Design, Synthesis, and Crystal Structures of 6-Alkylidene-2 - American

Sep 7, 2010 - Pathology, and Pharmacology, Case Western ReserVe UniVersity School of .... Here we present the design, synthesis, and characterization...
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Table 1. Kinetic Parameters

NCF

Imipenem

kcat (s-1)

Km (µM)

kcat/Km (µM-1s-1)

OXA-24 WT

538 ± 40

28 ± 3

19.2 ± 2.6

OXA-24Y112A

571 ± 55

238 ± 20

2.4 ± 0.3

OXA-24Y112A M223A

394 ± 40

143 ± 13

2.8 ± 0.4

OXA-24 WT

3.8 ± 0.4

2.3 ± 0.1

1.7 ± 0.2

OXA-24 Y112A

4.8 ± 0.5

12.0 ± 0.4

0.40 ± 0.04

OXA-24Y112A M223A

5.3 ± 0.2

22 ± 4

0.24 ± 0.04

SI Tables p1

Table 2a. IC50 CENTA

1 (nM)

3 (nM)

4 (nM)

5 (nM)

OXA-24 WT

127 ± 42

187 ± 17

192 ± 56

237 ± 7

OXA-24 Y112A

384 ± 13

975 ± 81

309 ± 55

648 ± 240

OXA-24 Y112A M223A

509 ± 100

500 ± 37

594 ± 64

898 ± 93

Table 2b. IC50 NCF

1 (nM)

2 (nM)

3 (nM)

4 (nM)

5 (nM)

17 ± 1

27 ± 1

22 ± 1

10 ± 1

55 ± 5

OXA-24 Y112A

142 ± 10

57 ± 5

105 ± 16

68 ± 7

297 ± 15

OXA-24 Y112A M223A

250 ± 44

180 ± 9

187 ± 8

185 ± 12

338± 20

OXA-24 WT

SI Tables p2

Table 3. Kinetic Parameters of Inhibition

1

2

3

4

5

Ki (µM)

kinact (s-1)

kinact/Ki (µM-1s-1)

OXA-24 Y112A

2.22 ± 0.88

0.08 ± 0.01

0.040 ± 0.008

OXA-24Y112A M223A

12.0 ± 0.7

0.09 ± 0.01

0.008 ± 0.002

OXA-24 Y112A

8.8 ± 0.3

0.03 ± 0.01

0.003 ± 0.001

OXA-24 Y112A M223A

22.6 ± 0.2

0.10 ± 0.01

0.0040 ± 0.0004

OXA-24Y112A

8.0 ± 0.9

0.22 ± 0.02

0.030 ± 0.005

OXA-24 Y112A M223A

12.0 ± 1.5

0.15 ± 0.01

0.013 ± 0.001

OXA-24 Y112A

5.0 ± 0.7

0.05 ± 0.01

0.010 ± 0.001

OXA-24Y112A M223A

3.0 ± 0.2

0.05 ± 0.01

0.020 ± 0.003

OXA-24 Y112A

11 ± 2

0.04 ± 0.01

0.004 ± 0.001

OXA-24 Y112A M223A

19 ± 2

0.07 ± 0.01

0.004 ± 0.001

Tazobactam Ki was determed by Drawz, et al and was 271 ± 37 M (ref).

SI Tables p3

Table 4. ESI-MS Analysis Expected Mass

Actual Mass (amu)

(amu)

± 3 amu

OXA-24WT

29073

29071

OXA-24Y112A

28980

28980

OXA-24Y112A M223A

28920

28919

OXA-24WT /1

29560

29560

OXA-24 Y112A /1

29467

29467

OXA-24 Y112A M223A /1

29407

29410

OXA-24 WT/3

29495

29496

OXA-24 Y112A /3

29402

29404

OXA-24 Y112A M223A/3

29342

29344

OXA-24 WT/4

29565

29565

OXA-24 Y112A/4

29472

29473

OXA-24 Y112A M223A/4

29412

29412

OXA-24 WT/5

29497

29496

OXA-24 Y112A/5

29404

29404

OXA-24 Y112A M223A/5

29344

29343

SI Tables p4

Supplementary Information FIGURE CAPTIONS

Schemes 1, 2, and 3. See text Figure 1.

Mass spectrometry. Intact mass spectrometry was performed to determine the

covalent intermediates in the inactivation pathway. Here we show the deconvoluted mass spectra of OXA-24 and variants Y112A and Y112A, M223A) before and after 15-min incubation with the inhibitors (1, 3, 4, 5). Spectra were obtained on a Q-STAR XL quadrupole-time-offlight mass spectrometer equipped with a nanospray source. A single mass shift was identified. OXA-24 and variants with 2 was not performed.

Figure 2. (A) Detail of the region at the lower border of the active binding cleft of the 1 complex. The catechol moiety runs almost parallel to the right side of the active site. (B), (C) Close-up view of the surface potential at the active binding site of OXA-24 in complex with 5 drawing in two different orientations. The molecular surface of the enzyme is depicted as a transparent solid surface coloured according the electrostatic potential. The 5 molecule is represented as sticks.

SI, Figures (text p1)

Scheme 1. Synthesis of compounds 4 and 5

S N

O

allocNH

1) Ph2CN2, CH2Cl2/MeOH, rt, 15 h, 40%

allocNH

SH

O

O

tol. reflux, 3 h, 82%

6-APA

N O

N

O

+

1.5 eq mCPBA, CH2Cl2, rt CH3

N

O CO 2CHPh2

14

CH2Cl

+

CH3

O

+ N

O2CCH2Cl

O

O CO 2CHPh2

15

O2 S

N

N

CO2CHPh2

O

O2 S

O

30 min, 33%

N

O

S

O2CCH2Cl

O

CO2CHPh2

13

N

N O

N

O2CCH2Cl

O CO 2CHPh2

N

CH3

N

12

O CH2Cl

+

N

CO2CHPh2

S

S

CH2Cl

O

11

PPh3

CH2Cl2/THF, -78 oC, 30 min, 33%

2) 0.0025 eq Rh2(OOct)4, propylene oxide, benzene rt, 30 min, 90% overall

O2CCH2Cl

CO 2CHPh2

S O

CH3

O

O

S

9 27% O

O

1) xs i-prONO, 0.05 eq TFA, EtOAc, rt, 30 min

N

10

N

CO2CHPh2

8 54% O

S

CH2Cl

+

N

O

CO2CHPh2

16 52%

17

CO 2CHPh2

5%

18

(separable by column chromatography)

N

N

O

O S

CH2Cl

O

N

O O2 S

1.2 eq mCPBA, rt

CH2Cl

O CO 2CHPh2

18

N

O O2 S

OPNP

O

2 eq

H2N

N

N N

O2 S

S

N H

O

S

py, rt, 10 min, 70%

2) aq NaHCO3 (51% overall)

O

N N H

NH2

Compound 4

S

N O CO 2Na

CO 2CHPh2

20

(PNP = p-nitrophenyl)

O O2 S

1) m-cresol, 50 oC, 3 h

NH2

O

N CO 2CHPh2

20

O

NH2

N O

CO 2CHPh2

19

N

OPNP

N

O CO 2CHPh2

16

O

py, rt, 5 h, 72%

N

O CO 2CHPh2

O O2 S

2 eq p-ClCO2C6H4NO2, 0.5 eq DMAP OH

EtOH, 60 oC, 20 min, 65%

N

O

N O2 S

5 eq thiourea O

CH2Cl2, 12 h, 85%

N

21

22

Ph

N

O O2 S O

H N OPNP

N

O

O2 S O

O CO 2CHPh2

py, rt, 10 min, 66%

N CO 2CHPh2

20

SI Figures p1

23

O O2 S

1) TFA - anisole

H N

O

NH O

O

N

Ph

O

2 eq H2N O

N

O2CCH2Cl

CO 2CHPh2

7 (Bt = benzothiazole) H 2N

S O

0.01 eq Pd(PPh3)4, CH2Cl2, rt, 30 min, 92%

CH3

N O

O H2N

1.1 eq (n-Bu)3SnH, 2.5 eq AcOH

+

CO 2CHPh2

6

S

CH2Cl

O

CH2Cl2, rt, 3 h, 81%

CO 2CHPh2

S

20 eq ClCH2CO2H, 2 eq AgOAc

N

N

allocNH

allocNH

S

N

2) ClCO2allyl, py, CH2Cl2, -10 oC, 30 min, 91% 3) 1 eq MeCO3H, -10 oC, 86%

CO 2-

1.0 eq

S

O

SBt

S

O

H3N+

O

2) aq NaHCO3 (37% overall)

N O CO 2-

24

NH

NH 3+

Compound 5

13%

CH2Cl

Scheme 2. Synthesis of compound 2

CH3

N

1) 2 eq KMnO4, H2O, 80 oC, 12 h 2) aq HCl, 30% overall

CH3

CO2H

1) SOCl2, cat. DMF, 80 oC, 2 h N

CO2allyl

CO2allyl

CH3

2) allyl-OH, Et3N, CH2Cl2, -78 oC to rt, 2 h, 71%

N

25

rt, 2 h, 93%

CH3

CO2allyl

CO2allyl

1.13 eq POCl3, 1.13 eq Et3N

1.4 eq mCPBA, CH2Cl2 N

CH3

1) 2 eq PPh3, reflux

CH2Cl2, rt to 40 oC, 2 h

2) KO-t-Bu, THF, rt, 2 h N

CH2Cl

O

26

27 allylO 2C

PPh3

N

28

29

allylO 2C

HO 2C

CO2allyl N H 2N

1) i-pr-ONO, cat. TFA

S

O

S N

2) propylene oxide, cat Rh2(OOct)4

N O

N

PPh3

-78 oC, CH2Cl2, THF, 37%

O

CO 2CHPh2

N

2.5 eq mCPBA

S

CH2Cl2, rt, 3 h, 65%

N O

N CO 2CHPh2

32

O

MeO

N

1) TFA - anisole

MeOH, 90 oC,

DMF, rt, 2 h

O2 S

1 h, 9% overall from 34

O2 S

2) aq NaHCO3 (25% overall)

N

N

N

O

O

O CO 2CHPh2

CO 2Na

CO 2CHPh2

35

SI Figures p2

H N O

N O2 S

34

MeO

H N O

1 eq DPPA, 1 eq Et3N,

CO 2CHPh2

33

N3

N

N

2) aq HCl, EtOAc, 70% O

CO 2CHPh2

31

O2 S

1) 1.1 eq p-TolSO2Na, 0.1 eq Pd(PPh3)4 THF/MeOH, 1 h, rt

O

CO 2CHPh2

30

N O2 S

36

Compound 2

Scheme 3. Synthesis of compound 3

allylO 2C

allyO 2C

CO2allyl

N

O O

O

S O N

CH 3

N

+

S

PPh3

N

N

CO 2CHPh2

-78 oC, CH2Cl2, THF, 32%

CO 2CHPh2

12

CH 3

N

+

O

O 2CCH 2Cl

O CO 2CHPh2

13

S

O 2CCH 2Cl

O

CO 2CHPh2

38

O2 S CH 3

N

+

O

CO 2CHPh2

37

CH2Cl

O

O

+ N O

CO 2CHPh2

39 54%

40

7%

CO 2CHPh2

41 16%

(separable by column chromatography)

allylO 2C

allylO 2C

N

N

O

O S O

2) aq NaHSO3 (79% yield)

N O

O2 S

EtOH, 60 oC, 20 min, 65%

O

CO 2CHPh2

43

42

O

O

O H2N

H2N N

O O2 S O

0.5 eq DMAP, py, rt, 2 h, 63%

N CO 2CHPh2

44

N

N OPNP

O

3.0 eq NH3, dioxane

O2 S

py, 30 min, rt 96%

O N

O

SI Figures p3

O CO 2CHPh2

41

3.0 eq p-ClCO2C6H4NO2

N

2) aq HCl, EtOAc, 84%

N O

CO 2CHPh2

PNPO

OH

OH

N

39

O2 S

1) 1.1 eq p-TolSO2Na, 0.1 eq Pd(PPh3)4 THF/MeOH, 1 h, rt

O2 S

5 eq thiourea

CH2Cl

O

CO 2CHPh2

N

N

O

1) 1.3 eq mCPBA CH2Cl2, 12 h, rt

CH2Cl

HO 2C

allylO 2C

O

1) TFA - anisole NH 2

O2 S

2) aq NaHCO3 (26% overall)

O N O

O

CO 2Na

CO 2CHPh2

45

46

NH 2

O

O2 S

N

30 min, 33%

N

O

O

1.5 eq mCPBA, CH2Cl2, rt

S

allylO 2C N

N

CH2Cl

O

O 2CCH 2Cl

O

O

N

O

S

CH2Cl

allylO 2C

allylO 2C

Compound 3

CH2Cl

OXA-24 (Y112A) 28980 +/- 3

LN-1-255 (200 uM) 15min

OXA-24WT:SA-4-17 1:5 15min

OXA-24 WT + JDB/SA-4-17 29496 +/- 3

OXA-24Tyr112:SA-4-17

OXA-24 (Y112A) + JDB/SA-4-17 29404 +/- 3 1:5 15min

Mass (amu)

Mass (amu) OXA-24 WT + JDB/SA-3-53 29496 +/- 3

OXA-24WT (40uM) SA-3-53 (200uM) 15min

SI Figures p4

Mass (amu)

Relative intensity

OXA-24 WT + JDB/SA-3-18 29565 +/- 3

Mass (amu) OXA-24 Double mutant:SA-4-17

OXA-24 (Y112A M223A) + JDB/SA-4-17 29344 +/- 3 1:5 15min

OXA-24Tyr112 (40uM)

Mass (amu)

SA-3-18 (200uM)

OXA-24 (Y112A) + JDB/SA-3-18 29473 +/- 3 15min

OXA-24 Double mutant (40uM)

OXA-24 (Y112A M223A) + JDB/SA-3-18 29412 +/- 3 SA-3-18 (200 uM) 15min

Mass (amu) Relative intensity

Relative intensity Relative intensity

15min

OXA-24 (Y112A M223A) + JDB/LN-1-255 29410 +/- 3

Mass (amu)

OXA-24WT SA-3-18

Mass (amu)

Mass (amu) Relative intensity

Relative intensity

Mass (amu)

Relative intensity

OXA-24 WT + JDB/LN-1-255 29560 +/- 3

15min

OXA-24Tyr112 (40uM)

OXA-24 (Y112A) + JDB/LN-1-255 29467 +/- 3

Relative intensity

LN-1-255 (200uM)

Relative intensity

OXA-24WT (40uM)

OXA-24 (Y112A M223A) 28919 +/- 3 OXA-24 Double mutant (40uM)

Mass (amu) Relative intensity

Relative intensity

Mass (amu)

Relative intensity

OXA-24Tyr112 (40uM)

OXA-24Tyr112 (40uM)

OXA-24 (Y112A) + JDB/SA-3-53 29404 +/- 3 SA-3-53 (200uM) 15min

Mass (amu)

Relative intensity

OXA-24 WT 29071 +/- 3

Relative intensity

Relative intensity

OXA-24WT (40uM)

Mass (amu) OXA-25 Double mutant (40uM)

OXA-24 (Y112A M223A) + JDB/SA-3-18 29343 +/- 3 SA-3-53 (200uM) 15min

Mass (amu)

Fig. 1

SI Figures p5

Fig. 2A

B

Y112 M223

C

SI Figures p6

Fig. 2 B, C