Tiacumicin Congeners with Improved Antibacterial Activity from a

Article Cite This: J. Nat. Prod. 2018, 81, 1219−1224

pubs.acs.org/jnp

Tiacumicin Congeners with Improved Antibacterial Activity from a Halogenase-Inactivated Mutant Haibo Zhang,† Xiaoxing Tian,‡ Xiaohui Pu,‡ Qingbo Zhang,† Wenjun Zhang,† and Changsheng Zhang*,† †

CAS Key Laboratory of Tropical Marine Bio-resources and Ecology, Guangdong Key Laboratory of Marine Materia Medica, RNAM Center for Marine Microbiology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road, Guangzhou 510301, People’s Republic of China ‡ Institute of Materia Medica, Pharmaceutical College of Henan University, Kaifeng 475004, People’s Republic of China S Supporting Information *

ABSTRACT: Tiacumicin B (1, also known as fidaxomicin or difimicin) is a marketed drug for the treatment of Clostridium dif ficile infections. The biosynthetic pathway of 1 has been studied in Dactylosporangium aurantiacum subsp. hamdenensis NRRL 18085 and has enabled the identification of TiaM as a tailoring dihalogenase. Herein we report the isolation, structure elucidation, and bioactivity evaluation of 14 tiacumicin congeners (including 11 new ones) from the tiaM-inactivated mutant. A new tiacumicin congener, 3, with a propyl group at C-7‴ of the aromatic ring was found to exhibit improved antibacterial activity.

T

trapping an open-clamp conformational state and preventing simultaneous interaction of promoter regions.13a Our studies on tiacumcin B (1) have led to the elucidation of its biosynthetic pathway in D. aurantiacum NRRL 18085 by characterizing eight biosynthetic enzymes and the discovery of more than 40 new tiacumicin analogues.14 Specifically, the halogenase TiaM was shown to be able to catalyze sequential chlorinations (first at C-4‴ and then at C-6‴) of dideschlorotiacumcin B (2) to furnish 1 (Figure 1). Herein we report the isolation, structure elucidation, and antibacterial activities of 14 tiacumicin congeners from the tiaM-inactivated mutant TCM50 (ΔtiaM), including 11 new ones (3−13). The congener 3 showed a 4-fold improvement in antibacterial activity over the parent compound tiacumicin B (1).

iacumicins belong to a family of 18-membered macrolide antibiotics that were discovered from Dactylosporangium aurantiacum subsp. hamdenensis NRRL 18085.1 Similar family members, such as lipiarmycins from Actinoplanes deccanensis ATCC 219832 and Catellatospora sp. Bp3323-813 and clostomicins from Micromonospora echinospora subsp. armeniaca,4 were found to have the same scaffold. Tiacumicin B, also known as lipiarmycin A3,5 fidaxomicin, or difimicin,6 has been marketed as Dificid since May 2011 after approval by the Food and Drug Administration (FDA) of the United States as a drug to treat Clostridium dif f icile infections (CDI).6 Tiacumicin B (1, Figure 1) was demonstrated to be an inhibitor of bacterial RNA polymerase to preclude the initial separation of DNA strands,7 displaying a unique mechanism distinct from that of other RNA polymerase inhibitors, such as streptolydigin (elongation inhibitors)8 and rifamycins (transcription initiation inhibitors).9 Currently, tiacumicin B (fidaxomicin) is thought to be the best marketed drug to treat CDI, given its lower relapse rate than vancomycin treatment and no reported clinical resistance.6b In addition, tiacumicins were effective against methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococci (VRE),10 and multidrug-resistant Mycobacterium tuberculosis clinical strains3 and also displayed antitumor activities.11 Therefore, the intriguing activities of tiacumicin B have motivated the total syntheses of tiacumicin B or its aglycone by several groups.12 Recently, two studies reported the cryo-electron microscopy (cryo-EM) structures of Mycobacterium tuberculosis RNAP (RNA polymerase) holoenzyme in complex with 1.13 These studies show that 1 inhibits transcription initiation by © 2018 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION The tiaM-inactivation mutant TCM50 (ΔtiaM) was previously reported to produce dideschlorotiacumicin B (2) in the production medium.14b When the TCM50 mutant was cultured in an alternative YMS media (yeast extract 0.4%, malt extract 1%, soluble starch 0.4%, CoCl2·6H2O 5 mg/L, XAD-16 resin 5%, pH 7.2−7.4, 200 mL), more tiacumicin-related congeners were discovered (Figure S1). Subsequent isolation efforts by multiple chromatographic steps yielded 14 tiacumicin congeners (2−15), including 11 new (3−13) congeners and three known tiacumicins (2, 14, and 15). Nomenclature of the new tiacumicin congeners is provided in the Experimental Section. Received: November 23, 2017 Published: April 20, 2018 1219

DOI: 10.1021/acs.jnatprod.7b00990 J. Nat. Prod. 2018, 81, 1219−1224

Journal of Natural Products

Article

Figure 1. TiaM functions as a tailoring dihalogenase to convert dideschlorotiacumicin B (2) to tiacumicin B (1) by sequentially adding two chlorines, first at C-4‴ and then at C-6‴.

Table 1. 1H NMR (500 MHz) Spectroscopic Data for Compounds 3−11 in CD3OD (δH, multi (J in Hz)) no. 3 4 5 6 7 9 10 11 13 15 16 17 18 19 20 21 22 23 24 25 1′ 2′ 3′ 4′ 5′ 6′ 7′ 1″ 2″ 3″ 4″ 6″ 7″ 4‴ 6‴ 8‴ 9‴ 10‴ 2⁗ 3⁗ 4⁗

3

4

5

6

7

8

9

10

11

7.24 (d, 11.5) 6.62 (dd, 11.5, 14.5) 5.97, m 2.50 m, 2.71 m 4.25, brs 5.17 (d, 10.0) 2.74, m 3.71 (d, 9.5) 5.85, s 5.59 (t, 8.0) 2.45 m, 2.74 m 4.73 m, overlap 4.04, m 1.21 (d, 6.5) 4.45 (d, 11.5); 4.64 (d, 11.5) 1.68, s 1.30, m; 2.03, m 0.90 (t, 7.5) 1.84, s 1.78, s 4.66, s 3.56, m, overlap 3.76 (dd, 3.0, 10.0) 5.14 (t, 10.0) 3.55, m, overlap 1.31 (d, 6.5)

7.23 (d, 11.5) 6.61 (dd, 11.5, 14.5) 5.97, m 2.51 m, 2.71 m 4.25, brs 5.16 (d, 10.0) 2.73, m 3.73 (d, 9.5) 5.86, s 5.59 (t, 8.0) 2.45 m, 2.75 m 4.74, m 4.04, m 1.20 (d, 6.0) 4.44 (d, 11.5); 4.64 (d, 11.5) 1.68, s 1.30, m; 2.04, m 0.90 (t, 7.5) 1.84, s 1.78, s 4.67, s 3.58, m, overlap 3.80 (dd, 3.5, 10.0) 5.13 (t, 10.0) 3.57, m, overlap 1.29 (d, 6.0)

7.42 (d, 11.5) 6.65 (dd, 11.5, 14.5) 6.09, m 2.56 m, 2.73 m 4.28, brs 5.21 (d, 10.0) 2.68, m 3.73 (d, 9.5) 5.88, s 5.31 (t, 8.0) 2.71 m, 2.83 m 5.16 (d, 3.5) 2.22, s 4.51 (d, 11.5); 4.58 (d, 11.5) 1.61, s 1.30, m; 2.02, m 0.89 (t, 7.5) 1.69, s 1.89, s 4.65, s 3.52 (d,3.5) 3.82 (dd, 3.0, 10.0) 5.14 (t, 10.0) 3.56, m, overlap 1.29 (d, 6.5)

7.25 (d, 11.5) 6.61 (dd, 11.5, 15.0) 5.98, m 2.52 m, 2.72 m 4.26, brs 5.19 (d, 10.5) 2.73, m 3.75 (d, 9.5) 5.84, s 5.57 (t, 8.0) 2.36 m, 2.60 m 4.90 m, overlap 1.73 m, 1.85 m 0.98 (t, 7.5) 4.43 (d, 11.5); 4.63 (d, 11.5) 1.65, s 1.32, m; 2.02, m 0.90 (t, 7.5) 1.82, s 1.73, s 4.66, s 3.58, m, overlap 3.78 (dd, 3.0, 10.0) 5.14 (t, 10.0) 3.56, m, overlap 1.30 (d, 6.5)

7.28 (d, 11.0) 6.62 (dd, 11.0, 14.0) 5.98, m 2.52 m, 2.71 m 4.26, brs 5.20 (d, 10.0) 2.73, m 3.75 (d, 9.5) 5.84, s 5.57 (t, 8.0) 2.35 m, 2.60 m 4.81 m, overlap 1.73 m, 1.85 m 0.99 (t, 7.5) 4.48 (d, 11.5); 4.62 (d, 11.5) 1.65, s 1.32, m; 2.02, m 0.91 (t, 7.5) 1.82, s 1.73, s 4.61, s 3.90 (d, 3.0) 3.76 (dd, 3.5, 10.0) 5.23 (t, 10.0) 3.58, m, overlap 1.32 (d, 6.0)



7.26 (d, 11.5) 6.61 (dd, 11.5, 15.0) 5.99, m 2.51 m, 2.71 m 4.26, brs 5.19 (d, 10.5) 2.72, m 3.73 (d, 9.5) 5.83, s 5.59 (t, 8.0) 2.35 m, 2.60 m 4.90 m, overlap 1.74 m, 1.86 m 0.99 (t, 7.5) 4.44 (d, 11.5); 4.64 (d, 11.5) 1.65, s 1.31, m; 2.02, m 0.90 (t, 7.5) 1.83, s 1.76, s 4.66, s 3.57, m, overlap 3.77 (dd, 3.0, 10.0) 5.15 (t, 10.0) 3.55, m, overlap 1.32 (d, 6.0)

3.56, s 4.73, s 3.94 (d, 3.0) 3.74 (dd, 3.5, 10.5) 5.04 (d, 10.5) 1.16, s 1.14, s 6.18 (d, 2.5) 6.23 (d, 2.5) 2.80, m 1.63, m 1.01 (t, 7.5) 2.61, s 1.20 (d, 6.5) 1.18 (d, 6.5)

3.58, s 4.73, s 3.94 (d, 3.0) 3.75 (dd, 3.5, 10.0) 5.03 (d, 10.0) 1.14, s 1.16, s 6.19 (d, 2.5) 6.23 (d, 2.5) 2.49, s

7.26 (d, 11.0) 6.67 (dd, 11.0, 14.0) 5.98, m 2.54 m, 2.70 m 4.25, brs 5.15 (d, 10.0) 2.72, m 3.72 (d, 9.5) 5.85, s 5.60 (t, 8.0) 2.44 m, 2.74 m 4.74, m 4.05, m 1.21 (d, 6.5) 4.48 (d, 11.5); 4.69 (d, 11.5) 1.68, s 1.32, m; 2.04, m 0.90 (t, 7.5) 1.84, s 1.78, s 4.68, s 3.59, m, overlap 3.83 (dd, 3.0, 10.0) 5.25 (t, 10.0) 3.54, m, overlap 3.70 (d, 6.5); 3.74 (d, 6.5) 3.58, s 4.73, s 3.94 (d, 3.0) 3.75 (dd, 3.5, 10.5) 5.04 (d, 10.5) 1.15, s 1.17, s 6.18 (d, 2.5) 6.25 (d, 2.5) 2.86, m 1.24 (t, 7.5)

3.59, s 4.72, s 3.93 (d, 3.0) 3.74 (dd, 3.5, 10.5) 5.02 (d, 10.5) 1.09, s 1.14, s 6.19 (d, 2.5) 6.25 (d, 2.5) 2.89, m 1.23 (t, 7.5)

3.58, s 4.74, s 3.94 (d, 3.0) 3.75 (dd, 3.0, 10.0) 5.04 (d, 10.5) 1.14, s 1.17, s 6.19 (d, 2.5) 6.25 (d, 2.5) 2.86, m 1.23 (t, 7.5)

4.74, s 3.94 (d, 3.0) 3.76 (dd, 3.5, 10.0) 5.04 (d, 10.0) 1.14, s 1.17, s 6.17 (d, 2.0) 6.24 (d, 2.0) 2.87, m 1.23 (t, 7.5)

3.58, s 4.73, s 3.95 (d, 3.0) 3.75 (dd, 3.0, 10.0) 5.06 (d, 10.5) 1.15, s 1.15, s 6.18 (d, 2.5) 6.26 (d, 2.5) 2.86, m 1.21 (t, 7.5)

3.56, s 4.70, s 3.92 (d, 3.0) 3.74 (dd, 3.5, 10.5) 5.02 (d, 10.0) 1.14, s 1.12, s 6.16 (d, 2.5) 6.23 (d, 2.5) 2.84, m 1.20 (t, 7.5)

3.58, s 4.68, s 3.90 (d, 3.0) 3.55, m, overlapped 3.50 (d, 10.0) 1.10, s 1.25, s 6.18 (d, 2.5) 6.25 (d, 2.5) 2.87, m 1.23 (t, 7.5)

2.61, m 1.20 (d, 6.5) 1.18 (d, 6.5)

2.61, m 1.20 (d, 6.5) 1.18 (d, 6.5)

2.62, m 1.20 (d, 6.5) 1.18 (d, 6.5)

2.40 (q, 7.5) 1.15 (t, 7.5)

2.07, s

2.61, m 1.20 (d, 6.5) 1.18 (d, 6.5)

2.61, m 1.20 (d, 6.5) 1.18 (d, 6.5)

The spectroscopic data of 2, 14, and 15 are identical to those reported.14b,c Comparison of spectroscopic data of 2−4 (Tables 1 and 2, Figures S2 and S3) showed that the three compounds differed

from each other only in the substituted groups at C-7‴ of the aromatic ring moiety (Figure 2). In 2, a C-7‴ ethyl group was found.14b COSY correlations of H3-10‴/H2-9‴/H2-8‴ and HMBC correlations from H2-8‴ to C-6‴/C-7‴/C-9‴ 1220



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Table 2. 13C NMR (125 MHz) Spectroscopic Data for Compounds 3−11 (δC, Type) in CD3OD no.

3

4

5

6

7

8

9

10

11

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 1′ 2′ 3′ 4′ 5′ 6′ 7′ 1″ 2″ 3″ 4″ 5″ 6″ 7″ 1‴ 2‴ 3‴ 4‴ 5‴ 6‴ 7‴ 8‴ 9‴ 10‴ 1⁗ 2⁗ 3⁗ 4⁗

169.2, C 125.7, C 146.4, CH 128.6, CH 143.9, CH 37.4, CH2 73.6, CH 137.0, C 124.7, CH 42.6, CH 94.4, CH 136.5 C 134.7, CH 136.5, C 126.9, CH 28.5, CH2 78.7, CH 68.3, CH 20.4, CH3 64.0, CH2 15.5, CH3 27.0, CH2 11.4, CH3 14.0, CH3 17.6, CH3 102.4, CH 82.6, CH 72.9, CH 76.0, CH 71.9, CH 18.2, CH3 62.3, CH3 97.3, CH 73.3, CH 70.6, CH 76.1, CH 74.6, C 28.8, CH2 18.8, CH3 172.1, C 106.3, C 165.4, C 101.9, CH 163.6, C 111.9, CH 148.8, C 39.6, CH2 26.4, CH2 14.8, CH3 178.5, C 35.5, CH 19.2, CH3 19.6, CH3

169.2, C 125.8, C 146.3, CH 128.6, CH 143.8, CH 37.4, CH2 73.3, CH 137.0, C 124.7, CH 42.6, CH 94.4, CH 136.5 C 134.7, CH 137.1, C 126.9, CH 28.4, CH2 78.7, CH 68.3, CH 20.4, CH3 64.0, CH2 15.5, CH3 27.0, CH2 11.4, CH3 14.0, CH3 17.6, CH3 102.4, CH 82.6, CH 73.6, CH 76.3, CH 71.9, CH 18.2, CH3 62.3, CH3 97.3, CH 73.0, CH 70.6, CH 76.0, CH 74.6, C 28.8, CH3 18.8, CH3 172.6, C 106.0, C 166.2, C 101.8, CH 164.0, C 112.7, CH 144.7, C 24.7, CH3

169.2, C 124.6, C 146.8, CH 128.7, CH 144.0, CH 37.4, CH2 73.6, CH 137.1, C 125.4, CH 42.6, CH 94.4, CH 136.5 C 134.7, CH 137.0, C 126.8, CH 28.4, CH2 78.7, CH 68.3, CH 20.4, CH3 63.8, CH2 15.5, CH3 27.0, CH2 11.4, CH3 14.1, CH3 17.7, CH3 101.9, CH 82.5, CH 73.3, CH 71.8, CH 76.6, CH 63.0, CH2 62.3, CH3 97.3, CH 73.1, CH 70.6, CH 76.0, CH 74.6, C 28.8, CH3 18.8, CH3 172.0, C 106.3, C 165.1, C 101.8, CH 163.9, C 111.0, CH 150.5, C 30.3, CH2 16.9, CH3

168.2, C 124.6, C 148.1, CH 128.4, CH 144.8, CH 37.7, CH2 73.2, CH 137.2, C 124.1, CH 42.5, CH 94.3, CH 137.7 C 133.9, CH 138.0, C 124.7, CH 30.0, CH2 78.9, CH 206.3, C 26.3, CH3 62.9, CH2 15.2, CH3 27.1, CH2 11.4, CH3 13.5, CH3 17.6, CH3 101.3, CH 82.8, CH 73.3, CH 76.2, CH 71.7, CH 18.2, CH3 62.3, CH3 97.3, CH 72.8, CH 70.6, CH 76.0, CH 74.6, C 28.8, CH3 18.8, CH3 172.2, C 106.0, C 165.5, C 101.9, CH 163.9, C 111.1, CH 150.7, C 30.5, CH2 17.0, CH3

169.2, C 125.7, C 146.4, CH 128.4, CH 143.8, CH 37.2, CH2 73.4, CH 137.1, C 124.5, CH 42.4, CH 94.5, CH 136.7 C 134.4, CH 136.8, C 125.9, CH 32.1, CH2 76.4, CH 27.3, CH2 10.6, CH3 63.9, CH2 15.3, CH3 27.1, CH2 11.4, CH3 13.8, CH3 17.6, CH3 102.3, CH 82.6, CH 72.9, CH 76.2, CH 71.8, CH 18.2, CH3 62.3, CH3 97.4, CH 73.3, CH 70.6, CH 76.0, CH 74.6, C 28.8, CH3 18.8, CH3 172.1, C 106.2, C 165.3, C 101.9, CH 163.9, C 111.1, CH 150.5, C 30.4, CH2 16.9, CH3


169.2, C 125.8, C 146.3, CH 128.6, CH 143.8, CH 37.4, CH2 73.6, CH 137.0, C 124.7, CH 42.6, CH 94.4, CH 136.5 C 134.7, CH 137.1, C 126.9, CH 28.4, CH2 78.7, CH 68.3, CH 20.4, CH3 64.0, CH2 15.5, CH3 27.0, CH2 11.4, CH3 14.0, CH3 17.6 CH3 102.4, CH 82.6, CH 72.9, CH 76.2, CH 71.8, CH 18.2, CH3 62.3, CH3 97.3, CH 73.3, CH 70.6, CH 76.2, CH 74.6, C 28.8, CH3 18.8, CH3 172.1, C 106.2, C 165.3, C 101.9, CH 163.9, C 111.1, CH 150.6, C 30.4, CH2 16.9, CH3

169.2, C 125.8, C 146.4, CH 128.7, CH 143.7, CH 37.4, CH2 73.6, CH 137.2, C 124.7, CH 42.7, CH 94.4, CH 136.5 C 134.8, CH 137.1, C 126.9, CH 28.5, CH2 78.8, CH 68.4, CH 20.4, CH3 64.1, CH2 15.5, CH3 27.1, CH2 11.5, CH3 14.1, CH3 17.7, CH3 102.5, CH 82.7, CH 72.9, CH 76.3, CH 71.8, CH 18.3, CH3 62.2, CH3 97.3, CH 73.3, CH 70.7, CH 76.5, CH 74.7, C 28.8, CH3 18.8, CH3 172.2, C 106.3, C 165.4, C 101.9, CH 163.9, C 111.1, CH 150.6, C 30.4, CH2 16.9, CH3


178.5, C 35.5, CH 19.2, CH3 19.6, CH3

178.6, C 35.5, CH 19.2, CH3 19.6, CH3

178.5, C 35.5, CH 19.2, CH3 19.6, CH3

178.5, C 35.5, CH 19.2, CH3 19.6, CH3

178.5, C 35.5, CH 19.2, CH3 19.6, CH3

176.0, C 28.6, CH2 9.6, CH3

172.7, C 21.2, CH3

(Figure S2) supported the presence of an n-propyl group at C-7‴ in 3. Similarly, HMBC correlations from H3-8‴ to C-2‴/C-6‴/ C-7‴ (Figure S3) confirmed the presence of a C-7‴ methyl group in 4. Compound 5 was found to possess a 2-O-methyl-Dmannose moiety compared to a 2-O-methyl-D-rhamnose moiety in 2 (Figure 2). This was confirmed by the presence of a

hydroxylated methylene group at C-6′ of 5 and supported by HMBC correlations from H-6′a/H-6′b to C-5′ and H-4′ to C-5′/C-1‴ (Tables 1 and 2, Figure S4). Analysis of the 1D and 2D NMR spectroscopic data of 6 (Figure 2) revealed the presence of a carbonyl group at C-18 (δC 206.3, Tables 1 and 2, Figure S5), which differed from the 1221



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Figure 2. Tiacumicins produced by the halogenase TiaM-inactivated mutant TCM50 and the proposed functions of the type I polyketide synthase TiaB and the acyltransferase TiaF. The stereochemistry of new compounds was tentatively assigned as the same as that of 1.

These assignments in 13 were confirmed by COSY correlations of H2-16/H-17/H-18/H3-19 and HMBC correlations from H3-3⁗/H-4″ to C-1⁗ (Figure S12). Antimicrobial activities of compounds 1−13 (excluding 5 as it is unstable) were evaluated by measuring minimal inhibition concentrations (MICs) against five bacterial strains, including methicillin-resistant Staphylococcus aureus shhs-A1, S. aureus ATCC 29213, Bacillus thuringensis SCSIO BT01, Micrococcus luteus SCSIO ML01, and Enterococcus faecalis ATCC 29212 (Table 4). Tiacumicin congener 3 was more potent than the parent compound 1 against Bacillus thuringiensis, Micrococcus luteus, and Enterococcus faecalis (Table 4). Consistent with previous observation,13 tiacumicins lacking the hydroxy group at C-18 or the isobutyryl group at C-4″ were less active. It should be also noted that the size of the alkyl substituents at C-7‴ exerts a large effect on the antibacterial activity of compounds. For example, compound 2 (C-7‴ ethyl) showed better inhibition activities than 4 (C-7‴ methyl), but was less active than 3 (C-7‴ propyl). This point should be notable in future development of better antibacterial agents of the tiacumicin family. Biosynthetically, the iterative type I polyketide synthase TiaB was proposed to be responsible for generating the aromatic ring moiety, which was in turn incorporated into tiacumicins by the acyltransferase TiaF.13a The variety in the functionality at C-7‴ (methyl, ethyl, or n-propyl) should be derived from the tolerance of TiaB toward different starter units and the flexibility of TiaF in recognizing slightly modified aromatic substrates (Figure 2). This discovery hints at an opportunity to engineer both TiaB and TiaF to produce tiacumicin congeners with varied aromatic rings.

C-18 hydroxy group in 2. The assignment of the carbonyl group at C-18 in 6 was supported by HMBC correlations from H3-19 to C-18/C-17 and from H-17 to C-15/C-16/C-18 (Figure S5). An additional methylene group (CH2-18, δC 27.3, δH 1.73 m, 1.85 m, Figure S6, Tables 1 and 2) was found in 7 (Figure 2) when compared to 2. The location of the methylene group at C-18 in 7 was deduced from COSY correlations of H2-16/H-17/ H2-18/H3-19 and was further supported by HMBC correlations from H-17 to C-16/C-1 and from H2-18 to C-17/C-19 (Figure S6). The NMR data of 8 (Figure S7) were similar to those of 7. A hydroxy group was found at C-2′ in 8 (Figure 2), which was supported by COSY correlations of H-1′/H-2′/H-3′/ H-4′ and HMBC correlations from H-2′ to C-1′/C-3′ (Figure S7), different from the C-2′ methoxy group in 7. Comparison of spectroscopic data of 9 and 10 (Tables 1 and 2, Figures S8 and S9) and 2 revealed that they differed from each other in the side acyl chains attached to C-4″ OH of the 5-methyl-D-rhamnose moiety (Figure 2). A propionyl group was esterified with the C-4″ hydroxy group in 9, which was confirmed by COSY correlations of H2-2⁗/H3-3⁗ and key HMBC correlations from H3-3⁗ to C-1⁗/C-2⁗ and from H-4″ to C-1⁗ (Figure S8). An acetyl group (H3-2⁗, δH 2.07) was esterified with the C-4″ OH in 10, supported by the HMBC correlations from H3-2⁗ to C-1⁗ and from H-4″ to C-1⁗ (Figure S9). No acyl group was observed at C-4″ of 11 (Figure 2). This was confirmed by COSY correlations of H-1″/H-2″/H-3″/ H-4″ and HMBC correlations from H-6″/H-7″ to C-4″/C-5″ (Tables 1 and 2, Figure S10). Meanwhile, COSY correlations of H3-19/H2-18/H-17 and HMBC correlations from H3-19 to C-17/C-18 indicated that a methylene group was present at C-18 in 11 (Figure S10). Comparison of NMR data showed that 12 (Table 3, Figure S11) was similar to the known compound 14 (Figure 2).14b,c The only difference was the presence of a hydroxy group at C-6′ in 12, which was supported by COSY correlations of H-1′/H-2′/H-3′/ H-4′/H-5′/H-6′ and HMBC correlations from H-6′/H-4′ to C-5′ (Figure S11). Analyses of the spectroscopic data of 13 and the known compound 15 clearly showed that 13 differed from 15 by the presence of an isobutyryl ester moiety at C-4″ in 13 and a hydroxy at C-18 (Table 3, Figure 2, Figure S12).

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EXPERIMENTAL SECTION

General Experimental Procedures. UV spectra were recorded on a UV-2600 spectrophotometer (Shimadzu). IR spectra were recorded on an Affinity-1 FT-IR spectrometer (Shimadzu). NMR spectra were recorded on a Bruker AV-500 NMR spectrometer (Bruker Biospin) with tetramethylsilane (TMS, δ 0.0 ppm) as the internal standard. Mass spectrometric data were obtained on a time-of-flight mass spectrometer (Bruker maxis 4G) for HRESIMS. Materials for column chromatography (CC) were silica gel (100−200 mesh; 300−400 mesh; Jiangyou Silica Gel Development Inc.), Sephadex LH-20 (40−70 μm; Amersham 1222



Article

Table 3. 1H (500 MHz) and 13C NMR (125 MHz) Spectroscopic Data for Compounds 12 and 13 in CD3OD 12 no.

δC, type

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

169.3, C 125.4, C 147.1, CH 128.5, CH 144.1, CH 37.2, CH2 73.5, CH 136.8, C 124.5, CH 42.5, CH 94.2, CH 137.0, C 134.4, CH 137.0, C 125.7, CH 30.8, CH2 76.5, CH 27.3, CH2 10.6, CH3 63.0, CH2

21 22 23 24 25 1′ 2′ 3′ 4′ 5′ 6′ 7′ 1″ 2″ 3″ 4″ 5″ 6″ 7″ 1⁗ 2⁗ 3⁗ 4⁗

15.3, CH3 27.1, CH2 11.4, CH3 13.8, CH3 17.6, CH3 100.6, CH 72.5, CH 75.4, CH 68.7, CH 78.5, CH 63.1, CH3 62.3, CH3 97.3, CH 73.2, CH 72.3, CH 74.9, CH 76.0, C 28.9, CH3 18.1, CH3

Table 4. Antibacterial Activities of Tiacumicins 1−13

13

δH, multi (J in Hz)

7.29 (d, 9.5) 6.65 (dd, 9.5, 12.0) 5.98, m 2.53, m; 2.72, m 4.26, brs 5.19 (d, 8.5) 2.72, m 3.73 (d, 8.0) 5.83, s 5.58 (t, 6.5) 2.36; m; 2.61; m 4.90, m, overlap 1.75, m; 1.86, m 0.99 (t, 7.5) 4.50 (d, 10.0); 4.61 (d, 10.0) 1.65, s 1.33, m; 2.04, m 0.90 (t, 7.5) 1.85, s 1.71, s 4.51, s 3.80 (d, 2.5) 3.42, m, overlap 3.56, m, overlap 3.24, m 3.75, m; 3.92,m 3.58, s 4.68, brs 3.90 (d, 3.0) 3.56, m, overlap 3.42 (dd, 3.0, 8.0) 1.09, s 1.25, s

δC, type 169.2, C 125.8, C 146.2, CH 128.6, CH 143.7, CH 37.4, CH2 73.6, CH 137.0, C 124.7, CH 42.6, CH 94.4, CH 136.5, C 134.7, CH 137.1, C 126.9, CH 28.4, CH2 78.7, CH 68.3, CH2 20.4, CH3 63.8, CH2 15.5, CH3 27.0, CH2 11.4, CH3 14.0, CH3 17.6, CH3 102.3, CH 82.6, CH 75.1, CH 74.0, CH 74.3, CH 18.1, CH3 62.2, CH3 97.3, CH 73.3, CH 70.6, CH 76.0, CH 74.6, C 28.8, CH3 18.8, CH3 178.5, C 35.5, CH 19.2, CH3 19.6, CH3

δH, multi (J in Hz) 1 2 3 4 6 7 8 9 10 11 12 13

7.22 (d, 11.0) 6.60 (dd, 8.5, 10.5) 5.96, m 2.50, m; 2.70, m 4.25, brs 5.15 (d, 7.5) 2.73, m 3.73 (d, 7.0)

Staphylococcus aureus ATCC 29213

MRSA shhs-A1 (clinical sample)

Micrococcus luteus SCSIO ML01

Bacillus thuringiensis SCSIO BT01

Enterococcus faecalis ATCC 29212

4 2 2 16 >128 >128 >128 8 128 >128 >128 >128

1 2 2 16 >128 >128 >128 8 >128 >128 >128 >128

4 2 1 16 >128 >128 >128 16 64 8 >128 >128

2 1 0.5 8 8 4 16 2 8 16 >128 >128

16 16 4 64 >128 >128 >128 32 >128 >128 >128 >128

5.85, s

YMS medium (yeast extract 0.4%, malt extract 1%, soluble starch 0.4%, CoCl2·6H2O 5 mg/L, macroporous resin XAD-16 5%, pH 7.2−7.4) in 1 L Erlenmeyer flasks (200 mL of media per flask) to afford a total of 14 L cultures. The fermentation broth was centrifuged at 4000 rpm for 10 min at 4 °C to recover the XAD-16 resin. Mycelia were spooned up carefully from the XAD-16 resin and were extracted with acetone−water (70%) three times. Subsequently, acetone were removed by a rotary evaporator, and the remaining water solution were extracted three times with EtOAc to get the crude extract I. The XAD-16 resin was soaked with 4 L of EtOH three times, and the extracts were concentrated under vacuum to get the crude extract II. The crude extracts I and II were combined (3.75 g) and chromatographed over a silica gel column (100− 200 mesh, 90 g) and eluted with a gradient of CHCl3−MeOH (100:0, 99:1, 98:2, 96:4, 92:8, 84:16, 68:32, and 0:100) to obtain 15 fractions. Subsequently, the 15 fractions were monitored by TLC (CHCl3− MeOH, 10:1) to be further fractioned to six subfractions, A−F. Fraction D was subjected to a YMC*GEL ODS-A column (30 × 250 mm, 12 nm, S-50 μm; YMC Company Ltd.) and was eluted with a MeCN−H2O system (a gradient elution 5/95 → 0/100, v/v, 20 mL/min) to obtain nine fractions (D1−D9). Fraction D7 was chromatographed on a silica gel column (300−400 mesh) and eluted with CHCl3−MeOH (100:0 → 0/100) to obtain eight subfractions. Fraction D7-8 was purified by reversed-phase MPLC, semipreparative reversed-phase HPLC, and preparative TLC (CHCl3−MeOH, 10:1) to afford 2 (730 mg), 4 (19.0 mg), 5 (1.8 mg), 9 (32.9 mg), 10 (15.2 mg), and 11 (5.0 mg). Similar purification efforts on fraction D-8 provided 3 (9.3 mg), 6 (10.2 mg), 7 (32.9 mg), and 8 (3.3 mg). Fraction E was chromatographed by reversed-phase MPLC to provide eight fractions, E-1−E-8. Tiacumicin analogues were enriched in three fractions, E-6, E-7, and E-8. Compound 12 (2.0 mg) was obtained from fraction E-6 by semipreparative reversedphase HPLC. Purification of fraction E-7 provided two compounds, 14 (7.8 mg) and 15 (2.0 mg). Compound 13 (6.0 mg) was purified from fraction E-8 by semipreparative reversed-phase HPLC. Dideschloro-7‴-n-propyltiacumicin B (3): pale white powder, UV (MeOH) λmax (log ε) 219 (4.37), 266 (4.28) nm; IR (KBr) νmax 3393, 2976, 2934, 1697, 1635, 1259, 1024 cm−1; 1H and 13C NMR data, Tables 1 and 2; HRESIMS m/z 1001.5136 [M − H]− (calcd for C53H77O18, 1001.5115). Dideschloro-7‴-methyltiacumicin B (4): pale white powder, UV (MeOH) λmax (log ε) 218 (4.45), 266 (4.38) nm; IR (KBr) νmax 3399, 2976, 2932, 1692, 1643, 1258, 1068, 1024 cm−1; 1H and 13C NMR data, Tables 1 and 2; HRESIMS m/z 973.4838 [M − H]− (calcd for C51H73O18, 973.4802). Dideschloro-6′-hydroxytiacumicin B (5): pale white powder, UV (MeOH) λmax (log ε) 218 (4.34), 266 (4.24) nm; IR (KBr) νmax 3379, 2920, 1695, 1643, 1258, 1072, 1024 cm−1; 1H and 13C NMR data, Tables 1 and 2; HRESIMS m/z 1003.4897 [M − H]− (calcd for C52H75O19, 1003.4908). Dideschloro-18-carbonyltiacumicin B (6): pale white powder, UV (MeOH) λmax (log ε) 218 (4.46), 267 (4.42) nm; IR (KBr) νmax 3393,

5.59 (t, 6.0) 2.44, m; 2.74, m 4.81, m, overlap 4.03, m 1.20 (d, 4.5) 4.40 (d, 8.0); 4.60 (d, 8.0) 1.68, s 1.30, m; 2.04, m 0.90 (t, 5.0) 1.83, s 1.77, s 4.55, s 3.48 (d, 2.5) 3.40 (dd, 2.5, 6.5) 3.24, m, overlap 3.24, m, overlap 1.33 (d, 4.0) 3.53, s 4.73, s 3.94 (dd,1.0,2.5) 3.75 (dd, 2.5, 7.5) 5.03 (d, 7.0) 1.14, s 1.16, s 2.61, m 1.20 (d, 5.0) 1.18 (d, 5.0)

Pharmacia Biotech), and YMC*GEL ODS-A (12 nm S-50 μm; YMC Company Ltd.). Thin-layer chromatography (TLC, 0.1−0.2 or 0.3− 0.4 mm) was conducted with precoated glass plates (silica gel GF254, 10−40 nm, Jiangyou Silica Gel Development, Inc.). Medium-pressure liquid chromatography (MPLC) was performed on a CHEETAH flash system (Bonna-Agela Technologies Inc.). Compounds were detected with a 1260 Infinity HPLC (Agilent Inc.). Semipreparative HPLC was carried out on a Gemini C18 column (5 μm; Phenomenex Company Ltd.). Macroporous resin XAD-16 was obtained from Amberlite (Rohm and Haas Company). Bacterial Material. The sources of the strain D. aurantiacum NRRL 18085 and its tiaM-inactivated mutant TCM50 (ΔtiaM) were previously described.14b Fermentation, Extraction, and Isolation. Fermentation of the D. aurantiacum mutant TCM50 was carried out at 28 °C for 1 week in 1223



Article

2976, 2934, 1697, 1636, 1259, 1065 cm−1; 1H and 13C NMR data, Tables 1 and 2; HRESIMS m/z 1021.4576 [M + Cl]− (calcd for C52H74ClO18, 1021.4569). Dideschloro-18-dehydroxytiacumicin B (7): pale white powder, UV (MeOH) λmax (log ε) 218 (4.41), 266 (4.34) nm; IR (KBr) νmax 3371, 2972, 2936, 1690, 1643, 1258, 1066, 1024 cm−1; 1H and 13C NMR data, Tables 1 and 2; HRESIMS m/z 1007.4804 [M + Cl]− (calcd for C52H76ClO17, 1007.4777). Dideschloro-2′-hydroxytiacumicin B (8): pale white powder, UV (MeOH) λmax (log ε) 218 (4.40), 266 (4.33) nm; IR (KBr) νmax 3431, 2972, 2931, 1690, 1643, 1258, 1063, 1030 cm−1; 1H and 13C NMR data, Tables 1 and 2; HRESIMS m/z 981.4809 [M + Na]+ (calcd for C51H74NaO17, 981.4818). Dideschloro-4″-propionyltiacumicin B (9): pale white powder, UV (MeOH) λmax (log ε) 219 (4.50), 266 (4.42) nm; IR (KBr) νmax 3416, 2920, 1694, 1643, 1258, 1067, 1026 cm−1; 1H and 13C NMR data, Tables 1 and 2; HRESIMS m/z 1009.4586 [M + Cl]− (calcd for C51H74ClO18, 1009.4569). Dideschloro-4″-acetyltiacumicin B (10): pale white powder, UV (MeOH) λmax (log ε) 218 (4.46), 266 (4.37) nm; IR (KBr) νmax 3370, 2974, 2934, 1690, 1643, 1256, 1063, 1022 cm−1; 1H and 13C NMR data, Tables 1 and 2; HRESIMS m/z 959.4649 [M − H]− (calcd for C50H71NaO18, 959.4846). Dideschloro-4″-hydroxytiacumicin B (11): pale white powder, UV (MeOH) λmax (log ε) 218 (4.39), 266 (4.29) nm; IR (KBr) νmax 3397, 2924, 1694, 1645, 1258, 1065, 1024 cm−1; 1H and 13C NMR data, Tables 1 and 2; HRESIMS m/z 937.4375 [M + Cl]− (calcd for C48H70ClO16, 937.4358). Tiacumicin G1 (12): pale white powder, UV (MeOH) λmax (log ε) 203 (4.08), 233 (4.01) nm; IR (KBr) νmax 3373, 2959, 2928, 1589, 1381, 1354, 1069, 1024 cm−1; 1H and 13C NMR data, Table 3; HRESIMS m/z 775.3686 [M + Cl]− (calcd for C38H60ClO14, 775.3677). Tiacumicin G2 (13): pale white powder, UV (MeOH) λmax (log ε) 203 (4.38), 237 (4.52) nm; IR (KBr) νmax 3349, 2974, 2922, 1589, 1352, 1065, 1033 cm−1; 1H and 13C NMR data, Table 3; HRESIMS m/z 847.4483 [M + Na]+ (calcd for C43H68NaO15, 847.4450). Biological Assays. In vitro antibacterial activity was evaluated by the Mueller Hinton broth microdilution method15 and was performed as previously described.14b The minimal inhibition concentration values were measured against six indicator strains including methicillinresistant Staphylococcus aureus, Staphylococcus aureus ATCC 29213, Bacillus thuringensis SCSIO BT01, Micrococcus luteus SCSIO ML 01, and Enterococcus faecalis ATCC 29212.

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Province (2016A030313158), and the Administration of Ocean and Fisheries of Guangdong Province (GD2012-D01-002). We are grateful to the analytical facility in South China Sea Institute of Oceanology for recording spectroscopic data.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b00990. 1D and 2D NMR and UV, IR, and MS spectroscopic data of compounds 3−13 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel/Fax: +86 20 89023038. E-mail: [email protected]; [email protected]. ORCID

Haibo Zhang: 0000-0003-3796-5095 Changsheng Zhang: 0000-0003-2349-3138 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported in part by the Chinese Academy of Sciences (QYZDJ-SSW-DQC004, XDA11030403), the National Natural Science Foundation of China (41406183, 31290233, 41106143), the Natural Science Foundation of Guangdong 1224


Tiacumicin Congeners with Improved Antibacterial Activity from a

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