DOI: 10.1002/chem.201406498
Full Paper
& Structure Elucidation
Novel Peptide Chemistry in Terrestrial Animals: Natural Luciferin Analogues from the Bioluminescent Earthworm Fridericia heliota Maxim A. Dubinnyi,[a] Aleksandra S. Tsarkova,[a, b] Valentin N. Petushkov,[c] Zinaida M Kaskova,[a, b] Natalja S. Rodionova,[c] Sergey I. Kovalchuk,[a] Rustam H. Ziganshin,[a] Mikhail S. Baranov,[a, b] Konstantin S. Mineev,[a] and Ilia V. Yampolsky*[a, b]
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Full Paper threonine, gamma-aminobutyric acid, homoarginine, and unsymmetrical N,N-dimethylarginine. These natural compounds represent a unique peptide chemistry found in terrestrial animals and rise novel questions concerning their biosynthetic origin.
Abstract: We report isolation and structure elucidation of AsLn5, AsLn7, AsLn11 and AsLn12: novel luciferin analogs from the bioluminescent earthworm Fridericia heliota. They were found to be highly unusual modified peptides, comprising either of the two tyrosine-derived chromophores, CompX or CompY and a set of amino acids, including
Introduction
Results and Discussion
Bioluminescence—emission of “cold light” by a live organism—generally results from oxidation of a small organic molecule called luciferin catalyzed by a specific enzyme luciferase.[1] Until recently, structures of only seven natural luciferins were known. In 2014 we have reported structure elucidation of a novel luciferin from the Siberian bioluminescent oligochaete earthworm Fridericia heliota.[2] Fridericia luciferin turned out to be an unusual peptide formed by the residues of oxalic acid, llysine, modified tyrosine, and g-aminobutyric acid. In contrast to other known earthworm bioluminescence mechanisms, which utilize hydrogen peroxide to oxidize N-isovaleryl-3amino-propanal in an ATP-independent manner,[3] Fridericia’s bioluminescent system was found to utilize ATP, Mg2 + ions, atmospheric oxygen, and a specific (although still not fully characterized) luciferase.[4, 5] In the course of the isolation and purification of Fridericia luciferin we encountered a number of highly unusual peptidic components of the worm biomass that showed chromatographic and UV spectral properties similar to luciferin. In our previous publications we reported structures of two such components, designated CompX[6, 7] and AsLn2[8, 9] (Figure 1). Here we present structure elucidation of four novel luciferin analogues from F. heliota: AsLn5, AsLn7, AsLn11, and AsLn12 which reveal an unprecedented peptide chemistry found in terrestrial animals (Figure 1).
Specimens of F. heliota were individually hand-collected in darkness from the forest soil near Krasnoyarsk (Russia) from June to November 2012. Anion-exchange chromatography of the extract prepared from 40 g of frozen homogenized worms followed by two rounds of RP-HPLC allowed isolation of four novel individual compounds, designated AsLn5, AsLn7, AsLn11, and AsLn12 ( 0.05, 0.04, 0.005 and 0.04 mg, respectively, Figure S1 in the Supporting Information). All four compounds showed UV/Vis absorption spectra similar to Fridericia luciferin. However, two distinct spectral types were observed: AsLn7 had UV absorption identical to luciferin, whereas AsLn5, AsLn11, and AsLn12 showed broader absorption peaks and the different line-shapes at short wavelengths, indicating a different chromophore (Figure 2). Structure elucidation of AsLn7 ESI-HRMS spectra of AsLn7 showed an [M+H] + molecular ion with m/z = 324.1068, to which the closest molecular formula is C15H18NO7 + (calcd m/z = 324.1078). (Figure S2 in the Supporting Information). Since the quantity of natural AsLn7 was low (ca. 40 mg), only 1H, DQF-COSY, and 1H,13C-HSQC NMR spectra could be obtained for its solution in D2O using a 700 MHz instrument equipped with a cryogenically cooled probe. Analysis of the NMR spectra (Figure 3, and Figure S3–S5 in the Supporting Information) revealed the presence of an aliphatic chain (CH2-CH2-CH2) with 1H and 13C chemical shifts characteristic for g-aminobutyric acid (GABA) and four protons with chemical shifts and multiplicities very close to those observed for the previously described CompX fragment.[2, 7, 8] This observation allowed us to presume that AsLn7 is the peptide formed by GABA’s amino group and one of the two carboxyls of CompX. The exact position of the peptide bond was determined by an NMR pH titration experiment, in which pH was gradually decreased from 5.0 to 3.0 and changes in 1H chemical shifts were monitored (Figure 3). The titration experiment revealed that H-3 proton is much more pH-sensitive than H-5 (Dd 0.31 vs. 0.06 ppm, respectively), implying that the C-1 carboxylic group is free, while the C10 carboxyl is occupied by the GABA residue. In order to verify the assumed structure of AsLn7 and to perform full characterization of this substance by NMR spectroscopy we performed its synthesis (see Supporting Information). NMR investigation of the synthetic sample (D2O, pH 5.0) demonstrated its exact coincidence in all chemical shifts and multiplicities with the
[a] M. A. Dubinnyi, A. S. Tsarkova, Z. M. Kaskova, S. I. Kovalchuk, R. H. Ziganshin, M. S. Baranov, K. S. Mineev, I. V. Yampolsky Institute of Bioorganic Chemistry, Russian Academy of Sciences Miklukho-Maklaya 16/10, Moscow 117997 (Russia) E-mail: [email protected] [b] A. S. Tsarkova, Z. M. Kaskova, M. S. Baranov, I. V. Yampolsky Pirogov Russian National Research Medical University Ostrovitianov 1, Moscow 117997 (Russia) [c] V. N. Petushkov, N. S. Rodionova Laboratory of Photobiology, Institute of Biophysics Siberian Branch of the Russian Academy of Sciences Akademgorodok, Krasnoyarsk 660036 (Russia) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201406498. It contains NMR and HRMS spectra and chromatographic data of natural and synthetic AsLn5, AsLn7, AsLn11 and AsLn12, synthetic procedures for both double bond isomers of CompY.
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Figure 1. Unusual peptides from Fridericia heliota. A) Previously identified compounds: Fridericia luciferin, CompX and AsLn2; B) Novel peptides, reported in this work: AsLn5, AsLn7, AsLn11, and AsLn12.
Table 1. 1H and 13C chemical shifts and proton multiplicities of synthetic AsLn7 (700 MHz, D2O, 30 8C, pH 5.0). Atom numbering according to Figure 3 D. Atom 1 2 3 4 5 6 7 8 9 10 11 g-GABA b-GABA a-GABA 1-GABA
Figure 2. UV/Vis absorption spectra of AsLn5, AsLn7, AsLn11, and AsLn12 compared to Fridericia luciferin (black) at pH 2.8 in water.
natural AsLn7 (Figure S6 and S11 in the Supporting Information). The full NMR data of AsLn7 are given in Table 1 and essential HMBC connectivities are shown in Figure 3 D.
dH [ppm] , mult. (J [Hz])
6.69, s 7.96, d (2.2)
7.00, d (8.5) 7.83, dd (8.5, 2.2) 3.69, 3.42, 1.90, 2.37,
s t (7.2) q (7.5) t (7.8)
dC 171.6 149.5 118.7 126.1 129.7 117.3 156.9 117.5 134.6 169.7 58.4 39.2 24.8 33.1 180.6
1
H-13C HSQC spectra outline coincident pattern of cross-peaks corresponding to the hydrogenated carbon atoms (Figure S13– S15 in the Supporting Information). The structure of the fragment associated with these signals was established by the full set of NMR spectra (1D 13C, 2D DQF-COSY, 2D 1H-13C HSQC, and 2D 1H-13C HMBC) acquired for the most abundant compound AsLn12. Analysis of HMBC connectivities identified that the signals above are part on an unusual tyrosine-derived
Identification of CompY as a building block of AsLn5, AsLn11, and AsLn12 Three natural products, AsLn5, AsLn11 and AsLn12, have the similar pattern of NMR signals (Figure 4 A–C): two aromatic doublets (d = 7.6 and 6.9 ppm, 2 H each), one aromatic singlet (d = 6.8 ppm, 1 H), and one methoxy singlet (d = 3.7 ppm, 3 H). Chem. Eur. J. 2015, 21, 1 – 7
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Figure 3. A) 1H NMR spectra of natural AsLn7 in D2O, pH 5.0 at 30 8C. B) The same sample at pH 3.0. C) Synthetic AsLn7 in the same conditions as in A). D) Essential HMBC connectivities in synthetic AsLn7.
Figure 4. 1H NMR spectra of natural and synthetic compounds comprising modified tyrosine residue, CompY. Conditions: 800 MHz (A,B,C) 700 MHz (D,E), D2O, 30 8C, pH 5.0. Chemical shift values of A)–C) are summarized inTable 2. A) AsLn5, dipeptide Thr-CompY. B) AsLn11, dipeptide NDMA-CompY contains nonsymmetric dimethylarginine (NDMA). C) AsLn12, dipeptide HomoArg-CompY, contains homoarginine (homoArg) residue. D) Synthetic CompY, double bond Z isomer, coincides with naturally occurring isomer of CompY in AsLn5, AsLn11 and AsLn12. E) Synthetic CompY, E isomer shows essentially different chemical shifts. Impurities are labeled with asterisks. F) Atom numbering in CompY and essential HMBC connectivities of CompY core in AsLn12. The absence of ROESY signal is shown. G) ROESY signal, observed in synthetic E isomer of CompY.
moiety designated CompY (Figure 4 F) To establish the configuration of the double bond in CompY, we synthesized both E and Z isomers (see Supporting Information) and acquired their ROESY spectra. The cross-peak from the methoxy group to the vinylic singlet was observed only for unnatural E isomer (Figure 4 F, G), similarly to the previously established CompX fragment.[2, 7, 8] Therefore, both CompX and CompY fragments in AsLns have the same Z configuration of the double bond.
threonine residue (C4H7NO2) as possible fragment in AsLn5. The threonine residue is identified in NMR spectra of AsLn5 (Figure 4 A, S13 in the Supporting Information), although no HMBC connetivity was observed between threonine and CompY fragments. The amide bond for threonine CompY is supported by the chemical shifts of threonine Ha proton (d = 4.287 ppm), which is incompatible with unsubstituted amino group of threonine, and the carbonyl group of CompY (d = 166.43 ppm) which deviates by 5 ppm from the chemical shift of free carboxylic group of CompY (d = 171.66 ppm in synthetic CompY under the same conditions).
Structure elucidation of AsLn5 The ESI-HRMS spectrum of AsLn5 showed molecular ion with m/z 296.1145, the closest formula being C14H18NO6 + (calcd m/z 296.1129). Extraction of CompY formula (C10H10O4) suggests &
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prise either of two different tyrosine-derived chromophores, CompX and CompY, differing from each other by carboxylation at the aromatic core. So far, CompX and its amides are unique natural products found only in F. heliota worms,[2, 7, 8] whereas the simpler CompY moiety has been reported earlier as a fragment of natural peptide-like products isolated from ascidians.[11–15] An interesting question is whether the F. heliota worms are responsible for the biosynthesis of AsLns, or do they obtain some key components, like CompX or CompY, through diet or from some unknown sybmionts. This question is tightly connected to the problem of biosynthesis of Fridericia luciferin. The structures of the newly identified compounds imply two possible pathways of Fridericia luciferin biosynthesis (Figure 5). The first pathway may proceed through consecutive linking of its four fragments: GABA, CompX, l-lysine, and oxalic acid by some specific or unspecific amino-acid ligase. As an example of such enzyme might be carnosine synthase, a 100 kDa protein present in human, oyster, mouse, and chicken genomes.[16] This pathway is strongly supported by the structure of AsLn7, since it comprises two of the four peptidic moieties of luciferin: GABA and CompX. On the other hand, the presence of AsLn12, HomoArg-CompY in the extract of Fridericia heliota suggests another possible biosynthetic pathway in which the CompY moiety of AsLn12 is converted to CompX by means of carboxylation, followed by linking the product with GABA and replacement of guanidinium fragment in homoArg by oxalic acid residue (Figure 5). At the present point we cannot distinguish between these two possibilities, although the first pathway seems to be more probable. Our further research will be focused on identification of the biochemical origin of CompX and CompY, as well as on identifying the alleged amide ligases, responsible of their further derivatization. We hope this approach will allow us to understand the whole set of Fridericia luciferin biosynthetic enzymes and intermediates.
The analysis of 1H and 2D HSQC NMR spectra of AsLn11 identified the chain of arginine residue with characteristic chemical shifts/multiplicities and an additional singlet chemical shift of possible N(Me)2 fragment (d = 2.954 ppm, 6 H), Figure 4 B, and Figure S14 in the Supporting Information). Dimethylarginine residues are present in nature in two forms, symmetric (SDMA) and nonsymmetric (NDMA) isomers, which are distinguished by MS/MS fragmentation with loss of either NH2Me (SDMA) or NHMe2 (NDMA) neutral fragments.[10] The MSMS fragmentation of AsLn11 showed loss of NHMe2 (45 Da) from molecular ion 379.1931 (Figure S16 in the Supporting Information). The closest chemical formula for AsLn11 molecular ion is C18H27N4O5 + (calcd m/z 379.1976), which is exactly NDMA+CompY H2O. The peptide bond between a-amino group of NDMA and CompY is supported by the chemical shift of Ha proton of NDMA (d = 4.321 ppm). Structure elucidation of AsLn12 The following NMR spectra were acquired for AsLn12: 1D 1H, 1D 13C, 2D DQF-COSY, 2D HSQC, and 2D HMBC (800 MHz). In addition to the previously identified CompY fragment, the combination of COSY and HSQC spectra unambiguously identified lysine residue with characteristic chemical shifts/multiplicities as the part of AsLn12 (Figure S15 in the Supporting Information). Surprisingly, the analysis of HMBC spectrum outlined unexpected connectivity between e-Lys to carbon atom with a characteristic guanidinium chemical shift 156.82 ppm (Table 2). The presence of a guanidinium fragment is supported by the ESI + HRMS molecular ion with m/z 365.1785, for which the closest formula is C17H25O5N4 + (calcd m/z 365.1819). Finally, the chemical structure of AsLn12 is homoarginine-CompY. The reported structures represent a very unusual peptide chemistry for terrestrial animals. These novel peptides comTable 2. 1H and 13C chemical shifts of AsLn5, AsLn11 and AsLn12. Atom CompY 1 2 3 4 5,9 6,8 7 10 amino acid a b g d e z
AsLn5 Thr-CompY dH [ppm] , mult. (J [Hz])
dC [ppm]
AsLn11 NDMA-CompY dH [ppm] , mult. (J [Hz]) dC [ppm]
– – 6.92, – 7.67, 6.94, – 3.72,
s
166.4 146.0 121.2 125.1 132.0 115.9 156.6 59.5
– – 6.88, – 7.66, 6.94, – 3.68,
4.29, d (3.7) 4.32, m 1.20, d (6.5)
60.3 68.0 19.2
4.32, 1.95, 1.63, 3.26, 2.95,
s d (9.0) d (9.0)
AsLn12 homoArg-CompY dH [ppm] , mult. (J [Hz]) dC [ppm]
s
n.o.[a] n.o. 120.9 n.o. 131.9 115.8 n.o. 59.5
– – 6.87, – 7.66, 6.93, – 3.68,
dd (5.2, 8.1) m; 1.80 m m dt (7.0, 2.4) s[b]
54.7 28.8 24.6 41.4 37.6
4.31, dd (4.8, 8.7) 1.90, m; 1.77 m 1.39 m 1.60 m 3.17 m –
s d (9.0) d (9.0)
s d (9.0) d (9.0) s
165.9 146.1 120.9 125.16 131.9 115.8 156.6 58.5
55.0 31.3 22.3 27.5 40.9 156.8
[a] n.o. = not observed. [b] Assigned to NMe2. Chem. Eur. J. 2015, 21, 1 – 7
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Full Paper Keywords: bioluminescence · Fridericia heliota · luciferin · peptides · structure elucidation
[1] O. Shimomura, Bioluminescence: Chemical Principles and Methods, World Scientific Publishing, Singapore, 2006. [2] V. N. Petushkov, M. A. Dubinnyi, A. S. Tsarkova, N. S. Rodionova, M. S. Baranov, V. S. Kublitski, O. Shimomura, I. V. Yampolsky, Angew. Chem. Int. Ed. 2014, 53, 5566 – 5568; Angew. Chem. 2014, 126, 5672 – 5674. [3] J. E. Wampler, B. G. M. Jamieson, Comp. Biochem. Physiol. Part B 1980, 66, 43 – 50. [4] V. N. Petushkov, N. S. Rodionova, V. S. Bondar, Dokl. Biochem. Biophys. 2003, 391, 204 – 207. [5] N. S. Rodionova, V. S. Bondar, V. N. Petushkov, Dokl. Biochem. Biophys. 2003, 392, 253 – 255. [6] S. M. Marques, V. N. Petushkov, N. S. Rodionova, J. C. G. Esteves da Silva, J. Photochem. Photobiol. B 2011, 102, 218 – 223. [7] V. N. Petushkov, A. S. Tsarkova, M. A. Dubinnyi, N. S. Rodionova, S. M. Marques, J. C. G. Esteves da Silva, O. Shimomura, I. V. Yampolsky, Tetrahedron Lett. 2014, 55, 460 – 462. [8] V. N. Petushkov, M. A. Dubinnyi, N. S. Rodionova, K. D. Nadezhdin, S. M. Marques, J. C. G. Esteves da Silva, O. Shimomura, I. V. Yampolsky, Tetrahedron Lett. 2014, 55, 463 – 465. [9] A. S. Tsarkova, M. A. Dubinnyi, M. S. Baranov, V. N. Petushkov, N. S. Rodionova, I. V. Yampolsky, Mendeleev Commun. 2015, in print. [10] E. Schwedhelm, R. Maas, J. Tan-Andresen, F. Schulze, U. Riederer, R. H. Bçger, J. Chromatogr. B 2007, 851, 211 – 219. [11] S. Kehraus, S. Gorzalka, C. Hallmen, J. Iqbal, C. E. Mller, A. D. Wright, M. Wiese, G. M. Kçnig, J. Med. Chem. 2004, 47, 2243 – 2255. [12] M. R. Rao, D. J. Faulkner, J Nat. Prod. 2004, 67, 1064 – 1066. [13] M. J. McKay, A. R. Carroll, R. J. Quinn, J Nat. Prod. 2005, 68, 1776 – 1778. [14] C. J. Henrich, R. W. Robey, K. Takada, H. R. Bokesch, S. E. Bates, S. Shukla, S. V. Ambudkar, J. B. McMahon, K. R. Gustafson, ACS Chem. Biol. 2009, 4, 637 – 647. [15] S. Yin, C. Cullinane, A. R. Carroll, R. J. Quinn, R. A. Davis, Tetrahedron Lett. 2010, 51, 3403 – 3405. [16] J. Drozak, M. Veiga-da-Cunha, D. Vertommen, V. Stroobant, E. V. Schaftingen, J. Biol. Chem. 2010, 285, 9346 – 9356.
Figure 5. Two possible pathways of Fridericia luciferin assembly, based on the structures of the newly found peptides from F. heliota.
Acknowledgements We thank Dr. K. V. Antonov for registration of LC-HRMS spectra. This work was supported by the Russian Science Foundation grant 14-50-00131.
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Received: December 16, 2014 Published online on && &&, 0000
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FULL PAPER & Structure Elucidation M. A. Dubinnyi, A. S. Tsarkova, V. N. Petushkov, Z. M. Kaskova, N. S. Rodionova, S. I. Kovalchuk, R. H. Ziganshin, M. S. Baranov, K. S. Mineev, I. V. Yampolsky* && – && What’s new in worms? The isolation and structure elucidation of AsLn5, AsLn7, AsLn11 and AsLn12, novel
luciferin analogues from the bioluminescent earthworm Fridericia heliota, are reported.
Novel Peptide Chemistry in Terrestrial Animals: Natural Luciferin Analogues from the Bioluminescent Earthworm Fridericia heliota
Novel Peptide Chemistry Unusual peptides revealing a novel class of natural products were found in the luminescent earthworms Fridericia heliota from Siberian taiga. The structures of these peptides give clue for understanding biosynthetic pathway for a recently described Fridericia luciferin. For more information, see the Full Paper by I. V. Yampolsky et al. on page && ff.
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Novel Peptide Chemistry in Terrestrial Animals: Natural Luciferin Analogues from the Bioluminescent Earthworm Fridericia heliota Maxim A. Dubinnyi,[a] Aleksandra S. Tsarkova,[a, b] Valentin N. Petushkov,[c] Zinaida M Kaskova,[a, b] Natalja S. Rodionova,[c] Sergey I. Kovalchuk,[a] Rustam H. Ziganshin,[a] Mikhail S. Baranov,[a, b] Konstantin S. Mineev,[a] and Ilia V. Yampolsky*[a, b]
Chem. Eur. J. 2015, 21, 1 – 7
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Full Paper threonine, gamma-aminobutyric acid, homoarginine, and unsymmetrical N,N-dimethylarginine. These natural compounds represent a unique peptide chemistry found in terrestrial animals and rise novel questions concerning their biosynthetic origin.
Abstract: We report isolation and structure elucidation of AsLn5, AsLn7, AsLn11 and AsLn12: novel luciferin analogs from the bioluminescent earthworm Fridericia heliota. They were found to be highly unusual modified peptides, comprising either of the two tyrosine-derived chromophores, CompX or CompY and a set of amino acids, including
Introduction
Results and Discussion
Bioluminescence—emission of “cold light” by a live organism—generally results from oxidation of a small organic molecule called luciferin catalyzed by a specific enzyme luciferase.[1] Until recently, structures of only seven natural luciferins were known. In 2014 we have reported structure elucidation of a novel luciferin from the Siberian bioluminescent oligochaete earthworm Fridericia heliota.[2] Fridericia luciferin turned out to be an unusual peptide formed by the residues of oxalic acid, llysine, modified tyrosine, and g-aminobutyric acid. In contrast to other known earthworm bioluminescence mechanisms, which utilize hydrogen peroxide to oxidize N-isovaleryl-3amino-propanal in an ATP-independent manner,[3] Fridericia’s bioluminescent system was found to utilize ATP, Mg2 + ions, atmospheric oxygen, and a specific (although still not fully characterized) luciferase.[4, 5] In the course of the isolation and purification of Fridericia luciferin we encountered a number of highly unusual peptidic components of the worm biomass that showed chromatographic and UV spectral properties similar to luciferin. In our previous publications we reported structures of two such components, designated CompX[6, 7] and AsLn2[8, 9] (Figure 1). Here we present structure elucidation of four novel luciferin analogues from F. heliota: AsLn5, AsLn7, AsLn11, and AsLn12 which reveal an unprecedented peptide chemistry found in terrestrial animals (Figure 1).
Specimens of F. heliota were individually hand-collected in darkness from the forest soil near Krasnoyarsk (Russia) from June to November 2012. Anion-exchange chromatography of the extract prepared from 40 g of frozen homogenized worms followed by two rounds of RP-HPLC allowed isolation of four novel individual compounds, designated AsLn5, AsLn7, AsLn11, and AsLn12 ( 0.05, 0.04, 0.005 and 0.04 mg, respectively, Figure S1 in the Supporting Information). All four compounds showed UV/Vis absorption spectra similar to Fridericia luciferin. However, two distinct spectral types were observed: AsLn7 had UV absorption identical to luciferin, whereas AsLn5, AsLn11, and AsLn12 showed broader absorption peaks and the different line-shapes at short wavelengths, indicating a different chromophore (Figure 2). Structure elucidation of AsLn7 ESI-HRMS spectra of AsLn7 showed an [M+H] + molecular ion with m/z = 324.1068, to which the closest molecular formula is C15H18NO7 + (calcd m/z = 324.1078). (Figure S2 in the Supporting Information). Since the quantity of natural AsLn7 was low (ca. 40 mg), only 1H, DQF-COSY, and 1H,13C-HSQC NMR spectra could be obtained for its solution in D2O using a 700 MHz instrument equipped with a cryogenically cooled probe. Analysis of the NMR spectra (Figure 3, and Figure S3–S5 in the Supporting Information) revealed the presence of an aliphatic chain (CH2-CH2-CH2) with 1H and 13C chemical shifts characteristic for g-aminobutyric acid (GABA) and four protons with chemical shifts and multiplicities very close to those observed for the previously described CompX fragment.[2, 7, 8] This observation allowed us to presume that AsLn7 is the peptide formed by GABA’s amino group and one of the two carboxyls of CompX. The exact position of the peptide bond was determined by an NMR pH titration experiment, in which pH was gradually decreased from 5.0 to 3.0 and changes in 1H chemical shifts were monitored (Figure 3). The titration experiment revealed that H-3 proton is much more pH-sensitive than H-5 (Dd 0.31 vs. 0.06 ppm, respectively), implying that the C-1 carboxylic group is free, while the C10 carboxyl is occupied by the GABA residue. In order to verify the assumed structure of AsLn7 and to perform full characterization of this substance by NMR spectroscopy we performed its synthesis (see Supporting Information). NMR investigation of the synthetic sample (D2O, pH 5.0) demonstrated its exact coincidence in all chemical shifts and multiplicities with the
[a] M. A. Dubinnyi, A. S. Tsarkova, Z. M. Kaskova, S. I. Kovalchuk, R. H. Ziganshin, M. S. Baranov, K. S. Mineev, I. V. Yampolsky Institute of Bioorganic Chemistry, Russian Academy of Sciences Miklukho-Maklaya 16/10, Moscow 117997 (Russia) E-mail: [email protected] [b] A. S. Tsarkova, Z. M. Kaskova, M. S. Baranov, I. V. Yampolsky Pirogov Russian National Research Medical University Ostrovitianov 1, Moscow 117997 (Russia) [c] V. N. Petushkov, N. S. Rodionova Laboratory of Photobiology, Institute of Biophysics Siberian Branch of the Russian Academy of Sciences Akademgorodok, Krasnoyarsk 660036 (Russia) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201406498. It contains NMR and HRMS spectra and chromatographic data of natural and synthetic AsLn5, AsLn7, AsLn11 and AsLn12, synthetic procedures for both double bond isomers of CompY.
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Figure 1. Unusual peptides from Fridericia heliota. A) Previously identified compounds: Fridericia luciferin, CompX and AsLn2; B) Novel peptides, reported in this work: AsLn5, AsLn7, AsLn11, and AsLn12.
Table 1. 1H and 13C chemical shifts and proton multiplicities of synthetic AsLn7 (700 MHz, D2O, 30 8C, pH 5.0). Atom numbering according to Figure 3 D. Atom 1 2 3 4 5 6 7 8 9 10 11 g-GABA b-GABA a-GABA 1-GABA
Figure 2. UV/Vis absorption spectra of AsLn5, AsLn7, AsLn11, and AsLn12 compared to Fridericia luciferin (black) at pH 2.8 in water.
natural AsLn7 (Figure S6 and S11 in the Supporting Information). The full NMR data of AsLn7 are given in Table 1 and essential HMBC connectivities are shown in Figure 3 D.
dH [ppm] , mult. (J [Hz])
6.69, s 7.96, d (2.2)
7.00, d (8.5) 7.83, dd (8.5, 2.2) 3.69, 3.42, 1.90, 2.37,
s t (7.2) q (7.5) t (7.8)
dC 171.6 149.5 118.7 126.1 129.7 117.3 156.9 117.5 134.6 169.7 58.4 39.2 24.8 33.1 180.6
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H-13C HSQC spectra outline coincident pattern of cross-peaks corresponding to the hydrogenated carbon atoms (Figure S13– S15 in the Supporting Information). The structure of the fragment associated with these signals was established by the full set of NMR spectra (1D 13C, 2D DQF-COSY, 2D 1H-13C HSQC, and 2D 1H-13C HMBC) acquired for the most abundant compound AsLn12. Analysis of HMBC connectivities identified that the signals above are part on an unusual tyrosine-derived
Identification of CompY as a building block of AsLn5, AsLn11, and AsLn12 Three natural products, AsLn5, AsLn11 and AsLn12, have the similar pattern of NMR signals (Figure 4 A–C): two aromatic doublets (d = 7.6 and 6.9 ppm, 2 H each), one aromatic singlet (d = 6.8 ppm, 1 H), and one methoxy singlet (d = 3.7 ppm, 3 H). Chem. Eur. J. 2015, 21, 1 – 7
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Figure 3. A) 1H NMR spectra of natural AsLn7 in D2O, pH 5.0 at 30 8C. B) The same sample at pH 3.0. C) Synthetic AsLn7 in the same conditions as in A). D) Essential HMBC connectivities in synthetic AsLn7.
Figure 4. 1H NMR spectra of natural and synthetic compounds comprising modified tyrosine residue, CompY. Conditions: 800 MHz (A,B,C) 700 MHz (D,E), D2O, 30 8C, pH 5.0. Chemical shift values of A)–C) are summarized inTable 2. A) AsLn5, dipeptide Thr-CompY. B) AsLn11, dipeptide NDMA-CompY contains nonsymmetric dimethylarginine (NDMA). C) AsLn12, dipeptide HomoArg-CompY, contains homoarginine (homoArg) residue. D) Synthetic CompY, double bond Z isomer, coincides with naturally occurring isomer of CompY in AsLn5, AsLn11 and AsLn12. E) Synthetic CompY, E isomer shows essentially different chemical shifts. Impurities are labeled with asterisks. F) Atom numbering in CompY and essential HMBC connectivities of CompY core in AsLn12. The absence of ROESY signal is shown. G) ROESY signal, observed in synthetic E isomer of CompY.
moiety designated CompY (Figure 4 F) To establish the configuration of the double bond in CompY, we synthesized both E and Z isomers (see Supporting Information) and acquired their ROESY spectra. The cross-peak from the methoxy group to the vinylic singlet was observed only for unnatural E isomer (Figure 4 F, G), similarly to the previously established CompX fragment.[2, 7, 8] Therefore, both CompX and CompY fragments in AsLns have the same Z configuration of the double bond.
threonine residue (C4H7NO2) as possible fragment in AsLn5. The threonine residue is identified in NMR spectra of AsLn5 (Figure 4 A, S13 in the Supporting Information), although no HMBC connetivity was observed between threonine and CompY fragments. The amide bond for threonine CompY is supported by the chemical shifts of threonine Ha proton (d = 4.287 ppm), which is incompatible with unsubstituted amino group of threonine, and the carbonyl group of CompY (d = 166.43 ppm) which deviates by 5 ppm from the chemical shift of free carboxylic group of CompY (d = 171.66 ppm in synthetic CompY under the same conditions).
Structure elucidation of AsLn5 The ESI-HRMS spectrum of AsLn5 showed molecular ion with m/z 296.1145, the closest formula being C14H18NO6 + (calcd m/z 296.1129). Extraction of CompY formula (C10H10O4) suggests &
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prise either of two different tyrosine-derived chromophores, CompX and CompY, differing from each other by carboxylation at the aromatic core. So far, CompX and its amides are unique natural products found only in F. heliota worms,[2, 7, 8] whereas the simpler CompY moiety has been reported earlier as a fragment of natural peptide-like products isolated from ascidians.[11–15] An interesting question is whether the F. heliota worms are responsible for the biosynthesis of AsLns, or do they obtain some key components, like CompX or CompY, through diet or from some unknown sybmionts. This question is tightly connected to the problem of biosynthesis of Fridericia luciferin. The structures of the newly identified compounds imply two possible pathways of Fridericia luciferin biosynthesis (Figure 5). The first pathway may proceed through consecutive linking of its four fragments: GABA, CompX, l-lysine, and oxalic acid by some specific or unspecific amino-acid ligase. As an example of such enzyme might be carnosine synthase, a 100 kDa protein present in human, oyster, mouse, and chicken genomes.[16] This pathway is strongly supported by the structure of AsLn7, since it comprises two of the four peptidic moieties of luciferin: GABA and CompX. On the other hand, the presence of AsLn12, HomoArg-CompY in the extract of Fridericia heliota suggests another possible biosynthetic pathway in which the CompY moiety of AsLn12 is converted to CompX by means of carboxylation, followed by linking the product with GABA and replacement of guanidinium fragment in homoArg by oxalic acid residue (Figure 5). At the present point we cannot distinguish between these two possibilities, although the first pathway seems to be more probable. Our further research will be focused on identification of the biochemical origin of CompX and CompY, as well as on identifying the alleged amide ligases, responsible of their further derivatization. We hope this approach will allow us to understand the whole set of Fridericia luciferin biosynthetic enzymes and intermediates.
The analysis of 1H and 2D HSQC NMR spectra of AsLn11 identified the chain of arginine residue with characteristic chemical shifts/multiplicities and an additional singlet chemical shift of possible N(Me)2 fragment (d = 2.954 ppm, 6 H), Figure 4 B, and Figure S14 in the Supporting Information). Dimethylarginine residues are present in nature in two forms, symmetric (SDMA) and nonsymmetric (NDMA) isomers, which are distinguished by MS/MS fragmentation with loss of either NH2Me (SDMA) or NHMe2 (NDMA) neutral fragments.[10] The MSMS fragmentation of AsLn11 showed loss of NHMe2 (45 Da) from molecular ion 379.1931 (Figure S16 in the Supporting Information). The closest chemical formula for AsLn11 molecular ion is C18H27N4O5 + (calcd m/z 379.1976), which is exactly NDMA+CompY H2O. The peptide bond between a-amino group of NDMA and CompY is supported by the chemical shift of Ha proton of NDMA (d = 4.321 ppm). Structure elucidation of AsLn12 The following NMR spectra were acquired for AsLn12: 1D 1H, 1D 13C, 2D DQF-COSY, 2D HSQC, and 2D HMBC (800 MHz). In addition to the previously identified CompY fragment, the combination of COSY and HSQC spectra unambiguously identified lysine residue with characteristic chemical shifts/multiplicities as the part of AsLn12 (Figure S15 in the Supporting Information). Surprisingly, the analysis of HMBC spectrum outlined unexpected connectivity between e-Lys to carbon atom with a characteristic guanidinium chemical shift 156.82 ppm (Table 2). The presence of a guanidinium fragment is supported by the ESI + HRMS molecular ion with m/z 365.1785, for which the closest formula is C17H25O5N4 + (calcd m/z 365.1819). Finally, the chemical structure of AsLn12 is homoarginine-CompY. The reported structures represent a very unusual peptide chemistry for terrestrial animals. These novel peptides comTable 2. 1H and 13C chemical shifts of AsLn5, AsLn11 and AsLn12. Atom CompY 1 2 3 4 5,9 6,8 7 10 amino acid a b g d e z
AsLn5 Thr-CompY dH [ppm] , mult. (J [Hz])
dC [ppm]
AsLn11 NDMA-CompY dH [ppm] , mult. (J [Hz]) dC [ppm]
– – 6.92, – 7.67, 6.94, – 3.72,
s
166.4 146.0 121.2 125.1 132.0 115.9 156.6 59.5
– – 6.88, – 7.66, 6.94, – 3.68,
4.29, d (3.7) 4.32, m 1.20, d (6.5)
60.3 68.0 19.2
4.32, 1.95, 1.63, 3.26, 2.95,
s d (9.0) d (9.0)
AsLn12 homoArg-CompY dH [ppm] , mult. (J [Hz]) dC [ppm]
s
n.o.[a] n.o. 120.9 n.o. 131.9 115.8 n.o. 59.5
– – 6.87, – 7.66, 6.93, – 3.68,
dd (5.2, 8.1) m; 1.80 m m dt (7.0, 2.4) s[b]
54.7 28.8 24.6 41.4 37.6
4.31, dd (4.8, 8.7) 1.90, m; 1.77 m 1.39 m 1.60 m 3.17 m –
s d (9.0) d (9.0)
s d (9.0) d (9.0) s
165.9 146.1 120.9 125.16 131.9 115.8 156.6 58.5
55.0 31.3 22.3 27.5 40.9 156.8
[a] n.o. = not observed. [b] Assigned to NMe2. Chem. Eur. J. 2015, 21, 1 – 7
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Full Paper Keywords: bioluminescence · Fridericia heliota · luciferin · peptides · structure elucidation
[1] O. Shimomura, Bioluminescence: Chemical Principles and Methods, World Scientific Publishing, Singapore, 2006. [2] V. N. Petushkov, M. A. Dubinnyi, A. S. Tsarkova, N. S. Rodionova, M. S. Baranov, V. S. Kublitski, O. Shimomura, I. V. Yampolsky, Angew. Chem. Int. Ed. 2014, 53, 5566 – 5568; Angew. Chem. 2014, 126, 5672 – 5674. [3] J. E. Wampler, B. G. M. Jamieson, Comp. Biochem. Physiol. Part B 1980, 66, 43 – 50. [4] V. N. Petushkov, N. S. Rodionova, V. S. Bondar, Dokl. Biochem. Biophys. 2003, 391, 204 – 207. [5] N. S. Rodionova, V. S. Bondar, V. N. Petushkov, Dokl. Biochem. Biophys. 2003, 392, 253 – 255. [6] S. M. Marques, V. N. Petushkov, N. S. Rodionova, J. C. G. Esteves da Silva, J. Photochem. Photobiol. B 2011, 102, 218 – 223. [7] V. N. Petushkov, A. S. Tsarkova, M. A. Dubinnyi, N. S. Rodionova, S. M. Marques, J. C. G. Esteves da Silva, O. Shimomura, I. V. Yampolsky, Tetrahedron Lett. 2014, 55, 460 – 462. [8] V. N. Petushkov, M. A. Dubinnyi, N. S. Rodionova, K. D. Nadezhdin, S. M. Marques, J. C. G. Esteves da Silva, O. Shimomura, I. V. Yampolsky, Tetrahedron Lett. 2014, 55, 463 – 465. [9] A. S. Tsarkova, M. A. Dubinnyi, M. S. Baranov, V. N. Petushkov, N. S. Rodionova, I. V. Yampolsky, Mendeleev Commun. 2015, in print. [10] E. Schwedhelm, R. Maas, J. Tan-Andresen, F. Schulze, U. Riederer, R. H. Bçger, J. Chromatogr. B 2007, 851, 211 – 219. [11] S. Kehraus, S. Gorzalka, C. Hallmen, J. Iqbal, C. E. Mller, A. D. Wright, M. Wiese, G. M. Kçnig, J. Med. Chem. 2004, 47, 2243 – 2255. [12] M. R. Rao, D. J. Faulkner, J Nat. Prod. 2004, 67, 1064 – 1066. [13] M. J. McKay, A. R. Carroll, R. J. Quinn, J Nat. Prod. 2005, 68, 1776 – 1778. [14] C. J. Henrich, R. W. Robey, K. Takada, H. R. Bokesch, S. E. Bates, S. Shukla, S. V. Ambudkar, J. B. McMahon, K. R. Gustafson, ACS Chem. Biol. 2009, 4, 637 – 647. [15] S. Yin, C. Cullinane, A. R. Carroll, R. J. Quinn, R. A. Davis, Tetrahedron Lett. 2010, 51, 3403 – 3405. [16] J. Drozak, M. Veiga-da-Cunha, D. Vertommen, V. Stroobant, E. V. Schaftingen, J. Biol. Chem. 2010, 285, 9346 – 9356.
Figure 5. Two possible pathways of Fridericia luciferin assembly, based on the structures of the newly found peptides from F. heliota.
Acknowledgements We thank Dr. K. V. Antonov for registration of LC-HRMS spectra. This work was supported by the Russian Science Foundation grant 14-50-00131.
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Received: December 16, 2014 Published online on && &&, 0000
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FULL PAPER & Structure Elucidation M. A. Dubinnyi, A. S. Tsarkova, V. N. Petushkov, Z. M. Kaskova, N. S. Rodionova, S. I. Kovalchuk, R. H. Ziganshin, M. S. Baranov, K. S. Mineev, I. V. Yampolsky* && – && What’s new in worms? The isolation and structure elucidation of AsLn5, AsLn7, AsLn11 and AsLn12, novel
luciferin analogues from the bioluminescent earthworm Fridericia heliota, are reported.
Novel Peptide Chemistry in Terrestrial Animals: Natural Luciferin Analogues from the Bioluminescent Earthworm Fridericia heliota
Novel Peptide Chemistry Unusual peptides revealing a novel class of natural products were found in the luminescent earthworms Fridericia heliota from Siberian taiga. The structures of these peptides give clue for understanding biosynthetic pathway for a recently described Fridericia luciferin. For more information, see the Full Paper by I. V. Yampolsky et al. on page && ff.
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