Patellamide studies

Three cyclic octapeptides, patellamides A-C (21-23) were isolated from L. patella and cytotoxicity data for these compounds and for ulicyclamide (15) and ulithiacyclamide (16) against L1210 murine leukaemia cells and the human acute lymphoblastic leukaemia (ALL) cell line CEM were reported [52]. The structures of the patellamides were later reassigned on the basis of synthetic studies. The proposed structures of patellamides B (22) and C (23) were synthesised and the products were shown to differ from the natural products. This led to new structures being proposed [53,54]. Separate syntheses of... 

Three cytotoxic peptides, patellamide D (31) and lissoclinamides 4-5 (32-33) were isolated from a Great Barrier Reef specimen of L. patella and identified by interpretation of spectral data. The peptides were found within the obligate algal symbiont of the genus Prochloron [65]. Another study of the same Australian L. patella reported lissoclinamide 6 (34), in addition to lissoclinamides 4-5 (32-33) and patellamide D (31). The structure of patellamide D (31) was obtained by X-ray crystallography and its conformation compared with those obtained by molecular modelling [66]. Patellamide D (31) has been reported to be a... 

Figure 1 What are natural products Tryptophan and two molecules derived from it are shown. Psilocin is a natural product, while serotonin s categorization depends on the context in which it is studied. Podophyllotoxin and camptothecin are natural products from trees and patellamide C is from an ascidian however, all three are actually produced by a microbial endosymbiont.

Ascidians are marine filter feeders with a rich natural products chemistry that five commonly associated with symbiotic bacteria (88, 89, 109). A well-studied symbiosis consists of photosynthetic Prochloron spp. cyanobacteria that occur in ascidians of the family Didemnidae (110). Prochloron spp. also can be found in bacterial mat structures of stromatoliths (111) but so far have not been detected outside of such structured environments. From didemnid ascidians, numerous cytotoxic cyclic peptides of the patellamide group (Fig. 5) were isolated (109, 112, 113). Mechanical separation of the Prochloron sp. symbiont from its host Lissoclinum patella and subsequent genome sequencing revealed a set of biosynthetic genes that after transfer into E. coli enabled this bacterium to produce two different patellamides (114). The genes also were identified in an independent study by screening a library of Prochloron sp. DNA... 

Circular dichroism (CD) is the ideal technique for the study of small changes in the chiral environment of UV chromophores. Previous studies have taken advantage of the conformational change concomitant with the binding of the patellamides to divalent metal ions. One obvious advantage of using CD is that only very small amounts of sample (<1 mg) are required making it an ideal technique for the small amounts of compounds isolated from L patella. 

MASS SPECTROMETRIC STUDIES OF THE METAL BINDING OF PATELLAMIDE A AND C... 

As was seen in the CD competition study (Section 3.4) copper displaced zinc from patellamide C and when a mixture of the two metals was added only copper species were formed. When copper was added to a solution containing predominantly the two zinc complex [PatCfij + Zn2Cl3] at m/z 995 formed by adding a large excess of zinc, this was displaced and the two copper complex [PatCHa + Cu2Cl2] at m/z 957 became the dominant species present. A further competition experiment was carried out with copper bound patellamide C in the presence of acid. The solution of copper and patellamide C was diluted with 2% formic acid and then introduced into the mass spectrometer. No copper species of any kind were detected because the amide NH s were fully protonated at low pH so no binding to Cu was possible. 

The study of these peptides and their copper and zinc complexes by mass spectrometry revealed much information about their composition and binding characteristics. The most interesting results were those from the study of patellamide C and its copper complexes. This revealed the formation of complexes, such as [PatCH2 + 2Cu + (CsHsOs)] which were not detected by CD. This species might be similar to the copper complex of ascidiacyclamide, which was shown by X-ray crystallography to have a bridging carbonate between the two copper atoms. ... 

In total 64 nOe restraints were obtained from the T-ROESY spectrum in CDCI3 of patellamide C (3), and the most important of these are indicated in Figure 8. Usually 8-10 nOe restraints per amino acid residue are required to model a peptide, but the cyclic nature of the compounds under study reduces the number of degrees of freedom and therefore the number of nOes required for the resulting conformation to be reliable. 

In all extractions of L. patella, the major compounds isolated are always cyclic peptides, with one of the patellamides usually the most abundant of these. " There has been only one reported case of L. patella which did not have Prochloron as its symbiont. When this sample was extracted no cyclic peptides were isolated. This supports the case for their being produced by Prochloron rather than the host. The production of these compounds, by either the host or symbiont, is costly to the internal metabolism of the producing organism and they must therefore play some ecological role. The selectivity and structural studies show these peptides are most suited to bind copper. Copper is therefore proposed to be the ecologically relevant metal for the patellamides. It is possible that the patellamide/copper complexes might be co-factors in enzymatic processes. We will discuss a few further possibilities here. 

Are they involved in the activation and mobilisation of CO2 Van den Brenk et al. suggested this from their studies of patellamide D and ascidiacyclamide, which formed carbonate bridged 2Cu complexes. They suggested that these complexes fix CO2 for use in the formation of CaCOs (used in the internal skeleton of the tunicate).. One argument against this is that marine photosynthetic organisms fix HCOs which is plentiful in seawater (0.002 M). The proposed process, CO2 -> HCOa is therefore the reverse of that expected. 

Are they a form of carbonic anhydrase Carbonic anhydrases (CAs) play an important role in photosynthetic carbon fixation, converting HC03 (aq) in seawater to CO2. It is possible that the copper complexes of these peptides perform this function. Evidence from the studies by van den Brenk et al show that patellamide D (4) forms [PatDH + Cu2 + C02]" and [PatDHa + Cui + COa] complexes in the mass spectrometer lending credence to this proposal. 

Do they have catalytic activity Type 3 copper proteins have two copper atoms in close proximity (3.6A), and have roles in oxygen transport and uptake, and as oxidising agents. In ascidiacyclamide/Cu2 the two copper atoms are 4.5 A apart. The formation of the C3Hs03 unit observed during our studies with patellamide C/Cu2 might be the result of catalytic activity of this system. 

Are they involved in carbon metabolism There are many candidates in the carbon metabolism of Prochloron that could be converted to the C3Hs03 moiety of the 2Cu patellamide C complex. Studies have shown that L. patella actively removes glycolate to encourage photosynthesis by prochloron Are the copper complexes of these cyclic peptides involved as enzymic cofactors in this process ... 

Other ESR studies include 2,2 -diamino-4,4 -bithiazole and 2,2 -bis(acetamido)-4,4 -bithiazole copper(ll) complexes <2000SRI1653>, patellamide A derivatives, and their copper(ii) compounds <2002CEJ1527>, endothelial nitric oxide synthase and its reaction with thiazole-containing ligand <1996JBC32563>. 

Metagenomic approaches have been used to study metabolites produced by bacterial symbionts of marine animals, insects, and nematodes. The literature now provides several examples where DNA-based strategies targeting specific genetic loci have enabled the isolation and cloning of biosynthetic gene clusters encoding for the biosynthesis of important symbiont-associated molecules such as pederin, onn amide A, bryostatins, trunkamide, theopalauamide, and the patellamides. These studies are the focus of another chapter in this series and therefore will not be discussed here. 

In a study of Lissodinum patella harvested in Palau, patellamides A, B and C were not present in the Prochloron sp. symbionts but were scattered in the tunic of the ascidian (Salomon and Faulkner, 2002 Piel, 2004). Later, genetic studies established that patellamides A and C were produced by symbiotic Prochloron (Prochloron didemni) that were isolated from the ascidian and were shown to contain the necessary genes for the formerly considered non-ribosomal but, actually, post-translationally modified ribo-somal biosynthesis of these peptides (Schmidt, Sudek, and Haygood, 2004 Schmidt et al., 2005 Milne et al., 2006 Oman and van der Donk, 2010). 

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