Supporting Information

Analogues with fluorescent leaving groups for screening and

selection of enzymes which efficiently hydrolyse organophosphorus
nerve agents
Luis Briseo-Roa,a Jim Hill,c Stuart Notman,c David Sellers,c Andy P. Smith,c
Christopher M. Timperley,*c Janet Wetherell,c Nichola H. Williams,c Gareth Rhys
Williams,c Alan R. Fersht,a and Andrew D. Griffiths b

a. Medical Research Council Centre for Protein Engineering, Hills Road, Cambridge,
CB2 2QH, UK.
b. Medical Research Council Laboratory of Molecular Biology, Hills Road,
Cambridge, CB2 2QH, UK.
c. Defence Science and Technology Laboratory, Porton Down, Salisbury, Wiltshire,
SP4 0JQ, UK.
* To whom correspondence should be sent: E-mail cmtimperley@dstl.gov.uk; Fax:
44 (0) 1980 613834; Tel: 44 (0) 1980 613566.

Contents
The effect of 1% v/v DMF on the hydrolysis kinetics of paraoxon

S2

Additional synthetic and NMR data for chemical intermediates

S2

LC-MS and LC-MSn experiments on the fluorogenic analogues

S3

Hydrolytic stability of fluorogenic analogues

S3

Hydrolytic stability dependence on pH

S3

Table S1. LC-MS data for the fluorogenic analogues

S5

S1

The effect of 1% v/v DMF on the hydrolysis kinetics of paraoxon

Enzyme DMF

kcat

KM

kcat/KM

(kcat/KM)/

(v/v)

(s-1)

(mM)

(M-1 s-1)

(kcat/KM)1%DMF

2453 33

0.018 0.001

1.35 (0.06) 108

1.8

1%

2380 60

0.031 0.002

7.60 (0.57) 107

9135 272

0.81 0.04

1.12 (0.06) 107

1%

9662 156

0.53 0.02

1.82 (0.08) 107

5.12 0.06

0.711 0.027

7.20 (0.28) 103

1%

5.23 0.09

0.888 0.042

5.89 (0.29) 103

19.58 0.49

1.124 0.076

1.73 (0.12) 104

1%

18.72 0.63

1.142 0.102

1.64 (0.16) 104

10.20 0.09

0.237 0.008

4.30 (0.15) 104

1%

10.59 0.08

0.273 0.008

3.88 (0.12) 104

PTE-wt

PTE-h5

PON1-wt

2.4PC

3.2PC

0.6

1.2

1.1

1.1

Additional synthetic and NMR data for chemical intermediates

Ethyl methylphosphonochloridate (f). H 4.41-4.17 (2H, m, OCH2), 1.98 (3H, d, J 18,
P-CH3), 1.41 (3H, t, J 7, CH3). C 63.2 (d, J 8, OCH2), 20.1 (d, J 130, P-CH3), 15.9
(d, J 8, CH3).
Isopropyl methylphosphonochloridate (g). H 5.04-4.86 (1H, dseptet, J 6 and 7,
OCH), 1.96 (3H, d, J 18, P-CH3), 1.42 (3H, d, J 4, CH3), 1.4 (3H, d, J 4, CH3). C
73.2 (d, J 8, OCH), 23.9 (d, J 5, CH3), 23.3 (d, J 5, CH3), 20.6 (d, J 131, P-CH3).
Isobutyl methylphosphonochloridate (h). H 3.88-3.68 (2H, m, OCH2), 2.03-1.84 (1H,
m, CH), 1.48 (3H, d, J 17, P-CH3), 0.95 (6H, d, J 7, CH3). C 73.2 (d, J 8, OCH2),
23.9 (d, J 5, CH), 23.3 (d, J 5, CH3), 20.6 (d, J 131, P-CH3).
Pinacolyl methylphosphonochloridate (i). H 4.56 (1H, dq, J 11 and 6, OCH), 4.43
(1H, dq, J 11 and 6, OCH), 1.97 (3H, d, J 18, P-CH3), 1.96 (3H, d, J 18, P-CH3), 1.39
(3H, d, J 7, OCHCH3), 1.37 (3H, d, J 7, OCHCH3), 0.95 (9H, s, CH3), 0.94 (9H, s,
CH3). C 84.5 (d, J 10, CHCH3), 83.7 (d, J 9, CHCH3), 35.0 (d, J 6, C-CH3), 34.8 (d,
J 6, C-CH3), 25.6 (s, CH3), 25.5 (s, CH3), 20.9 (d, J 132, P-CH3), 20.7 (d, J 132, PCH3), 17.1 (s, CHCH3), 16.2 (s, CHCH3).
Diisobutyl methylphosphonate. A solution of 2-methylpropanol (7 g, 95 mmol) in
ether (25 cm3) was added dropwise to a stirred suspension of sodium hydride (2.5 g,
105 mmol) in ether (25 cm3). The mixture was left to stir for 1 h. To this was added
over 30 min a stirred solution of methylphosphonic dichloride (12.6 g, 95 mmol) in
ether (50 cm3). The mixture was stirred for 90 min, precipitated sodium chloride
removed by filtration through CeliteTM, and the filtrate concentrated to give a liquid.
S2

Fractionation under reduced pressure gave diisobutyl methylphosphonate as a

colourless liquid (13.2 g, 67%). Bp 82-84C/4 mmHg. H 3.88-3.68 (4H, m, OCH2),
2.03-1.84 (2H, m, CH), 1.48 (3H, d, J 17, P-CH3), 0.95 (12H, d, J 7, CH3). C 71.4
(d, J 6, OCH2), 29.1 (d, J 6, CH), 18.6 (s, CH3), 10.6 (d, J 145, P-CH3). P 31.1.
LC-MS and LC-MSn experiments on the fluorogenic analogues
Material and sample infusion: Ammonium formate was purchased from Fluka
(Gillingham, UK) and HPLC grade methanol from Aldrich (Gillingham, UK). Water
from a Milli-Q system (Millipore, UK) was used for dilutions and the mobile phase.
Solutions (10 g cm-3) in 1:1 methanol-water were infused directly into a Finnigan
Deca XP+ ion trap MS using a 250 L syringe at a flow rate of 10 L min-1.
LC conditions: A Thermo Finnigan Surveyor LC system was fitted with a C18 column
(150 2 mm i.d., 5 m, 300 ) from Columbus (Phenomenex, Macclesfield, UK). A
linear gradient elution was performed at a flow rate of 0.2 mL min-1, with a mobile
phase of 20 mM ammonium formate in water (solvent A) and 20 mM ammonium
formate in methanol (solvent B). The gradient was 5% B (0-5 min), 5% to 90% B (510 min), held at 90% B (10-15 min), returned to the initial composition 5% B (15-20
min) and then equilibrated at 5% B (20-25 min).
MS conditions: MS analysis was performed using a Finnigan Deca XP+ ion trap MS
system fitted with an atmospheric pressure ionization (API) interface, operated in
positive APCI mode. The discharge current was set at 5 A, capillary temperature at
150C and vaporizer at 400C. The sheath gas flow was maintained at 60 (arbitrary
units) and the auxiliary gas at 20 (arbitrary units). Mass spectra were collected with a
scan range of m/z 50 to 500 and a source CID of 15 V. MS2 product ion spectra of
protonated molecular ions ( 2 m/z) were obtained with the ion trap collision energy
set at 35%, activation Q at 0.25, and the activation time at 60 ms. Results appear in
Table S1.
Hydrolytic stability of fluorogenic analogues
The hydrolytic stability was quantified by following the uncatalysed hydrolysis rate
for 24 h in 50 mM HEPES pH 8 at 25 C. The analogues exhibited different
uncatalysed rates (Figure S1a), the esters of alkylphosphonic acids (8 to 10) being
less stable than esters of phosphoric acid (e.g. 4 and 5). Nonetheless, even with the
least stable substrate, VX analogue 8, the hydrolysis rate was still 1010 lower than
the PTE-catalysed rate.
Hydrolytic stability dependence on pH
The dependence of hydrolytic stability upon pH was assessed by following the
uncatalysed hydrolysis rate for 24 h in 50mM Bis-Tris propane buffer with pH values
ranging from 6.5 to 9.0 at 25 C. All analogues exhibited similar profiles, in which
the stability decreased with increasing pH as shown for Russian-VX analogue 10
(Figure S1b). This profile is similar to that of the actual nerve agents.

S3

Figure S1. (a) Hydrolysis rates of substrates measured in 50 mM HEPES pH 8.0 at 25 C. (b) Stability
profile of Russian-VX analogue 10 between pH 6.5 and 9.0 in 50 mM Bis-Tris propane at 25 C.

S4

RT 19.45 0.02

12

RT 19.71 0.02

11

RT 18.70 0.01

10

RT 18.11 0.01

RT 17.52 0.01

RT 18.25 0.01

RT 17.99 0.01

RT 19.17 0.02

RT 18.27 0.01

RT 17.24 0.01

RT 20.17 0.02

CyO

PinO

BuO

PrO

EtO

EtO

Me2N

PrO

EtO

MeO

EtO

Me

Me

Me

Me

Me

Me2N

Me2N

PrO

EtO

MeO

EtO

MeO

MeO

RT 19.04 0.01

R2

R1

Compound

370

372

345

330

316

345

344

374

346

318

362

334

MW

370.7 (100), 371.9 (15),

372.8 (30), 373.8 (4)

372.9 (44), 374.1 (11),

374.9 (11), 375.9 (3)

345.9 (100), 346.0 (15),

346.9 (30), 347.9 (4)

330.8 (100), 331.9 (16),

332.9 (30), 333.9 (4)

317 (100), 318 (14), 319

(33), 320 (4)

346 (100), 347 (16), 348

(33), 349 (5)

345.1 (100), 346.1 (16),

347.0 (32), 348.0 (5)

374.7 (100), 375.9 (16),

376.8 (28), 377.8 (5)

347 (100), 348.1 (15), 349

(32), 350 (4)

319.1 (100), 320.1 (12),

321.1 (33)

363 (100), 364 (16), 365

(35), 366 (5)

335.1 (100), 336 (15), 337

(38), 338 (5)

[MH]+

S5

387.5 (30), 388.7 (7), 389.6

(9), 390.6 (1)

389.6 (50), 390.7 (9), 391.6

(13), 392.6 (3)

361.6 (22), 362.7 (3), 363.6

(7), 364.6 (1)

347.6 (17), 348.7 (3), 349.6

(5), 350.6 (1)

333.6 (20), 334.8 (4), 335.6

(5), 336.6 (1)

362.5 (7), 363.7 (1), 364.6

(2),

361.4 (6), 362.6 (1), 363.5 (2)

391.6 (12), 392.7 (3), 393.6

(4), 394.6 (1)

363.5 (11), 364.7 (2), 365.6

(3)

335.6 (23), 336.7 (4), 337.6

(7)

379.5 (7), 380.6 (1), 381.5

(2),

351.5 (40), 352.6 (5), 353.5

(12), 354.5 (2)

[M+NH4]+

289.0 (40), 290 (5), 291.0

(14), 292 (1)

289 (100), 290.1 (13), 291

(33), 392 (3)

289 (5)

289 (20), 290.1 (2), 291 (6)

Detected by LC-MS-MS (m/z

347)

Detected by LC-MS-MS (m/z

346)

Not detected

332.9 (15), 334 (2), 334.9 (4)

319 (13), 320 (2), 321 (4)

Detected by LC-MS-MS (m/z

319)

Detected by LC-MS-MS (m/z

363)

Detected by LC-MS-MS (m/z

335)

[MH-R]+

211.1 (9), 213.1 (2)

211.1 (12), 213.2 (4)

211.2 (5), 213.1 (1)

211.1 (8), 213.1 (3)

211.1 (11), 213.1 (3)

211.2 (3)

Not detected

211.1 (4), 213.1 (1)

211.1 (13), 212 (2)

211.3 (30), 213.2 (10)

211.1 (3), 213.1 (1)

211.2 (5), 213.2 (2)

[MH-O2R1R2PX]+

Table S1. LC-MS data for analogues R1R2P(X)Ocoumarin 1-12 (X = S or O) including m/z ratios and relative abundances; RT = retention time (min), Pin = pinacolyl.