Aquastatin C, a new glycoaromatic derivative from Sporothrix sp. FN611

Full text of DOI:10.1038/ja.2010.156

The Journal of Antibiotics (2011) 64, 213–216
2011 Japan Antibiotics Research Association All rights reserved 0021-8820/11 $32.00
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&
NOTE
Aquastatin C, a new glycoaromatic derivative from
Sporothrix sp. FN611
Yun-Ju Kwon, Mi-Jin Sohn and Won-Gon Kim
The Journal of Antibiotics (2011) 64, 213–216; doi:10.1038/ja.2010.156; published online 8 December 2010
Keywords: antibacterial; aquastatin; enoyl-ACP reductase; Sporothrix; Staphylococcus aureus
Bacterial fatty acid synthesis (FAS) is an attractive antibacterial target, ISOLATION
as FAS is organized differently in bacteria and mammals.1,2 Fatty acid The ethylacetate extracts from the culture broth (6 l) of Sporothrix sp.
biosynthesis in bacteria is crucial for the production of a number of AT 28 was subjected to silica gel (56ꢀ200 mm, Merck art no.
lipid-containing components, including the cell membrane. The 7734.9025, Merck, Darmstadt, Germany) column chromatography
bacterial fatty acid system (FAS II) uses discrete monofunctional followed by stepwise elution with CHCl3–MeOH (10:1, 5:1, 2:1,
enzymes, which operate in conjunction with acyl carrier protein 1:1). The active fractions eluted with CHCl3–MeOH (2:1) were pooled
(ACP)-associated substrates, whereas mammalian fatty acid synthase and concentrated in vacuo. The HPLC analysis of the residue dissolved
(FAS I) is mediated by a single multifunctional enzyme–ACP complex. in MeOH showed the presence of another peak with a similar UV
The differences in prokaryote and eukaryote fatty acid biosynthesis spectra with that of 2. The peak was purified by HPLC equipped with
provide an attractive opportunity for selective FAS II inhibition, which Waters 510 HPLC pump and Waters 996 Photodiode Array Detector
is a potential strategy for the development of antibacterial agents. (Waters, Milford, MA, USA). The reverse phase HPLC column
Bacterial enoyl-ACP reductase, which catalyzes the final and rate- (20ꢀ250 mm, YMC C18) was eluted with CH3CN–H2O (80:20) at a
limiting step in type II FAS, has been validated as a novel target for the flow rate of 8 mlminꢁ1 to afford 4.2 mg of 1 with a retention time of
development of antibacterial drugs.3,4 Three isoforms, FabI, FabK 49.2min as a white powder. Compound 2 was detected at a retention
and FabL, have been detected in enoyl-ACP reductase. Most of time of 7.5 min by HPLC in the same condition.
bacteria including Staphylococcus aureus contain FabI, but Streptococ-
cus pneumonia has FabK. Enterococcus faecalis and Pseudomonas STRUCTURE ELUCIDATION
aeruginosa were known to contain both FabI and FabK, whereas The molecular formula of 1 was determined to be C29H48O9 on the
Bacillus subtilis contains both FabI and FabL. InhA is the FabI basis of high-resolution ESI-MS ((M+Na)+ at m/z 563.3192
1
homolog from Mycobacterium tuberculosis. Indeed, the antibacterial (+0.1mmu error), (MꢁH)ꢁ at 539 m/z) in combination with H-
target of triclosan5 and isoniazid,6 which are currently used antibac- and 13C-NMR data. The IR data suggested the presence of a carbonyl
terial agents, have been determined to be the FabI and InhA, (1646 cmꢁ1) and a hydroxyl (3428 cmꢁ1) moiety (Table 1).
respectively. Therefore, the enoyl-ACP reductase inhibitors may
prove to be interesting lead compounds for the development of presence of two aromatic protons attributable to an 1, 2, 4, 6-
effective antibacterial drugs.
The 1H-NMR data (Table 2) with the COSY spectrum suggested the
tetrasubstituted benzene ring at d 6.48 (d, J¼2.4) and d 6.45 (d,
In the course of our screening for inhibitors of bacterial enoyl-ACP J¼2.4), protons attributable to a hexopyranoside moiety from d 4.89
reductase from microbial metabolites, we previously isolated aqua- (1¢-H) to d 3.76 and d 3.77, a methoxy proton at d 3.91 (s) and
statin A (2) from a fungal strain Sporothrix sp. AT 28.7 Aquastatin B protons attributable to a typical aliphatic chain from d 2.80 to d 0.86.
(3) was obtained by acid hydrolysis from aquastatin A. Further In the 13C-NMR spectrum, together with the HMQC spectrum, two
HPLC studies on minor and less polar metabolites with a similar olefinic carbons, 4 sp2 quaternary carbons, a carbonyl carbon, a
UV spectra have resulted in isolation of a new derivative of aquastatin methoxy carbon, an anomeric carbon, 4 hydroxylated methine car-
A, aquastatin C (1; Figure 1). In this paper, we report the bons, a hydroxylated methylene carbon, 14 aliphatic methylene
isolation and structure determination of 1, and the structure–activity carbons and an aliphatic methyl carbon were observed. The vicinal
relationship.
coupling constants together with the chemical shifts of 13C-NMR in
Korea Research Institute of Bioscience and Biotechnology, Yusong, Daejeon, Republic of Korea
Correspondence: Dr W-G Kim, Environmental Biotechnology Research Center, Korea Research Institute of Bioscience and Biotechnology, Yusong, Daejeon 305-806,
Republic of Korea.
E-mail: wgkim@kribb.re.kr
Received 4 June 2010; revised 11 October 2010; accepted 7 November 2010; published online 8 December 2010

Aquastatin C
Y-J Kwon et al
214
Figure 1 Chemical structures of aquastatins C (1), A (2), B (3) and related compounds.
Table 1 Physicochemical properties of 1
of 1 was determined to be a new glycoside of 2-hydroxy-6-pentadecyl
benzoic acid methyl ester as shown in Figure 1.
Appearance
White powder
Acid hydrolysis of compound 2
(a)D
ꢁ1.3 (c 0.35, MeOH)
Compound 2 (47 mg) dissolved in 0.4 ml MeOH was treated with HCl
(2.4ml) and then stirred overnight at room temperature. The resulting
hydrolysate was then extracted twice with ethyl acetate (20 ml) and the
extract was concentrated in vacuo. The residue dissolved in MeOH was
applied to SiO2 TLC developed with CHCl3–MeOH (7:1) to yield 5
(2.8mg) and 6 (2.9mg) at Rf of 0.80 and 0.50, respectively.
Compound 5: C23H38O4: a white powder. 1H-NMR (500MHz,
CD3OD) d: 6.18 (1H, d, 2.5), 6.14 (1H, d, 2.5), 3.88 (3H, s), 2.79 (2H,
t, 8.0), 1.52 (2H, m), 1.29 (24H, m), 0.89 (3H, t, 7.2). ESI-MS: 377.8
(MꢁH)ꢁ.
ESI-MS (m/z)
539 (MꢁH)ꢁ, 563 (M+Na)+
HRESI-MS (m/z)
Found.
563.3192
563.3191 (M+Na)+
C29H4809
Calcd.
Molecular formula
UV lmaxnm(log e) (MeOH)
IR (KBr)ncmꢁ1
214 (4.54), 257 (4.06), 302 (3.58)
3428, 2921, 1646, 1621, 1263, 1086
Abbreviation: HRESI-MS, high-resolution electron spray ionization-MS.
Compound 6: C7H8O2: a white powder. 1H-NMR (500MHz,
the sugar signals indicated the presence of the b-galactopyranoside. CD3OD) d: 6.11 (2H, d, 2.0), 6.05 (1H, d, 2.0), 2.17 (3H, s); 13C-
The methoxyl protons at d 3.91 (H-8) were long-range coupled with NMR (125MHz, CD3OD) d: 159.4 (C-1 and C-3), 141.2 (C-5), 108.7
the carbonyl carbon at d 172.3 (C-7) in the HMBC spectrum, (C-4 and C-6), 100.8 (C-2), 21.7 (C-7). ESI-MS: 123.4 (MꢁH)ꢁ.
indicating the carboxylic acid methyl ester group. In the 1H–1H
Aquastatin C is a new derivative of aquastatin A, in which the
COSY spectrum, a triplet methyl signal at d 0.86 (H-23) and two 2-hydroxy-6-methyl benzoic acid ring of aquastatin A was deleted.
multiplet methylene signals at d 2.80 (H-9) and d 1.50 (H-10) were Aquastatin C could be a direct fermentation product, not artifact that
correlated with a group of methylene signals at d 1.24 (H-11 to H-22). was hydrolyzed by some kind of esterase from the producing strain,
In the 13C-NMR data, a group of methylene carbons at d 30.0–d 37.1 because derivatives of aquastatin C with an unsaturated acyl group
together with the molecular formula indicated that the aliphatic chain instead of the pentadecyl were also detected even though the exact
is the pentadecyl group. These spectral data with the molecular position of the double bond was not determined. Aquastatin A and its
formula indicated that four substituents in the 1, 2, 4, 6-tetrasub- related compounds KS-501 and KS-502 have a common structure, in
stituted benzene ring are a b-galactopyranoside, a pentadecyl, a which two aromatic rings are linked by an ester bond, and a sugar and
carboxylic acid methyl ester and a hydroxyl. The position of the a pentadecyl group are attached with the ring.8–10 Aquastatin C is a
four substituents was determined by the HMBC and NOESY spec- rare metabolite incorporating one aromatic ring in its structure.
trum (Figure 2). The anomeric proton of the b-galactopyranoside at d Aquastatin B is a deglycosylated product derived from acid hydrolysis
4.89 (H-1¢¢) showed a long-range correlation with the sp2 quaternary of aquastatin A.8 Previously aquastatins A and B were reported to
carbon at d 162.1 (C-4), which was in turn long-range correlated with strongly inhibited bacterial enoyl-ACP reductases including S. aureus
the two meta-coupled aromatic protons at d 6.48 (H-3) and d 6.45 FabI and S. pneumoniae FabK in a dose-dependent manner.7 Also
(H-5). In addition, H-1¢ showed NOEs with both H-3 and H-5. This aquastatins A and B were reported to exert antibacterial activity
data clearly indicated that the sugar was attached with C-4. The against the Gram-positive pathogens, including S. aureus, methicil-
methylene protons at d 2.80 (H-9) of the pentadecyl group showed lin-resistant S. aureus and S. pneumonia. However, an active moiety
HMBC correlations with C-5 and two sp2 quaternary carbons at d for anti-enoyl-ACP reductases activity of aquastatins A and B has not
108.1 (C-1) and d 148.2 (C-6). In addition, the long-range correlations been determined yet.
from H-5 to C-1, C-3, C-4 and C-9 and from H-3 to C-1, C-2, C-4
Effect of aquastatin C against enoyl-ACP reductases and bacterial
and C-5 were observed. These spectral data indicated that the growth was investigated according to the previously reported
carboxylic acid methyl ester, the hydroxyl and the pentadecyl group method.11 Interestingly, aquastatin C did not inhibit S. aureus FabI
were attached with C-1, C-2 and C-6, respectively. Thus, the structure and S. pneumoniae FabK even at 100 mM (Table 3). Consistent with no
The Journal of Antibiotics

Aquastatin C
Y-J Kwon et al
215
Table 2 1H and 13C NMR spectral data for 1 and 2
1
2
Position
dH (multiplicity, JHH
)
dc
dH (multiplicity, JHH
)
dc
1
2
108.1 C
163.6 C
102.5 CH
162.1 C
112.0 CH
148.2 C
172.3 C
52.3 CH3
37.1 CH2
32.6 CH2
112.2 C
162.9 C
102.9 CH
164.0 C
109.5 CH
148.3 C
170.2 C
3
6.48 (1H, d, 2.4)
6.45 (1H, d, 2.4)
6.47 (1H, d, 2.1)
6.43 (1H, d, 2.1)
4
5
6
7
8
3.91 (3H, s)
9
2.80 (2H, m)
1.50 (2H, m)
1.24 (24H, m)
0.86 (3H, t, 6.6)
2.88 (2H, m)
1.56 (2H, m)
1.19 (24H, m)
0.81 (3H, t, 5.0)
37.2 CH2
33.3 CH2
33.0–30.5 CH2
14.4 CH3
118.1 C
10
11–22
23
1¢
2¢
3¢
4¢
5¢
6¢
7¢
8¢
100
200
300
400
500
600
32.6–30.0 CH2
14.2 CH3
164.7 C
6.51 (1H, d, 2.4)
6.39 (1H, d, 2.4)
2.59 (3H, s)
108.2 CH
153.0 C
115.5 CH
144.6 C
23.7 CH3
175.7 C
4.89 (1H, d, 7.5)
3.78 (1H, m)
101.4 CH
71.6 CH
74.2 CH
69.4 CH
76.3 CH
61.8 CH2
4.92 (1H, d, 7.2)
3.82 (1H, m)
102.0 CH
72.0 CH
3.57 (1H, dd, 9.6, 3.3)
3.92 (1H, d, 3.3)
3.68 (1H, t, 7.2)
Ha 3.77 (1H, m)
3.57 (1H, dd, 9.6, 3.2)
3.92 (1H, d, 3.2)
3.63 (1H, m)
74.7 CH
70.1 CH
77.0 CH
Ha 3.77 (1H, m)
Hb 3.76 (1H, m)
62.3 CH2
Hb 3.76 (1H, m)
1H- and 13C-NMR spectral data were measured at 500 and 125MHz, respectively, in CDCl3. The assignments were aided by 1H–1H COSY, DEPT, HMQC and HMBC.
Table 3 Comparison of inhibitory activity of compounds 1–3 and
related compounds against enoyl-ACP reductases and bacterial
viability
IC50(mM)
MIC (mg mlꢁ1
)
saFabI spFabK mtInhA S. aureus MRSA S. pneumoniae M. tuberculosis
1
2
3
4
5
6
4100 4100 4100
4128 4128
4128
128
NT
4128a
NT
3.2
3.4
9.2
9.6
32
32
16
32
10.6
18.4
64
Figure 2 Key HMBC and NOE correlations of 1.
4100 4100 4100
4100 4100 4100
4100 4100 4100
4128 4128
4128 4128
4128 4128
4128
4128
4128
NT
NT
NT
inhibition against the enoyl-ACP reductases, aquastatin C did not
prevent bacterial growth of S. aureus, methicillin-resistant S. aureus
and S. pneumonia. To more clearly examine the structure-activity
relationship, effects of 2-hydroxy-6-methyl-benzoic acid (4) and 2,4-
dihydroxy-5-pentadecyl-benzoic acid methyl ester (5) against enoyl-
ACP reductases and bacterial growth was investigated. Compound 5
Abbreviations: ACP, acyl carrier protein; M. tuberculosis, Mycobacterium tuberculosis H37Rv;
MRSA, methicillin-resistant Staphylococcus aureus CCARM 3167; mtInhA, M. tuberculosis
InhA; NT, not tested; saFabI, S. aureus FabI; S. aureu, S. aureus RN4220; spFabK,
Streptococcus pneumonia FabK, S. pneumonia KCTC 5412.
aCompound 2 inhibited the growth of M. tuberculosis by 17.9% at 128 mg mlꢁ1
.
was obtained from acid hydrolysis of 2. As 5-methyl-benzene-1,3-diol tadecyl-benzoic acid are important for its anti-enoyl-ACP reductase
(6) was made instead of 2,4-dihydroxy-6-methyl-benzoic acid from activity as well as antibacterial activity.
the acid hydrolysis, 4 was purchased (Johnson Matthey Company,
As InhA is the FabI homolog from Mycobacterium tuberculosis,
UK). Interestingly, all of 4–6 did not inhibited S. aureus FabI as well as effect of aquastatins A–C, and related compounds on M. tuberculosis
S. pneumoniae FabK even at 100 mM (Table 3). These compounds also InhA was examined. Aquastatins A and B inhibited M. tuberculosis
did not show antibacterial activity against S. aureus, methicillin- InhA with IC50 of 9.6 and 18.4mM, respectively, whereas aquastatin
resistant S. aureus and S. pneumonia. These results clearly indicated C and other compounds did not affect at 100 mM. Aquastatin A
that both 2-hydroxy-6-methyl-benzoic acid and 2,4-dihydroxy-5-pen- showed weak anti-bacterial activity against M. tuberculosis with
The Journal of Antibiotics

Aquastatin C
Y-J Kwon et al
216
inhibiting bacterial growth of M. tuberculosis by 17.9% at 128 mg mlꢁ1
The weak antibacterial activity of aquastatin A against M. tuberculosis
may be because of permeability barrier, as M. tuberculosis contains a
thick cell wall compared with S. aureus.
.
Korea. (A090598). We thanks the Korea Basic Science Institute for the NMR
measurements.
1
2
3
4
5
6
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In summary, aquastatin C is a rare metabolite incorporating one
aromatic ring with a sugar and an aliphatic chain, which was isolated
from liquid fermentation cultures of Sporothrix sp. FN611. Aquastatin
C is a new glycoaromatic derivative of aquastatin A in which the 2-
hydroxy-6-methyl benzoic acid ring of aquastatin A was deleted.
Aquastatin C completely lost inhibitory activity of aquastatin A
against bacterial enoyl-ACP reductases, such as S. aureus FabI,
S. pneumoniae FabK and M. tuberculosis InhA. Also aquastatin C
did not inhibited bacterial growth of S. aureus, methicillin-resistant S.
aureus and S. pneumonia. The 2-hydroxy-6-methyl benzoic acid, the
other moiety of aquastatin A, as well as the 2,4-dihydroxy-5-penta-
decyl-benzoic acid methyl ester also did not show the FabI-inhibitory
and antibacterial activity, indicating that both moieties are necessary
for its inhibitory activity against enoyl-ACP reductases and bacterial
growth. These results could provide new insights into the development
of a selective mechanism-based antibacterial agent with targeting FabI.
7
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11 Zheng, C. J., Sohn, M. J. & Kim, W. G. Vinaxanthone, a new FabI inhibitor from
Penicillium sp. J. Antimicrob. Chemother. 63, 949–953 (2009).
ACKNOWLEDGEMENTS
This work was supported by a grant of the Korea Healthcare Technology R
and D Project, Ministry for Health, Welfare and Family Affairs, Republic of
The Journal of Antibiotics