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1

ELECTRONIC SUPPLEMENTARY INFORMATION

Synthesis of a non-natural glucose-2-phosphate ester able to dupe the acc system of

Agrobacterium fabrum

Si-Zhe Li, a,# Armelle Vigouroux, b,# Mohammed Ahmar, a Abbas El Sahili, b Laurent Soulère,a

Laïla Sago, b David Cornu, b Solange Moréra, b * Yves Queneau a*

# Equal contribution

a Univ Lyon, Institut de Chimie et Biochimie Moléculaires et Supramoléculaires, CNRS,

Université Lyon 1, INSA Lyon, CPE Lyon, ICBMS, UMR 5246 ; Université Claude Bernard,

Bâtiment Lederer, 1 Rue Victor Grignard, 69622 Villeurbanne Cedex, France,

[email protected]

b Institute for Integrative Biology of the Cell (I2BC), CNRS CEA Université Paris-Sud,

Université Paris-Saclay. Avenue de la Terrasse, Gif-sur-Yvette 91198, France,

[email protected]

Data collection and processing statistics for the structure of AccA in complex with G2LP

Page 2

2D NMR Characterization of Glucose-2-lactate phosphate ammonium

page 3-6

1H and 13C NMR for compounds 2 to 6 and G2LP

page 7-19

Electronic Supplementary Material (ESI) for Organic & Biomolecular Chemistry.This journal is © The Royal Society of Chemistry 2019

2

Table 1. Crystallographic data and refinement parameters

AccA-G2LP

Space group Cell parameters (Å,°)

I222a = 78.3

b = 108.3c = 108.2β = 113.6

Resolution (Å) 42-1.8(1.91-1.8)

No. of observed reflections

274443(42603)

No. of unique reflections

45083(7249)

Rsym (%) 9(121.5)

Completeness (%) 99.5(98.2)

I/ 12.9(1.6)

CC1/2 99.8(58.6)

Rcryst (%) 17.1

Rfree (%) 19.5

rms bond deviation (Å) 0.01

rms angle deviation (°) 1.0

Average B (Å2)proteinligandsolvent

38.828.340

Values for the highest resolution shell are in parentheses

3

2D NMR Characterization of Glucose-2-lactate phosphate ammonium

Glucose-2-lactate phosphate ammonium salt was characterized by 2D-NMR. A mixture of α/β with 5/3 ratio was calculated from the integration of H-1 signals. H-1α at 5.36 ppm with J1,2 = 3.6 Hz, and H-1β at 4.68 ppm with J1,2 = 7.9 Hz, partially hidden by deuterium oxide. With the help of 1H-1H COSY, we found H-2α as a multiplet at 3.95 ppm and H-2β at 3.77 ppm, impaired by H-3α, H-5α. In addition, the α and β CH- group of lactic acid as multiplets were found at 4.5 ppm, and the corresponding α and β CH3- are doublet at 1.40 ppm. Decomposition of G2P lactate can be easily traced signals at 4.5 ppm and 1.4 ppm for free lactic acid (Fig 1).

Like G2P (ref 12), HSQC shows coupling with phosphorus for 13C-NMR signals. The carbon of α and β CH- group appears as doublet (δ=73.1 ppm, JC-P = 5.7 Hz), and the same splitting is also overserved for C-1, C-2 and C-3 (Fig 2). The differences are shown in the Table 2.

In comparison with glucose-2-phosphate (ref 12), the carbon shift of C-1, C-2 and C-3 of G2P lactate move to high fields slightly, and the α-anomer locates in a higher field than β-anomer in both cases. The coupling constant is almost the same except JC2-P of glucose-2-lactate phosphate which is stronger than that of glucose-2-phosphate due to the introduction of lactic acid. This could be explained by the conformation adjustment. Among those three carbons, the J-coupling of C-2 is the strongest one since the coupling of C-2 and phosphorus is the second-order coupling whereas others are third-order coupling. More interestingly, for α and β glucose phosphate, in both case, the J-coupling of C-1 and C-3 exhibit distinct differences.

2D NMR of the 31P-1H correlation shows the 31P-NMR of H-2α and CHα appears at -1.16 ppm, and H-2β and CHβ at -0.86 ppm. Theoretically, four phosphorus signals (C2α, C2β, CHα, CHβ) should be observed. The proximity of 31P-NMR chemical signals of CH and H-2 resulted in only two peaks, and the high resolution NMR spectroscopy could give more details (Fig 3).

Table 2. Partial 1H-NMR and 13C-NMR of G2P lactate

α-anomer β-anomerH-1 5.36 (J = 3.6 Hz) C-1 90.9 (J = 2.0 Hz) H-1 4.68 (J = 7.9 Hz) C-1 95.0 (J = 5.1 Hz)H-2 3.95 C-2 75.7 (J = 6.8 Hz) H-2 3.79 C-2 78.9 (J = 6.6 Hz)H-3 3.75 - 3.81 C-3 71.5 (J = 5.6 Hz) H-3 3.63 – 3.56 C-3 75.0 (J = 2.7 Hz)HCH 4.56 CCH 73.1 (J = 5.7 Hz) HCH 4.49 CCH 73.1 (J = 5.7 Hz)

4

1.01.52.02.53.03.54.04.55.05.5

δ(ppm)

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

f1 (ppm)

H- H- H-2 H-2

CH3

CH3

CH

CH

Fig 1. 1H-1H COSY of G2P lactate (400 MHz, D2O)

5

3.13.23.33.43.53.63.73.83.94.04.14.24.34.44.54.64.74.84.95.05.15.25.35.45.5

δ(ppm)

55

60

65

70

75

80

85

90

95

100

f1 (ppm)

C-

C-

C-2

C-2C-3

H-H- H-2H-2

CHC-3

C6-H

Fig 2. HSQC of G2P lactate (400 MHz, D2O)

6

0.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.5

δ(ppm)

-4

-3

-2

-1

0

1

2

3

4

f1 (ppm)

P-OCHα/P(α) H-2α/ P(α)

P-OCHβ/ P(β) H-2β/ P(β)

Fig 3. 31P-1H correlation of G2P lactate (400 MHz, D2O)

7

Benzyl (S)-2-((bis(diisopropylamino)phosphanyl)oxy)propanoate (2)

0.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.0

δ(ppm)

24.2

3

3.02

4.00

0.95

2.01

5.22

1.02

1.05

1.06

1.07

1.07

1.09

1.10

1.35

1.37

3.37

3.39

3.41

3.43

3.44

3.45

3.46

3.47

3.50

3.52

4.17

4.19

4.21

4.23

4.25

4.27

5.07

7.17

7.23

7.24

7.26

7.27

7.27

1H-NMR (300 MHz, Chloroform-d)

PN(iPr)2(iPr)2N

OOBn

O

2

8

-200-150-100-50050100150200250300

δ(ppm)

118.

79

31P-NMR (122 MHz, Chloroform-d)

PN(iPr)2(iPr)2N

OOBn

O

2

9

0102030405060708090100110120130140150160170180190200

δ(ppm)

20.2

020

.26

24.0

324

.11

24.1

924

.24

24.3

024

.35

44.5

444

.72

66.2

168

.83

69.1

0

128.

1012

8.43

135.

97

173.

5417

3.59

13C-NMR (75 MHz, Chloroform-d)

PN(iPr)2(iPr)2N

OOBn

O

2

10

Benzyl (2S)-2-(((benzyloxy)(diisopropylamino)phosphanyl)oxy)propanoate (3)

0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.5

δ(ppm)

12.0

2

3.06

1.98

0.99

1.99

2.00

9.85

1.16

1.18

1.19

1.20

1.48

1.50

3.63

3.64

3.67

3.68

3.69

3.71

3.73

4.42

4.44

4.45

4.48

4.50

4.51

4.66

4.69

4.71

4.72

4.73

4.75

4.75

4.77

5.14

5.17

5.17

5.19

7.24

7.26

7.27

7.31

7.32

7.34

7.35


PN(iPr)2BnO

OOBn

O

3

11

-200-150-100-50050100150200250300

δ(ppm)

148.

4714

8.63


PN(iPr)2BnO

OOBn

O

3

12

-100102030405060708090100110120130140150160170180190200210

δ(ppm)

19.9

620

.02

20.0

520

.10

24.3

824

.49

24.5

624

.66

43.0

343

.19

43.3

565

.23

65.4

765

.56

65.7

966

.51

66.6

068

.06

68.3

168

.77

69.0

1

126.

9012

6.96

127.

1112

7.26

128.

1412

8.21

128.

2412

8.49

128.

5113

5.74

139.

3213

9.42

139.

4513

9.55

172.

6717

2.74

173.

1117

3.14


PN(iPr)2BnO

OOBn

O

3

13

Benzyl (2S)-2-(((benzyloxy)(((2S,3R,4S,5R,6R)-2,4,5-tris(benzyloxy)-6-((benzyloxy)methyl)tetrahydro-2H-pyran-3-yl)oxy)phosphanyl)oxy)propanoate

(5)

0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.5

δ(ppm)

3.00

0.96

1.12

1.16

1.14

1.05

0.98

3.01

4.05

3.91

0.55

1.56

1.62

2.21

1.07

29.6

2

1.36

1.38

1.40

1.42

3.68

3.70

3.71

3.73

4.09

4.52

4.53

4.55

4.56

4.58

4.59

4.60

4.63

4.68

4.68

4.71

4.71

4.77

4.80

4.81

4.83

4.84

4.85

4.87

4.88

4.90

4.97

5.09

5.10

5.10

5.12

5.16

5.16

5.19

5.19

5.20

7.17

7.18

7.19

7.20

7.25

7.29

7.30

7.31

7.32

7.32

7.34

7.35

7.36

7.37

7.38

7.40

7.42

7.43

7.45


O

OBnO

BnO

OBn

PO

BnO OBn

COOBn

5

14

-200-150-100-50050100150200250300

δ(ppm)

138.

8813

9.12


O

OBnO

BnO

OBn

PO

BnO OBn

COOBn

5

15

-100102030405060708090100110120130140150160170180190200210

δ(ppm)

19.8

719

.91

19.9

563

.92

64.0

264

.05

64.1

266

.77

66.8

167

.29

67.3

667

.40

67.8

567

.97

68.4

168

.54

69.5

169

.59

70.5

670

.62

73.5

375

.13

75.4

677

.43

77.8

777

.97

81.3

997

.62

97.6

497

.75

97.7

712

7.16

127.

5012

7.57

127.

7212

7.75

127.

7912

7.79

127.

8512

7.96

128.

1112

8.20

128.

3512

8.61

128.

7213

5.53

135.

5513

7.35

137.

4813

8.01

138.

0713

8.17

138.

6713

8.76

171.

8717

1.89

172.

0117

2.05


O

OBnO

BnO

OBn

PO

BnO OBn

COOBn

5

16

-100102030405060708090100110120130140150160170180190200210

δ(ppm)

19.8

719

.91

19.9

563

.92

64.0

264

.05

64.1

266

.77

66.8

167

.29

67.4

067

.97

68.4

168

.54

69.5

169

.59

70.5

570

.62

73.5

273

.93

75.1

375

.46

77.8

777

.96

81.3

581

.39

81.4

3

97.6

297

.64

97.7

597

.77

127.

1612

7.50

127.

5412

7.57

127.

6312

7.72

127.

7512

7.76

127.

8212

7.85

127.

9212

7.95

127.

9712

8.11

128.

1612

8.20

128.

3012

8.35

128.

4212

8.57

128.

61

DEPT 135 13C-NMR (101 MHz, Chloroform-d)

O

OBnO

BnO

OBn

PO

BnO OBn

COOBn

5

17

Benzyl (2S)-2-(((benzyloxy)(((2S,3R,4S,5R,6R)-2,4,5-tris(benzyloxy)-6-((benzyloxy)methyl)tetrahydro-2H-pyran-3-yl)oxy)phosphoryl)oxy)propanoate

(6)

0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.0

δ(ppm)

1.23

1.79

1.01

0.99

1.08

1.03

1.03

0.43

0.61

3.63

2.09

2.09

1.55

3.09

1.04

0.41

0.60

2.12

29.1

7

1.33

1.34

1.37

1.39

1.40

3.58

3.63

3.65

3.66

3.67

3.97

4.43

4.45

4.45

4.47

4.48

4.52

4.55

4.57

4.63

4.65

4.66

4.67

4.68

4.72

4.73

4.75

4.77

4.82

4.85

4.92

4.93

4.95

4.97

4.99

5.00

5.03

5.05

5.08

5.10

5.11

5.21

5.22

5.35

5.36

7.06

7.07

7.08

7.09

7.18

7.19

7.21

7.22

7.23

7.24

7.26

7.27

7.29

7.30


O

OBnO

BnO

OBn

PO

BnO OBn

COOBn

O

6

18

-200-150-100-50050100150200250300

δ(ppm)

-2.6

1-2

.38


O

OBnO

BnO

OBn

PO

BnO OBn

COOBn

O

6

19

-100102030405060708090100110120130140150160170180190200210

δ(ppm)

19.0

919

.14

67.1

667

.21

68.2

568

.37

69.4

569

.55

69.6

169

.97

70.1

070

.52

70.6

072

.01

72.0

773

.51

73.5

475

.20

75.2

275

.45

75.5

477

.27

77.3

377

.52

77.6

777

.73

80.6

380

.70

96.5

196

.63

127.

5412

7.59

127.

7312

7.76

127.

8212

7.84

127.

8912

7.95

127.

9812

8.00

128.

0612

8.15

128.

2812

8.31

128.

3312

8.38

128.

4012

8.44

128.

5012

8.66

135.

1913

5.26

137.

1913

7.42

137.

9613

8.01

138.

0613

8.47

138.

49


O

OBnO

BnO

OBn

PO

BnO OBn

COOBn

O

6

20

-100102030405060708090100110120130140150160170180190200210

δ(ppm)

19.0

919

.14

67.1

667

.21

68.2

468

.37

69.4

069

.46

69.5

569

.61

69.9

770

.10

70.5

270

.60

72.0

272

.07

73.5

173

.53

75.2

075

.22

75.4

575

.54

77.3

377

.72

77.7

380

.63

80.7

096

.51

96.6

3

127.

5412

7.59

127.

7312

7.76

127.

8212

7.84

127.

8912

7.95

127.

9812

8.01

128.

0612

8.15

128.

2812

8.31

128.

3312

8.38

128.

4012

8.44

128.

5012

8.66

DEPT 135 13C-NMR (101 MHz, Chloroform-d)

O

OBnO

BnO

OBn

PO

BnO OBn

COOBn

O

6

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