3), 24.6 (4), 22.9 (5), 21.3 (5').

Example 9 Aberrant Biosynthesis of Cryptophvcin-213

To each of three carboys (20L) of Nostoc sp. GSV 224 growing under standard conditions was added 5-3-aminobutanoic acid (180mg) as described in the general procedure. After harvesting, the freeze-dried alga (31g) was extracted with 1.5L of 5:1 CH₃CN/CH₂CL, for 24 hours, and the extract (lg) was concentrated in vacuo to give a dark green solid. The crude extract was applied to an ODS-coated silica column and subjected to flash chromatography with 25:75 CH₃CN/H₂O (250mL), 50:50 CH₃CN/H₂O (250mL), 65:35 CH₃CN/H₂O (250mL), 80:20 CH₃CN/H₂O (250mL), CH₃OH (500mL), and CH₂C1₂ (500mL). The fractions that were eluted with 65:35 CH₂CN/H₂O (205mg) and 80:20 CH₃CN/H₂O (108mg) were evaporated and further separated by reversed-phase HPLC (Econosil C-18, 10 μ, 250 X 22mm column, 65:35 MeCN/H₂O, 6mL/min). The fraction (192mg) containing Cryptophycin-213 eluted at 52min< t_R < 69min. Further purification by normal phase HPLC (Econosil silica, 5μ, 250 x 4.6mm column, 50:50 EtOAc/hexane, 2.5mL/min) afforded <0.1mg of the pure compound (t_R = ??min).

To facilitate the isolation and identification of Cryptophycin-213, [methyl-²H₃]-5-3-aminobutanoic acid was fed to the alga. The ²H₃-labeled Cryptophycin-213 displayed the following spectral properties: ²H NMR (500MHz, CHC1₃) δ (carbon position, multiplicity) Unit C 1.11 (CΗ₃ on C-2).

Example 10 Aberrant Biosynthesis of Crvptophvcin-214 and Crvptophvcin-21

To each of three carboys (20L) of Nostoc sp. GSV 224 growing under standard conditions was added 5-2-aminobutanoic acid (180mg) as described in the general procedure. After harvesting, the freeze-dried alga (41g) was extracted with 1.7L of 5:1 CH₃CN/CH₂CL₂ for 24 hours, and the extract (lg) was concentrated in vacuo to give a dark green solid. The crude extract was applied to an ODS-coated silica column and subjected to flash chromatography with 25:75 CH₃CN/H₂O (250mL), 50:50 CH₃CN/H₂O (250mL), 65:35 CH₃CN/H₂O (250mL), 80:20 CH₃CN/H₂O (250mL), CH₃OH (600mL), and CH₂C1₂ (500mL). The fractions that were eluted with 65:35 CH₂CN/H₂O (529mg) and 80:20 CH₃CN/H₂O (59mg) were evaporated and further separated by reversed-phase HPLC (Econosil C-18, 10μ, 250 X 22mm column, 65:35 MeCN/H₂O, 6mL/min). The fraction containing Cryptophycin-214 eluted at 87min<t_R and the fraction containing Cryptophycin-215 eluted at 53min<t_R<68min. Further separation was achieved by normal phase HPLC to afford < lmg of impure Cryptophycin-214 (Econosil silica, 5μ, 250 x 4.6mm column, 45:55 EtOAc/hexane, 2.5mL/min) (17min<t_R< 19.5min) and < lmg of impure Cryptophycin-215 (Econosil silica, lOμ, 250 x 10mm, 60:40 EtOAc/hexane, 3mL/min) (10mm<t_R< 12min).

To facilitate the isolation and identification of Cryptophycin-214 and Cryptophycin-215, [methyl-²H₃]-5-2-aminobutanoic acid was fed to the alga. The ²H₃-labeled Cryptophycin-214 displayed the following spectral properties: ²H NMR (500MHz, CHC1₃) Unit D δ (assignment) 0.76 (4-Η₃). The Η₃-labeled Cryptophycin-215 displayed the following spectral properties: ²H NMR (500MHz, CHC1₃) Unit D δ (assignment) 0.76 (4-Η₃).

Example 11 Cytotoxicity of Cryptophycins Produced bv Aberrant Biosynthesis

Cryptophycin-52 (IC₅₀ 43pM) and Cryptophycin- 115 (IC₅₀ 39pM) are slightly less cytotoxic than Cryptophycin- 1 (IC₅₀ 9-29pM) against the human tumor cell line KB. Cryptophycin-110 (IC₅₀ 4.6nM) has a cytotoxicity against KB comparable to that of Cryρtophycin-3 (IC₅₀ 3.1-4.6nM).

The cytotoxicities (MIC's) of the novel sytrene-type Cryptophycins 208, 210 and 214 against KB are in the range of 10-lOOnM. The cytotoxicities of Cryptophycins 189, 190, 209, 211, 213 and 215 against KB are < lnM.

All publications and patent applications cited in this specification are hereby incorporated by reference as if they had been specifically and individually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be apparent to those of ordinary skill in the art in light of the disclosure that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Claims

Claims:

1. A method for producing a cryptophycin compound comprising culturing at least one strain of bacteria capable of producing cryptophycins in the presence of a substimted amino acid under conditions sufficient to produce the desired cryptophycin compound.

2. A method according to claim 1 wherein said bacteria is a cyanobacteria.

3. A method according to claim 2 wherein said bacteria is at least one species from the genus Nostoc.

4. A method according to claim 3 wherein said bacteria is at least one strain selected from the group consisting of Nostoc sp. strain GSV 224 (ATCC

Accession No. 55483) and strain MB 5357 (ATCC Accession No. 53789).

5. A method according to claim 1 wherein said substimted amino acid is an α-amino acid.

6. A method according to claim 1 wherein said substimted amino acid is at least one amino acid selected from the group consisting of substimted phenylalanine, substimted tyrosine, substituted O-methyltyrosine and substimted β-alanine.

7. A method according to claim 1 wherein said substimted amino acid is substimted with at least one halogen moiety.

8. A method according to claim 1 wherein said substimted amino acid is substimted with at least one substiment selected from the group consisting of halo- substituted aryl compounds.

9. A method according to claim 1 wherein said substimted amino acid is substimted with at least one substiment selected from the group consisting of alkyl, alkoxy, alkaryl and alkoxyaryl compounds and halo-substituted derivatives thereof.

10. A cryptophycin compound obtained by culturing at least one strain of bacteria capable of producing a native cryptophycin compound in the presence of a substimted amino acid under conditions sufficient to produce at least one cryptophycin compound which is distinct from any such native cryptophycin compound produced by the bacteria under standard culmre conditions.

11. A cryptophycin compound according to claim 10 wherein said bacteria is a cyanobacteria.

12. A cryptophycin compound according to claim 10 wherein said bacteria is at least one species from the genus Nostoc.

13. A cryptophycin compound according to claim 10 wherein said bacteria is at least one strain selected from the group consisting of Nostoc sp. strain GSV 224 (ATCC Accession No. 55483) and strain MB 5357 (ATCC Accession No. 53789).

14. A cryptophycin compound according to claim 10 wherein said substimted amino acid is an α-amino acid.

15. A cryptophycin compound according to claim 10 wherein said substimted amino acid is at least one amino acid selected from the group consisting of substimted phenylalanine, substimted tyrosine, substimted O-methyltyrosine and substimted β-alanine.

16. A cryptophycin compound according to claim 10 wherein said substimted amino acid is substimted with at least one halogen moiety.

17. A cryptophycin compound according to claim 10 wherein said substimted amino acid is substimted with at least one substiment selected from the group consisting of halo-substituted aryl compounds.

18. A cryptophycin compound according to claim 10 wherein said substimted amino acid is substimted with at least one substiment selected from the group consisting of alkyl, alkoxy, alkaryl and alkoxyaryl compounds and halo- substituted derivatives thereof.