Supplementary Material for The C-glycosyltransferase ...

[Pages:6]Supplementary Material for

The C-glycosyltransferase UrdGT2 is unselective towards D- and L-configurated nucleotide-bound rhodinoses

Dirk Hoffmeister , Gerald Dr?ger, Koji Ichinose?, J?rgen Rohr? and Andreas Bechthold

Institute for Pharmaceutical Biology, Albert-Ludwigs-University, Stefan-Meier-Strasse 19, 79104 Freiburg, Germany, Institute for Organic Chemistry, University of Hannover, Schneiderberg 1b, 30167 Hannover,

Germany, ?Graduate School of Pharmaceutical Science, The University of Tokyo, Bunkyo-ku, 113-0033 Tokyo, Japan, and ?College of Pharmacy, University of Kentucky, 907 Rose Street, Lexington, KY 40536-0082

1. Experimental procedures

Fermentation. Streptomyces fradiae RN-435 was cultivated in NL111V liquid medium (2% lab lemco meat powder, 1% CaCO3, 10% malt extract, pH adjusted to 7.2) dispensed into single baffled erlenmeyer flasks and kept at 27?C and 150 rpm. With 100 ml seed culture, incubated for 65 h, a 10 liter main culture was inoculated and fermented for 55 h. Isolation of urdamycin derivatives. Upon harvest, the culture broth was treated with Celite (2%, w/v) and passed through a filter press to separate mycelia from the aequous phase. The latter was extracted three times with an equal volume of ethyl acetate, the organic phase was evaporated to dryness, redissolved in 90% methanol, defatted by extracting with n-pentane, and evaporated again. The crude extract was split in equal parts and applied to two Sephadex LH-20 columns (each 100 ? 6 cm, solvent was methanol). Further urdamycin purification was done using the preparative HPLC-system described in the next section. Final work-up was done on miniature silica gel 60 columns (150 ? 6 mm) with methylene chloride/methanol (19:1, v/v) as solvent. HPLC systems and conditions. Analytical HPLC runs were performed on a Tosoh HPLCsystem equipped with a Tosoh TSK-gel ODS-80TM column (5? particle size, 150 ? 4.6 mm), maintained at 40?C. Detection wavelength range of the diode-array was set to 250-500 nm. The gradient profile was: Solvent A: 0.5 % acetic acid in H2O, Solvent B 0.5% acetic acid in acetonitrile. An initial hold for 5 min with 45% B was followed by a linear gradient 45-95% B within 25 mins, held at that relation for further 5 min. The solvent flow rate was 0.8 mL/min. LC/MS was performed on a Thermoquest LCQ equipped with a Hewlett Packard HP1000 series LC under identical conditions described for analytical HPLC, by atmospheric pressure chemical ionization (APCI) and detection in the positive and negative mode. As preparative HPLC a Waters system and Macherey&Nagel RP-18 Hypersil column (5?, 250 ? 4.6 mm) was deployed. Solvent A was 0.5 % acetic acid in H2O, solvent B was 0.5 % acetic acid in acetonitrile/methanol (5:1, v/v). The gradient was 30% B from 0-2 min, 30-40% B from 2-8 min, 40-50 % B from 8-11 min, 50-100 % B from 11-19 min, held at 100% B from 19-22 min, at a flow rate of 1.2 mL/min. NMR measurements. 1H-NMR spectra were recorded at 500 MHz (compound (6) at 400 MHz), 13C-spectra at 125 MHz using JEOL JNM alpha 500, Bruker Avance 400 and DRX 500 spectrometers. The samples were dissolved in acetone-d6, and 1H-chemical shifts were referenced to acetone-d5 at 2.04 ppm. Hydrolysis of urdamycin S (5). Hydrolysis was performed by treatment with methanolic HCl (5 mL, 0.1 M) for 5 min at room temperature. The solvent was removed under reduced pressure, and the residue was purified by column chromatography on silica gel (methylene chloride/methanol 9:1, v/v) and Sephadex LH-20 (methanol), yielding 12b,4?-diderhodinosylurdamycin S (6) C-glycoside as an orange solid.

S1

2. NMR spectral data - NMR analysis of urdamycin R (4)

HO

CH3 6???

6??

H3C

H

6?

H3C

H O

5?

1?

H

O

O

4?

2?

?3?

1??

H

11 10 9

8

OH

O

1???

H O O O

12

11a

12a

6a 7 7a

2 1

3-CH3

CH3

3

OH

12b

4

4a

OH

5

6

O

HO

selected HMBC-couplings

1H-NMR (500 MHz, acetone-d6, acetone-d52?!#?? )

proton #

chem. shift [ppm]

multiplicity

2-Hax

2.82

d

2-Heq

2.53

dd

3-CH3

1.14

s

4-Hax

2.27

d

4-Heq

1.97

dd

5-H

6.46

d

6-H

6.86

d

10-H

7.92

d

11-H

7.62

d

1?-H

4.83

br d

2?-Hax

1.86-1.88

m

2?-Heq

2.16-2.12

m

3?-Hax

*

3?-Heq

*

4?-H

3.54

br s

5?-H

3.77

br q

6?-H

1.23

d

1??-H

4.82

br s

2??-Hax

*

2??-Heq

*

3??-Hax

*

3??-Heq

*

4??-H

3.50

br s

5??-H

3.96

br q

6??-H

1.06

d

1???-H

5.36

br d

2???-Hax

*

2???-Heq

*

3???-Hax

*

3???-Heq

*

4???-H

3.34

br s

5???-H

3.66

br q

6???-H

0.50

d

coupling J [Hz] 12.7 12.7, 3.0 / 15.0 15.0, 3.0 10.0 10.0 7.7 7.7 9.0 / /

/ 6.5 6.5 /

/ 6.5 6.5 3.2

/ 6.5 6.5

integral 1H 1H 3H 1H 1H 1H 1H 1H 1H 1H 1H 1H

1H 1H 3H 1H

1H 1H 3H 1H

1H 1H 3H

S2

13C-NMR (125 MHz, acetone-d6) :

Carbon #

chemical shift [ppm]

1

202.5

2

54.7

3

76.1

4

44.1

4a

82.5

5

146.4

6

117.3

6a

138.0

7

189.8

7a

115.0

8

158.4

9

140.2

10

134.4

11

120.0

11a

131.7

12

183.5

12a

141.2

12b

81.7

3-CH3

30.2

1?

74.3

2?

30.8

3?

*

4?

75.1

5?

76.7

6?

18.2

1??

100.5

2??

*

3??

*

4??

67.2

5??

67.8

6??

17.6

1???

95.1

2???

*

3???

*

4???

67.1

5???

67.8

6???

16.8

* The CH2-signals could not be specifically assigned

chem. shift multiplicity chem. shift multiplicity

[ppm] 13C

[ppm] 1H

23.8

t

2.00-1.95

m

24.6

t

2.07-2.02

m

26.2

t

2.00-1.95

m

26.9

t

2.05-2.00

m

27.9

t

1.90-1.80

m

multiplicity s t s t s d d s s s s s d d s s s s q d t

d d q d

d d q d

d d q

chem. shift [ppm] 1H 1.70-1.65 1.60-1.55 1.55-1.50 1.65-1.60 1.90-1.80

multiplicity

m m m m m

S3

NMR analysis of urdamycin S (5)

HO

CH3 6???

6??

H3C

H3C 6?

H

O

O

1??

H O

5?

1?

4?

2?

?3?

11 10 9

8

OH

O

1???

O

12 11a

7 7a

H O O

12a 6a

2 1

3-CCHH33

3

OH

12b

4

4a

OH

5

6

O

HO

selected HMBC-couplings

1H-NMR (500 MHz, acetone-d6, acetone-d52?!#?? )

proton #

chem. shift [ppm] multiplicity

2-Hax

2.85

d

2-Heq

2.65

dd

3-CH3

1.14

s

4-Hax

2.31

d

4-Heq

1.96

dd

5-H

6.46

d

6-H

6.86

d

10-H

7.91

d

11-H

7.60

d

1?-H

5.06

br dd

2?-Hax 2?-Heq 3?-Hax 3?-Heq 4?-H

1.42-1.32 2.17 1.95-1.90 1.95-1.90 3.95

m dddd m m ddd

5?-H

4.41

dq

6?-H

1.31

d

1??-H

4.92

br s

2??-Hax

1.42-1.32

m

2??-Heq

2.06-2.04

m

3??-Hax

1.59-1,50

m

3??-Heq

1.72-1.64

m

4??-H

3.51

br s

5??-H

3.95

q

6??-H

1.11

d

1???-H

5.29

br d

2???-Hax

1.72-1.64

m

2???-Heq

1.88-1.85

m

3???-Hax

1.42-1.32

m

3???-Heq

1.59-1.50

m

4???-H

3.34

br s

5???-H

3.65

br q

6???-H

0.55

d

coupling J [Hz] 12.6 12.6, 2.8 14.8 14.8, 2.8 9.7 9.7 7.8 7.8 11.0, 1.6 13.2, 4.0, 3.6, 1.6 10.4, 5.8, 5.2 5.8 (d), 6.7 (q) 6.7 6.6 6.6 2.8 6.6 6.6

integral 1H 1H 3H 1H 1H 1H 1H 1H 1H 1H 3H 1H 2H 2H 1H 1H 3H 1H 3H 1H 2H 2H 1H 1H 3H 1H 2H 1H 3H 2H 1H 1H 3H

S4

13C-NMR (125 MHz, acetone-d6? )

Carbon #

chemical shift [ppm]

1

202.5

2

54.7

3

76.2

4

44.1

4a

82.5

5

146.4

6

117.2

6a

137.9

7

191.9

7a

114.9

8

158.6

9

139.9

10

134.2

11

120.0

11a

131.8

12

138.4

12a

141.3

12b

81.6

3-CH3

30.2

1?

74.3

2?

32.5

3?

26.2

4?

73.1

5?

71.6

6?

11.5

1??

96.7

2??

24.6

3??

26.6

4??

67.2

5??

67.5

6??

17.5

1???

95.0

2???

23.8

3???

26.0

4???

67.1

5???

67.8

6???

16.9

multiplicity s t s t s d d s s s s s d d s s s s q d t t d d q d t t d d q d t t d d q

S5

NMR analysis of 12b,4?-diderhodinosyl-urdamycin S (6)

6?

H3C HO

O

5?

1?

4?

2?

?3?

11 10

9 8

O O

HO

12

11a

12a

6a 7 7a

3-CH3

2

1

3

OH

12b

4

4a

OH

5

6

OH

O

1H-NMR (400 MHz, acetone-d6, acetone-d5=2.04 ppm) )

proton #

chem. shift [ppm] multiplicity

2-Hax

3.00

d

2-Heq

2.69

dd

3-CH3

1.24

s

4-Hax

2.27

d

4-Heq

2.06

dd

5-H

6.46

d

6-H

6.84

d

10-H

7.91

dd

11-H

7.57

dd

1?-H

5.05

dd

2?-Hax

1.42-1.33

m

2?-Heq 3?-Hax

2.17 1.90-1.84

dddd m

3?-Heq

1.90-1.84

m

4?-H

3.95

ddd

5?-H

4.29

dq

6?-H

1.33

d

coupling J [Hz] 12.8 12.8, 3.2 14.8 14.8, 3.2 9.8 9.8 7.8, 0.8 7.8, 0.3 11.2, 2.4 13.2, 4.0, 3.6, 2.4 10.6, 5.6, 5.2 5.6 (d), 6.8 (q) 6.8

integral 1H 1H 3H 1H 1H 1H 1H 1H 1H 1H 2H 1H 2H 1H 1H 1H 3H

Further signals: 5.24 (bs, 1H, OH), 4.75-4.74 (m, 1H, OH), 4.64 (m, 1H, OH), 4.03 (d, J= 4.6 Hz, 1H, OH), 3.33-3.31 (m, 1H, OH) ppm.

The following data were extracted from an HSQC-spectrum. 13C-NMR (125 MHz, acetone-d6, HSQC-Edit) )

Carbon #

chemical shift [ppm]

2

52.3

3-CH3

29.6

4

44.0

5

145.8

6

117.1

10

133.7

11

119.1

1?

64.5

2?

31.9

3?

27.2

4?

67.9

5?

73.7

6?

10.6

multiplicity t q t d d d d d t t d d q

S6

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