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Letters Cyclodextrin-Mediated Hydrolyses of Novel Phosphotriesters† Robert A. Moss* and Paul K. Gong Department of Chemistry, Rutgers, The State University of New Jersey, New Brunswick, New Jersey 08903 Received February 14, 2000. In Final Form: May 29, 2000 The basic cleavages (pH 10) of p-nitrophenyl diphenyl phosphate (1, PNPDPP), p-nitrophenyl 1,8naphthyl phosphate (2, PNPNP), and p-nitrophenyl biphenyl phosphate (3, PNPBPP) were mediated by R-, β-, and γ-cyclodextrins. Derived kinetic parameters revealed substantial selectivity for the β-CD/ PNPNP system, with efficient cyclodextrin-catalyzed cleavage characterized by a high value of kcat/Kdiss.
Introduction The specter of chemical weapons includes the horrific Monday morning rush hour sarin attack in Tokyo (20 March, 1995) in which 12 people died and more than 4700 others required hospital treatment.1 The need for rapid, efficient chemical agent detoxification and decontamination is urgent and accentuated by the Chemical Weapons Convention, which requires the destruction of stockpiled chemical weapons. The high toxicity of the phosphorus nerve agents (sarin, soman, and VX) requires that most laboratory decontamination research employs model compounds or simulants in place of actual agents. Since its introduction in 1969, p-nitrophenyl diphenyl phosphate (1, PNPDPP) has been the most widely used simulant for the fluorophosphate toxins, sarin and soman; PNPDPP has been the substrate in dozens of hydrolytic and phosphorolytic reactions.2-5
Recently, we described p-nitrophenyl 1,8-naphthyl phosphate (2, PNPNP), an analogue of PNPDPP, in which † Part of the Special Issue “Colloid Science Matured, Four Colloid Scientists Turn 60 at the Millennium”.
(1) Kristof, N. D. The New York Times 1995, 144 (Tuesday, March 21), 1. (2) Bunton, C. A.; Robinson, L. J. Org. Chem. 1969, 34, 773.
“fusion” of the phenyl residues of 1 into the naphthyl moiety of 2 led to substantial reactivity enhancements with many of the nucleophiles deployed in phosphorolytic reactions.6 PNPNP is thus a very sensitive nerve agent simulant. Similarly, we have prepared p-nitrophenyl biphenyl phosphate (3, PNPBPP) by the reaction of 2,2′biphenol with p-nitrophenyl phosphorodichloridate in the presence of triethylamine. Substrate 3 is more reactive than PNPDPP toward various nucleophiles, but about 10 times less reactive than PNPNP.7 Cyclodextrins occupy a dominant position among the many reagents employed to model the enzymatic cleavage of various substrates. Their ability to recognize, bind, and catalyze the scission of numerous size-complementary substrates has been described in many reviews and reports.8 However, there are few examples of the cyclodextrin-mediated lysis of phosphorus esters. Both R- and β-cyclodextrins (R-CD, β-CD) accelerate the cleavage of p-nitrophenyl dimethyl phosphate in 0.0094 M aqueous NaOH, but the kinetic advantage (kcat/kun) is only a factor of ∼2.9 The β-CD-mediated cleavage of PNPDPP, however, is more dramatic; a 50-fold acceleration is induced by excess β-CD at pH ) 11.2.10 Bender demonstrated that CDs cleaved (e.g.) diphenyl methylphosphonate by a two-step mechanism in which substrate binding was followed by (3) Moss, R. A.; Kotchevar, A. T.; Park, B. D.; Scrimin, P. Langmuir 1996, 12, 2201. (4) Scrimin, P.; Ghirlanda, G.; Tecilla, P.; Moss, R. A. Langmuir 1996, 12, 6235. (5) See (a) Fendler, J. H.; Fendler, E. J. Catalysis in Micellar and Macromolecular Systems. New York, Academic Press: 1975; pp. 150161. (b) Moss, R. A.; Ihara, Y. J. Org. Chem. 1983, 48, 588. (6) Moss, R. A.; Bose, S.; Ragunathan, K. G.; Jayasuriya, N.; Emge, T. J. Tetrahedron Lett. 1998, 39, 347. (7) Bose, S. Ph.D. Dissertation, Rutgers University, New Brunswick, NJ, 1998. PNPBPP was fully characterized, including the determination of an X-ray crystal structure. (8) (a) Breslow, R.; Dong, D. Chem. Rev. 1998, 98, 1997. (b) D’Souza, V. T.; Lipkowitz, K. B.; Ed. Chem. Rev. 1998, 98, 8, Number 5, “Cyclodextrins.” (c) Kirby, A. J. Angew. Chem., Int. Ed. Engl. 1996, 35, 707 (esp. pp 714-716). (d) Tee, O. S. Adv. Phys. Org. Chem. 1994, 29, 1. (e) Breslow, R. Acc. Chem. Res. 1991, 24, 317. (f) Zejtli, J.; Osa, T.; Ed. Comprehensive Supramolecular Chemistry, Vol. 3, Cyclodextrins, Oxford, Pergamon: Elsevier: 1996. (g) Bender, M. L.; Komiyama, M. Cyclodextrin Chemistry, New York, Springer, 1978. (h) Rekharsky, M. V.; Inoue, Y. Chem. Rev. 1998, 98, 1875. (9) Mochida, K.; Matsui, Y.; Ota, Y.; Arakawa, K.; Date, Y. Bull. Chem. Soc. Jpn. 1976, 49, 3119.
10.1021/la000200o CCC: $19.00 © 2000 American Chemical Society Published on Web 07/13/2000
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Table 1. Rate Constants for Cyclodextrin-Mediated Cleavagesa conditions and rate constants substrate PNPDPP, 1 PNPNP, 2 PNPBPP, 3
kunb
(s-1) 10-5
(4.5 ( 0.1) × (6.0 ( 0.7) × 10-3 4.0 × 10-4 e
R-CD, k2 (M-1 s-1) c
0.13 ( 0.02 0.78 ( 0.05 0.21 ( 0.01
β-CD, kcatd (s-1) (4.1 ( 0.4) × 0.31 ( 0.06 2.2 ( 0.2f
10-3
γ-CD, kcatd (s-1) (1.5 ( 0.2) × 10-3 0.19 ( 0.02 0.0103 ( 0.0004
a At pH 10.05, 25 °C. For other conditions and concentrations, see text. b Pseudo-first-order rate constant at pH 10 in the absence of catalyst. c Second-order rate constant with [R-CD] varied. d Catalytic rate constant from Michaelis-Menten analysis of kobs vs [CD]. e Single determination. f Second-order rate constant from linear dependence of kobs vs [β-CD].
nucleophilic phosphorolytic attack of a secondary CD hydroxyl group (in its anionic form) on the bound phosphonate.11 More reactive phosphonates [bis(p-nitrophenyl) methylphosphonate] exhibited CD-catalyzed general base hydrolysis of the substrate in competition with the twostep mechanism.11 With phosphodiester substrates, the two-step mechanism was again dominant, albeit that the cleavages of these refractory substrates were very slow.12 Most appositely, an o-iodosobenzoate-β-CD covalent conjugate is able to bind the nerve agent soman (4) in the CD cavity and then efficiently cleave the bound fluorophosphonate at its P-F bond; turnover and catalysis were observed.13,14 R-CD is also known to stereoselectively cleave the enantiomers of sarin (isopropyl methylfluorophosphonate).15 In the present report, we combine the dual themes of the cleavage of nerve agent simulants and the CD mediation of these cleavages. On the basis of the reported binding of pyrene to β-CD,16 as well as many naphthalene derivatives8h and modeling studies,17 we imagined that PNPNP (2) would bind well to β-CD and that this phosphotriester simulant would be readily cleaved by the cycloheptaamylose. Here we describe comparative kinetic studies of the cleavages of substrates 1-3 by R-, β-, and γ-cyclodextrins. The β-CD/PNPNP system is found to display significant selectivity. Results and Discussion The cleavage kinetics of 2.5 × 10-5 M substrates 1-3 were determined in aqueous solutions of 0-20 mM R-, β-, or γ-cyclodextrins in 10 mM CHES buffer, 10 mM KCl. The reaction medium, which contained 5 vol % of acetonitrile (to ensure homogeneity), was maintained at pH 10.05 ( 0.02 and 25.0 ( 0.5 °C. Kinetics were followed spectrophotometrically, monitoring the release of pnitrophenylate ion at 405 nm. Reactions were followed for at least 7 half-lives, with stopped-flow methodology employed for faster reactions. The observed and extracted rate constants appear in Table 1, where we also indicate the reproducibilities of multiple experiments. (10) Kausar, A. R.; Younas, M.; Sadiq, M. Z., Wahid, T. Indian J. Chem. 1984, 23B, 476. (11) Brass, H. J.; Bender, M. L. J. Am. Chem. Soc. 1973, 95, 5391. (12) Hengge, A. C.; Cleland, W. W. J. Org. Chem. 1991, 56, 1972. (13) Seltzman, H. H.; Szulc, Z. M. Proc. 1996 Medical Defense Biosci. Rev. Army Medical Research Institute of Chemical Defense, Vol. 1, 16 May 1996; pp. 339-346. Private communication from Dr. John E. Walker, Natick RDEC, 19 December, 1997. See also: Hoskin, F. C. G.; Steeves, D. M.; Walker J. E. Biol. Bull. 1999, 197, 284. (14) For leading references to the iodosobenzoate-catalyzed hydrolysis of phosphotriesters and nerve agents, see ref 3. (15) van Hooidonk, C.; Breebart-Hansen, J. C. A. E. Rec. Trav. Chim. 1970, 89, 289. van Hooidonk, C.; Groos, C. C. Rec. Trav. Chim. 1970, 89, 845. (16) Udachin, K. A.; Ripmeester, J. A. J. Am. Chem. Soc. 1998, 120, 1080. (17) With Macromodel, version 5.0. For example, computer docking simulations demonstrate a good fit of substrate 2 (with COOH replacing NO2) into the β-CD cavity. The naphthalene ring tilts in such a way that the substrate’s PdO group is in close proximity and appropriate orientation to a sec-OH group on the more open face of the CD. We thank Dr. Z. Yang and Professor R. Breslow for assistance with the modeling.
The data reflect both the intrinsic activities of substrates 1-3 and an impressive sensitivity to substrate/CD pairing in these CD-mediated cleavages. In the absence of CD (kun, Table 1), the substrate reactivity order (at pH 10) is clearly PNPNP > PNPBPP > PNPDPP, with ∼1 order of magnitude separating each substrate. We have noted that PNPNP is ideally constructed for nucleophilic displacement: The 1,8-naphthalenedioxy unit is “tied back,” so that nucleophile access to PNPNP at P is less hindered than for PNPDPP with its 2 “floppy” phenoxy groups.6 Additionally, there will be no phenyl/phenyl nonbonded interactions in the trigonal bipyramidal transition state (or short-lived intermediate)18 derived from OH- and PNPNP, in contrast to PNPDPP, where such interactions will be present and energetically costly.19 The composite result is a reactivity advantage of ∼130 for PNPNP over PNPDPP. The related PNPBPP substrate (3) is of intermediate reactivity. Its X-ray crystal structure7 reveals an internal O-P-O bond angle of 105.4°, which is unstrained. However, the additional flexibility of the seven-membered P-containing ring provides a less accessible trajectory of attack for an incoming nucleophile, and probably leads to deleterious nonbonded interactions at the TBP stage of the cleavage. (In the crystal structure, there is a dihedral angle of 40° between the phenyl groups of PNPBPP.) Substrate 3 is therefore ∼15 times less reactive than PNPNP, but still ∼9 times more reactive than the acyclic PNPDPP. The addition of R-CD accelerates the cleavages of all three substrates, but binding-saturation kinetics are not observed: the rate constants for substrate cleavage (kobs) are linear in [R-CD] over CD concentration ranges of ∼220 mM; cf. Figures 1-3. From the correlations of kobs with [R-CD], second-order rate constants (k2) were derived for the R-CD cleavage of each substrate; these appear in Table 1. The 2 > 3 > 1 substrate reactivity ordering is again observed with R-CD, although it is much compressed in comparison to the unmediated pH 10 hydrolyses. To estimate the R-CD acceleration, we can compare the pseudo-first-order rate constant, kψ, for substrate cleavage in the presence of 10 mM R-CD (R-CD/substrate ) 400) with kun for the unassisted hydrolysis, with all reactions at pH 10. The kψ values are 0.155 s-1 (PNPNP), 0.0297 s-1 (PNPBPP), and 0.0021 s-1 (PNPDPP).20 Note that the original substrate reactivity order is reasserted; compared to the kun values in Table 1, the large excess of R-CD (18) For discussions of TBP intermediates vs TBP SN2(P) transition states in phosphotriester cleavage, see (a) Yang, Y.-C.; Berg, F. J.; Szafraniec, L. L.; Beaudry, W. T.; Bunton, C. A.; Kumar, A. J. Chem. Soc., Perkins Trans. 2 1997, 607. (b) Thatcher, G. R. J.; Kluger, R. Adv. Phys. Org. Chem. 1989, 25, 99. (c) Williams, A. Adv. Phys. Org. Chem. 1992, 27, 1. (d) Gorenstein, D. G.; Chang, A.; Yang, J.-C. Tetrahedron 1987, 43, 469. (19) The internal O-P-O bond angle of PNPNP is unstrained at 105.8°, and some strain will develop as this angle expands toward 120° during nucleophilic attack. The structural/architectural features described in the text provide compensation. (20) The PNPDPP experiment employed 11 mM R-CD.
Letters
Figure 1. Observed rate constants for the cleavage of PNPDPP (1) in the presence of R-CD (b), β-CD (0), and γ-CD (]) as a function of [CD]. See text for conditions and concentrations.
Figure 2. Observed rate constants for the cleavage of PNPNP (2) in the presence of R-CD, β-CD, and γ-CD; symbols as in Figure 1. See text for conditions and concentrations.
Figure 3. Observed rate constants for the cleavage of PNPBPP (3) in the presence of R-CD, β-CD, and γ-CD; symbols as in Figure 1. See text for conditions and concentrations.
produces rate accelerations in substrate cleavages of 26, 74, and 46, respectively. The absence of saturation behavior with increasing [R-CD] suggests that inclusive substrate binding, followed
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by CD-OH nucleophilic attack at P,11 does not operate here; the R-CD cavity is too small to productively bind PNPNP or PNPBPP.21 Rather, R-CD catalysis of these hydrolyses probably occurs via the cyclodextrin hydroxyls acting as general bases to activate water for attack at the substrate P atom.11,12,22 In keeping with this suggestion, acyclic polyhydroxy compounds also elicit rate increases in the hydrolysis of (e.g.) PNPNP. Mannitol and R-Dglucose afford k2 ) 0.10 and 0.17 M-1 s-1, respectively, for PNPNP cleavage under the conditions of Table 1. These accelerations, however, are inferior to that produced by R-CD (k2 ) 0.78 M-1 s-1). With added β-CD, the correlations of kobs vs [CD] for PNPDPP and PNPNP each exhibited saturation due to substrate binding (Figures 1 and 2), but the correlation for PNPBPP remained linear, affording k2 (Table 1). In contrast, added γ-CD provided saturation curves of kobs vs [γ-CD] for all three substrates (Figures 1-3). MichaelisMenten analyses, using Eadie plots of (kobs - kun) vs (kobs - kun)/[CD], gave values of kcat, the rate constant for cleavage of the complexed substrate, and Kdiss, the dissociation constant of the substrate-CD complex.23 Values of kcat, Kdiss, kcat/Kdiss, and kcat/kun for β- and γ-CD appear in Tables 1 and 2. β-CD clearly exhibits selectivity toward substrate PNPNP: kcat is ∼76 times greater for PNPNP than PNPDPP, and kcat/Kdiss (which relates to the specificity of the CD) is ∼84 times greater for PNPNP. Curiously, Kdiss is the same for both PNPNP and PNPDPP, but the tighter, more restrictive fit into the β-CD cavity of the PNPNP naphthyl moiety, relative to the phenyl unit of PNPDPP, apparently leads to a more efficient ensuing reaction of the PNPNP with a β-CD secondary hydroxyl (anionic form).24 That kcat/kun is actually 1.7 times larger for PNPDPP vs PNPNP, reflects the very high value of kun for the latter (133 times greater than that of PNPDPP, Table 1). PNPBPP is too large25 to be strongly bound by β-CD. Its cleavage modality probably involves the hydroxyl general base mechanism displayed by R-CD; a linear kobs vs [β-CD] correlation is obtained, with k2 ) 2.2 M-1 s-1.26 For comparison, at 10 mM β-CD, the cleavage of PNPNP is ∼6.4 times faster than that of PNPBPP. Overall, the kinetic data reveal a clear selectivity of β-CD for PNPNP. With γ-CD, all three substrates display saturation and binding behavior (Figures 1-3). The various kinetic parameters can be readily extracted and are collected in Tables 1 and 2. PNPNP is again the most reactive substrate; in terms of kcat, it is ∼19 times more reactive than PNPBPP and 127 times more reactive than PNPDPP. A similar sequence prevails for kcat/Kdiss, where PNPNP: (21) For the CD’s, top and bottom cavity diameters (in Å) are given as R-CD, 4.7 and 5.3; β-CD, 6.0 and 6.5; and γ-CD, 7.5 and 8.3.8h The “widths” of PNPNP and PNPBPP are discussed below; cf., refs 24 and 25. (22) Komiyama, M. J. Am. Chem. Soc. 1989, 111, 3046. (23) VanEtten, R. L.; Sebastian, J. F.; Clowes, G. A.; Bender, M. L. J. Am. Chem. Soc. 1967, 89, 3242. (24) Although the width of PNPNP (from H2 to H8 or H3 to H7 is ∼ 6.7 Å, whereas the wider diameter of β-CD is given as 6.5 Å,8h, 21 modeling studies17 reveal an inclusive fit of PNPNP into β-CD. Additionally, numerous naphthyl derivatives are bound by β-CD.8h A referee has suggested that the phosphate group of PNPNP may reside closer to the β-CD hydroxyls than the phosphate of PNPDPP in their respective bound complexes. (25) The p-p H width across the biphenyl system is 9.2 Å; the shorter H-H distance across the bottom of the substrate is 6.8 Å.7 (26) However, k2 for PNPBPP and β-CD is ∼10 times greater than the analogous k2 with R-CD, suggesting that some kind of weak but productive binding (perhaps “face-to-face”)22 does occur between PNPBPP and β-CD. This conclusion is supported by the inhibition experiments described below.
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Table 2. Extracted Kinetic Parameters for Cyclodextrin-Mediated Cleavagesa β-CD substrate
Kdiss (M)
PNPDPP, 1 PNPNP, 2 PNPBPP, 3
0.011 ( 0.003 0.010 ( 0.003 c
γ-CD b
kcat/Kdiss (M-1 s-1)
kcat/kun
0.37 31
89 52
k2 (M-1 s-1)
Kdiss (M)
kcat/Kdiss (M-1 s-1)
kcat/kun
k2b (M-1 s-1)
0.10 15 2.2d
0.011 ( 0.003 0.006 ( 0.001 0.0022 ( 0.0003
0.14 32 4.7
33 31 2.5
0.04 11 0.6
a Kinetic parameters were extracted by analysis of k obs vs [CD] correlations; see Figures 1-3 and text. See Table 1 and text for reaction conditions. b Approximate second-order rate constant based on initial portions of kobs vs [CD] correlations. c A linear dependence of kobs on [β-CD] was observed; saturation behavior was absent. d Second-order rate constant from linear dependence of kobs vs [β-CD].
Figure 4. Inhibition of PNPBPP cleavage in β-CD by added naphthalene 2-carboxylate; kobs (s-1) vs [5] (M). See text for conditions and concentrations.
PNPBPP:PNPDPP is 228:34:1. Interestingly, however, PNPBPP is now the most tightly bound substrate, with the Kdiss ordering PNPDPP > PNPNP > PNPBPP. Values of kcat/kun (∼30) are similar for PNPNP and PNPDPP, again due to the high kun of PNPNP, and both are ∼12 times greater than kcat/kun for PNPBPP. A binding/hydroxyl cleavage mechanism for β-CD with these phosphotriesters is also supported by competitive inhibition studies in which addition of naphthalene 2-carboxylate (5) retarded the cleavages.27 With [β-CD] fixed at either 1 or 3 mM, under the previously cited reaction conditions, additions of 1-18 mM 5 led to decreasing kobs with increasing [5] for all three substrates. A representative example of this behavior (for PNPBPP, β-CD, and 5) is shown in Figure 4. Plots of (kcat - kobs)/(kobs - kun) vs [5] were linear for PNPDPP and PNPNP (see Figure 5) affording23 Ki and Kdiss:28 for PNPNP, Ki ) 8 mM and Kdiss ) 7 mM; for PNPDPP, Ki ) 12 mM and Kdiss ) 8 mM. The Ki values are larger than the inverse of Kbind calorimetrically determined for 5;27 however, the Kdiss values agree well with those determined from the direct cleavage studies (Table 2).
It should be noted that the CDs provide rate enhancements for the initial cleavage of the activated phosphotriester substrates. However, the phosphorylated CDs formed in this step do not turn over rapidly.8a,11,12 The CDs therefore are not true catalysts. Conclusions R-, β-, and γ-cyclodextrins mediate the basic cleavages of PNPDPP, PNPNP, and PNPBPP at pH 10. Binding
Figure 5. Concentration of inhibitor 5 vs (kcat - kobs)/ (kobs kun) for the cleavage of PNPNP by β-CD (conditions as above). The slope (Ki[CD]/Kdiss) and intercept (-Ki) afford Ki ) 8 mM and Kdiss ) 7 mM.
followed by cleavage (saturation kinetics) is exhibited by β-CD with PNPDPP and PNPNP and by γ-CD with all three substrates. Cleavages by R-CD do not show saturation kinetics; substrates are not strongly bound, and their cleavages are probably mediated by hydroxyl group general base catalysis. The most efficient kinetic system is β-CD/PNPNP, where good complementarity between host and substrate, as well as intrinsic substrate reactivity, leads to high values of kcat, kcat/Kdiss, and kcat/kun.29 Acknowledgment. P.K.G. acknowledges support from a NIH Biotechnology Training Grant (GM08339). We thank Dr. Tom Emge for the X-ray structure of PNPBPP. We are grateful to Professor Ronald Breslow for a helpful discussion, and to both the U.S. Army Edgewood Research, Development, and Engineering Center, and the U.S. Army Research Office for financial support. LA000200O (27) Naphthalene 2-carboxylate has Kbind ) 320 ( 20 for β-CD by flow calorimetry in pH 8.6 aqueous 0.1M NaCl at 298 K: Godinez, L. A.; Schwartz, L.; Criss, C. M.; Kaifer, A. E. J. Phys. Chem. B 1997, 101, 3376. (28) This analysis could not be applied to PNPBPP because kcat is unknown for β-CD.; see above. However, the observation of competitive inhibition with 5/PNPBPP/β-CD indicates that there is some kind of productive binding between PNPBPP and β-CD. (29) A referee has commented on the low kcat values of γ-CD, particularly with PNPBPP, pointing out that all 3 substrates have at least 2 complexation sites, and that, in aqueous solutions, loose fitting is usually preferred.30 Therefore, substrates carrying both phenyl and naphthyl moieties may preferentially bind the former group within the cavity, leading to nonproductive complex geometries. We thank the referee for these perceptive comments. (30) Schneider, H.-J.; Blatter, T.; Simova, S.J. Am. Chem. Soc. 1991, 113, 1996. Garel, L.; Lozach, B.; Dutasta, J.-P.; Collet, A. J. Am. Chem. Soc. 1993, 115, 11652.
