A. J. Kirby, F. Nome et al.
products as a function of time, 1H NMR spectra were recorded in D2O
on a Varian Mercury Plus 400 MHz instrument operating at 400 MHz
with sodium 3-(trimethylsilyl) propionate (TSP) as internal reference.
Chemical shifts are reported as d (ppm). Mass spectrometry was per-
formed on a Shimadzu GCMS-QP5050A instrument. The injector and in-
terface temperatures were maintained at 280 and 3008C, respectively,
and the oven temperature for 5 min at 808C before being raised, at a
constant rate of 108C minꢀ1, to 3008C, for a further 5 min. UV spectra
were obtained by using Varian Cary 50 and Cary 1 Bio UV/Vis spectro-
photometers.
ditions (Table S4 in the Supporting Information), as might be expected
ꢀ
for C O cleavage involving a better, alkyl aryl phosphate, leaving group.
However, NMR spectroscopy product studies show unambiguously (Fig-
ure S5 in the Supporting Information) that the only hydrolysis products
ꢀ
are 2-pyridone and diethyl phosphate, as expected for P O cleavage. We
conclude that at the pH-rate minimum the kH +kOH reactions are togeth-
er faster than k0, which is too slow to contribute significantly to the ob-
served rate (or product). Curve fits, with and without k0 as an independ-
ent variable, do not differ convincingly.
Hydrolysis in mixed solvents: Reactions were performed in solutions con-
taining 5–50% (v/v) of acetonitrile in water at pH 7 (0.01m BISTRIS).
Final concentrations of substrate needed to be in the range of 1ꢀ10ꢀ6–2ꢀ
10ꢀ5 m, depending on the concentration of acetonitrile, to prevent precipi-
tation.
Synthesis: Procedures for the preparation of triaryl phosphate, bis-tri-
fluoroethylaryl phosphate and 2-pyridyl phosphate triesters are detailed
in the Supporting Information.
Kinetic methods: Reactions were initiated by adding to a buffered aque-
ous solution (1 or 3 mL) at the appropriate pH, either 10 or 30 mL of a
stock solution of the substrate (5ꢀ10ꢀ3 m: in acetonitrile, or in dioxane
for the bis-trifluoroethyl compounds and compounds studied in 30% di-
oxane). Kinetics were followed at constant temperature and ionic
strength 1.0m (KCl or NaCl), for at least five half-lives, by monitoring
the appearance of the pyridone at 294 nm, and of other leaving groups at
the appropriate wavelength (2,4-dinitrophenolate at 360 nm, 4-chlorophe-
nolate at 300 nm, 4-nitro- and 3-fluoro-4-nitrophenolate at 400 and
320 nm for the acid form), on Varian spectrophotometers equipped with
thermostatted cell holders. Activation parameters were calculated by
using the Eyring equation, from rate constants obtained in the range of
25 to 558C. The pH of the reaction mixture was measured at the end of
each run, by using a Hanna Instruments model pH 200 pH meter. Ob-
served first order rate constants (kobs) were calculated by non-linear
least-squares fitting of the absorbance versus time curves: correlation co-
efficients were 0.998 or better. Buffers used were HCl (pH <2), chloroa-
cetate (pH 2.0–3.5), formate (pH 3.0–4.0), acetate (pH 4.0–5.5), phos-
phate (pH 5.5–7.0), 2-(N-morpholino)ethanesulfonic acid (MES) (pH 5–
7), 3-[4-(2-hydroxyethyl)piperazin-1-yl]propane-1-sulfonic acid (HEPPS)
and N-methylmorpholine (pH 7–8), BISTRIS (pH 7.0–9.0), borate
(pH 8.0–9.0) and 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS)
(pH 10–11). Ionic strength was maintained at 1.0m with KCl (NaCl in
Sheffield: with no significant differences in rate). Full data from kinetic
measurements are summarised in Tables S1–S6 in the Supporting Infor-
mation.
Acknowledgements
We are grateful to FAPESC (Fundażo de Apoio ꢄ Pesquisa Cientꢁfica e
Tecnolꢂgica do Estado de Santa Catarina), CNPq (Conselho Nacional de
Desenvolvimento Cientꢁfico e Tecnolꢂgico) and INCT-Catꢅlise (Instituto
Nacional de CiÞncia e Tecnologia; Brazil), to EPSRC (Engineering and
Physical Sciences Research Council; EP/E01917X; UK), and to The
Libyan Ministry of Higher Education (to AA), for support of this work.
[1] A. J. Kirby, M. Medeiros, P. S. Oliveira, T. A. S. Brand¼o, F. Nome,
[2] A. J. Kirby, F. Hollfelder, From Enzyme Models to Model Enzymes,
RSC, Cambridge, 2009.
[7] U. Costas-Costas, C. Bravo-Dꢁaz, H. Chaimovich, I. M. Cuccovia,
Hydrolysis of DEPP: Reactions were initiated by adding a stock solution
of the substrate DEPP (30 mL, 0.01m in acetonitrile) to buffered aqueous
solution (3 mL) at the appropriate pH. Rates were measured at 25, 45
and 558C to allow reliable estimates of the slowest rates near pH 7. A
full pH-rate profile was obtained at 458C, by using the initial rate
method near pH 7, a partial one at 258C and limited further data at
558C, to allow extrapolation, through Arrhenius plots, to 258C. The full
data set appears in Table S4 in the Supporting Information, and pH-rate
profiles at both 45 and 258C are shown in Figure S4 in the Supporting In-
formation. Other triesters with two or three 2-pyridyl groups are hydro-
lysed at rates close to those expected for compounds with aryl leaving
groups of the same pKa, so DEPP stands out as an exception. A possible
[10] J. A. A. Ketelaar, H. R. Gersmann, K. Koopmans, Rec. Trav. Chim.
Pays-Bas 1952, 71, 1253.
[13] H. Lçnnberg, R. Strçmberg, A. Williams, Org. Biomol. Chem. 2004,
2, 2165.
[15] A. J. Kirby, D. W. Tondo, M. Medeiros, B. S. Souza, J. P. Priebe,
[16] A. J. Kirby, M. Medeiros, P. S. M. Oliveira, T. A. S. Brand¼o, A.
Amer, N. H. Williams, F. Nome, unpublished results.
[17] E. P. Lyznickij, Jr., K. Oyama, T. S. Tidwell, Can. J. Chem. 1974, 52,
1066.
ꢀ
explanation is that the hydrolysis of this ester involves C O cleavage.
The spontaneous hydrolysis of triethyl phosphate (at 1018C) is known to
involve 100% C O cleavage, with a rate constant k0 =8.35ꢀ10ꢀ6 sꢀ1 and
ꢀ
[17]
DH° =23.4 kcalmolꢀ1
.
This allows the calculation of rates of 2.3ꢀ
10ꢀ9 sꢀ1 and 3.0ꢀ10ꢀ8 sꢀ1 at 25 and 458C, respectively. The observed rates
Received: June 22, 2011
for the hydrolysis of DEPP are a few (2–7) times faster under these con-
Published online: November 21, 2011
15004
ꢃ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2011, 17, 14996 – 15004