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
