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28th seminar SMU: «Synthesis luciferin luminescent worm Fridericia heliota and its analogs»

Bioluminescence - a phenomenon of light emission by living organisms. The main role in this process is played by the enzyme - luciferase and a substrate called luciferin, which takes place during the oxidation of education oxyluciferin in an excited state, followed by the emission of visible light. This phenomenon is quite common in nature - today there are about 700 genera of living beings belonging to 17 types with 4 kingdoms, whose representatives are capable of bioluminescence. It is known that there are about 30 different chemical mechanisms by which the action of living beings can emit light. And yet, scientists still open more and more new species of bioluminescent systems.

One of them - a unique bioluminescent system oligochaetes Fridericia heliota - has been described by members of the Total Synthesis Lab Institute of Bioorganic Chemistry of the Shemyakin and Ovchinnikov of the Russian Academy of Sciences (IBCh RAS) in cooperation with the Laboratory biomolecular NMR-spectroscopy . On how scientists were able to do it, said one of the participants in this study, graduate student Alexander Tsar'kova the 28th seminar SMU IBCh: «Synthesis of luciferin luminescent worm Fridericia heliota and its analogs».

«Despite the fact that according to current estimates, there are about 30 different chemical mechanisms of bioluminescence», - says Alexandera Tsar'kova – «until recently was known only seven luciferin, and the structure of the last of them was deciphered about 25 years ago - it was a luminous substance armored Dinoflagellata.

If we talk in general about the shining representative of the class Oligochaeta, then for a long time there was the concept of a unified nature of the luminescence of these creatures. It was based on the results of a comparative study of the biochemistry of twelve species of oligochaetes. All of them are secreted bioluminescent mucus. It is interesting that all investigated oligochaete biolyuminiestsentsiya characterized by one common feature - it takes an active part hydrogen peroxide. Also note that the only luciferase and luciferin Oligochaeta, whose structure is currently installed - an enzyme and a substrate worm Diplocardia longa - enter into cross reaction with other eleven luciferin and luciferase Oligochaeta.

With regard to the phenomenon of bioluminescence soil oligochaetes from the family Enchytraeidae, limited delivery Henlea and Fridericia, until recently it remained virtually unexplored. It all started with the fact that recently our colleagues from Krasnoyarsk, Petushkov Valentin and Natalia Rodionova discovered a new species from the genus Fridericia - this is the Fridericia heliota. This is a small white and yellow worms that glow blue light in response to mechanical stimulation. Note that bioluminescence Fridericia heliota differ from those of previously studied Oligochaeta - it is localized in the epidermal cells, and no luminous mucus is not released. If after mechanical stimulation to look at this worm in the dark, it becomes a point of light throughout the body, which itself remains dark.

Fridericia heliota fluorescent system has five components is a very luciferin, luciferase, ATP, magnesium ions and oxygen. It should be noted that the isolation and identification of all the components of this system has been very difficult due to the fact that the data has been difficult to search worms, and because of the small amount of luciferin in the biomass found instances. Hearts had to search for the winter in the Siberian taiga at night - when you consider that the size of the oligochaetes is 10-15 mm in length, one can imagine how difficult the task was. Search is as follows - the ground was collected in bags, then it was screened, then sorted the found species (because of place and home to members of the genus Henlea). Each collection was giving about 30 g of raw biomass worm. The content of luciferin is about 0.1 micrograms per gram of biomass. Thus, after treatment with 150 g of biomass was obtained worm only 5 mcg of luciferin. With such a small amount of a substance has been very difficult to work with.

However, fortunately for us, in the process of allocation lyutsiferinovoy faction we stumbled upon the substance contained indeterminate composition, which possessed a chromatographic mobility and spectral properties were very similar to luciferin. We hypothesized that these substances can be either peers or predecessors luciferin - or its degradation products. It should be noted that the number of these compounds are 20-30 times superior to those of luciferin. We call these substances and CompX AsLn2.

After that we decided to get ahead of CompX structure and then synthesize this substance. According to NMR, which provided us with Maxim Dubinnyi, the substance proved modified tyrosine. It results from three modifications: deamination enol methylation, carboxylation and then into an aromatic ring. NMR spectral data were sufficient to determine the configuration of the double bond of the compound, and therefore the synthesis was undertaken both isomers - in order to then compare the NMR data of the synthetic and natural substances. A key step in the synthesis is the reaction of CompX olefination, which allowed us to obtain two isomers in a ratio of 2: 1 (Z to E). Major Isomer Z-CompX was absolutely identical to natural compounds in their spectral data. But other isomer dramatically different from the natural counterpart, and, in addition, he did not have fluorescent properties.

Further NMR studies revealed the presence of characteristic natural CompX pattern of aromatic protons in the spectra of the luciferin and its analogues. Thus, we were able to establish that the substance is a structural fragment of luciferin.

Further, on the basis of further NMR and mass spectrometric studies, we found out we discovered the structure of another substance, AsLn2. It was found that the modified tripeptide in which the carboxyl groups CompX involved in the formation of peptide bonds with lysine and tyrosine. This hypothesis is also confirmed by counter synthesis in which the methyl ester CompX was sequentially introduced into condensation with L-lysine and L-tyrosine. As a result of this five-step synthesis it was obtained AsLn2 with access to 14 percent. NMR spectral data of the product of the last stage of the synthesis of natural samples were identical, suggesting that the identity of the order of chemical bonds in the compound AsLn2.

Unfortunately, at our disposal we were few natural counterpart AsLn2, so we were not able to determine its stereochemistry. To do this, synthetic and natural AsLn2 were driven by a chiral column and found that the retention times of both substances were completely identical. These data have suggested L-configuration of the two stereocenters in the molecule. Thus it was established stereochemical structure of the obtained contact AsLn2.

Finally, we have begun work to determine the structure of the luciferin. However, due to the fact that it was very small, with 5 micrograms, we were able to obtain only partial proton NMR spectra. However, these data indicated that the molecule contains three fragments luciferin - CompX residue, L-lysine, and the residue gamma-aminobutyric acid (GABA), which together give us the material of the following composition: C₂₁H₂₉N₃O₈. But this formula is contradicted by mass spectrometry of high resolution according to which the matter was to have the formula C₂₃H₂₉N₃O₁₁. In order to understand what happened to the missing atoms, as if we subtract one from the other formula and obtained a fragment C₂O₃, which corresponds to the oxalate. Thus, we have identified four missing pieces of luciferin.

So, it became clear that the luciferin molecule consists of 4 pieces: CompX, L-Lysine, GABA and oxalate. However, these four fragments were obtained four patterns corresponding to the data obtained by NMR and mass spectrometry - they differ only in the order of the peptide bonds that connect these fragments. We decided to synthesize all four options and compare their NMR spectra with natural luciferin.

Based on the structure of luciferin analog AsLn2, we hypothesized that the luciferin, also having a lysine moiety, the amino acid is attached to the C10 carboxy group CompX fragment, whereas GABA is likely attached to a C1-carboxyl group. This was the first isomer which we synthesized. Thereafter, its NMR spectra were compared to the spectra of natural luciferin, but the chemical shifts of protons synthetic isomer were very similar but not identical to those of natural substances. Moreover, when mixed with luciferase Fridericia heliota (in the presence of ATP and magnesium ions) the substance is not light. Next, we synthesized the other isomers, which do the same. And only one of these synthetic substances NMR coincided with that of natural luciferin.
It is also interesting that only this isomer is entered in the bioluminescent reaction with the luciferase worm. In addition, the intensity of luminescence of synthetic luciferin concentration was similar to that characteristic of natural counterpart.

In addition to the unique and CompX AsLn2 lyutsiferinovoy fraction of the worm Fridericia heliota been allocated one analogue - a dipeptide AsLn7. According to NMR spectroscopy, we have found that it consists of CompX residue and GABA. But we were not clear how these two fragments joined to each other. To understand this, we resorted to the experimental NMR titration. Who led Konstantin Mineev. This experiment allowed us to determine that GABA is attached to the C10 carboxy group CompX, while C1-carboxyl group is free. The same experiment allowed us to see that the carboxyl group of GABA is also free.

In this regard, we have assumed that AsLn7, most likely, is the immediate precursor luciferin Fridericia heliota. This assumption is based on the fact that it has two of the four structural fragments of this substance, which are also connected in the correct order. We also confirmed the structure AsLn7 using counter-synthesis, and then compared the data NMR spectra of natural and synthetic analogs. These spectra coincided.

It is said that the presence of the worm in the biomass AsLn7, the structure of which includes two of the four pieces of luciferin, which suggests that the pathway Fridericia heliota bioluminescent substance is through a serial connection to its four fragments. All these four components - CompX, Lysine, GABA and oxalate - connected by specific (or non-specific) amino acid ligases, which we are not yet known. Now we are working to decipher the immediate succession of the luciferin biosynthesis mechanisms».

After the report by Alexandera Tsar'kova, answering questions from the audience at the seminar formulated another problem that should be solved in the near future. The fact that the structure of Fridericia heliota luciferase is still unknown. According to the rapporteur, it would do well to study the structure of this enzyme and find the gene responsible for synthesis of luciferase and decipher its structure. These data could significantly accelerate the process of studying the mechanism of luminescence Fridericia heliota.

In addition, at the end of the seminar there was an interesting discussion on the question of why is this little worm that lives permanently in the dark, bioluminescence need? Among oligochaetes known cases of using this phenomenon to attract a mate during breeding - as do the adult beetles, fireflies (Lampyridae). During the discussion, Alexandera Tsar'kova suggested that, perhaps, bioluminescence helps these tiny creatures to scare away predators, that is about to do the same as cuttlefish Lycoteuthis diadema, using their glowing symbiotic bacteria in these purposes. According to Alexandera, in favor of this version is the fact that Fridericia heliota lights up in response to mechanical stimulation, which is the normal touch. Thus, we can assume the following scheme: a predator touches the body of the worm, the back lights and the aggressor, frightened flash of bright light, hurriedly removed.

The other, no less interesting version that explains this phenomenon, invited to attend the seminar leading researcher of the Department of Microbiology, Lomonosov Moscow State University  Doctor of Biological Sciences Anvar  Dzhuraevich Ismailov. He noted that the bioluminescence is often characteristic of the organisms that live there, where there is a lack of oxygen. In such conditions often formed during oxidation of hydrogen peroxide, which is a source of active oxygen and thus poses a risk for living beings. The emitted light by living organisms contributes to the rapid decomposition of hazardous substances for them. According to Anwar Dzhuraevich, bioluminescence can be used by soil oligochaetes for such purposes.

Discuss this version, the participants agreed that this could very well be. However, he noted Alexandera Tsar'kova, all these arguments is that they remain purely theoretical, as there is currently no way to experimentally verify some of the proposed versions.The fact that none of the attempts to create a worm population Fridericia heliota in an artificial environment was not a success - taken from the nature of the soil Oligochaeta not reproduced in the lab and quickly died. This occurred even when the conditions for them to recreate, as closely as possible to those in their natural habitat. Thus, it is possible that the riddle of worm bioluminescence Fridericia heliota can unravel only when it is decoded the entire mechanism of this phenomenon, and also determined the structure of the luciferase gene and its encoding.

november 1, 2015

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