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Structure and Biosynthesis of the Jamaicamides, New Mixed Polyketide-Peptide Neurotoxins from the Marine Cyanobacterium Lyngbya Majuscula

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Structure and Biosynthesis of the Jamaicamides, New Mixed Polyketide-Peptide Neurotoxins from the Marine Cyanobacterium Lyngbya Majuscula Chemistry & Biology, Vol. 11, 817–833, June, 2004, 2004 Elsevier Ltd. All rights reserved. DOI 10.1016/j.chembiol.2004.03.030 Structure and Biosynthesis of the Jamaicamides, New Mixed Polyketide-Peptide Neurotoxins from the Marine Cyanobacterium Lyngbya majuscula Daniel J. Edwards,1 Brian L. Marquez,1 While the biological activities reported for marine cy- Lisa M. Nogle,1 Kerry McPhail, anobacterial metabolites vary widely, three major trends Douglas E. Goeger, Mary Ann Roberts, emerge. A number of these metabolites target either the and William H. Gerwick* polymerization of tubulin (e.g., dolastatin 10 [4], curacin College of Pharmacy A [5]) or the polymerization of actin (e.g., hectochlorin [6], Oregon State University majusculamide C [7]). Additionally, a growing number Corvallis, Oregon 97331 of potently bioactive metabolites from cyanobacteria target the mammalian voltage-gated sodium channel, either as blockers (kalkitoxin [8]) or activators (antilla- Summary toxin [9]). Hence, we have employed a simple cell-based screen of marine cyanobacterial extracts and com- A screening program for bioactive compounds from pounds for detecting new neurotoxins that modulate marine cyanobacteria led to the isolation of jamai- the activity of this important ion channel [10]. This ap- camides A–C. Jamaicamide A is a novel and highly proach has been fruitful, and we report here the results functionalized lipopeptide containing an alkynyl bro- that followed from initial detection of neurotoxic activity mide, vinyl chloride, ␤-methoxy eneone system, and in a Jamaican collection of Lyngbya majuscula. Because pyrrolinone ring. The jamaicamides show sodium of the unusual structures of the isolated compounds, channelblocking activity and fish toxicity. Precursor jamaicamides A–C (Figure 1), we have been motivated feeding to jamaicamide-producing cultures mapped to examine their biosynthetic origins in considerable out the series of acetate and amino acid residues and detail. Isotope-labeling experiments with producing lab- helped develop an effective cloning strategy for the oratory cultures identified the component building biosynthetic gene cluster. The 58 kbp gene cluster is blocks, whereas their assembly has been partially eluci- composed of 17 open reading frames that show an dated by a molecular genetics approach. In this work, exact colinearity with their expected utilization. A we report the discovery, structure elucidation, biological novel cassette of genes appears to form a pendent properties, biosynthetic subunits, and sequence of the carbon atom possessing the vinyl chloride functional- gene cluster encoding for the biosynthesis of jamai- ity; at its core this contains an HMG-CoA synthase- camides A–C. like motif, giving insight into the mechanism by which this functional group is created. Results and Discussion Introduction Isolation and Structure Elucidation of the Marine life forms continue to provide some of the most Jamaicamides A, B, and C structurally intriguing and biologically active natural A dark green strain of Lyngbya majuscula was found products, several of which are contributing to a very growing in low abundance in Hector’s Bay, Jamaica active pipeline of pharmaceutical agents [1]. Among (designated strain JHB). A small sample preserved at the these organisms, marine cyanobacteria (blue-green al- time of collection was extracted and shown to possess gae) are quite possibly the most exciting in their produc- potent brine shrimp toxicity. Additional effort ultimately tion of new chemotypes with important biological activ- was successful in adapting this organism to laboratory ity [2]. The major metabolic theme employed by marine culture. Pan cultures yielded approximately 10 g/l bio- cyanobacteria to construct these natural products inte- mass after 6 weeks, and this was extracted by standard grates two modular biosynthetic systems, nonribosomal methods for lipid natural products. Again, the extract peptide synthetases (NRPSs), which are responsible for was strongly bioactive in the brine shrimp model, and assembling amino acids, and polyketide synthases this was used to direct the isolation of hectochlorin (1), (PKSs), for linking together acetate as the primary build- a novel cyclic peptide with potent actin-polymerizing ing block. Inherent to this mixed NRPS/PKS biosynthe- properties [6]. Further evaluation of the extract and its sis is great structural diversity, and this is further com- primary vacuum liquid chromatographic (VLC) fractions pounded by a truly extraordinary number of secondary identified a midpolarity mixture with activity in a cellular tailoring manipulations, including oxidation, methyla- assay designed to detect mammalian voltage-gated so- tion, and diverse forms of halogenation. The study of dium channel-blocking substances [10]. This fraction these cyanobacterial metabolites and their biosynthetic was also identified as possessing potentially novel sub- origins has great value not only for drug discovery but stances by TLC and 1H NMR analysis. Subsequent HPLC also for harnessing these pathways to create new mole- of this fraction led to the isolation of three new lipopep- cules of biomedical utility [3]. tides with low micromolar levels of channel-blocking activity. The HRFABMS of jamaicamide A (2) yielded an *Correspondence: [email protected] ϩ ϩ 1 These authors contributed equally to this work (D.J.E., cloning and [M H] for a molecular formula of C27H37N2O4ClBr (m/z biochemistry; B.L.M., isolation and structure; L.M.N., biosynthetic 567.1625, Ϫ0.7 mmu dev.). The isotope peaks at m/z precursor studies). 567/569/571 (4:5:1.5 ratio) were consistent with the pres- Chemistry & Biology 818 Figure 1. Structures of Metabolites Isolated from Jamaican Strain of Lyngbya majuscula and Assembly of Partial Structures A–F of Jamaica- mide A (A) Structures of hectochlorin and jamaicamide A–C, four metabolites isolated from this Jamaican strain of Lyngbya majuscula. (B) Partial structures A–F of jamaicamide A (2) derived from 1H-1H COSY, 1H-13C HSQC, HSQC-COSY, and 1H-13C HMBC data. 3 (C) Assembly of partial structures A–F in jamaicamide A (2). Double-headed arrows represent JHH couplings, single-headed arrows represent 2,3 1 15 important JCH couplings from HMBC, dashed arrows represent key H- N HMBC correlations, and bolded arrow represents a key ACCORD-1, 1-ADEQUATE correlation (H-18 to C-19). The boxed letters indicate the original partial structure designations. ence of one chlorine and one bromine atom in the mole- the latter of which was located proximately, as revealed cule, and the molecular formula indicated 10 degrees by a correlation observed by Accordion 1,1-ADEQUATE of unsaturation. Comparison of the 13C NMR and DEPT [13]. This variant of the ADEQUATE experiment, created spectra identified jamaicamide A to possess 3 methyl in the course of the present investigation, comprehen- groups, 9 methylenes, 8 methines, and 7 quaternary sively detects 1H-13C-13C spin systems for a wide range carbon atoms. of 13C-13C coupling values. Moreover, when this data set Analysis of the HSQC [11] and HSQC-COSY (HSQC- is used in conjunction with that obtained from HMBC, TOCSY with 12 ms DIPSI mixing time) [12] spectra al- it allows for unequivocal discrimination between 2- and lowed construction of six partial structures (A–F, Figure 3-bond HMBC correlations. The shifts for this highly 1). By 1H-1H COSY, partial structure A was composed polarized and substituted system closely match those of a modestly deshielded doublet methyl group (␦1.46) for a similar arrangement of atoms observed in the cyan- adjacent to three contiguous methine protons. The first obacterial metabolite barbamide [14]. Selective irradia- of these was at a shift (␦4.87) consistent with its being tion of the protons at ␦3.75 (H3-25) using the DPFGSE attached to a carbon bearing a heteroatom (␦C 58.1) and 1D NOE experiment resulted in a strong NOE enhance- showed both vinylic and allylic couplings to an adjacent ment of H-18 (␦6.73), indicating 17(E) geometry in jamai- polarized double bond (␦7.22 and 6.08), the shifts of camide A (2, Figure 1) [15, 16]. which were indicative of an enone functionality. This Partial structure C was composed of two mutually was confirmed by HMBC, which identified an adjacent coupled and therefore adjacent midfield methylenes, carbonyl carbon at ␦170.0, a chemical shift consistent one of which was also a portion of partial structure B with an amide or ester group. Partial structure B was (H2-16), and the other (␦3.49 and 3.53) was coupled to composed of a deshielded singlet proton (H-18, ␦H 6.73; an amide NH proton (␦6.68). A series of contiguous meth- ␦C 95.0), which showed reciprocal HMBC correlations ylene and methine groups, plus one branching methyl to an O-methyl group (␦H 3.75; ␦C 56.1) and an allylic group, delineated partial structure D. A triplet-appearing methylene group (␦H 2.84, 3.00). This proton, H-18, also methylene group at ␦2.18, a chemical shift consistent showed an HMBC correlation to a deshielded olefinic with an adjacent carbonyl, was sequentially adjacent to carbon (␦C 175.3), and this latter signal showed HMBC an allylic methylene (␦2.28), a trans disubstituted double correlations from both the O-methyl group and H2-16 bond (J ϭ 15.1 Hz), a methine (␦2.02) possessing a methylene. The extreme level of double bond polariza- branching doublet methyl group (␦0.94), and then two tion was explained by the combination of vinyl methoxy additional methylene groups (␦1.34 and 2.00).
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