开题报告Opening Report
论文标题Title of thesis： Cyclic peptides produced by an actinobacterium associated with Tenebrio molitor
论文研究的目的、意义Objective amp; Meaning：
Find a new compound and figure out its planar structure (and 3D structure if possible) which have the bioactivity to kill cancer cells.
文献综述Previous Researches：
Actinobacteria may be one of the most abundant sources to find natural bioactive products. Due to the fact that most of the actinobacteria strains have been studied for a long time, we focus on finding actinobacteria strains from deep ocean or gut of insects. In the gut of Tenebrio molitor, which is in the order of Coleoptera, an actinobacterium strain, GG23, was isolated. After incubating this strain for several days, some special compounds, Lydiamycin and its derivatives came out.
The cyclodepsipeptides lydiamycins A, B, C and D were obtained as white solids by standard chromatographic procedures from a 300-L fermentation of the strain. High-resolution ESIMS of the Na adduct of lydiamycin A (1)indicated a molecular formula of C31H49NaN7O9 ([M Na] , m/z: found 686.3495, calcd. 686.3489). Like the IR spectrum with characteristic absorption bands of amide carbonyls at 1663 and 1539 cm-1 and ester carbonyls at 1745 cm-1 , the NMR data showed typical characteristics of peptides, for example, resonances for amide carbonyls in the 13C NMR spectrum and for amide and a protons in the 1 H NMR spectrum. Signals for five amino acid a carbons (delta;C = 50.5, 50.7, 51.4, 52.7, 55.6 ppm)and seven carbonyls, either of amide or carboxylic acid origin (delta;C = 169.1, 169.3, 169.7, 170.9, 174.3, 175.9, 177.3 ppm)were found. As fragment ion peaks commonly observed for acyclic peptides were not noted in the CID ESIMS/MS data, a cyclic oligopeptide structure was proposed for 1.
Based on analysis of the 2D NMR data, 1 was determined to contain alanine (Ala), leucine (Leu), Serine (ser), PBB (piperazic acid building block 1, PBB1), dehydropiperazic acid (piperazic acid building block 2, PBB2), and 2-pentylsuccinic acid (PSA)moieties. The two unusual heterocyclic amino acids were confirmed by comparison to literature data.[2,3] The presence of long-range couplings (nJC,H, HMBC)suggested amide linkages between the leucine carbonyl and the alanine amine, between the alanine carbonyl and the piperazic acid d-amine, between the serine carbonyl and the leucine amine, and between the dehydropiperazic acid carbonyl and the serine amine. This data, combined with indication of an ester linkage between the piperazic acid carbonyl and the serine beta;-OH, established the structure as a thirteen-membered cyclic depsipeptide, with the dehydropiperazic acid moiety located peripheral to the ring.
For the determination of the absolute configurations, an acidic hydrolysate was generated according to the Marfey protocol.[4-6] Subsequent HPLC analysis indicated the presence of L-alanine, D-leucine, and L-serine. However, piperazic acid derivatives could not be detected in the hydrolysate. Instead, the stereochemistry of the piperazic acid moiety PBB1 was determined through conformational analysis and computation with an MM force field. The assignment of H-2 (delta;H = 5.15 ppm, br d, 3.1 Hz in [D4]methanol)in the equatorial position, as well as discrimination between Hax and Heq at C-5 was possible through their 3JH,H coupling data and NOE correlations of H-2 with Heq-3 (delta;H = 2.35 ppm)and Hax-3 (delta;H = 1.77 ppm)and between Hax-5 (delta;H = 2.75 ppm, br dq, 3.8 and 13.5 Hz in CDCl3)and Hax-3 (delta;H = 1.77 ppm). Based on this information along with X-ray results of other piperazic acid containing depsipetides, we assumed a chair conformation for PBB1 in 1.[7] Molecular dynamic force field calculations and subsequent ab initio calculations were carried out for the peptide core (including PBB2)for both possible configurations of C-2 in PBB1.[8] The lowest energy conformation for each diastereomer was confirmed after intermediate annealing processes. Owing to the small size of the peptide ring, the two three-dimensional structures are almost planar, which is consistent with the lack of NOEs between nonvicinal amino acids. The two diastereomeric structures can be distinguished by noting the significant difference in the distance between 5- Hax and Ala-CH3 : 4.06 E in the case of the 2R of PBB1 and 2.65 E in the case of 2S. A rough estimate of the distance between Hax-5 (delta;H = 2.75 ppm)and Ala-CH3 (delta;H = 1.43 ppm)by the ROESY peak volumina, when calibrated against the correlation between H-2 and CH3 of the alanine residue, yielded a distance of 4.1 E (5% intensity). Thus, the 2R configuration was suggested for PBB1. The absolute configuration at C-2 of PBB2 could not be assigned. The configuration cannot be rationalized by biosynthetic relationships, as related compounds include two piperazic acid building blocks with different configurations.[2]
Structure determinations of lydiamycins B–D (2–4)were conducted by analogy to that of 1. Absolute configurations were assumed to be identical to those in lydiamycin A (1). Thus, in the following section, we discuss only the elucidation of altered structural features. The molecular formula C31H49N7O10 of lydiamycin B (2)differed from that of 1 by one additional oxygen atom. The analogous fragment ion peaks in the CID ESIMS/MS indicated that this additional oxygen was located in the depsipeptide core of 2. 1 H and 13C NMR data of 2 were very similar to those of 1 except for a missing methylene group (delta;C= 21.4 ppm in 1)and an additional oxymethine group (delta;C= 61.3 ppm)in PBB1. TOCSY and HMBC NMR data revealed the piperazic acid of 1 to be replaced by 4-hydroxypiperazic acid in 2. As the remaining methine proton (H-4)appears as the broad singlet of an equatorial proton in the 1H NMR data (delta;H = 3.95 ppm, Table 2), the hydroxy substituent was assigned an axial position. Thus, PBB1 of 2 is suggested to be 2R,4R-configured. The molecular formula of lydiamycin C (3), C31H47N7O9, suggested that it is a dehydro analogue of 1. Consistent with this, in the 13C spectra of lydiamycin C (3) one CH2 signal is replaced by a signal for an sp2 -hybridized carbon (delta;C = 146.9 ppm). An additional double bond between C-5 and the neighboring nitrogen of the piperazic acid moiety (PBB1)could readily be assigned based on the altered spin systems detected in COSY and TOCSY spectra. Compounds 3 and 1 show identical fragmentation sequences in the CID ESIMS/MS data, which confirms the position of the double bond in 3. As for 2, the molecular formula of C31H49N7O10 indicated that lydiamycin D (4)is an oxygenated analogue of 1. Since the fragment-ion peaks of 4 in the CID ESIMS/MS spectra were identical to those of 1, the oxygenation site had to be located in the 2-pentylsuccinic acid moiety. The regiochemistry of the hydroxylation was determined by HMBC and COSY experiments.
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