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Phosphorolytic Cleavage of Phosphate and Phosphonoformate Diesters by Cetyltrimethylammonium Iodosobenzoate Robert A. Moss,* Saketh Vijayaraghavan, and Suseela Kanamathareddy Department of Chemistry and Chemical Biology, Rutgers, The State University of New Jersey, New Brunswick, New Jersey 08903 Received November 5, 2001. In Final Form: January 10, 2002 Micellar cetyltrimethylammonium iodosobenzoate (4) cleaves P-O ester linkages of bis(p-nitrophenyl) phosphate (3), methoxycarbonyl phenyl phosphonoformate (6), and hexyloxycarbonyl phenyl phosphonoformate (7). Kinetic advantages of several orders of magnitude are obtained relative to the unassisted hydrolyses.
o-Iodosobenzoate (1, IBA) is a potent catalyst for the cleavage of neutral P-O substrates,1,2 including phosphotriesters,3 thiophosphates and phosphonothioates,4 phosphonofluoridate nerve agents,5 and phosphate or thiophosphate pesticides, such as paraoxon or parathion.6 For these applications, IBA is often deployed in cetyltrimethylammonium (CTA) micellar solutions, where the cationic micelles solubilize both the anionic IBA and the neutral substrate, concentrating the reactants in the micellar phase and providing a significant kinetic advantage.3,7
Although IBA in micellar (CTA)Cl is an exceptional facilitator of the hydrolysis of neutral, activated phosphotriesters such as bis(p-nitrophenyl) methyl phosphate (2), it is much less reactive toward anionic phosphodiester substrates: even the “activated” bis(p-nitrophenyl) phosphate 3 (BNPP) is not readily cleaved.3 Thus, 1 × 10-4 M IBA in 2.5 mM aqueous (CTA)Cl at pH 8 cleaved 1 × 10-5 M phosphotriester 2 with k ) 0.074 s-1, whereas phosphodiester 3 initially appeared not to cleave with IBA at various (CTA)Cl concentrations under similar conditions.3,8 Presumably, attack of the anionic IBA is inhibited at the phosphorus center of the anionic O-P-O triad of BNPP. (1) (a) Moss, R. A.; Alwis, K. W.; Bizzigotti, G. O. J. Am. Chem. Soc. 1983, 105, 681. (b) Moss, R. A.; Awis, K. W.; Shin, J.-S. J. Am. Chem. Soc. 1984, 106, 2651. (2) On the 1-oxido-1,2-benziodoxol-3(1H)-one structure of IBA, see: Moss, R. A.; Vijayaraghavan, S.; Emge, T. J. Chem. Commun. 1998, 1559 and references therein. (3) Moss, R. A.; Kotchever, A. T.; Park, B. D.; Serimin, P. Langmuir 1996, 12, 2200. (4) (a) Moss, R. A.; Morales-Rojas, H.; Zhang, H.; Park, B.-D. Langmuir 1999, 15, 2738. (b) Moss, R. A.; Morales-Rojas, H. Langmuir 2000, 16, 6485. (5) Hammond, P. S.; Forster, J. S.; Lieske, C. N.; Durst, H. D. J. Am. Chem. Soc. 1989, 111, 7860. (6) Moss, R. A.; Kanamathareddy, S.; Vijayaraghavan, S. Langmuir 2001, 17, 6108. (7) Bunton, C. A.; Nome, F.; Quina, F. H.; Romsted, L. R. Acc. Chem. Res. 1991, 24, 357. (8) Our reinvestigation (see below) shows that IBA/(CTA)Cl does cleave BNPP, albeit slowly.
Recently we reported that cetyltrimethylammonium iodosobenzoate9 (4, (CTA)IBA) is 20-30% more reactive than a 1:1 (CTA)Cl-IBA blend in the micellar hydrolyses of paraoxon and parathion.6 In holomicellar (CTA)IBA, where there are no chloride ions to compete for the cationic CTA binding sites, the reactive IBA anions may bind more strongly at the CTA cationic sites, affording more efficient reaction with the hydrophobically bound (neutral) substrate.
These results led us to restudy the reaction between IBA and BNPP. We imagined that in holomicellar (CTA)IBA the CTA cations might support stronger electrostatic interactions with the (O-P-O)- units of nBNPP, mitigating the negative charge and potentiating the attack of IBA on what would effectively be CTA-BNPP ion pairs. We now find that (CTA)IBA, and (CTA)Cl/IBA, lyse the P-O bond of BNPP. This is the initial report of phosphodiester hydrolysis mediated by IBA. Additionally, we describe the cleavage of several phosphonoformate diester substrates by (CTA)IBA, noting two further instances of P-O scission. Results and Discussion Substrates. The substrates we employed included BNPP (3), dimethyl phosphonoformate (5, DMPF), methoxycarbonyl phenyl phosphonoformate (6), hexyloxycarbonyl phenyl phosphonoformate (7), phenoxycarbonyl methyl phosphonoformate (8), and diphenyl phosphonoformate (9, DPPF). Substates 3 and 5-9 were used as sodium salts. BNPP was commercially available from Sigma Chemical Co. DMPF (5) was prepared by the NaI demethylation of commercial trimethyl phosphonoformate.10 Methoxycarbonyl phenyl phosphonoformate (6) was obtained from trimethyl phosphonoformate by conversion (9) Bunton, C. A.; Mhala, M. M.; Moffatt, J. R. J. Phys. Chem. 1989, 93, 854. (10) Moss, R. A.; Morales-Rojas, H. J. Am. Chem. Soc. 2001, 123, 7457.
10.1021/la0116488 CCC: $22.00 © 2002 American Chemical Society Published on Web 02/21/2002
Letters
first to methoxycarbonyl phosphonodichloridate (Me3SiBr, then PCl5), then to methoxycarbonyl diphenyl phosphonoformate (phenol, DBU), and finally to 6 by hydrolysis (aqueous NaHCO3). For the preparation of hexyloxycarbonyl phenyl phosphonoformate (7), the P-anion derived from diphenyl phosphite (NaH/THF) was reacted with hexyl chloroformate, yielding hexyloxycarbonyl diphenyl phosphonoformate. Hydrolysis of the latter with NaHCO3 in aqueous MeCN gave 7. Phenoxycarbonyl methyl phosphonoformate (8) was obtained from trimethyl phosphite by initial conversion to phenoxycarbonyl dimethyl phosphonoformate (with phenyl chloroformate), followed by NaI demethylation in refluxing acetone. Diphenyl phosphonoformate (9) was prepared by the NaHCO3-mediated hydrolysis of triphenyl phosphonoformate in aqueous MeCN. The triphenyl ester was itself obtained by the reaction of the P-anion derived from diphenyl phosphite with phenyl chloroformate. All phosphonoformate substrates were characterized by 1H and 31P NMR spectroscopy and by acceptable elemental analyses. Kinetics and Products. The critical micelle concentration of (CTA)IBA is ∼8 × 10-4 M, as determined in 0.01 M pH 9 Bis-Tris [bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane] buffer (in the presence of 1 × 10-5 M paraoxon).6 We can therefore be confident that (CTA)IBA micelles will be present under the kinetics conditions described below. Reaction of 5 mM BNPP (3) with 33 mM (CTA)IBA (4) in 0.1 M Bis-Tris buffer at pH 9.0 (25 °C) led to the loss of substrate (31P NMR at δ -11.95)11 and the appearance of product p-nitrophenyl phosphate (δ 0.15), confirmed by spiking with authentic material. The pseudo-first-order rate constant for BNPP cleavage, followed by 31P NMR was 2.30 (( 0.05) × 10-6 s-1 (t1/2 ) 83.7 h) from two separate experiments. Taking k ∼ 1.1 × 10-11 s-1 for the unassisted hydrolysis of BNPP at pH 7 and 25 °C,12 micellar (CTA)IBA at pH 9 brings about an acceleration of ∼2.1 × 105. (At pH 9 in 0.1 M aqueous Bis-Tris and 33 mM NaCl, BNPP did not hydrolyze over a period of 10 days.) In the presence of micellar (CTA)Cl, IBA also cleaves BNPP. Three conditions were examined: (a) 5 mM BNPP was reacted with 33 mM added iodosobenzoic acid in 33 mM aqueous (CTA)Cl with 0.1 M Bis-Tris buffer at pH 9.0 and 25 °C; (b) 5 mM BNPP was reacted with 33 mM added sodium iodosobenzoate in 33 mM aqueous (CTA)Cl under similar conditions; and (c) 5 mM BNPP was reacted with 33 mM (CTA)IBA to which had been added 33 mM NaCl (again, under similar buffer conditions). Reactions a-c were followed for 112-141 h, monitoring the disappearance of substrate by 31P NMR; p-nitrophenyl phosphate was the only product observed. BNPP cleavage rate constants were similar in each case, and slightly smaller than 2.30 × 10-6 s-1 observed with micellar (CTA)IBA. The rate constants were (a) 1.94 × 10-6 s-1, (b) 1.68 × 10-6 s-1, and (c) 1.85 × 10-6 s-1. Clearly, IBA supplied as (CTA)IBA, or with (CTA)Cl under conditions (a)-(c), cleaved BNPP with a very significant phosphorolytic rate advantage (∼105). The presence of Cl- counterions resulted in a 16-27% rate depression relative to holomicellar (CTA)IBA, similar to our findings in the (CTA)IBA cleavage of paraoxon or parathion.6 (11) All 31P NMR chemical shifts are relative to external 85% H3PO4. Methyl methylphosphonate (δ 28.78) was added to samples as an integration standard. (12) Takasaki, B. K.; Chin, J. J. Am. Chem. Soc. 1995, 117, 8582. The rate constant at pH 7 was extrapolated from the rate measured at 0.1 M NaOH.
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Given the phosphorolytic reactivity of IBA toward BNPP, we turned our attention to phosphonoformate substrates 5-9. Monoanionic phosphonoformate diesters are more labile than activated phophodiesters such as BNPP. For example, diethyl phosphonoformate (5, Et in place of Me) cleaves with k ) 5 × 10-8 s-1 in Tris buffer at pH 7,13 >103 times faster than BNPP.12 However, the hydrolysis of diethyl phosphonoformate occurs at the carboxy ester, not the phosphonate ester.13 The reaction of DMPF (5) with aqueous micellar (CTA)IBA at pH 8.414 proceeded very slowly with k ) 7.64 × 10-7 s-1, and solely with C-O cleavage as manifested by the 31P NMR signal11 of the P-OMe phosphonoformate monoester product at δ 3.11. To “load the dice” in favor of P-O scission, we next prepared unsymmetrical substrate 6, in which the considerably more reactive phenoxy leaving group constituted the phosphonoformate’s P-ester, whereas a methoxy group was retained as the C-ester. Cleavage of 6 with (CTA)IBA at pH 8.4,14 monitored by 31 P NMR, proceeded with ∼90% of P-O Ph scission; the substrate’s 31P signal at δ -8.76 decreased as that of product methoxycarbonyl phosphonoformate dianion (10) grew in at δ -3.09. A spiking experiment confirmed this product’s identity.10 Minor cleavage (∼10%) was also observed at the C-OMe site of substrate 6, leading to product 11 at δ -1.95. Time-dependent NMR monitoring of substrate decay afforded k ) 2.88 × 10-5 s-1 (t1/2 ) 6.7 h). Allowing for differences in leaving group and pH, the IBA mediated phosphorolytic cleavage of 6 is at least 10 times faster than that of BNPP, confirming the greater reactivity of phosphonoformate diesters vs phosphate diesters.
Three control experiments were performed to gauge the effectiveness of (CTA)IBA in the cleavage of 6: (a) 5 mM substrate was cleaved by 33 mM NaIBA (no micelles) in 0.1 M Bis-Tris buffer at pH 8.4. We observed k ) 6.09 × 10-6 s-1, so that the micellar advantage of (CTA)IBA vs IBA alone is about 4.7. (b) 10 mM 6 was hydrolyzed in 0.1 M (CTA)Cl and 0.1 M Bis-Tris at pH 8.4 (no IBA). Remarkably, C-OMe rather than P-OPh scission was observed: the 31P resonance of product 11 slowly grew in (k ) 2.34 × 10-7 s-1) at δ -1.87 as the substrate signal decreased at δ -8.76. (c) 10 mM 6 was hydrolyzed in 0.1 M Bis-Tris at pH 8.4 (no IBA, no (CTA)Cl). Again, exclusive C-O cleavage to 11 was observed, with k ) 3.0 × 10-7 s-1. Spiking experiments verified the identity of product 11, which was prepared by basic hydrolysis of substrate 6 with 1 equiv of aqueous NaOH. Controls b and c reveal the absence of (CTA)Cl micellar catalysis for the C-O cleavage of substrate 6,15 but the more striking observation is the regioselectivity of IBA. IBA and (CTA)IBA cleave 6 mainly at P-OPh, whereas OH- at pH 8.4 leads to slower scission at C-OMe. To learn if enhanced substrate hydrophobicity affected (CTA)IBA reactivity, we examined hexyloxycarbonyl (13) Ferguson, C. G.; Thatcher, G. R. J. Org. Lett. 1999, 1, 829. (14) Standard reaction conditions: 5 mM substrate, 33 mM (CTA)IBA, 0.1 M Bis-Tris buffer, 25 °C. (15) This result contrasts with the well-known acceleration of phosphate triester basic hydrolysis by cationic micelles: Fendler, J. H.; Fendler, E. J. Catalysis in Micellar and Macromolecular Systems; Academic Press: New York, 1975; pp 130f.
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phenyl phosphonoformate, 7. Under standard conditions14 at pH 8.4, (CTA)IBA cleaved 7 only by P-OPh scission with k ) 2.60 × 10-5 s-1 (t1/2 ) 7.4 h), comparable to the rate constant for the cleavage of 6. The additional hydrophobicity of 7 is therefore unimportant, suggesting that both 7 and 6 are “fully” bound by the (CTA)IBA micelles at 33 mM surfactant. Importantly, only 4% of P-OPh cleavage occurred after 12 days of reaction between substrate 7 and 33 mM (CTA)Cl at pH 8.4 under otherwise standard conditions.14 Obviously, IBA is essential to the phosphorolytic cleavage of phosphonoformate 7. As expected, the C-OPh isomer of 7, phenoxycarbonyl methyl phosphonoformate (8) underwent extremely rapid C-OPh cleavage with (CTA)IBA under standard conditions.14 The reaction was complete within the time required to obtain the 31P NMR spectrum (∼3 min), and the appearance of (only) C-OPh cleavage product 12 at δ 3.0211 was verified by spiking with authentic material.10 The ethyl analogue of 8 (Et in place of Me) is cleaved at C-OPh by R-aminocyclodextrins.13 The phosphonoformate cleavage experiments described thus far indicate that (CTA)IBA splits substrate 5 (very slowly) at C-OMe, 6 at P-OPh, and 7 at P-OPh. In these examples, the leaving group superiority of phenoxide over methoxide determines the regiochemistry and dominates the innate higher reactivity of the C-ester relative to the P-ester site of the phosphonoformate diesters. As a further test of this conclusion, we studied diphenyl phosphonoformate (9). The basic hydrolysis of 5 mM 9 was examined with 5-50 mM aqueous NaOH (pH 11.7-13.4), monitored by 31P NMR. At 5 mM NaOH, no reaction occurred over 1 week, but, with 10 mM NaOH, slow cleavage (k ) 1.52 × 10-6 s-1) to C-OPh scission product 11 was observed at δ -1.37.16 Between 10 and 14 mM NaOH, the C-OPh cleavage of 9 was first order in both substrate and OH-, with k2 ) 8.8 × 10-2 M-1 s-1. At 15 mM NaOH, the scission of 9 was complete within 5 min. At 50 mM NaOH, further cleavage of 11 to the phosphonoformate trianion was apparent after 7 h. These experiments demonstrate intrinsically greater reactivity of 9 at its carbonyl-OPh site, relative to its phosphonate-OPh center. With 5 mM 9 and equimolar (CTA)IBA in 0.1 M Bis-Tris buffer at pH 8.4, only C-OPh cleavage is observed, complete within 3 min. Obviously, (CTA)IBA strongly potentiates the C-OPh scission: 5 mM NaOH brings about no reaction over 7 days, but 5 mM (CTA)IBA affords complete cleavage in 3 min! More precise measurement of the rate of (CTA)IBA cleavage of 9 is possible using an insufficiency of the reagent. With 5 mM 9 and 1 mM (CTA)IBA in 0.1 M BisTris at pH 8.4, C-OPh scission proceeds with k ) 1.32 × (16) Product 11 was confirmed by a spiking experiment. The 31P NMR chemical shift of 11 varies between (δ) -1.37 and -1.95 depending on the concentrations of hydroxide and (CTA)X.
Letters Table 1. Kinetics of Cleavages Mediated by (CTA)IBAa substrate
cleavage site
BNPP (3) DMPF (5) 6 7 8 9
P-O C-O P-O P-O C-O C-O
k (s-1) 2.30 × 10-6 b 7.64 × 10-7 2.88 × 10-5 2.60 × 10-5 v. fastc 4.7 × 10-3 d
a All reactions on 5 mM substrate with 33 mM (CTA)IBA in pH 8.4 0.1 M. Bis-Tris buffer (25 °C), unless otherwise noted. b At pH 9. c See text. d 2 mM (CTA)IBA.
10-3 s-1 (t1/2 ∼ 9 min). Most importantly, the (CTA)IBA “turns over” in this reaction; it is a true catalyst for the C-OPh hydrolysis of 9. At 2 mM (CTA)IBA, k increases to 4.76 × 10-3 s-1 (t1/2 ∼ 2.4 min).17 Clearly, (CTA)IBA very strongly potentiates the C-OPh hydrolysis of both 8 and 9. For calibration, 5 mM 9 in 20 mM (CTA)Cl (other conditions as above) gives largely C-OPh cleavage18 with k ) 1.57 × 10-5 s-1. The kinetic advantage provided by (CTA)IBA at pH 8.4 is therefore ∼300-3800, depending on whether an insufficiency or excess17 of (CTA)IBA is used. A concise summary of the kinetics of the principal (CTA)IBA-mediated esterolysis reactions reported in this work appears in Table 1. (CTA)IBA is there seen to cleave aryl phosphate and phosphonate esters 3, 6, and 7 at moderate rates. Although these IBA-mediated esterolyses are several orders of magnitude slower than analogous reactions of phosphotriester substrates, they are also several orders of magnitude faster than the unassisted hydrolyses of 3, 6, and 7. The catalytic potential of IBA toward anionic phosphate and phosphonoformate diesters is thus apparent. Conclusions IBA, most efficiently in the form of aqueous micellar (CTA)IBA, mediates the phosphorolytic cleavages of bis(p-nitrophenyl) phosphate (3), methoxycarbonyl phenyl phosphonoformate (6), and hexyloxycarbonyl phenyl phosphonoformate (7) with kinetic advantages of several orders of magnitude over unassisted hydrolysis. The C-ester centers of the phosphonoformate substrates are intrinsically more reactive than the P-ester sites, but reaction at the latter can be directed by the employment of more reactive leaving groups at these sites. LA0116488 (17) The reaction of 0.2 mM 9 with 1 mM (excess) (CTA)IBA in 0.1 M Bis-Tris at pH 8.4 could be followed by UV spectroscopy, monitoring phenoxide release at 270 nm. We observed k ) 6.07 × 10-2 s-1. (18) Minor product signals at δ -7.59 and δ -8.26 probably correspond to Bis-Tris cleavage of 9 (and product formation including the Bis-Tris moiety); the same signals appeared with 0.1 M buffer alone, where k ) 1.3 × 10-4 s-1 for the disappearance of 9. Cleavage is slower in 20 mM (CTA)Cl/0.1 M Bis-Tris than in the buffer alone due to micellar binding of the substrate by the (CTA)Cl, which lacks a reactive counterion.
