Natural Products from Three Nudibranchs: Nembrotha kubaryana, Hypselodoris infucata and Chromodoris petechialis

Nudibranchs are shell-less molluscs that are often brightly colored and seemingly defenseless against predation. However, these beautiful animals usually contain large amounts of diet-derived natural products that help defend them against predation. We have isolated a blue tetrapyrrole from Nembrotha kubaryana, the known nakafuran-8 and -9 from Hypselodoris infucata and spongiane-16-one from Chromodoris petechialis. These compounds have previously been found in other marine organisms, thus supporting a link between diet and natural products in the nudibranchs.


Introduction
It has long been recognized that nudibranchs are infrequently preyed upon even though they have no obvious morphological defense against predation [1]. In particular, the families Chromodoridae and Polyceridae are generally conspicuously colored and have invariably been found to contain compounds identical or similar to those found in their diet [2]. These compounds are accumulated on a functional basis, and it is possible that nudibranchs can be used by chemists to fractionate the best chemical defense compounds from their diet. This biorationale approach suggests that the potent defense compounds may have other uses.

Results and Discussion
As part of our continuing study of natural products from marine invertebrates [3] we have isolated the known though unusual blue pigment 1 from the nudibranch Nembrotha kubaryana (Fig. 1A) † . This pigment has previously been isolated from a mutant strain of the bacterium Serratia marcescens [4], blue marine ascidians [5,6] and a blue bryozoan [7]. Since nudibranchs of the family Nembrothidae are known to feed on either ascidians or bryozoans [8], presumably the metabolite is diet-derived. (3)

Cl
The rather uncommon nudibranch, Chromodoris petechialis ( Fig. 1B) † , yielded three diterpenes. The major was identified as spongian-16-one (2) previously isolated from both New Zealand and Australian specimens of the sponge Chelonaplysilla violacea [9]. A species of Chelonaplysilla occurs in the nudibranch's habitat [10] and this may be the source of 2. The same compound has also been isolated from another chromodorid nudibranch, Chromodoris obsoleta [11].
Compound 1 was found to be a potent antimicrobial agent; active against B. subtilus at 5 µg/disc. Nakafuran-8 and -9 were not found to be antimicrobial, though they have previously been reported as having fish antifeedant properties [12]. Spongian-16-one (2) has been reported as cytotoxic [11]. Too little material was available to perform antimicrobial assays.

Conclusions
Three nudibranchs that have not previously been chemically investigated yielded compounds which appear to be derived from their diets. The two chromodorids yield terpenoids potentially derived from their sponge diet and the nembrothid yielded a blue tetrapyrrole, most likely also derived from its ascidian diet.
These data support the dietary link between nudibranchs and their natural products. High concentrations of the compounds were found and it may be possible to use nudibranchs to indicate which compounds from their diet are most likely to be biologically active.

General
All solvents were reagent grade and redistilled from appropriate drying agents before use. Analytical thin-layer chromatography (TLC) separations were carried out on Merck silica gel 60 F-254 (0.2 mm) precoated aluminum plates. Once developed, plated were visualized by spraying with 5% vanillin in sulfuric acid followed by gentle heating. High-performance liquid chromatography (HPLC) was done on a Waters 6000A solvent delivery system equipped with UV detection (254 nm). Normal phase separations were performed on an Alltech Adsorbosphere® 5 mm column (25 x 0.46 cm) and † Figure 1 that  reverse phase separations on an Adsorbosphere® HS 5 mm (25 x 0.46 cm) column. Nuclear magnetic resonance spectra (NMR) were recorded in CDCl 3 ("100%", Aldrich) on a GE-400 400MHz instrument using the solvent signal as internal standard. Electron impact mass spectrometry (EIMS) was performed on a MAT 311 instrument.

Sample Collection:
One specimen of N. kubaryana [8] was collected from the lagoon of Pohnpei in the Federated States of Micronesia. Seven specimens of H. infucata [13] were collected from Kaneohe Bay, Oahu, Hawaii and one specimen of C. petechialis [13] from Koko Head, Oahu.
One specimen of C. petechialis was extracted in a similar fashion to H. infucata and the hexane extract filter-chromatographed on reverse phase silica (BondElute® 50-100% methanol/water). The fraction eluting with 100% methanol was subject to normal phase HPLC (10% ethyl acetate/hexane) to yield three compounds, the major being identified as 2 (0.