Charles F. Hirsch

Pharmaceuticals from cultured algae

SummaryAn algae screening program, including cultured macroalgae, cultured cyanobacteria and cultured eukaryotic microalgae has been undertaken. Methods for the isolation, purification, preservation and cultivation of axenic cyanobacteria and eukaryotic cultures have been developed. Screening of these groups for biologically active components has lead to the isolation of pachydictyol and caulerpenyne from cultured macroalgae, while a series of hapalindoles and an antifungal depsipeptide have been isolated from cyanobacteria.

Bioconversion of indene to cis (1S,2R) indandiol and trans (1R,2R) indandiol by Rhodococcus species

Abstract cis (1 S ,2 R ) indandiol or trans (1 R ,2 R ) indandiol are both potential precursors to (−)- cis (1 S ,2 R )-1-aminoindan-2-ol, a key chiral synthon for Crixivan ® (Indinavir), a leading HIV protease inhibitor. Enrichment and isolation studies yielded two Rhodococcus sp. strain B 264-1 (MB 5655) and strain I-24 (MA 7205) capable of biotransforming indene to cis (1 S ,2 R ) indandiol and trans (1 R ,2 R ) indandiol respectively. Isolate MB 5655 was found to have a toluene dioxygenase, while isolate MA 7205 was found to harbor both toluene and naphthalene dioxygenases as well as a naphthalene monooxygenase. When scaled up in a 14- l bioreactor, MB 5655 produced up to 2.0 g/ l of cis (1 S ,2 R ) indandiol with an enantiometric excess greater than 99%. MA 7205 cultivated under similar conditions produced up to 1.4 g/ l of trans (1 R ,2 R ) indandiol with an enantiomeric excess greater than 98%. Process development studies yielded titers greater that 4.0 g/ l of cis indandiol for MB 5655. Due to their resistance to indene toxicity and easy cultivation in bioreactors, both Rhodococcus sp. strains appeared as good candidates for future strain engineering and process development work.

Use of polymerase chain reaction (PCR) fingerprinting to differentiate bacteria for microbial products screening.

PCR fingerprinting offers a practical molecular means to quickly and reliably differentiate bacteria for microbial products screening. A combination of low resolution and high resolution PCR fingerprinting provides a hirarchical system which allows the discrimination of bacteria at species and subspecies level within 7 h. DNA was extracted from cells by incubating them in water at 95°C for 30 min. A sample of 1 μl of the cell-free aqueous extract then was used as a source of template DNA in the PCR. The PCR products were separated by electrophoresis on an acrylamide gel and visualized by ethidium bromide staining. The band patterns generated for each different culture were unique, reproducible, and independent of cultivation conditions. Band patterns may be compared visually or by using imaging and pattern matching software. In our laboratory, bacteria such as actinomycetes, Gram-negative and Gram-positive soil eubacteria, and photosynthetic non-sulfur bacteria have been differentiated using PCR fingerprinting.

Actinoplanic acids A and B as novel inhibitors of farnesyl-protein transferase

Actinoplanic acids A and B are macrocyclic polycarboxylic acids that are potent reversible inhibitors of farnesyl-protein transferase. Actinoplanic acids A and B were isolated from Actinoplanes sp. MA 7066 while actinoplanic acid B was isolated from both MA 7066 and Streptomyces sp. MA 7099. Actinoplanic acids A and B are competitive with respect to farnesyl diphosphate and are selective inhibitors of farnesyl-protein transferase because they do not inhibit geranylgeranyl-protein transferase type 1 or squalene synthase. MA 7066 is believed to be a novel species of actinomycetes while MA 7099 is believed to be a novel strain of Streptomyces violaceusniger on the basis of morphological, biochemical and chemotaxonomic characteristics as well as its production of actinoplanic acids.

Bioconversion of the sodium salt of Simvastatin (MK-733) to 6-desmethyl-6-α-hydroxymethyl Simvastatin

SummaryAn actinomycete (MA 6474, ATCC 53828) isolated from a soil sample (Mutare, Zimbabwe) was found to biotransform the sodium salt of Simvastatin (MK-733) to 6-α-hydroxymethyl MK-733, 6-β-hybroxymethyl MK-733, and 6-ring-hydroxy MK-733. The bioconversion efficiency to the desired compound, 6-α-hydroxymethyl MK-733, was enhanced by optimizing the physico-chemical parameters of the process. In shake flask cultures, addition of magnesium (0.125 mg/l Mg SO4·7H2O) to the medium resulted in a five-fold increase in the rate of bioconversion to the α diastereomer. The ratio of bioconversion products (6-α-hydroxymethyl, 6-β-hydroxymethyl, and 6-ring-hydroxy MK-733) was regulated by pH. Process improvements and scale up in 23-1 fermentors, which consisted of a controlled addition of substrate (MK-733), resulted in a 2-fold increase in alpha diastereomer Production (42 vs. 79 U/ml) and a 23-fold rate increase in the formation of α-diastereomer. A high diastereomeric ratio (α: β=9∶1) facilitated downstream processing.

Serratiomycin, a new antibacterial peptolide from an eubacterium culture, MB 5691

Abstract A novel peptolide antibiotic, serratiomycin, 1 , was isolated from the culture broth of a eubacterium by solvent extraction and column chromatography. The structure of 1 was determined to be cyclo( d -β-hydroxydecanoyl- l -leucyl- l -seryl- l - allo threonyl- d -phenylalanyl- d -isoleucyl). 1 exhibited a weak and mostly Gram-positive antibacterial spectrum in vitro .