Kerr, Russell G.

9,11-Secogorgosterol, a secondary metabolite from the gorgonian Pseudopterogorgia americana, exhibits inhibitory activity against protein kinase C, and potent anti-proliferative and anti-inflammatory activity. An efficient method for the production of 9,11-secogorgosterol has been developed and optimized using an enzyme extract from the gorgonian P. americana. The gorgonian also produces two other 9,11-secosteroids which have marked differences in their side chains and nuclei, which suggested that the enzymes responsible for their production were likely relatively nonspecific. Novel 9,11-secosteroids have been synthesized using the enzyme extract from the gorgonian.

Cephalostatin 1 has been isolated from the colonial marine worm Cephalodiscus gilchristi (Phylum Hemichordata) in the Indian Ocean. It has shown biomedical potential in preclinical trials as a cell growth inhibitor, active against U. S. murine P388 lymphocyte leukemia ED5010^-7-10^-9 μ/ml and a potent growth inhibitor against solid tumor types. Our research is focusing on developing methodology to produce disteroidal pyrazine and the introduction of delta 14, 15 unsaturation and C-16 hydroxyl groups in Cephalostatin 1A. The synthetic intermediates may be useful for biosynthetic studies and also analogues can be used to study structure-activity relationships.

Ecteinascidin 743 is a trace secondary metabolite isolated from the marine tunicate, Ecteinascidia turbinata. Ecteinacidin 743 a most potent antitumor agent, is currently in Phase II clinical trials in Europe and in the USA. A cell-free extract of Ecteinascidia turbinata was used to investigate the biogenetic origin of the ecteinascidins. Incubation experiments with radiolabeled diketopiperazines indicated that the diketopiperazine of tyrosine is the first committed intermediate in the biosynthesis of ecteinascidins. Phenylalanine diketopiperazine was not transformed into the ecteinascidins indicating that this cyclic dipeptide is not an intermediate in the biosynthesis of ecteinascidins. The diketopiperazine of DOPA was used as a cold carrier demonstrating that the diketopiperazine of tyrosine is oxidized to DOPA diketopiperazine and then further transformed to the ecteinascidins.

The ecteinascidins are a family of marine derived alkaloids with potent antitumor activity. Ecteinascidins 743 and 729 are the prime targets due to their superior antitumor activity. beta-Mercaptopyruvic acid is a proposed intermediate in the biosynthesis of ecteinascidins 743/729. A novel synthesis of beta-mercaptopyruvic acid using an enzymatic system was achieved. This was accomplished by trapping the equilibrium between beta-mercaptopyruvic acid and 2,5-dihydroxy-1,4-dithiane-2,5-dicarboxylate using oxidative deamination of cysteine by amino acid oxidase.

9,11-Secosteroids are biologically active secondary metabolites from the marine invertebrate, Pseudopterogorgia americana. They are produced in vitro through an incubation of the steroid substrate with a cell-free extract of P. americana containing the necessary enzymes. Further optimization of this incubation was attempted through adjustment of incubation reagents and cofactors. The enzymes responsible for secosteroid production were partially purified through chromatography in an effort to isolate and purify these enzymes. Finally, experiments with radiolabeled gorgosterol led to the elucidation and isolation of a key secosteroid precursor, 9,11-dihydroxygorgosterol.

Stevensine is a biologically active secondary metabolite produced by several marine sponges, including Teichaxinella morchella. Two in vitro methods were used to investigate the biosynthetic origin of stevensine in the sponge T. morchella. A cell-free enzyme extract was developed to test for the incorporation of the potential radiolabeled metabolic precursors histidine, arginine, ornithine, and proline. Secondly, cell cultures of T. morchella were used to determine the biosynthetic origin of stevensine from the radiolabeled amino acids histidine, arginine, ornithine, and proline. Histidine and ornithine/proline were converted to stevensine in the cell culture system. This represents the first study involving the use of cell cultures of a marine invertebrate to investigate the biosynthesis of a biologically active natural product. An understanding of the biosynthetic process leading to the production of this compound in the source organism could lead to the development of more efficient and environmentally safer production methods.

Pseudopterosin C is a novel diterpene glycoside isolated from the Caribbean gorgonian coral, Pseudopterogorgia elisabethae. Pseudopterosin C is non-steroidal and has been shown to exhibit promising anti-inflammatory and analgesic properties. The in vitro production of pseudopterosin C through biosynthetic methods has been examined. A cell-free system as well as an enzyme precipitate capable of transforming terpene precursors to the product has been developed. The parameters toward optimizing the enzymatic production through cofactor requirements, differing terpene precursors and sugars were investigated.

The fatty acid synthetase (FAS) from the marine invertebrate Bugula neritina was isolated using cold ethanol precipitation followed by the sequence of G-100 and G-200 size exclusion columns. Native gel analysis indicates the isolation of the FAS and the elution volume from the G-100 column suggests the FAS to be ~282kd. One band with a molecular weight of 66 kDa appeared on the SDS gel of the G-200 sample (F17-30) that eluted at 52.5 mL. The G-200 sample that eluted at 19.2 mL (F1-6) displayed two predominant bands on the SDS gel corresponding to molecular weights 66 kDa and 97 kDa. Both F1-6 and F17-30 showed FAS activity displaying de novo production of myristic and palmitic acids. From the sequence of purification starting from the cell-free extract (CFE) to the F17-30 sample, a 240 fold increase in specific activity was observed. The Type II FAS experiments showed no substantial evidence of activity, namely of the beta-Hydroxybutyryl acyl-dehydrase and the enoyl reductase enzymes.

9,11-Secosteroids exhibit novel biological activity and interesting pharmacological properties. A biosynthetic study of 9,11-secogorgosterol and other novel 9,11-secosteroids using an enzyme extract from the gorgonian Pseudopterogorgia americana demonstrated that this system is an efficient catalyst for a wide range 9,11-secosteroid production. Two methods to recover the enzyme from 9,11-secosteroid synthesis have been developed: (1) membrane-enclosed enzymatic catalysis, which combines certain of the advantages of soluble enzymes and immobilized enzymes, shows high activity, simple recovery and at least 5 cycles or 23 days; (2) covalently bonded enzyme-agarose gel, attached by cyanogen bromide via the amino groups of enzymes, show 80% activity of free enzymes and at least 7 cycles or 46 days. Based on stability and kinetic studies of these immobilized enzymes in 9,11-secosteroid synthesis, a continuous flow column reactor for scale-up of 9,11-secosteroid production has been developed with great efficiency, which represents the first enzymatic production of a bioactive compound from a marine invertebrate on a large scale.

A family of tetrahydroisoquinoline alkaloids, the ecteinascidins, are a group of biologically active secondary metabolites produced by the marine tunicate Ecteinascidia turbinata. Ecteinascidins have shown in vivo anti-tumor activity against P388 lymphoma, B16 melanoma, M5076 ovarian sarcoma, Lewis lung carcinoma, and LX-1 human lung and MX-1 human mammary carcinoma xenografts in laboratory mice. Because ecteinascidins are produced in low yields, 1x10^-4%, supply for clinical development is a significant problem. The ultimate goal of this study is to develop an enzyme-based synthesis of the ecteinascidins. In this regard, the biosynthesis of these alkaloids has been investigated. Optimal conditions for in vitro ecteinascidin biosynthesis were found. The origin of the C22-C1 two-carbon unit was identified as pyruvate and the tyrosine and DOPA diketopiperazines were identified as key intermediates. Methods were developed for an in-the-sea aquaculture of the colonal marine ascidian Ecteinascidia turbinata.