Rdc-enhanced NMR Spectroscopy in Structure Elucidation of the Natural Product Sucro-neolambertellin

Supporting Information

© Wiley-VCH 2008

69451 Weinheim, Germany

S1

Rdc-enhanced NMR Spectroscopy in Structure Elucidation of the Natural Product Sucro-neolambertellin Anne Schuetz, Takanori Murakami, Noboru Takada, Jochen Junker, Masaru Hashimoto*, Christian Griesinger*

Contents

1. General S2

2. Isolation of Sucro-neolambertellin S2

3. Synthesis of Sucro-neolambertellin-peracetate 2 S2

4. Photodiode array UV spectra of 1 and neolambertellin 3 S3

5. Procedure for establishing the absolute stereochemistry of the glucose moiety

5.1. Derivation of the natural product S3

5.2. Preparation of the authentic sample S3

5.3. 1H-NMR of 1,2,3,4,6-O-benzoyl-α-D-glucopyranose S4

5.4. CD-specta S5

6. NMR spectra of Sucro-neolambertellin (1) S6

7. NMR spectra of Sucro-neolambertellin peracetate (2) S17

8. NOE analysis and data S24

9. Alignment media S26

10. Residual dipolar couplings S27

11. Structure calculation S28

12. References S29

S2

1. General

1H-NMR spectra were measured on a JEOL ALPHA 400 spectrometer (400 MHz), except for the NOESY build-up series and HSQC spectra for rdc extraction, which were recorded on a BRUKER Avance 700 spectrometer (700 MHz) with TXI cryoprobe.The chemical shifts are expressed in ppm downfield from the signal of trimethylsilane used as an internal standard in the case of CDCl3. When CD3OD or DMSO-d6 were employed, the remaining proton signals in the deuterated solvents CHD2OD (3.30 ppm), or CHD2SOCD3 (2.49 ppm) were used as the internal standards. Splitting patterns are designated as s (singlet), d (doublet), t (triplet), m (multiplet), and br (broad). 13C-NMR spectra were recorded also on a JEOL ALPHA 400 spectrometer (100 MHz). The isotope 13C in the solvents were used as the internal standard (13CDCl3; 77.0 ppm, 13CD3OD; 49.0 ppm, or 13CD3SOCD3; 39.5 ppm). IR spectra were obtained with a HORIBA FT-720 Fourier transform infrared spectrometer on a KBr cell. Measurements of fast atom bombardment (FAB) mass spectra were performed on a JEOL JMS AX500 or JEOL JMS AX102A spectrometers in Hokkaido University. When MS spectra were measured by negative mode, “negative mode” is mentioned. Analytical and preparative thin-layer chromatographies were carried out using precoated silica gel plates, Merck silica gel 60F254 (Art. 1.05715). Silica gel used for column chromatography was Merck silica gel 60 (Art. 1.07734). Medium-pressure column chromatographies were performed employing YAMAZEN ULTRA PACK SI-40B or Merck Lobar® LiChroprep® RP-18 Type A) equipped with FMI LAB PUMP RP-SY. HPLC was performed on a Waters 600 pump system with a Waters 996 photodiode array detector. CD spectra were recoreded on a JASCO J725 spectropolarlmeter.

2. Isolation of Sucro-neolambertellin The culture broth (5.0 L) of Lambertella sp. 1346, obtained after culturing with potato-sucrose medium (prepared by boiling with diced potato (1.0 kg) in water (5.0 L) for 30 min, and following filtration with cotton, adding sucrose (100 g), and sterilaization with autoclave) at 20°C for 14 days, was filtrated. The filtrate was washed with AcOEt (1.0 L). After the aqueous layer was concentrated in vacuo until the whole volume became ca. 2 L, the aqueous solution was loaded on ODS Sep-Pak (10 g). The fraction eluted with CH3OH/H2O (1:1) was collected and it was concentrated in vacuo to give a crude material. Medium pressured ODS column chromatography (YAMAZEN ULTRA PACKTM, ODS-S-50A, φ11 ×300mm) of the crude material was performed by eluting with CH3CN/H2O (15/85 containing 0.1% TFA) gave Sucro-neolambertellin 1 as pale yellow powder. [α]D 25 - 15.0 (c = 0.28, MeOH), UV (nm, in MeOH, 120 µmol/L) 286 (ε 10600) 276 (ε 10100), 250 (ε 5200), 380 (e 2200) 315 (ε 2400), The IR spectrum was not measured because this compound was soluble in only polar solvent. NMR spectra, see the table, FABMS (negative mode): 581(2.5, [M-H]-), 148(100), 146(99), HRFABMS (negative mode): found m/z 581.1525.calced for C26H29O15 [M-H]- 581.1506.

3. Synthesis of Sucro-neolambertellin-peracetate 2 A solution of Sucro-neolambertellin 1 (2.0 mg, 3.4 µmol) in pyridine was stirred with N,N-dimethyl-4-aminopyridine (2.0 mg) and acetic anhydride (100 µl) at room temperature for 12 hr. After concentration of the mixture, the residue was purified with silica gel column chromatography acetone/CH2Cl2 (12:88). The peracetate (2.4 mg, 74%) 2 was obtained as an oil. [α]D25 -19.7 (c = 0.29, CHCl3), IR: (film) 2920, 2850, 1751, 1369, 1223, 1053 cm-1. FAB-MS (3-nitrobenzylalcohol) m/z = 983 (M+Na)+, FAB-HRMS (3-nitorobenzyl alcohol) found m/z 983.2408.calced for C44H48O24Na [M+Na]+ 983.2433.

S3

4. Photodiode array UV spectra of 1 and Neolambertellin 3 Analytical HPLC with photodiode array detector showed the almost identical UV spectra below. Conditions: column: Merck LiChrosper 100 RP18e (5 µm) φ4.0 × 150mm eluent: CH3CN/H2O containing 0.1% TFA, 20-100% linear gradient for 15 min flow rate: 1.0 mL/min retention time: 4.6 min for 1, 8.1 min for 3

5. Procedure for establishing the absolute stereochemistry of the glucose moiety

5.1. Derivation of the natural product A solution of Sucro-neolambertellin 1 (2.0 mg) in 1:1 mixture of trifluoroacetic acid and H2O (2.0 mL) was stirred at 50°C for 3 hr. After concentration, the mixture was stirred with benzoyl chloride (100 µL) in pyridine at room temperature for 12 hr. Methanol (500 µL) was added to decompose excess benzoyl chloride. After further stirring for 2 hr, the mixture was poured into water (30 mL) and extracted with AcOEt (20 mL × 3). The combined organic layer was washed with brine, dried over MgSO4, and concentrated in vacuo. Silica gel column chromatography of the residue with AcOEt:hexane (20:80) and the following preparative silica gel TLC (AcOEt:benzene = 3:97) to give 1,2,3,4,6-O-benzoyl-α-D-glucopyranose (Rf = 0.35), which involved its β-isomer (α:β = 3:4). Preparative HPLC (Develosil C30-UG-5, φ4.6 × 250mm; CH3CN/H2O (25:75, containing 0.1% TFA), 1.0 ml/min flow) allowed to remove the β-isomer to give 1,2,3,4,6-O-benzoyl-α-D-glucopyranose (tR = 15.9 min) in a pure form (200 µg). The 1H-NMR data in CDCl3, the CD spectrum in CH3CN, the Rf value in silica gel TLC, and tR value in HPLC of this sample were identical with those of the reference sample prepared from D-glucose. The amount of this sample was estimated by comparison of its UV absorption at 233 nm in CH3CN with that of reference sample. The β-isomer appeared at 16.7 min under the same HPLC conditions. But the sample even after the HPLC still contained small amount of the α-isomer due to insufficient separation.

nm

1

3

S4

5.2. Preparation of the reference sample To a solution of commercial D-glucose (200 mg, 1.1 mmol) in pyridine (10 mL), benzoyl chloride (1.2 mL, excess) was added at 0°C. The mixture was stirred and allowed to warm to room temperature. After stirring for 12 hr, methanol (1.0 mL) was added to decompose the excess benzoyl chloride. After additional stirring for 30 min, the mixture was poured into 1M aqueous HCl solution (100 mL) and extracted with diethyl ether (150 mL). The ethereal solution was washed with brine, dried over MgSO4, and then concentrated in vacuo to give crude solid. Recrystallization from AcOEt:hexane (3:7) gave pure reference sample (620 mg, 80%) of 1,2,3,4,6-O-benzoyl-α-D-glucopyranose. The ε value of this compound in CH3CN was measured using this sample (ε 73000 at 233 nm, 8000 at 275? nm)?.

5.3. 1H-NMR of 1,2,3,4,6-O-benzoyl-α-D-glucopyranose (400 MHz, CDCl3) δ 4.47(1H, m, C6HH), 4.61(2H, m, C6HH, C5H), 5.66(1H, dd, 9.8, 3.9 Hz, C2H), 5.85(1H, t, 9.86 Hz, C4H), 6.30 (1H, t, 9.8 Hz, C3H), 6.84 (1H, d, 3.9 Hz, ), 7.27-7.55 (14H, aromatic protons), 7.65 (1H, tt, 7.6, 1.2 Hz, aromatic protons), 7.87 (4H, brdd, 8.1 Hz, aromatic protons), 7.93 (2H, dd, 8.3, 1.2 Hz, aromatic protons), 8.01 (2H, dd, 8.5, 1.2 Hz, aromatic protons), 8.15 (2H, dd, 8.5, 1.5 Hz, aromatic protons)

reference

from natural product satellite

imp (CH2Cl2)

S5

5.4. CD-specta

The reference sample from D-glucose (2.0 mg, 2.9 µmol) as described under 5.2 was dissolved in MeOH (100 mL) and then measured employing a quartz cell (2.0 mm thickness). Since the sample prepared from 1 as described in 5.1. could not be weighed due to small amount available, its methanol solution with the same concentration according to UV absorption was prepared .

sample from natural product

-10

0

10

20

30

40

200 220 240 260 280 300

(nm)

∆ ε

authentic sample

-10

0

10

20

30

40

200 220 240 260 280 300

(nm)∆

ε

reference

S6

6. NMR spectra of Sucro-neolambertellin (1)

10 4

5

8 9

HOD

31 24 23

35

26 36

21

33 36

32

34

3-Me

1H-NMR (400MHz, CD3OD)

S7

7 6 10b

4 9

10a 3 6a

4a 8

10 5

25 22 31 23

24 33 35

32 34 21 36

26 2

3-Me

O

O

OH

HOHOHO O

O

OH

HO

OH

O O

CH3OHHO

1 in CD3OD

1

36

7

10

2226

24

36

3133

13C-NMR (100MHz, CD3OD)

S8

Concentration 4 mg/mL, 128 (F1) and 1024 (F2) complex points, 8 scans per increment.

4, 3-Me

10, 9 8,10

31, 32

24,23

35,34

33,34

33,32

O

O

OH

HOHOHO O

O

OH

HO

OH

O O

CH3OHHO

1 in CD3OD

1

36

7

10

2226

24

36

3133

COSY

S9


4

10

9

8

31

24

23 3533 32

3421

36 26

3-Me

solv.

O

O

OH

HOHOHO O

O

OH

HO

OH

O O

CH3OHHO

1 in CD3OD

1

36

7

10

2226

24

36

3133

S10

Concentration 4 mg/mL, 256 (F1) and 1024 (F2) complex points, 160 scans per increment, mixing time 70 ms.

4-C2

4-C5

10-C8

10-C6a

10-C10b

4-C10b

5-C6

5-C10b

5-C4

5-C6a

9-C10a

9-C7 8-C7

8-C6a

8-C10

31-C33

4-C3-Me

31-C22

24-C23

23-C24

26-C24

33-C34

34-C33,35

33-C32

21-C22

26-C25 3-Me-C3

3-Me-C4

3-Me-C2

O

O

OH

HOHOHO O

O

OH

HO

OH

O O

CH3OHHO

1 in CD3OD

1

36

7

10

2226

24

36

3133

S11

1H-NMR (400MHz, DMSO-d6)

4

7-OH

10

5,9

8 31 32- OH

24- OH

21- OH

34-OH, 33-OH

26- OH

36- OH

23 24

32

21 33 35 36

35 34

3-Me 23-OH

26 26 21

21

O

O

OH

HOHOHO O

O

OH

HO

OH

O O

CH3OHHO

1 in DMSO-d6

1

36

7

10

2226

24

36

3133

S12

2 6 7

10b 4 9 3

6a 4a

8

10 5 25 22 31

23 24

35 33

32 34

36

21 26 3-Me

13C-NMR (100MHz, DMSO-d6)

O

O

OH

HOHOHO O

O

OH

HO

OH

O O

CH3OHHO

1 in DMSO-d6

1

36

7

10

2226

24

36

3133

S13


4-C3-Me

23OH-23

31-32 32OH-32

34OH-34

21OH-21 33OH-33

24OH-24 26OH-26

36OH-36

23-24

35-36

35-34 33-34

33-32

10-9

9-8

O

O

OH

HOHOHO O

O

OH

HO

OH

O O

CH3OHHO

1 in DMSO-d6

1

36

7

10

2226

24

36

3133

S14


7OH-23OH

4-3Me

4-

31-

10-9

9-8

NOESY

O

O

OH

HOHOHO O

O

OH

HO

OH

O O

CH3OHHO

1 in DMSO-d6

1

36

7

10

2226

24

36

3133

S15


5

89

10

31

2423

35 36

3332

34

2126

3-Me

O

O

OH

HOHOHO O

O

OH

HO

OH

O O

CH3OHHO

1 in DMSO-d6

1

36

7

10

2226

24

36

3133

S16


7OH-C6a

7OH-C8

7OH-C7

10-C8

4-C10b

4-C5

10-C6a

10-C10b

5-C3

5-C6a

5-C4

5-C10b 5-C6

9-C7

8-C6a

8-C10

31-C22 23-C22

26-C25

24-C23

3Me-C3

3Me-C4

3Me-C2

4-C3Me

4-C2

O

O

OH

HOHOHO O

O

OH

HO

OH

O O

CH3OHHO

1 in DMSO-d6

1

36

7

10

2226

24

36

3133

HMBC

S17

7. NMR spectra of Sucro-neolambertellinperacetate (2)

10

4 9

5

8

24 23

31 33 34

32

21 21 26 26

36

35

1H-NMR (400MHz, CDCl3)

O

O

OAc

AcOAcOOAc O

O

OAc

OAc

OAc

O O

CH3OAc

AcO

2 in CDCl3

1

36

7

10

2226

24

36

31

33

S18

Acetyl-CO ×9

10b 7

2 6

4 10a 6a

9 8

10

4a 5 25

22 31 24

23 32 33

35 34

21 36 26

3-Me

Acetyl-Me ×9

3

13C-NMR (100MHz, CDCl3)

O

O

OAc

AcOAcOOAc O

O

OAc

OAc

OAc

O O

CH3OAc

AcO

2 in CDCl3

1

36

7

10

2226

24

36

31

33

S19


O

O

OAc

AcOAcOOAc O

O

OAc

OAc

OAc

O O

CH3OAc

AcO

2 in CDCl3

1

36

7

10

2226

24

36

31

33

10-9

9-8

4-3Me

34-3531-32 33-32

33-34

24-23

21 (AB)26 (AB)

35-36

S20


10

4

9

8

5

31

23

2433

34

3235

36

212126

O

O

OAc

AcOAcOOAc O

O

OAc

OAc

OAc

O O

CH3OAc

AcO

2 in CDCl3

1

36

7

10

2226

24

36

31

33

S21


10-C10b

10-C84-C3

9-C5

9-C7 4-C10b

4-C2

8-C75-C10b

5-C6

5-C4 8-C6q

8-C10

5-C6a

31-C22

24-C23

23-C24

31-C32

33-C32

34-C35 21-C23

26-C24

21-C22

26-C25

4-C3Me

O

O

OAc

AcOAcOOAc O

O

OAc

OAc

OAc

O O

CH3OAc

AcO

2 in CDCl3

1

36

7

10

2226

24

36

31

33

HMBC

S22

O

O

OAc

AcOAcO

AcO O

O

OAc

AcO

OAc

O O

CH3OAcAcO

2 in DMSO

1

36

7

6a

26

24

21

2733

36

2330

NOESY 700 MHz, mixing time 700 ms 2 at 15 mM concentration, 8 scans per increment, spectral widths 7001 Hz and 7003 Hz, sampled with 512 (F1) and 4096 (F2) complex points, respectively. Both dimensions were apodized with a p/2-shifted squared sine-bell function, zero fillingwas applied to 1024×8192 complex points.

S23

NMR spectral data of Sucro-neolambertellin 1 and its nona-acetate 2 Sucro-neolambertellin 1 nona-acetate 2

in CD3OD in DMSO-d6 in CDCl3 position δH (J Hz)

δC HMBC

(H) δH (J Hz) δC HMBC (H) NOESY δH (J Hz) δC

2

- 164.16 4, 3-Me - 161.28 4, 3-Me - - 161.78 3 - 126.77 3-Me - 125.16 5, 3-Me - - 125.80 4 7.81 (q, 1.0) 141.87 3-Me, 5 7.90 (1H, q, 1.0) 140.32 3-Me 5, 3-Me 7.60 (q, 1.0) 139.27 4a - 116.02 - - 114.39 - - - 114.41 5 7.56+-

(s) 111.39 4 7.47 (1H, s) 109.92 4 4 7.39 (s)

112.14

6 - 148.7 5 - 147.13 5 - - 145.20 6a - 118.79 5, 8, 10 - 117.19 5, 8, 10, 7-OH - - 122.51 7 - 155.79 8, 9 - 154.56 7-OH - - 146.21

7-OH - - - 10.28 (1H, s) - - 23-OH - - 8 6.96 (dd, 0.9, 7.8) 115.04 10 6.91 (1H, brd, 7.6) 113.4 10, 7-OH 9 7.22 (dd, 1.0, 7.8) 122.18 9 7.49 (t, 7.8) 129.85 - 7.47 (1H, t, 8.0) 128.69 - 8, 10 7.60 (t, 7.8) 127.44 10 7.89 (dd, 0.9, 8.2) 113.4 8 7.74 (1H, brd, 8.3) 111.49 8 9 8.46 (dt, 1.0, 7.8) 120.67 10a - 127.11 9 - n. d. - - - 127.68 10b - 147.12 4, 5, 10 - 144.84 4, 5, 10 - - 146.39

neol

ambe

rtelli

n

3-Me 2.22 (d, 1.0) 16.97 4 2.14 (s, 1.0) 16.54 4 4 2.28 (d, 1.0) 17.13

21 3.82 (2H, not assignable ) 65.25 - 3.49-3.65 (2H) 60.63 - 23 4.04 (d, 12.1) 4.45 (d, 12.1) 62.21

21-OH - - - 4.86 (1H, brt, 6.5) - - - - - 22 - 105.39 21, 31 - 103.94 23, 31 - - 102.12 23 4.38 (d, 8.8) 77.73 24 4.33 (1H, brdd, 5.4,

9.2) 75.67 24 5.84 (d, 9.7) 72.22

23-OH - - - 6.35 (1H, brd, 5.4) - - 7-OH - - 24 4.76 (d, 8.8) 76.44 23, 26 4.20 (1H, brdd, 8.5,

9.2) 75.05 23 - 5.94 (d, 9.7) 73.82

24-OH - - - 4.96 (1H, brd, 8.5) - - - - - 25 - 108.9 26 - 107.03 26 - - 104.45

26 3.87(2H, not assignable) 61.17 - 3.68-3.81 (2H) 60.18 - - 4.19 (d, 12.6) 4.41 (d, 12.6) 61.13

hexo

diul

ose

26-OH - - - 4.67 (1H, brdd, 4.2, 6.7)

- - - - 31 5.59 (d, 3.4) 94.16 - 5.33 (1H, d, 3.6) 92.40 - 32 5.75 (d, 3.5) 89.91 32 3.49 (dd, 3.4, 9.8) 73.37 33 3.24 (1H, m) 71.66 32-OH 31 4.99 (dd, 3.5, 10.5) 69.97

32-OH - - - 5.08 (1H, brd, 6.95) - - - - - 33 3.77 (not assignable) 74.73 31, 32, 34 3.52 (1H) 72.86 - - 5.48 (dd, 9.8, 10.5) 69.64

33-OH - - 4.83 (1H) - - - - - 34 3.34 (dd, 9.6, 10.1) 71.45 33 3.11 (1H, m) 69.89 33 - 5.12 (t, 9.8) 68.05

34-OH - - 4.83 (1H) - - - - - 35 3.94 (ddd, 2.2, 5.1, 10.1) 74.57 34 3.79 (1H, m) 73.03 - - 4.38 (m) 68.73

3.70 (dd, 5.1, 11.7), 4.31 (dd, 4.5, 12.5) 36 3.83 (not assignable) 62.4 - 3.58-3.66 (2H) 62.31 - - 4.22 (dd, 2.1, 12.5) 61.56

gluc

ose

36-OH - - - 4.45 (1H, brt, 5.1) - - - - -

CH 3CO - - - - - - - 1.62, 1.75, 2.04, 2.05, 2.05, 2.15, 2.23, 2.29, 2.53

19.70, 20.39, 20.55, 20.58, 20.59, 20.65, 20.69, 20.78, 21.39

acet

ate

CH3CO - - - - - - - - 169.08, 169.45, 169.74, 169.77, 169.88, 170.07, 170.09, 170.52,

170.60

S24

8. NOE analysis and data

Quantitative NOE build-up curves of Sucro-neolambertellin peracetate (2) for eight different

mixing times varying from 55 to 1000 ms were derived from a NOESY experiment that

suppresses zero-quantum-artifacts almost completely.[1] The spectra were recorded on a

Bruker Avance 700 MHz NMR spectrometer equipped with a cryo-probe. The concentration of 2

in DMSO was 15 mM. The experiment was recorded at 298 K, with 8 scans per increment,

spectral widths 7001 Hz and 7003 Hz, sampled with 512 (F1) and 4096 (F2) complex points,

respectively. Both dimensions were apodized with a p /2-shifted squared sine-bell function, zero

filling was applied to 1024 × 8192 complex points. Integration was performed using the CARA

software.[2] Distances were referenced to H4/H5. A total of 49 NOEs were collected. Due to

signal overlap and strong coupling between H23 and H24, 22 could be treated as unambiguous

and the rest as ambiguous restraints. There are 18 specific unambiguous NOEs between the

three moieties of the molecule.

Proton 1 Proton 2 Distance [?] Error [?]

H4 H211 4.9 2

H4 H261 4.7 1

H4 H361/H212 5.5 0.2

H4 H362/H262 5.0 2

H5 H4 2.4 -

H5 H211 3.4 0.2

H5 H23 3.9 1

H5 H24 4.5 1

H5 H261 2.55 0.2

H5 H31 4.0 0.2

H5 H361/H212 3.8 0.3

H5 H362/H262 2.9 0.2

H8 H9 2.4 0.2

H10 H8 4.4 1

H10 H9 2.45 0.2

H211 H212 1.75 0.2

H23 H211 3.0 0.2

H23 H261 3.6 0.2

H23 H35 3.35 0.2

H23 H361/H212 2.8 0.2

S25

H23 H362/H262 2.85 0.2

H24 H211 3.7 0.2

H24 H261 3.2 0.2

H24 H33 4.5 2

H24 H35 2.8 0.2

H24 H361/H212 3.35 0.2

H24 H362/H262 2.5 0.2

H261 H262 1.75 0.2

H31 H211 2.5 0.2

H31 H261 4.3 1

H31 H33 4.2 0.4

H31 H34 4.1 0.4

H31 H35 3.7 0.4

H31 H361/H212 2.6 0.2

H31 H362/H262 3.9 0.4

H32 H211 4.5 0.4

H32 H31 2.3 0.2

H32 H361/H212 4.5 1

H32 H362/H262 4.9 1

H33 H211 4.7 1

H33 H32 2.6 0.2

H33 H34 2.6 0.4

H33 H35 2.6 0.2

H33 H361/H212 4.5 2

H33 H362/H262 4.1 1

H34 H35 2.8 0.4

H34 H361/H212 3.1 0.2

H34 H362/H262 2.8 0.2

H35 H32 4.3 1

S26

9. Alignment media

Polymer Characteristics 2D splitting

[Hz]

Medium A Poly(acryloamide) preparation following [3], but uncharged

polymer made of n-isopropylacrylamide

polymerized in DMSO; monomer

concentration 2.0 mol/L; linker N,N'-

methylene-bis-acrylamide at 0.7%; gel

cylinder diameter 5.4 mm

7.8

Medium B Poly(acrylonitrile) preparation following [4], monomer

concentration 210 g/L, irradiation with

480 kGy, polymer molecular weight

231000 Da

15.6

Medium C Poly(acryloamide) preparation following [3], but uncharged

polymer made of N,N-

dimethylacrylamide; monomer

concentration 1.0 mol/L; linker N,N'-

methylene-bis-acrylamide at 2%; gel

cylinder diameter 7.0 mm

6.2

S27

10. Residual dipolar couplings Residual dipolar couplings were measured for CH-vectors of Sucro-neolambertellin peracetate

(2) in three alignment media. The rdcs were taken from t2-coupled 1H,13C-HSQC-spectra by

superimposing and fitting ? 2 traces from isotropic and anisotropic spectra. Concentrations of 2

in DMSO were 15 mM (isotropic sample), 8 mM (medium A), 5 mM (medium B) and 8 mM

(medium C). All experiments were performed at 298 K with 64 scans per increment, spectrall

widths 28170 Hz and 8389 Hz, sampled with 512 (F1) and 4096 (F2) complex points. Both

dimensions were apodized with a p /2-shifted squared sine-bell function, zero filling was applied

to 1024 × 8192 complex points. Adding noise to the ? 2 traces and re-extracting the couplings

did not introduce errors larger than 0.2 Hz. Errors of approximately 0.2 to 0.5 Hz were

determined from the different ways that various scientists in the group superimposed the

submultiplets manually.

CH-vector rdc [Hz]

medium A

rdc [Hz]

medium B

rdc [Hz]

medium C

error [Hz]

C4-H4 -14.6 -5.9 -7.3 0.2

C5-H5 -14.5 -6.2 -5.9 0.2

C8-H8 -7.1 -3.0 0.0 0.2

C9-H9 -13.4 -7.0 -7.2 0.2

C10-H10 -5.2 -11.2 -5.6 0.2

C21-H211 -3.1 - 3.6 0.5

C21-H212 -3.1 - 0.0 0.5

C23-H23 6.2 7.0 0.0 0.2

C24-H24 4.7 4.5 0.0 0.2

C26-H261 2.0 - 4.9 0.5

C26-H262 - - - -

C31-H31 1.4 -5.0 0.0 0.2

C32-H32 8.0 - 4.1 0.2

C33-H33 9.3 21.2 5.5 0.2

C34-H34 7.8 20.4 3.6 0.2

C35-H35 7.8 20.0 3.7 0.2

C36-H361 -6.8 - -3.0 0.5

C36-H362 8.0 - 3.9 0.5

S28

11. Structure calculation .Structure calculations were performed in X-PLOR NIH [5a,5b] with a simulated annealing

protocol [6a,6b] modified specifically for calculations with small molecules. Floating chirality was

applied to all centers in the furanose ring and the diastereotopic methylene protons at C22,

C26, C36. The stereocenters of the a-D-glucopyranoside were fixed. Starting from randomized

atom coordinates, calculations were performed using the above mentioned 49 NOE-derived

distance restraints including error boundaries, 27 of which had to be treated as ambiguous. The

final force constant or the NOEs was set to 50 kcal mol-1 Å-2, standard value also used for

protein structure calculations. For each of the 16 possible configurations, the ten structures with

the lowest NOE energy penalties were selected. These were of comparable energies and

similar conformation. Rdc cross-validation for three sets of alignment media was accomplished

using the SANI module [7a,7b] in X-PLOR, which treats diplolar couplings as angular restraints.

The force constant was left at the default value of 50 kcal mol-1 Hz-2. This value produced nicely

distinct energy values, allowing the use of a grid search to find the optimal axial tensor

component and rhombicity for each configuration.[8] The implemented grid search optimizes Da

and Rh only, using the previously determined conformations as frame for the determination of

the tensor orientation. NOE and rdc derived energy penalties for all 16 configurations are given

in the following. The x indicates that for this configuration, the grid search and following rdc

energy minimization failed.

Configuration NOE energy Rdc energy

SSSS 62.1 1191.7

SSSR 62.3 5676.3

SSRS 61.0 10451.8

SSRR 74.3 185.3

SRSS 107.5 1098.1

SRSR 48.2 0.4

SRRS 53.6 2156.9

SRRR 58.3 7.7

RSSS 35.6 217.0

RSSR 29.9 0.0

RSRS 105.8 384.0

RSRR 77.2 5635.3

RRSS 112.2 x

RRSR 49.1 3878.5

RRRS 53.0 3705.9

RRRR 44.4 174.9

S29

12. References

[1] M. J. Thrippleton, J. Keeler, Angew. Chem. Int. Ed. 2003, 42, 3938-3941. [2] R. Keller, PhD thesis, ETH Zürich (CH), 2004. [3] P. Haberz, J. Farjon, C. Griesinger Angew. Chem Int. Ed. 2005, 44, 427-429 [4] G. Kummerloewe, J. Auernheimer, A. Lendlein, B. Luy, J. Am. Chem. Soc. 2007, 129,

6080-6081. [5a] C. D. Schwieters, J. Kuszewski, N. Tjandra, G. M. Clore, J. Magn. Reson. 2003, 160, 66-

74. [5b] C. D. Schwieters, J. Kuszewski, G. M. Clore, Progr. NMR Spectroscopy 2003, 48, 47-62. [6a] M. Nilges, G. M. Clore, A. M. Gronenborn, FEBS Lett. 1988, 239, 129-136. [6b] M. Nilges, J. J. Kuszewski, A. T. Brunger in Computational Aspects of the Study of

Biological Macromolecules by NMR (Ed.: J. C. Hoch), Plenum Press, New York, 1991. [7a] G. M. Clore, A. M. Gronenborn, N. Tjandra, J. Magn. Reson. 1998, 131, 159-162. [7b] G. M. Clore, A. M. Gronenborn, A. Bax, J. Magn. Reson. 1998, 133, 216-221. [8] J. Klages, C. Neubauer, M. Coles, H. Kessler, B. Luy, ChemBioChem 2005, 133, 1672-

1678.

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Rdc-enhanced NMR Spectroscopy in Structure Elucidation of the Natural Product Sucro-neolambertellin