Lenize F. Maia

Raman spectroscopy as a tool in differentiating conjugated polyenes from synthetic and natural sources.

This work presents the Raman spectroscopic characterization of synthetic analogs of natural conjugated polyenals found in octocorals, focusing the unequivocal identification of the chemical species present in these systems. The synthetic material was produced by the autocondensation reaction of crotonaldehyde, generating a demethylated conjugated polyene containing 11 carbon-carbon double bonds, with just a methyl group on the end of the carbon chain. The resonance Raman spectra of such pigment has shown the existence of enhanced modes assigned to ν₁(CC) and ν₂(CC) modes of the main chain. For the resonance Raman spectra of natural pigments from octocorals collected in the Brazilian coast, besides the previously cited bands, it could be also observed the presence of the ν₄(CCH₃), related to the vibrational mode who describes the vibration of the methyl group of the central carbon chain of carotenoids. Other interesting point is the observation of overtones and combination bands, which for carotenoids involves the presence of the ν₄ mode, whereas for the synthetic polyene this band, besides be seen at a slightly different wavenumber position, does not appear as an enhanced mode and also as a combination, such as for the natural carotenoids. Theoretical molecular orbital analysis of polyenal-11 and lycopene has shown the structural differences which are also responsible for the resonance Raman data, based on the appearance of the (CH3) vibrational mode in the resonant transition only for lycopene. At last, the Raman band at ca. 1010 cm(-1), assigned to the (CH₃) vibrational mode, can be used for attributing the presence of each one of the conjugated polyenes: the resonance Raman spectrum containing the band at ca. 1010 cm(-1) refers to the carotenoid (in this case lycopene), and the absence of such band in resonance conditions refers to the polyenal (in this case the polyenal-11).

Raman spectroscopic study of antioxidant pigments from cup corals Tubastraea spp.

Chemical investigation of nonindigenous Tubastraea coccinea and T. tagusensis by Raman spectroscopy resulted in the identification of carotenoids and indolic alkaloids. Comparison of Raman data obtained for the in situ and crude extracts has shown the potential of the technique for characterizing samples which are metabolic fingerprints, by means of band analysis. Raman bands at ca. 1520, 1160, and 1005 cm(-1) assigned to ν1(C═C), ν2(C-C), and ρ3(C-CH3) modes were attributed to astaxanthin, and the band at 1665 cm(-1) could be assigned to the ν(C-N), ν(C-O), and ν(C-C) coupled mode of the iminoimidazolinone from aplysinopsin. The antioxidant activity of the crude extracts has also been demonstrated, suggesting a possible role of these classes of compounds in the studied corals.

Identification of Shell Colour Pigments in Marine Snails Clanculus pharaonius and C. margaritarius (Trochoidea; Gastropoda)

Colour and pattern are key traits with important roles in camouflage, warning and attraction. Ideally, in order to begin to understand the evolution and ecology of colour in nature, it is important to identify and, where possible, fully characterise pigments using biochemical methods. The phylum Mollusca includes some of the most beautiful exemplars of biological pigmentation, with the vivid colours of sea shells particularly prized by collectors and scientists alike. Biochemical studies of molluscan shell colour were fairly common in the last century, but few of these studies have been confirmed using modern methods and very few shell pigments have been fully characterised. Here, we use modern chemical and multi-modal spectroscopic techniques to identify two porphyrin pigments and eumelanin in the shell of marine snails Clanculus pharaonius and C margaritarius. The same porphyrins were also identified in coloured foot tissue of both species. We use high performance liquid chromatography (HPLC) to show definitively that these porphyrins are uroporphyrin I and uroporphyrin III. Evidence from confocal microscopy analyses shows that the distribution of porphyrin pigments corresponds to the striking pink-red of C. pharaonius shells, as well as pink-red dots and lines on the early whorls of C. margaritarius and yellow-brown colour of later whorls. Additional HPLC results suggest that eumelanin is likely responsible for black spots. We refer to the two differently coloured porphyrin pigments as trochopuniceus (pink-red) and trochoxouthos (yellow-brown) in order to distinguish between them. Trochopuniceus and trochoxouthos were not found in the shell of a third species of the same superfamily, Calliostoma zizyphinum, despite its superficially similar colouration, suggesting that this species has different shell pigments. These findings have important implications for the study of colour and pattern in molluscs specifically, but in other taxa more generally, since this study shows that homology of visible colour cannot be assumed without identification of pigments.

Conjugated polyenes as chemical probes of life signature: use of Raman spectroscopy to differentiate polyenic pigments.

Polyenes, which are represented by carotenes, carotenoids and conjugated polyenals, are some of the most important targets for astrobiology, because they can provide strong evidence of the presence of organic compounds in the most extreme environments, such as on Mars. Raman spectroscopy has been used as the main analytical tool in the identification of such compounds, for the greatest variety of living species, from microorganisms to animals and plants. However, using only the position of the characteristic Raman bands can lead to errors in tentatively identifying chemicals. In this work, we present a series of observations that can provide a more complete and robust way to analyse the Raman spectrum of a polyenal, in which the position, the intensity, the use of various laser lines for excitation, and the combination of more than one pigment can be considered in the complete analysis.

Chapter 10 - New Strategies for Identifying Natural Products of Ecological Significance from Corals: Nondestructive Raman Spectroscopy Analysis

Abstract Coral tissues commonly contain high concentrations of secondary or special metabolites. These chemicals are identified by spectroscopic analysis after extraction and purification procedures. A new alternative to the conventional techniques is the use of Raman spectroscopy, which is a nondestructive method of analysis suitable to in situ studies of biomaterials as well as chemical compounds. This approach has been successfully demonstrated with corals from the Brazilian coast. Diverse ecological roles have been investigated in crude extracts and pure compounds from the indigenous species Phyllogorgia dilatata , Leptogorgia punicea , L. violacea , and Carijoa riisei and the nonindigenous species Chromonephthea braziliensis , Tubastrea coccinea , and T. tagusensis . Compounds with possible roles in these bioactivities have been quickly characterized by Raman macro- and microspectroscopy: a feeding deterrent sterol from C. braziliensis ; carotenoids from L. violacea , C. riisei , P. dilatata , and Tubastrea spp.; and polyenals from Leptogorgia spp. and C. braziliensis and from purple spots in P. dilatata affected by purpling . Most of the chemical constituents could be identified in tissues and sclerites without the need for any previous treatment. Several Raman vibrational modes are used to correlate chemical species to a chemical substance. The polyenal biochromes could be identified by two “fingerprint” vibrational bands. A complementary analysis can be performed by Raman microimaging, which is a sensitive and selective method of investigating the distribution of compounds in both in situ and in vivo samples. The results obtained by the Raman microimaging studies of chemical compounds present in the surface of healthy and necrotic tissues of P. dilatata may be helpful in evaluating different stages and the extent of infection that causes purpling. Competition for space, antifouling defense, and chemical deterrence are the three main roles traditionally attributed to the chemically mediated bioactivity in corals. Raman spectroscopy may become a valuable nondestructive technique for monitoring the accumulation or production of metabolites during a biological interaction. It also allows information about the distribution and concentration as well as molecular information to be obtained in a very short period of time compared to the usual time and solvent-consuming separation procedures.