1

ELECTRONIC SUPPORTING INFORMATION (ESI)

Chrysomycins A-C, antileukemic naphthocoumarins from Streptomyces

sporoverrucosus

Shreyans K. Jain,ab Anup S. Pathania,c Rajinder Parshad,d Chandji Raina,d Asif Ali,ab Ajai P. Gupta,e

Manoj Kushwaha,e Subrayashastry Aravinda,f Shashi Bhushan,bc Sandip B. Bharate,bf* Ram A.

Vishwakarmaabf*

aNatural Products Chemistry Division, Indian Institute of Integrative Medicine (CSIR), Canal Road, Jammu-180001,

India.

bAcademy of Scientific and Innovative Research (AcSIR), Anusandhan Bhawan, 2 Rafi Marg, New Delhi-110001, India

cCancer Pharmacology Division, Indian Institute of Integrative Medicine (CSIR), Canal Road, Jammu-180001, India

dFermentation Division, Indian Institute of Integrative Medicine (CSIR), Canal Road, Jammu-180001, India.

eQuality control and quality assurance division, Indian Institute of Integrative Medicine (CSIR), Canal Road, Jammu-

180001, India.

fMedicinal Chemistry Division, Indian Institute of Integrative Medicine (CSIR), Canal Road, Jammu-180001, India

*E-mail: ram@iiim.ac.in; sbharate@iiim.ac.in

¤ IIIM Publication number IIIM/1556/2013

Fax: +91-191-2569333; Tel: +91-191-2569111

Electronic Supplementary Material (ESI) for RSC AdvancesThis journal is © The Royal Society of Chemistry 2013

2

CONTENTS

S1 . 1H -NMR Spectrum of chrysomycin A (1) in CD3OD

S2. HRMS Spectrum of chrysomycin A (1)

S3. 1H -NMR Spectrum of chrysomycin B (2) in CD3OD

S4. HRMS Spectrum of chrysomycin B (1)

S5. 1H -NMR Spectrum of chrysomycin C (3) in CD3OD

S6. 13C-NMR Spectrum of chrysomycin C (3) in CD3OD

S7. DEPT 135 Spectrum of chrysomycin C (3) in CD3OD

S8. COSY Spectrum of chrysomycin C (3) in CD3OD

S9. HSQC Spectrum of chrysomycin C (3) in CD3OD

S10. HMBC spectrum of chrysomycin C (3) in CD3OD

S11. HRMS of chrysomycin C (3)

S12. HRMS of chrysomycin D (4)

S13. LCMS of active fraction

S14. Screenshot of DNP database search during dereplication

S15. Screenshot of DNP substructure search using basic naphthocoumarin skeleton

Electronic Supplementary Material (ESI) for RSC AdvancesThis journal is © The Royal Society of Chemistry 2013

3

S1 1H -NMR Spectrum of chrysomycin A (1) in CD3OD

-0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.0f1 (ppm)

0

500

1000

1500

2000

2500

3000

3500

4000

4500

500029-jul-shreyan-h1SS-Peak-II

2.49

2.91

1.62

1.21

5.01

0.92

2.48

0.52

1.03

1.03

0.77

0.94

0.77

1.02

0.26

0.43

0.53

1.02

1.00

1.20

1.21

1.22

1.26

1.28

1.29

1.29

1.30

1.31

1.45

3.30

3.30

3.31

3.31

3.31

3.37

3.93

3.94

3.95

3.96

3.97

3.98

4.72

4.74

4.91

5.37

5.39

5.89

5.93

6.21

6.22

6.23

6.24

6.71

6.91

6.92

6.93

6.94

7.17

7.29

7.82

7.89

7.89

7.90

7.91

8.17

8.23

Electronic Supplementary Material (ESI) for RSC AdvancesThis journal is © The Royal Society of Chemistry 2013

4

S2. HRMS Spectrum of chrysomycin A (1)

Electronic Supplementary Material (ESI) for RSC AdvancesThis journal is © The Royal Society of Chemistry 2013

5

S3 1H -NMR Spectrum of chrysomycin B (2) in CD3OD

-0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.5f1 (ppm)

-500

0

500

1000

1500

2000

2500

3000

3500

4000

4500

29-jul-shreyan-h1SS-Peak-I

3.18

3.04

3.01

1.11

3.05

0.95

1.14

1.07

0.98

1.23

1.00

1.10

1.12

1.18

1.35

2.29

3.27

3.89

6.11

6.13

6.83

6.84

7.02

7.53

7.80

7.81

8.12

Electronic Supplementary Material (ESI) for RSC AdvancesThis journal is © The Royal Society of Chemistry 2013

6

S4. HRMS Spectrum of chrysomycin B (2)

Electronic Supplementary Material (ESI) for RSC AdvancesThis journal is © The Royal Society of Chemistry 2013

7

S5 1H -NMR Spectrum of chrysomycin C(3) in CD3OD

0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.5f1 (ppm)

-500

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

5500

Jul22-2013SS-P-III

2.8

93.4

52.6

2

1.9

5

0.8

5

5.8

6

1.0

2

0.9

2

0.9

6

1.0

1

0.9

5

1.0

0

1.0

0

1.2

01.2

11.2

71.4

5

2.6

82.7

0

3.3

03.3

03.3

13.3

13.3

13.3

7

3.9

23.9

33.9

4

4.7

24.7

3

6.2

26.2

4

6.9

06.9

27.0

9

7.6

3

7.8

87.9

08.1

5

1.11.21.31.41.51.6f1 (ppm)

0

1000

2000

3000

4000

5000

Jul22-2013SS-P-III 1

.20

1.2

1

1.2

7

1.4

5

Electronic Supplementary Material (ESI) for RSC AdvancesThis journal is © The Royal Society of Chemistry 2013

8

S6. 13C NMR Spectrum of chrysomycin C (3) in CD3OD

0102030405060708090100110120130140150160170f1 (ppm)

-2000

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

20000

22000

24000

26000

28000

30000Jul22-2013SS-P-III

15.2

217.2

5

24.1

0

29.6

4

56.6

356.6

956.8

356.8

6

73.0

674.6

575.1

276.6

577.7

8

102.9

8

113.0

7115.3

2116.5

3118.4

2121.0

5122.9

1126.8

4128.3

1130.5

3

143.5

9

147.8

0

153.2

7155.4

2158.6

2162.5

5

Electronic Supplementary Material (ESI) for RSC AdvancesThis journal is © The Royal Society of Chemistry 2013

9

S7. DEPT 135 Spectrum of chrysomycin C (3) in CD3OD

0102030405060708090100110120130140f1 (ppm)

-40000

-35000

-30000

-25000

-20000

-15000

-10000

-5000

0

5000

10000

15000

20000

25000

30000

35000

40000

45000

50000Jul22-2013SS-P-III

-0.0

0

15.2

217.2

7

24.1

1

29.6

4

56.6

656.8

4

73.0

674.6

076.6

677.7

9

102.9

4

113.0

6

118.3

7

121.0

2

130.5

3

Electronic Supplementary Material (ESI) for RSC AdvancesThis journal is © The Royal Society of Chemistry 2013

10

S8. COSY Spectrum of chrysomycin C (3) in CD3OD

-0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.0f2 (ppm)

0

1

2

3

4

5

6

7

8

9

10

f1 (

ppm

)

Jul22-2013SS-P-III

Electronic Supplementary Material (ESI) for RSC AdvancesThis journal is © The Royal Society of Chemistry 2013

11

S9. HSQC Spectrum of chrysomycin C (3) in CD3OD

-10123456789f2 (ppm)

-20

-10

0

10

20

30

40

50

60

70

80

90

100

110

120

130

140

150

160

f1 (

ppm

)

Jul22-2013SS-P-III

Electronic Supplementary Material (ESI) for RSC AdvancesThis journal is © The Royal Society of Chemistry 2013

12

S10. HMBC spectrum of chrysomycin C (3) in CD3OD

-0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.5f2 (ppm)

10

20

30

40

50

60

70

80

90

100

110

120

130

140

150

160

170

180

190

f1 (

ppm

)

Jul22-2013SS-P-III

Electronic Supplementary Material (ESI) for RSC AdvancesThis journal is © The Royal Society of Chemistry 2013

13

S11. HRMS of chrysomycin C (3)

Electronic Supplementary Material (ESI) for RSC AdvancesThis journal is © The Royal Society of Chemistry 2013

14

S12. HRMS of chrysomycin D (4)

Electronic Supplementary Material (ESI) for RSC AdvancesThis journal is © The Royal Society of Chemistry 2013

15

13. LCMS of active fraction 13.1 LC chromatogram at 280 nm

AU

0.00

0.10

0.20

0.30

0.40

Minutes0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 65.00 70.00 75.00

13.2. LCMS chromatogram in TIC mode

5x10

0

0

0

0

0

0

0

0

0

0

1

1

1

1

1

1

+/-ESI BPC Scan Frag=135.0V SP0004.d Smooth

CH

RY

SO

MY

CIN

-D

CH

RY

SO

MY

CIN

-E CH

RY

SO

MY

CIN

-B

CH

RY

SO

MY

CIN

-A

CH

RY

SO

MY

CIN

-C

Counts vs. Acquisition Time (min)

2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60 62 64 66 68 70 72 74

13.3. MS spectras of chrysomycins A-E

3x10

0

2

-ESI Scan:2 (53.3 min) Frag=135.0V SP0004.d

525.0

480.9592.9510.8 560.9 651 0 684 8

3x10

0

5

+ESI Scan:1 (53.3 min) Frag=135.0V SP0004.d

549.0

509.0409.1

527.0465.0425.2352.8 564 9 615 0 658 3 694 9 737 9

Counts vs. Mass-to-Charge (m/z)360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 700 720 740 760

Electronic Supplementary Material (ESI) for RSC AdvancesThis journal is © The Royal Society of Chemistry 2013

16

3x10

0

0

1

-ESI Scan:2 (58.8 min) Frag=135.0V SP0004.d

523.0

590.8558.8

3x10

0

2

4

+ESI Scan:1 (58.8 min) Frag=135.0V SP0004.d

547.0

407.0 507.1599.0377.0 432 7 568 9 710 3633 3 679 3 743 1

Counts vs. Mass-to-Charge (m/z)360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 700 720 740 760

4x10

0

0

1

-ESI Scan:2 (64.9 min) Frag=135.0V SP0004.d

495.0

563 0376 7 990 7606 94x10

0

2

4

+ESI Scan:1 (64.9 min) Frag=135.0V SP0004.d

379.0

479.1

519.0 764.2434.9 993 1563 1

Counts vs. Mass-to-Charge (m/z)400 450 500 550 600 650 700 750 800 850 900 950 1000

3x10

0

5

-ESI Scan:2 (70.8 min) Frag=135.0V SP0004.d

507.0

574 9174 8 257 0 642 74x10

0

1

2

+ESI Scan:1 (70.7 min) Frag=135.0V SP0004.d

391.0491.0

531.0361.0 782.6447.0273.9141 0 593 0213 0 710 5

0.0

Counts vs. Mass-to-Charge (m/z)100 150 200 250 300 350 400 450 500 550 600 650 700 750 800

Electronic Supplementary Material (ESI) for RSC AdvancesThis journal is © The Royal Society of Chemistry 2013

17

S14. Screenshots of DNP database search during dereplication

Electronic Supplementary Material (ESI) for RSC AdvancesThis journal is © The Royal Society of Chemistry 2013

18

Electronic Supplementary Material (ESI) for RSC AdvancesThis journal is © The Royal Society of Chemistry 2013

19

Electronic Supplementary Material (ESI) for RSC AdvancesThis journal is © The Royal Society of Chemistry 2013

20

S15. Screenshots of DNP substructure search using basic naphthocoumarin skeleton

Electronic Supplementary Material (ESI) for RSC AdvancesThis journal is © The Royal Society of Chemistry 2013