Venturicidin C, a new 20-membered macrolide produced by Streptomyces sp. TS-2-2.

Venturicidin C (1), a new 20-membered macrolide along with the known venturicidins A (2) and B (3) were isolated from the crude extract of the Appalachian bacterial strain Streptomyces sp. TS-2-2. Additionally, nine other known compounds namely nocardamine, dehydroxynocardamine, desmethylenylnocardamine, ferrioxamine E, adenosine, riboflavin, cyclo(D)-trans-4-OH-Pro-(D)-Phe, cyclo(D)-Pro-(D)-Phe and N-(2-phenylethyl)-acetamide were also isolated and identified. The structure of the new macrolide 1 was elucidated by the cumulative analyses of NMR spectroscopy and HR-MS data. Complete NMR assignments for the known venturicidins A (2) and B (3) are also provided, for the first time, in this report. Venturicidins A-C did not inhibit the proliferation of A549 lung cancer cell line but all displayed potent antifungal activity.

INTRODUCTION

As a part of our ongoing natural product discovery initiative program, we are investigating soil actinomycetes collected near thermal vents emanating from a range of underground coal mine fire sites throughout Appalachia.[1, 2] Notable alteration of soil composition in and around the vents due to the fire-related emission of greenhouse gases and the dispersion of volatile organic and inorganic species through surface vents of the mine, in conjunction with the natural biodiversity of Applachian Mountain, supports the contention that such collection sites present unique ecological environments soil actinobacteria-based natural product discovery.[3-11] Located in the upper Elkhorn No. 3 coal bed of Appalachian Mountain of Eastern Kentucky, the Truman Shepherd coal bed was last mined in 1930s and the corresponding Truman Shepherd coal mine fire first observed in 2009.[8] More than 70 pure bacterial cultures isolated from the soil samples collected around the vents of the Truman Shepherd fire displayed actinobacteria-like morphology on solid agar media. Comparison of HPLC-High Resolution Mass Spectrometry (HPLC-HR-MS) molecular ions of the Streptomyces sp. TS-2-2 culture extracts to Antibase[12] revealed the potential presence of novel metabolites, with the corresponding MS fragmentation patterns suggestive of glycosylated natural products. In this report, we describe the isolation, structure elucidation and biological activity of a new 20-membered glycosylated macrolide venturicidin C (1), along with the two known counterparts venturicidin A (2) and B (3), from a mycelial extract of Streptomyces sp. TS-2-2 (Figure 1). Also reported herein for the first time are the full NMR spectral datasets for venturicidins A (2) and B (3) to complement the previously published isolation/characterization data.[13-20] Nine additional known compounds namely nocardamine,[21, 22] dehydroxynocardamine,[21] desmethylenlnocardamine,[21] ferrioxamine E (FOE),[23, 24] adenosine, riboflavin, cyclo(d)-trans-4-OH-Pro-(d)-Phe,[25] cyclo(d)-Pro-(d)-Phe,[25] and N-(2-phenylethyl)-acetamide[26] were also isolated during the culture extract work up.

Figure 1

Chemical structure of venturicidins C (1), A (2), and B (3), along with the related macrolides 4-8

RESULTS AND DISCUSSION

Streptomyces sp. TS-2-2 was fermented in liquid medium for both small scale (50 mL) screening and large scale (8L) production of metabolites. Screening of metabolites in low resolution LC-ESI-MS revealed three compounds which displayed similar retention time, UV profile and MS-fragmentation pattern. A difference of 231 amu between the base MS ion and the fragmentation MS ion for compounds 1 and 2, and 170 amu for compound 3 suggested a parental glycoside. The extract did not reveal any major UV-visible spots (at 254 or 365 nm) on TLC and treatment with anisaldehyde/sulfuric acid followed by heating revealed a spot with an intensive blue coloration which turned dark-green within a few minutes. This suggested a predominance of non-aromatic compounds within the extract. Subsequent purification of compounds from a large scale fermentation extract using various chromatographic techniques led to the isolation of one new macrolide, venturicidin C (1, 3.2 mg l-1), along with the two known antifungal macrolide antibiotics venturicidin A (2; 40.0 mg l-1) and B (3; 10.8 mg l-1) (see experimental and supporting information for details). The structure of these molecules were determined by a cumulative consideration of 1D and 2D NMR spectroscopy and high resolution mass spectrometry (ESI) data.

Structure elucidation

The physicochemical properties of compounds 1~3 are summarized in Table 1. Compound 1 was isolated as white powder (3.2 mg l-1) from the mycelial extract using various chromatographic techniques (Figure S3). The molecular formula of 1 was deduced as C42H69NO11 on the basis of HR-ESI-MS (m/z 786.4799 [M + Na]+, Calcd. 786.4763 for C42H69NO11Na) and 1H and 13C NMR. The HRESI-MS spectrum of 1 also revealed a fragmentation ion (m/z 555.4067) consistent with the cleavage of a glycosidic bond and the elimination of two water molecules (Figure 2). The proton NMR spectrum of 1 in CD3OD (Table 2) was rich in aliphatic proton signals with four additional olefinic signals. The triplet signal observed at δ 0.98, five doublets at δ 1.27, 0.98, 0.91, 0.88 and 0.82 along with two singlets at δ 1.43 and 1.48, indicated the presence of nine methyl groups in the molecule. When the solvent was changed to DMSO-d6, an additional broad signal comprising two protons were observed at δ 6.48, suggesting the presence of an amino group (-NH2).

Table 1

Physico-chemical properties of macrolides 1-3

	Venturicidin C (1)	Venturicidin A (2)	Venturicidin B (3)
Molecular Formula	C42H69NO11	C41H67NO11	C40H66O10
Appearance	White powder, UV non-absorbing (254 nm)	White powder, UV non-absorbing (254 nm)	White powder, UV non-absorbing (254 nm)
Rf	0.24a,0.34b, 0.28c	0.23a,0.33b	0.23a,0.31b
HPLC-Rta)	17.01 (min)	16.25 (min)	17.01 (min)
Anisaldehyde/H2SO4 reagent	Pink then dark-blue and few minutes later turned to dark-green	Pink then dark-blue and few minutes later turned to dark-green	Pink then dark-blue and few minutes later turned to dark-green
(+)-ESI-MS: m/z	786 [M + Na]+, 555 [(M – 3’-O-carbamoyl-β-d-olivose – 2H2O) + H]+ – 2H2O) + H]+,537 [(M – 3’-O-carbamoyl-β-d-olivose – 3H2O) + H]+	772 [M + Na]+, 541 [(M – 3’-O-carbamoyl-β-d-olivose – 2H2O) + H]+, 523 [(M – 3’-O-carbamoyl-β-d-olivose – 3H2O) + H]+	729 [M + Na]+, 541 [(M – β-d-olivose –2H2O) + H]+, 523 [(M – β-d-olivose – 3H2O) + H]+
(+)-HRESI-MS: m/z	786.4799 [M + Na]+, 555.4067 [(M – 3’-O-carbamoyl-β-d-olivose – 2H2O) + H]+	772.4676 [M + Na]+, 559.4016 [(M – 3’-O-carbamoyl-β-d-olivose – H2O) + H]+, 541.3908 [(M – 3’-O-carbamoyl-β-d-olivose – 2H2O) + H]+, 523.3795 [(M – 3’-O-carbamoyl-β-d-olivose – 3H2O) + H]+	1435.9323 [2M + Na]+, 729.4594 [M+ Na]+, 559.4022 [(M – β-d-olivose – H2O) + H]+, 541.3915 [(M – β-d-olivose – 2H2O) + H]+, 523.3806 [(M – β-d-olivose – 3H2O) + H]+
Calcd.	786.4763 for C42H69NO11Na [M + Na]+ and 555.4044 for C35H55O5 [(M – 3’-O-carbamoyl-β-d-olivose – 2H2O) + H]+	772.4606 for C41H67NO11Na [M + Na]+, 559.3993 for C34H55O6 [(M – 3’-O-carbamoyl-β-d-olivose – H2O) + H]+, 541.4044 for C34H53O5 [(M –3’-O-carbamoyl-β-d-olivose – 2H2O) + H]+ and 523.3782 for C34H51O4 [(M – 3’-O-carbamoyl-β-d-olivose – 3H2O) + H]+	1435.9204 for C80H132O20Na [2M+Na]+, 729.4548 for C40H66O10Na [M + Na]+, 559.3993 for C34H55O6 [(M – β-d-olivose – 2H2O) + H]+, 541.4044 for C34H53O5 [(M – β-d-olivose – 2H2O) + H]+ and 523.3782 for C34H51O4 [(M – β-d-olivose – 3H2O) + H]+

For HPLC purification, see figures S3-S6, S22-S23, S34-S35;

CH2Cl2/5% MeOH;

CH2Cl2/50% EtOAc;

CH2Cl2/7% MeOH

Figure 2

ESI/MS fragmentation patterns of venturicidins C (1), A (2), and B (3)

Table 2

1H NMR spectroscopic data of venturicidins C (1), A (2), and B (3) (δ in ppm, mult., J in [Hz])

Position	1 a)	1 b)	1 c)	2 a)	2 b)	3 a)	3 b)
	δH d)	δH e)	δH d)	δH d)	δH e)	δH d)	δH e)
2	2.75-2.60 (m)	2.95-2.50 (m)	2.70-2.45 (m)	2.70 (d, 16.0), 2.61 (d, 16.0)	2.90-2.40 (m)	2.70 (d, 16.0), 2.60 (d, 16.0)	2.78-2.46 (m)
3-OH	-	5.44 (brs)	-	-	5.43 (brs)
4	2.10 (m), 1.45 (m)	2.08 (m), 1.55 (m)	2.10-2.25 (m)	2.10 (m), 1.50 (m)	2.08 (m), 1.50 (m)	2.10 (m), 1.50 (m)	2.08 (m), 1.50 (m)
5	5.49 (brm)	5.50-5.40 (brm)	5.47 (brm)	5.49 (brm)	5.48 (brm)	5.48 (m)	5.46 (m)
6-CH3	1.48 (s)	1.41 (s)	1.46(s)	1.48 (s)	1.41 (s)	1.48 (s)	1.41 (s)
7	4.41 (brs)	4.32 (brs)	4.43 (brs)	4.42 (brs)	4.32 (brs)	4.42 (brs)	4.32 (brs)
8-CH3	1.43 (s)	1.36 (s)	1.38 (s)	1.44 (s)	1.36 (s)	1.44 (s)	1.36 (s)
9	5.44 (m)	5.50-5.40 (brm)	5.38 (m)	5.44 (dd, 10.5, 5.5)	5.41 (m)	5.44 (m)	5.42 (m)
10	2.10 (m), 1.80 (m)	2.10 (m), 1.70 (m)	2.15 (m), 1.80 (m)	2.10 (m), 1.89 (m)	2.10 (m), 1.70 (m)	2.15 (m), 1.90 (m)	2.15 (m), 1.90 (m)
11	1.48-1.25 (m)	1.60-1.20 (m)	2.12 (m), 1.80 (m)	1.50-1.20 (m)	1.50-1.20 (m)	1.50-1.20 (m)	1.50-1.20 (m)
12	1.50-1.40 (m)	2.05 (m)	1.30-1.20 (m)	1.50-1.46 (m)	2.10 (m)	2.10 (m)	2.10 (m)
13	3.94 (m)	3.86 (m)	3.82 (m)	3.94 (m)	3.86 (m)	3.91 (m)	3.84 (m)
14	5.38 (dd, 15.0, 8.0)	5.38-5.20 (m)	5.38 (m)	5.39 (dd, 15.0, 7.5)	5.31 (dd, 15.2, 7.6)	5.38 (dd, 15.5, 8.5)	5.30 (dd, 15.2, 7.6)
15	5.33 (dd, 15.5, 8.0)	5.38-5.20 (m)	5.26 (m)	5.34 (dd, 15.0, 8.5)	5.22 (dd, 15.2, 8.4)	5.30 (dd, 15.0, 8.5)	5.21 (dd, 15.2, 8.4)
16	2.17 (m)	2.07 (m)	2.12 (m)	2.17 (m)	2.10 (m)	2.10 (m)	2.10 (m)
16-CH3	0.98 (d, 7.0)	0.83 (d, 7.2)	1.07 (d, 7.0)	0.97 (d, 7.0)	0.83 (d, 6.8)	0.96 (d, 7.0)	0.85 (d, 7.2)
17	1.24 (m), 1.10 (m)	1.30 (m), 0.96 (m)	1.22 (m), 0.95 (m)	1.24 (m), 1.09 (m)	1.30 (m), 0.96 (m)	1.30 (m), 1.12 (m)	1.30 (m), 1.15 (m)
18	1.89 (m)	2.20-1.65 (m)	1.83 (m)	1.89 (m)	2.20-1.70 (m)	1.95-1.80 (m)	1.90-1.70 (m)
18-CH3	0.88 (d, 7.0)	0.78 (d, 6.4)	0.91 (d, 6.5)	0.89 (d, 7.5)	0.78 (d, 6.0)	0.88 (d, 7.5)	0.78 (d, 6.4)
19	4.68 (m)	4.48 (m)	4.82 (m)	4.68 (dd, 8.0, 4.5)	4.49 (m)	4.69 (m)	4.57 (m)
20	1.88 (m)	1.80 (m)	1.70 (m)	1.89 (m)	1.80 (m)	1.85 (m)	1.80 (m)
20-CH3	0.91 (d, 7.5)	0.81 (d, 6.4)	0.82 (d, 6.5)	0.91 (d, 7.0)	0.80 (d, 6.8)	0.89 (d, 7.0)	0.81 (d, 6.8)
21	1.40 (m), 1.0 (m)	1.55 (m), 0.95 (m)	1.20 (m), 0.95 (m)	1.41 (m), 1.0 (m)	1.55 (m), 0.95 (m)	1.50 (m), 1.0 (m)	1.55 (m), 0.95 (m)
22	1.69 (m)	1.80 (m)	1.55 (m)	1.68 (m)	1.70 (m)	1.70 (m)	1.72 (m)
22-CH3	0.82 (brd, 5.0)	0.71 (brd, 6.0)	0.83 (d, 6.4)	0.82 (d, 6.5)	0.70 (d, 6.4)	0.82 (d, 7.0)	0.70 (d, 6.4)
23	3.56 (brdd, 9.5, 2.0)	3.18 (m)	3.51 (m)	3.56 (dd, 9.5, 2.0)	3.15 (m)	3.56 (dd, 10.0, 1.5)	3.38-3.20 (m)
23-OH	-	5.03 (brd, 6.2)	-	-	5.03 (d, 6.8)	-	4.84 (brs)
24	2.70 (m)	2.90 (m)	2.73 (m)	2.78 (m)	3.15 (m)	2.78 (m)	3.03 (m)
24-CH3	-	-	-	0.96 (d, 7.0)	0.88 (d, 6.4)	0.97 (d, 7.0)	0.88 (d, 6.4)
24-CH2CH3	1.42 (m), 1.30 (m)	1.35 (brm), 1.23 (brm)	1.70-1.50 (m)	-	-	-	-
24-CH2CH3	0.98 (t, 7.5)	0.89 (t, 7.2)	0.84 (t, 6.5)	-	-	-	-
26	2.61 (m)	2.90-2.40 (m)	2.50 (m)	2.60 (m)	2.90-2.40 (m)	2.58 (m)	2.78-2.46 (m)
27	1.00 (t, 7.0)	0.89 (t, 8.0)	1.02 (t, 7.5)	1.00 (t, 7.0)	0.89 (t, 6.4)	1.01 (t, 7.0)	0.89 (t, 7.2)
1’	4.61 (dd, 10.0, 1.5)	4.59 (m)	4.53 (brd, 8.0)	4.61 (dd, 10.0, 1.5)	4.59 (brd, 6.4)	4.56 (brdd, 10.0, 1.0)	4.46 (dd, 9.2, 1.2)
2’	2.20 (m), 1.40 (m)	2.08 (m), 1.30 (m)	2.25 (m), 1.56 (m)	2.24 (m), 1.50 (m)	2.13 (m), 1.30 (m)	2.15 (m), 1.40 (m)	2.13 (m), 1.30 (m)
3’	4.56 (m)	4.47 (m)	4.63 (m)	4.56 (m)	4.43 (m)	3.48 (brdd, 7.5, 4.5)	3.38-3.20 (m)
3’-CONH2	-	6.48 (brs)	-	-	6.46 (brs)	-	-
3’-OH	-	-	-	-	-	-	4.84 (brs)
4’	3.07 (dd, 9.5, 9.0)	3.36 (m)	3.22 (m)	3.10 (dd, 9.5, 9.0)	3.36 (brm)	2.88 (dd, 9.0, 9.0)	3.38-3.20 (m)
5’	3.26 (m)	3.20 (m)	3.22 (m)	3.26 (m)	3.36 (m)	3.17 (m)	3.38-3.20 (m)
5’-CH3	1.27 (d, 6.0)	1.14 (d, 7.2)	1.30 (d, 6.0)	1.27 (d, 6.0)	1.13 (d, 6.4)	1.24 (d, 6.5)	1.11 (d, 6.0)

CD3OD;

DMSO-d6;

CDCl3;

500 MHz;

400 MHz;

100 MHz;

100 MHz; See supporting information (Figures S10, S19, S31, S36, S43) for NMR spectra.

The 13C NMR/HSQC spectra of 1 (Table 3) displayed 42 signals which corresponded to nine methyl groups, ten methylene groups, six quaternary carbons, and seventeen methine groups of which eight were oxygenated. In addition, carbon signals pertaining an acetal (δ 99.8), a ketal (δ 95.3), six olefinic (δ 140.1~118.3) an amide (δ 159.7), a lactone/carboxylic acid/amide (δ 174.0), and a ketone (δ 218.0) functionality appeared in the downfield region of the spectrum. The presence of 3’-O-carbamoyl-β-d-olivose sugar moiety in compound 1 was confirmed through both the MS fragmentation pattern and the cumulative analyses of 1H-1H-COSY/HMBC spectra (Figure 3). The 3J HMBC correlation observed between H-3’ and the quaternary carbon at δ 159.7, confirmed the attachment of the C-3’ sugar carbamoyl group. A substructure search of AntiBase 2012[12] using the 3’-O-carbamoyl-β-d-olivose moiety as a prompt revealed only four 20-membered macrolides - venturicidin A (2),[27-29] X-14952B (4),[30] 17-hydroxyventuricidin A (5),[14] and irumamycin (6).[31] Comparison of the 13C NMR chemical shifts of 1 with that reported for X-14952B (4) revealed both compounds to share an identical glycosylated macrolactone core with structural divergence at C-17 (Table 3). Specifically, compared to 4, 1 lacks a C-17 hydroxyl as indicated by a notable C-17 13C NMR chemical shift (δ 43.8 in 1 compared to δ 78.2 in 4). The 3J HMBC cross peaks observed between H-13 and C-1’ (δ 99.8), and between H-1’ and C-13 (δ 84.2) are consistent with the attachment of 3’-O-carbamoyl-β-d-olivose sugar moiety at C-13 position of 1 as previously observed for 2 and 4-6.[14, 30, 31] All of the remaining HMBC correlations (Figure 3) and NMR data (Table 2 and 3) are in full agreement with structure 1. The relative configuration at the stereocenters were indirectly established through the analyses of NOESY correlations (Figure 4) and their comparison to those of the reported analogues 2-9. [16, 32] Thus, thorough analyses of 1H, 13C, HSQC, 1H-1H-COSY, TOCSY, HMBC and NOESY spectra cumulatively established the structure of compound 1 as depicted in Figure 3 and was subsequently named as venturicidin C.

Table 3

13C NMR spectroscopic data of venturicidins C (1), A (2), and B (3) in comparison with the literature data of X-14952B (4)[30], and 17-hydroxyventuricidin A (5), (δ in ppm)

Position	1 a)	1 b)	1 c)	2 a)	2 b)	3 a)	3 b)	4 c)	5 c)
	δC d)	δC d)	δC d)	δC e)	δC d)	δC e)	δC d)	δC f)	δC
1	174.0, C	171.9, C	173.2, C	174.3, C	171.9, C	174.8, C	171.9, C	173.7, C	173.8, C
2	44.8, CH2	43.8, CH2	43.8, CH2	44.8, CH2	43.6, CH2	44.8, CH2	43.6, CH2	43.6, CH2	43.2, CH2
3	95.3, C	93.4, C	94.0, C	95.3, C	93.4, C	95.3, C	93.4, C	94.3, C	93.9, C
4	36.2, CH2	34.6, CH2	35.3, CH2	36.2, CH2	34.6, CH2	36.2, CH2	31.1, CH2	35.3, CH2	35.0, CH2
5	118.3, CH	117.5, CH	117.0, CH	118.3, CH	117.5, CH	118.3, CH	117.5, CH	117.0, CH	116.7, CH
6	134.1, C	131.8, C	133.3, C	134.1, C	131.8, C	134.1, C	131.8, C	133.7, C	132.7, C
6-CH3	19.5, CH3	18.9, CH3	19.5, CH3	19.5, CH3	18.9, CH3	19.5, CH3	19.5, CH3	19.1, CH3	19.1, CH3
7	81.8, CH	79.2, CH	80.3, CH	81.7, CH	79.2, CH	81.8, CH	79.2, CH	80.3, CH	79.9, CH
8	135.8, C	134.1, C	136.2, C	135.8, C	134.1, C	135.8, C	134.1, C	135.4, C	134.7, C
8-CH3	11.2, CH3	10.9, CH3	11.0, CH3	11.2, CH3	10.7, CH3	11.2, CH3	10.8, CH3	11.0, CH3	10.6, CH3
9	131.1, CH	129.9, CH	129.7, CH	131.0, CH	129.9, CH	131.3, CH	130.0, CH	129.7, CH	129.3, CH
10	28.4, CH2	26.9, CH2	27.3, CH2	28.4, CH2	26.9, CH2	28.4, CH2	26.9, CH2	27.3, CH2	27.0, CH2
11	27.3, CH2	25.5, CH2	26.1, CH2	27.3, CH2	25.5, CH2	27.4, CH2	25.5, CH2	26.2, CH2	25.9, CH2
12	36.1, CH2	34.3, CH2	35.5, CH2	36.1, CH2	34.3, CH2	36.8, CH2	34.3, CH2	35.5, CH2	34.5, CH2
13	84.2, CH	82.3, CH	83.3, CH	84.1, CH	82.3, CH	84.1, CH	82.3, CH	82.6, CH	82.2, CH
14	131.2, CH	129.1, CH	134.8, CH	131.2, CH	129.1, CH	131.1, CH	129.2, CH	134.8, CH	134.3, CH
15	140.1, CH	137.4, CH	134.6, CH	140.0, CH	137.4, CH	139.9, CH	137.2, CH	134.6, CH	133.9, CH
16	36.8, CH	34.8, CH	42.0, CH	36.8, CH	34.8, CH	36.8, CH	34.7, CH	42.3, CH	42.0, CH
16-CH3	20.2, CH3	19.4, CH3	18.0, CH3	20.2, CH3	19.4, CH3	20.2, CH3	18.9, CH3	17.4, CH3	17.1, CH3
17	43.2, CH2	41.3, CH2	43.8, CH2	43.1, CH2	41.3, CH2	43.2, CH2	41.3, CH2	78.2, CH	77.7, CH
18	33.2, CH	31.2, CH	35.0, CH	33.2, CH	31.2, CH	33.2, CH	34.6, CH	34.9, CH	34.5, CH
18-CH3	14.2, CH3	13.4, CH3	14.6, CH3	14.2, CH3	12.8, CH3	14.2, CH3	12.8, CH3	5.7, CH3	5.5, CH3
19	84.8, CH	80.8, CH	82.9, CH	84.7, CH	80.8, CH	84.8, CH	80.6, CH	82.2, CH	81.8, CH
20	33.5, CH	31.5, CH	33.6, CH	33.4, CH	31.5, CH	33.5, CH	31.2, CH	33.5, CH	33.5, CH
20-CH3	16.6, CH3	15.8, CH3	16.2, CH3	16.6, CH3	15.8, CH3	16.6, CH3	15.8, CH3	16.0, CH3	15.8, CH3
21	38.6, CH2	36.2, CH2	37.0, CH2	38.1, CH2	36.2, CH2	38.2, CH2	37.5, CH2	37.2, CH2	35.9, CH2
22	33.1, CH	31.1, CH	32.7, CH	33.0, CH	31.1, CH	33.1, CH	31.5, CH	32.8, CH	32.4, CH
22-CH3	11.5, CH3	11.7, CH3	13.0, CH3	11.5, CH3	10.9, CH3	11.5, CH3	10.9, CH3	12.9, CH3	14.0, CH3
23	78.8, CH	76.3, CH	78.0, CH	78.7, CH	76.3, CH	78.8, CH	76.6, CH	77.0, CH	77.6, CH
24	58.5, CH	56.6, CH	55.3, CH	50.8, CH	48.9, CH	50.9, CH	49.0, CH	55.4, CH	48.3, CH
24-CH3	-	-	-	13.9, CH3	13.4, CH3	13.9, CH3	13.4, CH3	-	14.1, CH3
24-CH2CH3	23.2, CH2	21.4, CH2	23.0, CH2	-	-	-	-	22.9, CH2	-
24-CH2CH3	12.2, CH3	12.8, CH3	12.0, CH3	-	-	-	-	12.0, CH3	-
25	218.0, C	214.5, C	217.1, C	217.9, C	214.5, C	218.0, C	214.5, C	217.5, C	216.8, C
26	37.2, CH2	35.4, CH2	37.0, CH2	37.2, CH2	35.4, CH2	37.2, CH2	35.4, CH2	37.4, CH2	35.9, CH2
27	7.9, CH3	7.4, CH3	7.8, CH3	7.9, CH3	7.4, CH3	7.9, CH3	7.4, CH3	7.6, CH3	7.4, CH3
1’	99.8, CH	97.6, CH	98.5, CH	99.8, CH	97.6, CH	100.3, CH	98.1, CH	98.4, CH	98.1, CH
2’	38.9, CH2	37.5, CH2	38.2, CH2	38.6, CH2	37.5, CH2	41.0, CH2	36.2, CH2	37.0, CH2	36.8, CH2
3’	75.4, CH	72.7, CH	75.4, CH	75.3, CH	72.7, CH	72.5, CH	70.5, CH	75.2, CH	74.8, CH
3’-CONH2	159.7, C	156.4, C	157.6, C	159.6, C	156.5, C	-	-	157.7, C	157.5, C
4’	75.6, CH	73.5, CH	75.8, CH	75.6, CH	73.5, CH	78.6, CH	76.3, CH	74.7, CH	75.0, CH
5’	73.4, CH	71.7, CH	72.2, CH	73.4, CH	71.7, CH	73.4, CH	71.5, CH	72.2, CH	71.9, CH
5’-CH3	18.5, CH3	18.0, CH3	18.0, CH3	18.5, CH3	18.0, CH3	18.5, CH3	18.1, CH3	17.7, CH3	17.7, CH3

CD3OD;

DMSO-d6;

CDCl3;

100 MHz;

125 MHz;

75 MHz; See supporting information (Figures S11-S21, S25-S33, S37-S45) for the NMR Spectra.

Figure 3

Selected 1H,1H-COSY (▬) and HMBC (→) correlations of venturicidin C (1)

Figure 4

Selected NOESY ( ) couplings of venturicidin C (1)

The physicochemical properties of compound 2 (40.0 mg l-1) were similar to those of 1. Low resolution ESI MS revealed a 749-Dalton-base peak suggesting its molecular weight as 14 amu less than that of 1. The molecular formula of compound 2 was deduced as C41H67NO11 based on the molecular ion peak observed at m/z 772.4676 [M + Na]+ in HR-ESI-MS spectrum. The presence of an additional fragment peak at m/z 541.3908 was consistent with generation of an aglycone fragment via the elimination of the sugar moiety and two water molecules (Figure 2). Like 1, the proton NMR spectrum of 2 indicated nine methyl groups. The methyl triplet at δ 0.98 in 1 (24-CH2CH) was missing in case of 2 and instead, a methyl doublet was displayed at δ 0.96 in 2 (24-CH). The 13C NMR spectrum revealed 41 signals. The chemical shift in the sugar residue and the lactone part of the aglycone moiety in 2 were nearly identical to those of 1 in both CD3OD and DMSO-d6 solvents (Tables 2 and 3). Thorough analyses of NMR spectra indicated 2 and 1 to share an identical 20-membered lactone core with the sole structural difference C-24 (-CH2CH3 in 1 versus -CH3 in 2). A substructure search in AntiBase 2012 revealed the identity of 2 as venturicidin A, also known as aabomycin A1 (2). Although this molecule was isolated previously from several Streptomyces species (including Streptomyces sp. No. 325-17, Streptomyces griseolus, Streptomyces xanthophaeus and Streptomyces aureofaciens) and its structure subsequently determined through chemical degradation and through partial NMR analyses, we report here for the first time the complete physicochemical and NMR spectroscopic data (Figures 5-6 and Tables 1-3).[27-29]

Figure 5

Selected 1H,1H-COSY (▬) and HMBC (→) correlations of venturicidin A (2)

Figure 6

Selected NOESY ( ) couplings of venturicidin A (2)

Likewise, compound 3 was isolated as white powder (10.8 mg l-1) and displayed similar physicochemical properties to 1 and 2. ESI-MS spectrum of 3 revealed a molecular weight of 706, 43 amu less than that of 2. The molecular formula of 3 was deduced as C40H66O10 (m/z 729.4594 [M + Na]+ with an identical fragment peak at m/z 541.3915 as 2, indicating the same aglycon in both compounds (Figure 2). This implicated structural divergence of the appended sugar wherein the 43 amu molecular weight difference between 3 and 2 could correlate to the sugar C-3’ amide group (-CONH2). Consistent with this, the carbamoyl carbon signal observed at δ 159.6 in the 13C NMR spectrum of compound 2 was absent in the spectrum for 3. Thus, through the cumulative analysis of NMR data and relevant precedent in the literature, the structure of compound 3 was confirmed as depicted in Figures 7-8 – the known molecule venturicidin B (aabomycin A2). While venturicidin B was previously reported as a metabolite of Streptomyces aureofaceins,[13] we report here for the first time the complete physicochemical and NMR spectroscopic data.

Figure 7

Selected 1H,1H-COSY (▬) and HMBC (→) correlations of venturicidin B (3)

Figure 8

Selected NOESY ( ) couplings of venturicidin B (3)

Including ventruricidin C reported herein, the venturicidins make up 9 of the 17 naturally-occurring 20-membered macrolide glycosides. The closely related congeners venturicidins A,[13] B,[13] X,[16] X-14952B,[30] 17-hydroxyventuricidin A,[14] irumamycin,[31] 3′-decarbamoylirumamycin[14] and irumanolide II[32] are summarized in Figure 1. Their remarkable potency against different fungal strains[14, 15] and their anti-trypanosomal activities[18] are notable. Consistent with this precedent, the antifungal activities of venturicidins A, B, and C against Cladosporium cucumerium ATCC 26212 were comparable in disc diffusion assays (supporting information, figure S48). Mechanistically, venturicidins inhibit F0F1-ATPase and are also known to inhibit ATP synthesis in both fungi and in bacteria.[17,19,33] However, unlike their structurally/mechanistically similar cytotoxic counterparts (ossamycin, cytovaricin and apoptolidin),[34] venturicidins A, B or C did not exhibit significant cytoxoxicity against non-small cell carcinoma cell line A549 (supporting information, figure S47). This distinction is noteworthy in the context of considering the further development of non-toxic anti-infective members of this unique family. None of these molecules exhibited antibacterial activities against bacterial test strains S. aureus ATCC6538 and S enterica ATCC10708 up to 124 μM concentrations.

EXPERIMENTAL SECTION

General Experimental Procedures

UV spectra were recorded on an Ultrospec 8000 spectrometer (GE, Pittsburgh, USA). NMR spectra were measured on Varian VnmrJ 500 (1H, 500 MHz; 13C, 125.7 MHz) and VnmrJ 400 (1H, 399.8 MHz; 13C, 100.5 MHz) spectrometers; the δ-values were referenced to the respective solvent signals. ESI mass spectra were recorded on a Finnigan LCQ ion trap mass spectrometer. HRESI mass spectra were recorded on AB SCIEX Triple TOF® 5600 System. HPLC-MS analyses were carried out in Waters 2695 LC module (Waters corp. Milford, MA, USA) using a Symmetry Anal C18 5 μm column (4.6 × 250 mm, (Waters corporation, Milford, MA 01757) and a gradient elution profile (solvent A: H2O, solvent B: acetonitrile; flow rate: 0.5 mL min-1; 0-4 min 90% A and 10% B, 4-22 min, 90-0% A, 22-27 min 0% A and 100% B, 27-29 min 0-90% A, 29-35 min 90% A and 10 % B). For preparative scale separation, Phenomenex (Torrance, CA 90501-1430) C18 column (10 × 250 mm, 5 μm) was used on a Varian (Varian, Palo Alto, CA, USA) ProStar Model 210 equipped with a photodiode diode array detector and a gradient elution profile (solvent A: H2O, solvent B: acetonitrile; flow rate: 5.0 mL min-1; 0-2 min 75% A and 25% B, 2-15 min, 75-0% A, 15-17 min 0% A and 100 % B, 17-18 min 0-75% A, 18-19 min 75% A and 25% B). All solvents used were of ACS grade and purchased from the Pharmco-AAPER (Brookfield, CT). Silica gel (230–400 mesh) for column chromatography was purchased from Silicycle (Quebec City, Canada). Rf values were measured on Polygram SIL G/UV254 (Macherey-Nagel & Co., Dueren, Germany). Amberlite XAD-16 was obtained from Sigma-Aldrich (St. Louis, MO, USA). Size exclusion chromatography was performed on Sephadex LH-20 (25 ~ 100 μm; GE Healthcare, Piscataway Township, NJ, USA).

Isolation of Streptomyces sp. TS-2-2 and its Taxonomy

A soil sample containing TS-2-2 was collected from the Truman Shepherd underground mine fire, Floyd County, KY (coordinates: N 37° 28.218’ and W 83° 51.132’). Streptomyces sp. TS-2-2 was isolated following previously reported methods.[35] Genomic DNA was isolated from a fully grown colony using InstaGene Matrix (Biorad, Hercules, CA, USA). DNA was purified using QIAquick PCR purification kit (Qiagen, Valencia, CA, USA). The partial 16S rRNA gene fragment was amplified using universal primers (27F, 5′-AGAGTTTGATCMTGGCTCAG-3′; 1492R, 5′-GGTTACCTTGTTACGACTT-3′) and Advantage GC2 DNA polymerase (Clontech, Mountain View, CA, USA).[36] PCR conditions were as follows: initial denaturation for 95 °C for 3 min followed by 30 cycles at 94 °C for 30 sec, 48 °C for 30 sec, 68 °C for 1 min 30 sec, followed by a final extension temperature at 68 °C for 5 min. QIAquick gel extraction kit (Qiagen) was used to gel-purify the amplified product. The amplified fragment was sequenced (1,256 bp) and the sequence was used to search the NCBI 16S rRNA library for bacteria and archaea by Basic Local Alignment Search Tool (BLAST). This comparison revealed a 99% identity to the 16S rRNA gene sequence of Streptomyces bingchenggensis BCW-1. The sequence of 16S rRNA has been deposited in the NCBI nucleotide database under the accession number KC526988.

Cell Viability Assay

Conversion of resazurin (7-hydroxy-10-oxido-phenoxazin-10-ium-3-one) to its fluorescent product resorufin was monitored to assess viability of human lung non-small cell carcinoma cell line A549. DMEM/F-12 Kaighn’s modification and MEM/EBSS media (Thermoscientific, Rockford, IL, USA) were used to grow A549 (ATCC, Manassas, VA, USA) with 10% heat-inactivated fetal bovine serum (FBS), 100 U ml-1 penicillin, 100 μg ml-1, streptomycin, 2 mM L-glutamine. Cells were seeded at a density of 2 × 103 cells per well in 96-well clear bottom culture plates (Corning, NY, USA), incubated 24 hours at 37 °C in a humidified atmosphere containing 5% CO2 and were subsequently exposed to known toxin (1.5 mM hydrogen peroxide, 10 μg ml-1 actinomycin D) and test compounds for two days. To assess cell viability, 150 μM of resazurin (Sigma, St. Louis, MO, USA) was added to each well, plates were shaken briefly for 10 seconds and incubated for another 3 hours (A549 cells) at 37 °C to allow viable cells to convert resazurin into resorufin. The fluorescence intensity for resorufin was detected on a scanning microplate spectrofluorometer FLUOstar Omega (BMG Labtech, Cary, NC, USA) using an excitation wavelength of 560 nm and an emission wavelength of 590 nm. The assay was repeated in three independent experiment replications. In each replication, the resorufin values of treated cells were normalized to, and expressed as percent of, the mean resorufin values of untreated, metabolically active cells (100%, all cells are viable). In addition, the CellTox Green Cytotoxicity Assay (Promega, Madison, WI) was applied for assessing the level of dead cells, or cytotoxicity, in the same A549 cell culture. Before assaying viability with resazurin, 20μl of CellTox™ Green Dye (a proprietary asymmetric cyanine dye) reagent was added to each well of plate with cells, plate was mixed on shaker for 1 minute, incubated for 15 minutes at room temperature and fluorescent intensity of dye intercalated with dead cells DNA was measured at an excitation wavelength of 485 nm and an emission wavelength of 520 nm on a scanning microplate spectrofluorometer FLUOstar Omega, then resazurin was added and viability was assayed as described above. Cytotoxicity values were converted to corresponding viability values and plotted on the same graph with resazurin assay data.

Media, Fermentation and Isolation

A-Medium

Glucose (10.0 g), yeast extract (5.0 g), soluble starch (20.0 g), peptone (5.0 g), NaCl (4.0 g), K2HPO4 (0.5 g), MgSO4·7H2O (0.5), and calcium carbonate (2 g) were dissolved in 1 liter of demineralized water. The suspension (pH 7.0) was sterilized by autoclaving for 33 min at 121 °C.

M2-agar plates

Glucose (4.0 g), malt extract (10.0 g), yeast extract (4.0 g), and agar (15.0 g) were dissolved in 1 liter of demineralized water. The suspension (pH 7.2) was sterilized by autoclaving for 33 min at 121 °C.

Fermentation, Extraction and Isolation

The terrestrial Streptomyces sp. TS2-2 was cultivated on M2-agar plates at 28 °C for 3 days. To prepare the seed culture, chuncks of grown agar plate were used to inoculate three-250 ml baffled flasks, each containing 100 ml of A-medium, and the cultures grown at 28 °C with shaking (210 rpm) for 3 days. An aliquot of seed culture (3 ml) was subsequently used to inoculate 80 250 ml baffled flasks, each containing 100 ml of A-medium. Fermentation was continued at 28°C with shaking (210 rpm) for five days. The obtained reddish-brown culture broth was centrifuged and filtered over celite. The supernatant was mixed with XAD-16 (4%) resin overnight, followed by filtration. The resin was washed with water (4 × 800 ml) and then extracted with methanol (3 × 600 ml). The methanol extract was subsequently evaporated in vacuo at 38 °C to afford 15.1 g of reddish-brown solid crude extract. The biomass (mycelium) was extracted with methanol (3 × 500 ml) followed by acetone (1 × 800 ml) and then evaporated in vacuo at 38 °C to yield 8.8 g of reddish-brown solid crude extract. Both extracts revealed different sets of metabolites based upon HPLC and TLC analyses wherein the targeted metabolites were found to be mostly present in the mycelium-extract. Thus, this extract was subjected to the following work up and isolation procedure.

The mycelial extract (8.8 g) was dissolved in MeOH/50% H2O (40 ml) and was fractionated with a gradient of H2O/0-100% CH3CN using RP-18 column (column 3 × 30 cm, 75 g) chromatography. This resulted in the generation of following fractions: 0.25 ml 0% CH3CN and 0.25 ml 10% CH3CN → fraction FI (1.6 g); 0.25 L 20% CH3CN → fraction FII (0.8 g); 0.25 L 30% CH3CN → fraction FIII (0.6 g); 0.25 L 40% CH3CN → fraction FIV (0.25 g); 0.25 L 60% CH3CN, 0.25 ml 50% CH3CN, 0.25 ml 25% CH3CN (combined based on the HPLC and TLC similarity) → fractions FV-FVII (1.2 g); 0.5 L 100% CH3CN → fraction FVIII (0.92 g). TLC analysis followed by anisaldehyde sulfuric acid development indicated that the target dark-green band was present mainly in fraction FVIII (0.92 g) (see supporting information, Figure S4). Further purification of fraction FVIII using sephadex LH-20 (CH2Cl2/40% MeOH, 2.5 × 50 cm) and then by HPLC (see supporting information Figure S6, for HPLC conditions) afforded venturicidins A (2; 40.0 mg ml-1), B (3; 10.8 mg ml-1) and C (1; 3.2 mg ml-1) as white powders. Fractions FI, FII and FV-FVII were avoided based on the HPLC/MS and TLC analysis, as no related compounds were observed. Further purifications of fractions FIII and FIV using Sephadex LH-20 (MeOH, 2.5 × 40 cm) led to the isolation of nocardamine-Fe-complex (240.0 mg, red solid, molecular weight 654 daltons) and ferrioxamine E (45.8 mg, orange solid), respectively.

Similarly, the XAD-extract (15.1 g) was dissolved in MeOH/50% H2O (60 ml) and then subjected to fractionation using RP-18 column (column 3 × 30 cm, 75 g) chromatography and gradients of H2O/0-100% CH3CN systems. This resulted in the generation of nine fractions as follows: 0.25 ml 0% CH3CN and 0.25 ml 10% CH3CN → fraction FI (5.5 g); 0.15 L 20% CH3CN → fraction FII (1.3 g); 0.25 L 20% CH3CN → fraction FIII (1.25 g); 0.25 L 30% CH3CN → fraction FIV (0.65 g); 0.25 L 40% CH3CN → fraction FV (2.5 g); 0.25 L 50% CH3CN → fraction FVI (0.20 g); 0.25 L 60% CH3CN → fraction FVII (0.15 g); 0.25 L 80% CH3CN → fraction FVIII (0.55 g); 0.5 L 100% CH3CN → fraction FIX (0.21 g). Fractions FI and FIX were discarded as no significant metabolites were observed by TLC or HPLC/MS. Fractions FII, FIII and FIV were combined based on their HPLC/MS and TLC similarities. Further purification of the combined fractions using Sephadex LH-20 and HPLC afforded adenosine (131.2 mg, white powder), riboflavin (120.8 mg, yellowish-green solid) and cyclo(d)-trans-4-OH-Pro-(d)-Phe (28.3 mg, colorless solid).[25] In a similar manner, fractions FVI and FVII were combined and purified to afford cyclo(d)-Pro-(d)-Phe[25] (29.8 mg, colorless solid) and N-(2-phenylethyl)-acetamide[26] (10.8 mg, colorless solid) in pure forms. HPLC purification of fraction FIII (0.55 g) led to the isolation of nocardamine[21, 22] (28.3 mg), dehydroxynocardamine[21] (4.8 mg) and desmethylenlnocardamine[21] (3.7 mg) as white solids. Fine crystals were observed in fraction FV (2.5 g) when it was kept overnight in H2O/40% CH3CN. Filtration of fraction of crystals followed by washing with water afforded nocardamine (1.6 g, white needles). The filtrate was not further analyzed as it mainly contained an unknown nocardamine-Fe-complex[23] (1.31 g, red solid, molecular weight 654 Daltons), (Figure S3).

Antibacterial and Antifungal Activity Tests

The fungal strain S. cerevisiae (ATCC 204508), and the two bacterial strains S. aureus (ATCC 6538) and S. enterica (ATCC 10708) were used as model strains for susceptibility assays. S. cerevisiae, S. enterica and S. aureus were grown in liquid or on agar plates using YAPD (ATCC medium number 1069), nutrient broth (BD 234000) and in tryptic soy broth (BD211825) media, respectively. Individual strains were grown in 5 ml medium for 16 h at 37 °C with shaking (200 rpm). An aliquot of a fully grown culture (100 μL) was diluted to 30 ml using sterile liquid medium. Aliquots (160 μL) of each diluted culture were then transferred into 96 well plates supplied with 2 μL of venturicidins (final concentration of range 1-125 μm). Negative controls tested in parallel include vehicle alone (DMSO) while positive controls ampicillin and kanamycin sulfate (200 μg each for S. enterica and S. aureus) or cycloheximide (50 μg for S. cerevisiae) were employed. The culture plate was incubated at 37°C for 16 h with shaking (200 rpm) and the OD600 was subsequently measured using a scanning microplate spectrofluorometer FLUOstar Omega (BMG Labtech, Cary, NC, USA). The acquired OD600 values were normalized considering the negative control wells have 100% viability.

The fungal strain Cladosporium cucumerinum was used in disc diffusion assays. Solutions of amphotericin B and test compounds were made in DMSO. Each sterile paper disc was loaded with 20 μL solution and was allowed to dry in the biosafety cabinet for 4 hours. The dried discs were then placed on the V8 agar plate following the homogenous distribution of Cladosporium cucumerinum spores. DMSO was used as a negative control. The plates were then incubated at 24°C for three days under the dark condition. Inhibition zone were then measured.

V8-medium

6 gram of calcium carbonate was added into 360 mL V8 juice and stirred the mixture for 30 minutes. Supernatant liquid was collected following centrifugation at 4000 × g for 10 minutes. pH of the solution was adjusted to 7.0 using 1 M KOH solution. The solution was then diluted to four fold with water. Bacto agar was added to 1.5% (W/V) and then autoclaved to prepare V8 agar plates.