4 Terrestrial Streptomyces sp
4.8 Terrestrial Streptomyces sp. ANK26
4.8.4 seco-Decarboxy-cromycin
Compound 136 was isolated as colourless UV absorbing oil, which on spraying with anisaldehyde/sulphuric acid turned to violet. The molecular weight was estab-lished as 324 Dalton by ESI MS, and the corresponding molecular formula C19H32O4, delivered four double bond equivalents.
The 1H NMR spectrum exhibited two methyl triplet at δ 1.06, while the triplet methyl described in the macrolides above (~0.92) disappeared. The spectra displayed additionally two trans olefinic protons (J~15.5) at δ 6.48 and 6.40, while that of the β-proton (H-5) found in the macrolides 133 and 135) was shifted to δ 6.24 (J ~10.2) confirming the existence of the same -CH=C(CH3)-CO fragment. The shift of the conjugated carbonyl signal was however at δ 204.0, at a higher chemical shift (∆δ ~8 ppm) than those for 133 and 135. With this C-3 carbonyl, a terminal ethyl group was connected based on the HMBC correlations, between the methyl triplet (δH 1.06, δC
8.5) and the 2J coupling of the methylene carbon at δ 30.5 (δH 2.65) with CO-3. Ad-ditionally, a connection of a pentyl group with the β-olefinic carbon C-5 was recog-nized. In a similar way, a C5 fragment from 6-Me via C-6,7,8 to 8-Me was derived from COSY and HMBC couplings; atom C-5 connects both parts to give the partial structure A (Figure 68).
The 13C NMR spectra of 136 displayed no carbons at ~ 173, confirming the missing lactonized carbonyl. The trans-olefinic protons at δ 6.48 (δC 148.5) and 6.40 (δC 127.0) showed 3J and 2J correlations with a carbonyl which appeared at δ 202.1, establishing an additional α,β-conjugated enone system. The methyl triplet at δ 1.06 (δC 11.1) exhibited a 3J correlation with a quaternary oxymethine carbon visible at δ 80.3, and the latter was stronger downfield shifted (∆δ∼ +4) than that in the lac-tones 133, 134 and 135. The methine proton H-13 (δ 3.36) displayed a 3J coupling with the olefinic β-carbon (δC 148.5) and the methyl singlet of CH3-12 (δC 24.5), confirming the attachment between the enone system and the residual partial
struc-ture (CH3CH2-CH(OH)-C(OH)(CH3)-) through C-11 (δ 148.5) and C-12 (δ 76.5).
This was further confirmed by two 3J correlations from the methyl singlet (CH3-12, δ 1.29) at the olefinic β-carbon (C-11, δ 148.5) and C-13 (δ 80.3), and hence the par-tial structure B was fixed (Figure 68).
Figure 67: 13C NMR spectrum (CDCl3, 125.7 MHz) of seco-Decarboxy-cromycin
Figure 68: Selected HMBC correlations of the constituent partial structures, A & B of compound 136.
Both fragments, A and B were connected through C-8 and C-9 based on the fol-lowing observations: the α-methine proton (H-10, 6.40) in fragment B displayed a 3J correlation towards the methine carbon C-8 (44.3) in A besides the cross signal
be-tween the methine proton at δ 2.65 (H-8 of A) and the carbonyl (C-9, 202.1) of B (Figure 69). This established compound 136 as seco-decarboxy-cromycin, which according to the search in different databases (AntiBase, DNP and Chemical Ab-stracts) it as new compound. It seems plausible to assume that the configuration is (6S,8R,12S,13R) as in the cromycin analogues.
O
Table 17: 13C and 1H NMR assignments of 10,11-Dihydro-cromycin (135) and seco-Decarboxy-cromycin (136) in CDCl3.
Position 10,11-Dihydro-cromycin (135) seco-Decarboxy-cromycin (136) δCa δH (J in [Hz]) b δCc δH (J in [Hz])b
4.8.5 (4E,8E)-4,8-Dimethyl-12-oxo-trideca-4,8-dienoic acid
Compound 137 was isolated as a low polar colourless solid, which showed no UV absorbance on TLC, but was detected by spraying with anisaldehyde/sulphuric acid as yellow spot, which turned later to green. The molecular weight was deduced from (+)-ESI MS as 252 Dalton, and its molecular formula as C15H24O3, containing four double bond equivalents.
The 1H NMR/HMQC spectra (Table 18) displayed three methyl signals, one of them at δ 2.12 (δC 30.0) as singlet, most likely of an acetyl group, while the other two methyls were seen as doublets (J~ 1Hz) at δ 1.66 (δC 23.0) and 1.65 (δC 23.4), re-spectively, bound to sp2 carbons of olefinic systems. Two olefinic methine signals at δ 5.14 (δC 126.1) and 5.04 (δC 123.3) were according to their triplet splitting (J~7 Hz) connected to methylene groups one side, while the neighbouring olefinic carbons could be attached to any of the above methyl groups. This resulted in two olefinic fragments of structure CH2-CH=C(CH3)-. Moreover, four methylene groups were displayed as multiplets; one of them with an intensity of 4H appeared at δ 2.35 (δC
33.2 and δC 27.3), and the residual signals were each of 2H at δ 2.04 (δC 26.2) and 2.02 (δC 32.1), respectively.
The 13C NMR spectrum displayed four quaternary carbons: a ketone carbonyl at 210.0, and an ester or carboxylic acid signal at δ 177.9, while the remaining two car-bons (136.4 and 133.6) were corresponding to two olefinic double bonds. In the HMBC spectrum, the methyl singlet at δ 2.12 showed a 2J correlation with the car-bonyl C-12 (210.0), which confirmed the acetyl group. The latter was additionally proven by the downfield shift of the connected methyl carbon (30.0) as well as the 3J cross-signal with the methylene carbon (C-11) at δ 44.0, and vice versa. The latter methylene (CH2-11, δ 44.0) showed in turn a 3J coupling from the olefinic methine proton (H-9) at δ 5.04, and displayed two correlations (2J and 3J) at δ 123.3 and 136.4. The latter quaternary carbon must be attached to the methyl at δ 1.65 (23.4) due to the displayed 2J coupling. The methylene group in between, (CH2-10; δH 2.22, δC 22.3) was established by H,H COSY, constructing the hept-5-en-2-one partial structure C (Figure 70).
The downfield shift and the HMBC correlations of the carboxyl group (177.9) indicated its attachment to the methylene groups C-2 and C-3. Both methylene groups showed correlations with the two olefinic carbons, C-4 (δ 133.6) and C-5 (δ 126.1). The absent COSY correlation between the methine proton (H-5) at δ 5.14 and H-3 (δ 2.35) excluded a neighbouring of such methylene group. The remaining two methylene groups appeared at δ 2.04 and 2.02 were vicinal due to the 3J coupling seen in the H,H COSY spectrum. The direct attachment of the methylene carbon C-6 (δ 26.2) to C-5 (δ 126.1) was derived from H,H COSY and the 3J correlation with the methyl group 4-CH3 (23.0, δH 1.66). The other methylene (H2-7) showed a 3J cou-pling with the olefinic methine carbon C-5 (126.1), confirming the second partial structure, D (Figure 70).
C H3
O CH3
H H H
H H
13 11
2.12 210.0 44.0 22.3 9
123.3
136.4 23.4 1.65
5.04 2.43
2.22
30.0 O
O CH3
H H
H H
H H H
H H
4 1
2.02 32.1 26.2
2.04
23.0
126.1
5.14 133.6
27.3 33.2 2.35
2.35
177.9
C D
Figure 70: Selected H,H COSY ( ) and HMBC( ) correlations of the constituent partial structures, C and D of compound 137.
Figure 71: 13C NMR spectrum (CDCl3, 125 MHz) of (4E,8E)-4,8-Dimethyl-12-oxo-trideca-4,8-dienoic acid (137).
The only way to connect the partial structures, C and D is via the quaternary sp2 carbon C-8 (δ 136.4) and the methylene one of C-7 (δ 32.1). This was confirmed by
the HMBC correlation between the methyl doublet of CH3-8 (δ 1.65) with the me-thylene carbon C-7 (δ 32.1), and additionally by correlations from the methylene protons (H2-7, δ 2.02) toward CH3-8 and the two carbons (C-8 and C-9) of the ole-finic bond (Figure 72). This resulted in 4,8-dimethyl-12-oxo-trideca-4,8-dienoic acid (137), where the missing NOE couplings of the two methyls CH3-4/-8 with their me-thine partners indicated the trans configuration of both double bonds, pointing to 137 as (4E,8E)-4,8-dimethyl-12-oxo-trideca-4,8-dienoic acid. A search for this acid in diverse databases proved its novelty.
C H
3O
OH O CH
3CH
31 5 3
9 7 11
13
Figure 72: H,H COSY ( ) and HMBC ( ) correlations of 137.
Table 18: 13C NMR and 1H NMR of (4E,8E)-4,8-Dimethyl-12-oxo-trideca-4,8-dienoic acid (137) in CDCl3.
Position (4E,8E)-4,8-Dimethyl-12-oxo-trideca-4,8-dienoic acid (137) δC(125.7 MHz) δH (J in [Hz]) (599.7 MHz)
1 177.9 -
2 33.2 2.35 (m)
3 27.3 2.35 (m)
4 133.6 -
4-CH3 23.0 1.66 (d, 0.9)
5 126.1 5.14 (t, 6.2)
6 26.2 2.04 (m)
7 32.1 2.02 (m)
8 136.4 -
8-CH3 23.4 1.65 (d, 1.2)
9 123.3 5.04 (t, 7.1)
10 22.3 2.22 (q, 7.1)
11 44.0 2.43 (t, 7.4)
12 210.0 -
13 30.0 2.12 (s)