Metallocyclodextrin catalysts for hydrolysis of phosphate triesters

Article abstract of DOI:10.1016/S0040-4039(02)01757-4

The copper complexes of 6^A-(2-aminoethylamino)- and 6^A-(3-aminopropylamino)-6^A-deoxy-β-cyclodextrin catalyse the hydrolysis of 4-tert-butyl-2-nitrophenyl dimethyl phosphate, with k_inc=3.1×10^-2 and 2.3×10^-2 s^-1, and K_d=4.3×10^-4 and 1.2×10^-3 M, respectively, in 0.05 M pH 7.0 HEPES buffer at 298 K. This corresponds to rate accelerations of more than 95 000 and 70 000 times for reaction of the cyclodextrin-bound species.

Full text of DOI:10.1016/S0040-4039(02)01757-4

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 43 (2002) 7797–7800
Metallocyclodextrin catalysts for hydrolysis of
phosphate triesters
Lorna Barr,a Christopher J. Easton,a,* Kitty Lee,a Stephen F. Lincolnb and Jamie S. Simpsona
aResearch School of Chemistry, Institute of Advanced Studies, Australian National University, Canberra, ACT 0200, Australia
bDepartment of Chemistry, University of Adelaide, Adelaide, SA 5005, Australia
Received 8 August 2002; accepted 22 August 2002
Abstract—The copper complexes of 6A-(2-aminoethylamino)- and 6A-(3-aminopropylamino)-6A-deoxy-b-cyclodextrin catalyse the
hydrolysis of 4-tert-butyl-2-nitrophenyl dimethyl phosphate, with kinc=3.1×10−2 and 2.3×10−2 s−1, and Kd=4.3×10−4 and 1.2×10−3
M, respectively, in 0.05 M pH 7.0 HEPES buffer at 298 K. This corresponds to rate accelerations of more than 95 000 and 70 000
times for reaction of the cyclodextrin-bound species. © 2002 Elsevier Science Ltd. All rights reserved.
Cyclodextrins have been studied extensively as catalysts
and enzyme mimics.1 Much of this work has been
focussed on reactions of carboxylate esters2 but rela-
tively little has been concerned with the hydrolysis of
phosphate triesters. Natural3 and modified4 cyclodex-
trins are known to either inhibit, or only modestly
accelerate, breakdown of these organophosphates in
aqueous solutions. Even when reaction occurs, in some
cases the cyclodextrins are thereby phosphorylated, so
they are not acting as true catalysts. Organophosphates
such as paraoxon 1 are important as insecticides and
nerve agents.5 There is considerable interest in methods
for the catalytic breakdown of these compounds, for
the bioremediation of contaminated soil and water, and
the destruction of stockpiles of chemical warfare
agents.6 Therefore, we set out to develop cyclodextrin-
based catalysts for hydrolysis of phosphate triesters.
diaminoethane increases the rates of hydrolysis of
paraoxon 1 and the related compounds 2 and 3 by 2–3
orders of magnitude.9 Accordingly, in our studies we
employed the Cu(II) complexes of the modified b-
cyclodextrins 4-7,10 to exploit the catalytic properties of
the metal and the binding characteristics of the
cyclodextrins. The principal substrate chosen for this
investigation was the triester 8, in which the tert-butyl
group on the aromatic ring promotes complexation by
the cyclodextrin11 and the nitro group facilitates detec-
tion of the hydrolysis product 9 (Scheme 1).
The organophosphate 8 was produced by phosphoryla-
tion of 4-tert-butylphenol with dimethyl phosphoro-
chloridate,12 followed by nitration.13 Hydrolysis of this
compound was monitored at 435 nm, which corre-
Various metal complexes are known to catalyse the
reactions of phosphate triesters.7–9 For example, the
Cu(II)
complex
of
N,N,N%,N%-tetramethyl-1,2-
* Corresponding author. Tel.: +61-2-6125-8201; fax: +61-2-6125-8114;
e-mail: easton@rsc.anu.edu.au
Scheme 1.
0040-4039/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(02)01757-4

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L. Barr et al. / Tetrahedron Letters 43 (2002) 7797–7800
sponds to the umax of the phenoxide 9 (m=3700 M−1
cm−1). At this wavelength, the triester 8 shows negligi-
ble absorption. At 298 K, in 0.05 M HEPES buffer
containing 1% methanol, at pH 8.0 and 7.0, and in 0.05
M MES buffer containing 1% methanol, at pH 6.0,
hydrolysis of a 3×10−4 M solution of the triester 8
occurred in a pseudo-first order manner, with rate
constants (kun) of 2.3×10−6, 3.2×10−7 and <1×10−7 s−1,
respectively.14 By comparison, in the presence of the
Cu(II) complex of 6A-(2-aminoethylamino)-6A-deoxy-b-
cyclodextrin 4 (1×10−3 M),15 but under otherwise identi-
Figure 1. Hydrolysis of the phosphate triester 8 by hydroxide
bound to copper in the complex with 6A-(3-aminopropyl-
amino)-6A-deoxy-b-cyclodextrin 5.
cal conditions, pseudo-first order rate constants (kobs
)
of 4.6×10−2, 2.9×10−2 and 3.4×10−3 s−1 were observed
for the hydrolysis, corresponding to rate increases of
ca. 20 000, 91 000 and >34 000, respectively. For the
Cu(II) complex of 6A-(3-aminopropylamino)-6A-deoxy-
b-cyclodextrin 5, the corresponding values for kobs were
found to be 2.1×10−2, 9.9×10−3 and 1.0×10−3 s−1, show-
ing increases in hydrolysis rates of ca. 9000, 31 000 and
>10 000. By contrast, the Cu(II) complexes of the
cyclodextrins 6 and 7 had much less effect. When used
at a concentration of 1×10−3 M, at pH 7.0 they
increased the rate of hydrolysis of the triester 8 (kobs) by
factors of only 2800 and 380, respectively. While the
Cu(II) complexes of the cyclodextrins 4 and 5 were
found to be effective catalysts, their components were
not. At pH 7.0, 1×10−3 M solutions of b-cyclodextrin,
6A-(2-aminoethylamino)-6A-deoxy-b-cyclodextrin 4, 6A-
almost a factor of ten when the pH is increased from
6.0 to 7.0, but only by a factor slightly above two when
the pH is increased from 7.0 to 8.0.
Multiple turnover (>10) of the substrate 8 by the cop-
per complex of 6A-(3-aminopropylamino)-6A-deoxy-b-
cyclodextrin
5 was observed. Thus, the metallo-
cyclodextrin is a true catalyst for the hydrolysis. It was
also observed to be substrate selective. For example, it
had very little effect on the rate of hydrolysis of
paraoxon 1. It also displayed a low degree of stereose-
lectivity. The racemate 10 was prepared by sequential
treatment of phosphorus oxychloride with 4-tert-
butylphenol, 2-propanol and methanol, in the presence
of triethylamine, followed by nitration.17 Using the
triester 10 (3×10−4 M) and the Cu(II)–cyclodextrin 5
complex (either 1.0×10−3 or 1.5×10−4 M), in 0.05 M
HEPES buffer containing 1% methanol at pH 7.0 and
298 K, the rates of hydrolysis (kobs) of the enantiomers
differed by a factor of 1.5. Practical limitations pre-
vented an analysis of this stereoselectivity in terms of
the separate contributions of binding and reaction of
the bound species.
(3-aminopropylamino)-6A-deoxy-b-cyclodextrin
5,
Cu(II) and the Cu(II) complexes of 1,2-diaminoethane15
and 1,3-diaminopropane increased the rate of hydroly-
sis of the triester 8 by factors of only 2, 2, 2, 20, 10 and
40.
When ranges of concentrations of the triester 8 and the
Cu(II) complexes of the cyclodextrins 4 and 5 were
used, saturation kinetics were observed, as is typical of
enzyme-catalysed reactions and characteristic of dis-
crete substrate binding and reaction processes. Under
these conditions, the rate constant for reaction of the
complexed triester 8 (kinc or kcat) and the equilibrium
constant for complexation of the triester 8 by the
metallocyclodextrin (Kd or Km) were determined to be
5.0×10−2 s−1 and 3.8×10−3 M at pH 8.0, and 3.1×10−2 s−1
and 4.3×10−3 M at pH 7.0, with the cyclodextrin 4, and
4.8×10−2 s−1 and 1.2×10−3 M at pH 8.0, and 2.3×10−2 s−1
and 1.2×10−3 M at pH 7.0, with the cyclodextrin 5. It
was not practical to determine the corresponding values
at pH 6.0 for two reasons. Firstly, at this pH the rates
of hydrolysis of the phosphate 8 are too slow to mea-
sure accurately when only low concentrations of metal-
locyclodextrin are used. Secondly, the conjugate acid of
the phenoxide 9 has a pKa of 7.48 at 303 K,16 so only
a small percentage of the hydrolysis product is
detectable as the chromophoric species 9 at pH 6.0.
In summary, the Cu(II) complexes of the cyclodextrins
4 and 5 are effective catalysts for the hydrolysis of the
phosphate triester 8. At pH 7.0 and 298 K, they
increase the rate of reaction by more than 95 000 and
70 000 times (kinc/kun), respectively.
References
1. (a) Breslow, R.; Dong, S. D. Chem. Rev. 1998, 98, 1997;
(b) Rizzarelli, E.; Vecchio, G. Coord. Chem. Rev. 1999,
188, 343; (c) Easton, C. J.; Lincoln, S. F. Modified
Cyclodextrins. Scaffolds and Templates for Supramolecu-
lar Chemistry; Imperial College: London, 1999; pp. 1–
293.
2. (a) Tee, O. S. In Advances in Physical Organic Chemistry;
Bethell, D., Ed.; Academic: London, 1994; Vol. 29, pp.
1–85; (b) VanEtten, R. L.; Sebastian, J. F.; Clowes, G.
A.; Bender, M. L. J. Am. Chem. Soc. 1967, 89, 3242; (c)
VanEtten, R. L.; Clowes, G. A.; Sebastian, J. F.; Bender,
M. L. J. Am. Chem. Soc. 1967, 89, 3253.
Hydrolysis of the triester 8 by the metallocyclodextrins
is most probably brought about by metal-bound
hydroxide (Fig. 1).8,9 This is consistent with the pH
dependence observed in the kobs and kinc values
reported above. Water bound to copper in the complex
of the cyclodextrin 5 is reported to have a pKa of 7.84,10
and accordingly, with this system kobs increases by

L. Barr et al. / Tetrahedron Letters 43 (2002) 7797–7800
7799
3. (a) Moss, R. A.; Gong, P. K. Langmuir 2000, 16, 8551;
(b) Mochida, K.; Matsui, Y.; Ota, Y.; Arakawa, K.;
Date, Y. Bull. Chem. Soc. Jpn. 1976, 49, 3119.
under reduced pressure to give an oil (0.5 g). This oil was
added slowly to an ice-cooled mixture of HNO3 (1 mL)
and H2SO4 (1 mL). The resulting solution was stirred on
ice for 12 min, poured into ice-water (20 mL) and
extracted with ethyl acetate (2×30 mL). The combined
ethyl acetate extracts were washed with brine (2×30 mL)
followed by saturated NaHCO3 solution (2×30 mL),
dried (MgSO4) and evaporated to give a yellow oil.
Chromatography of the oil on silica, eluting with 50%
ethyl acetate/hexane, gave the title compound (8) as a pale
yellow oil (0.36 g, 24%), which was further purified by
bulb to bulb distillation (oven temperature 200°C, 40
mmHg). IH NMR (CDCl3): l 1.25 (s, 9H), 3.82 (d,
4. (a) Mochida, K.; Nakamura, T.; Yamamoto, M.; Yanai,
K.; Shiotsuki, T.; Matsui, Y. Bull. Fac. Agr. Shimane
Univ. 1983, 17, 152; (b) Hu, C.-C.; Chen, W.-H.; Liu,
C.-Y.; Chen, J.-L. J. Inclusion Phenom. Mol. Recognit.
Chem. 1996, 23, 289; (c) Mochida, K.; Ozoe, Y.;
Miyazaki, H.; Matsui, Y. Bull. Fac. Agr. Shimane Univ.
1980, 14, 158; (d) Breslow, R.; Zhang, B. J. Am. Chem.
Soc. 1994, 116, 7893; (e) Akkaya, E. U.; Czarnik, A. W.
J. Phys. Org. Chem. 1992, 5, 540.
5. (a) Eto, M. In Handbook of Organophosphorus Chem-
istry; Engel, R., Ed.; Marcel Dekker: New York, 1992;
pp. 807–873; (b) Holstege, C. P.; Kirk, M.; Sidell, F. R.
Critical Care Clin. 1997, 13, 923.
JP–H=9.6 Hz, 6H), 7.46 (d, J=8.7 Hz, 1H), 7.62 (dd,
J=8.7, 2.4 Hz, 1H), 7.92 (d, J=2.4 Hz, 1H). 13C NMR
(CDCl3): l 30.8, 34.5, 55.1 (d, JP–C=6.5 Hz), 120.2 (d,
6. (a) Grimsley, J. K.; Rastogi, V. K.; Wild, J. R. Bioreme-
diation 1998, 2, 577; (b) Furlong, C. E.; Humbert, R.;
Withington, A. P.; Richter, R. J.; Chadsey, M. S.; McE-
wan, N.; Gray, J.; Lara, J. C.; Staley, J. T.; Li, W.-F.;
Costa, L. G.; Harrington, M. J.; Thomas, L. C.; Herring-
ton, R. T.; Sayles, G. D.; Haines, J. R. Bioremediation
1998, 2, 521.
7. (a) Ketelaar, J. A. A.; Gersmann, H. R.; Beck, M. M.
Nature 1956, 177, 392; (b) Smolen, J. M.; Stone, A. T.
Environ. Sci. Technol. 1997, 31, 1664.
8. (a) Kazankov, G. M.; Sergeeva, V. S.; Efremenko, E. N.;
Alexadrova, L.; Varfolomeev, S. D.; Ryabov, A. D.
Angew. Chem., Int. Ed. 2000, 39, 3117; (b) Kuo, L. Y.;
Perera, N. M. Inorg. Chem. 2000, 39, 2103; (c) Tsao, B.
L.; Pieters, R. J.; Rebek, J. J. Am. Chem. Soc. 1995, 117,
2210; (d) Morrow, J. R.; Trogler, W. C. Inorg. Chem.
1989, 28, 2330; (e) Menger, F. M.; Gan, L. H.; Johnson,
E.; Durst, D. H. J. Am. Chem. Soc. 1987, 109, 2800; (f)
Gellman, S. H.; Petter, R.; Breslow, R. J. Am. Chem.
Soc. 1986, 108, 2388.
J
P–C=6.2 Hz), 121.7 (d, JP–C=2.6 Hz), 125.3, 131.2 (d,
J
P–C=1.4 Hz), 140.6 (d, JP–C=5.7 Hz), 148.8 (d, JP–C
=
1.4 Hz); EI MS (70 eV) m/z: 303 (M+, 15%), 288 (100),
257 (65), 227 (10), 163 (10), 127 (23), 109 (21), 77 (8).
Anal. calcd for C12H18NO6P: C, 47.53; H, 5.98; N, 4.62.
Found: C, 47.25; H, 5.87; N, 4.79.
14. Results obtained from repeating each kinetic experiment
were reproducible to within 10%.
15. The Cu(II) complexes of the cyclodextrins 4–7, 1,2-
diaminoethane and 1,3-diaminopropane were prepared in
situ using equimolar quantities of Cu(II) perchlorate and
amine. Under the conditions used, the Cu(II) is effec-
tively fully complexed since the binary association con-
stants for such complexes are typically >107 M−1. For
example, values of 5.7×109 and 6.9×107 M−1, have been
reported for the cases of 1,3-diaminopropane (du Preez,
J. G. H.; van Brecht, B. J. A. M. J. Chem. Soc., Dalton
Trans. 1989, 253) and the modified cyclodextrin 5,10
respectively.
16. Seguchi, K. Yukagaku 1979, 28, 20.
9. Scrimin, P.; Ghirlanda, G.; Tecilla, P.; Moss, R. A.
17. 4-tert-Butyl-2-nitrophenyl isopropyl methyl phosphate
(10). To a solution of 4-tert-butylphenol (3.0 g, 20 mmol)
in benzene (10 mL) was added triethylamine (3.06 mL, 22
mmol) followed by phosphorus oxychloride (1.87 mL, 20
mmol). As a precipitate formed, further benzene (20 mL)
was added and the mixture was stirred at 90°C for 60
min. 2-Propanol (1.5 mL, 20 mmol) and triethylamine
(3.06 mL, 22 mmol) were added and the resulting solu-
tion was stirred at 90°C for 60 min. Methanol (0.8 mL,
20 mmol) and triethylamine (3.06 mL, 22 mmol) were
then added and the mixture was stirred for a further 60
min at 90°C, then poured into ethyl acetate (150 mL).
The solution was washed with aqueous HCl (1N, 3×100
mL), then saturated NaHCO3 (2×100 mL), dried
(MgSO4) and evaporated to give an oil (4.78 g). Column
chromatography of the oil on silica eluting with a gradi-
ent of hexane and ethyl acetate gave an oil (1.89 g). A
fraction of the oil (0.25 g) was slowly added to an
ice-cooled mixture of HNO3 (1 mL) and H2SO4 (1 mL).
The resulting solution was stirred on ice for 12 min, then
poured into ice-water (20 mL). The mixture was extracted
with ethyl acetate (2×30 mL). The combined ethyl acetate
extracts were washed with brine (2×30 mL), followed by
saturated NaHCO3 solution (2×30 mL), dried (MgSO4)
and evaporated to give a yellow oil. Chromatography of
the oil on silica eluting with a gradient of hexane and
ethyl acetate gave the title compound (10) as a pale yellow
Langmuir 1996, 12, 6235.
10. (a) Brown, S. E.; Coates, J. H.; Easton, C. J.; Lincoln, S.
F. J. Chem. Soc., Faraday Trans. 1994, 90, 739; (b)
Brown, S. E.; Coates, J. H.; Easton, C. J.; van Eyk, S. J.;
Lincoln, S. F.; May, B. L.; Stile, M. A.; Whalland, C. B.;
Williams, M. L. J. Chem. Soc., Chem. Commun. 1994, 47;
(c) Brown, S. E.; Haskard, C. A.; Easton, C. J.; Lincoln,
S. F. J. Chem. Soc., Faraday Trans. 1995, 91, 1013; (d)
Haskard, C. A.; Easton, C. J.; May, B. L.; Lincoln, S. F.
Inorg. Chem. 1996, 35, 1059; (e) May, B. L.; Kean, S. D.;
Easton, C. J.; Lincoln, S. F. J. Chem. Soc., Perkin Trans.
1 1997, 3157; (f) Sandow, M.; Easton, C. J.; Lincoln, S.
F. Aust. J. Chem. 1999, 52, 1151.
11. Matsui, Y.; Nishioka, T.; Fujita, T. Top. Curr. Chem.
1985, 128, 61.
12. Rossi, R. A.; Bunnett, J. F. J. Org. Chem. 1973, 38, 2314.
13. 4-tert-Butyl-2-nitrophenyl dimethyl phosphate (8). 4-tert-
Butylphenol (0.75 g, 5.0 mmol) was dissolved in toluene
(4 mL) and ether (2 mL) cooled to 5–10°C. Sodium
hydroxide solution (25%, 3 mL) and dimethyl phospho-
rochloridate (0.64 mL, 6.0 mmol) were added simulta-
neously over 30 min. The resulting suspension was stirred
for 4 h at room temperature, then poured into ethyl
acetate (40 mL). The organic solution was separated and
washed with 10% sodium hydroxide solution (2×30 mL)
and water (30 mL), then dried (MgSO4) and evaporated

7800
L. Barr et al. / Tetrahedron Letters 43 (2002) 7797–7800
oil (0.21g, 24%), which was further purified by bulb to
bulb distillation (oven temperature 200°C, 40 mmHg). H
EI MS m/z (70 eV): 331 (M+, 25%), 316 (50), 290 (43),
1
274 (82), 243 (100), 180 (70), 105 (15), 91 (19), 77 (22).
Anal. calcd for C14H22NO6P: C, 50.76; H, 6.69; N, 4.23.
Found: C, 50.74; H, 6.41; N, 4.17. Chiral HPLC analysis
of the oil [Chromtech AB Chiral AGP 100×4 mm
column, eluting with 7.5% 2-propanol in 0.01 M aqueous
NMR (CDCl3): l 1.25 (s, 9H), 1.26 (d, J=5.7 Hz, 3H),
1.30 (d, J=6.0 Hz, 3H), 3.80 (d, JP–H=11.7 Hz, 6H), 4.73
(m, 1H), 7.39 (dd, J=8.7, 1.2 Hz, 1H), 7.52 (dd, J=8.7,
2.4 Hz, 1H), 7.82 (dd, J=2.4, 1.2 Hz, 1H). 13C NMR
(CDCl3): l 30.9, 34.7, 53.3, 55.1 (d, JP–C=6.6 Hz), 74.6
(d, JP–C=6.3 Hz), 121.2 (d, JP–C=2.6 Hz), 122.2, 131.2,
140.6 (d, JP–C=6.8 Hz), 140.8 (d, JP–C=5.7 Hz), 148.7;
phosphate buffer (pH 7.0), flow rate 0.9 mL min−1
]
showed two enantiomers with retention times of 45.6 and
51.8 min.