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Dissociation and phosphorylation constants for the inhibition of acetylcholinesterase by 2-fluoro, 2-oxo-1,3,2-dioxaphosphorinanes
PESTICIDE
BIOCHEMISTRY
AND
Dissociation
(1976)
and Phosphorylation Constants for the of Acetylcholinesterase by 2-Fluoro, 2-0x0-1,3,2-dioxaphosphorinanes J. HART AND RICHARD
GEOFFREY Section
6, 464470
PHYSIOWGY
of Neurobiology
and Behavior,
D. S. MILBRATH Department
oJ Chemistry,
Iowa
Cornell
Inhibition
D. O’BRIEN
University, Ithaca,
New
York
14863
AND J. G. VERKADE State
Unive+sity,
Ames,
Iowa
60010
Received September 24, 1975; accepted January 28, 1976 Phosphorylation, dissociation and bimolecular reaction constants were determined for a number of fluoro 1,3,2-dioxaphosphorinane a-oxide derivatives in the presence of substrate. The data are discussed in terms of the steric effects of dioxaphosphorinane ring substituents on the kinetic and dissociation constants for the inhibition reaction.
Interpretation of studies on the structureactivity relationships of organophosphorus esters may be hampered by the conformational flexibility of substituents. This may present a major obstacle to deducing the topography of the active zone of the enzyme by extrapolating from the known structures and conformations of the inhibitors. ln the majority of enzymological studies using organophosphorus esters, the pentavalent phosphorus atom has carried substituents which are not sterically constrained in ring structures. However, the work of Westheimer (1) and others [see (2) for review], on organic reaction mechanisms involving phosphorus ring systems, has shown that such structures may provide a very useful basis for stereochemical interpretations. In view of this evidence we decided to undertake a study using a suitable ring structure to provide a fairly rigid framework upon which various substituents could be located with some certainty as to their spatial orientation. We chose a six-membered, unstrained dioxa464 Copyright All rights
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
phosphorinane ring system which provides a number of carbon positions at which to introduce substituents of known orientation. The dioxaphosphorinane compounds were :
Ashani et al. (3) have reported on compound A. In the present paper we report our findings using methyl and dimethyl derivatives (B-E) of the parent compound, A. The most generally useful kinetic scheme for describing the progressive inhibit,ion of acetylcholinesterase by organophosphorus esters is : E+P&
k-l
EPXEEP
+ X
[l-J
2-FLUORO,
2-0X0-1,3,2-DIOXAPHOSPHORINANES
where E represents the enzyme, PX the organophosphorus ester with its leaving group X, EPX the reversible enzyme/inhibitor complex, and EP the covalently inhibited enzyme. The dissociation constant, and the phosphorylation Kd (= Wh), constant, kz, were determined for each of the dioxaphosphorinane derivatives in the presence of substrate, using the methods described previously (4-7). EXPERIMENTAL
Materials
and Methods
Because the preparations of compounds B-E are similar, the procedure will be outlined in detail only for B. All NMR spectra were run in CDCII. The 3rP and lgF spectra were run with protons decoupled on a Bruker HX-90 spectrometer operating in the FT mode using 850/, HsP04 and CFCla as external standards, respectively. Preparation of 2-chloro-1,3,2-phosphorinanes. These compounds were prepared from PCl, and the appropriate diols as has been previously described (8). Preparation of W-Jluoro-6,6-dimethyl-1,3,2dioxaphosphorinane. The procedure used is a modification of Schmutzler’s method (9). Antimony trifluoride, 8.6 g (0.04 mol), was added to 2-chloro-5,5-dimethyl-1,3,2-dioxaphosphorinane, 5.0 g (0.032 mol), in a 50-ml round bottom flask under a positive pressure of nitrogen. The heterogeneous mixture was heated to 50°C with stirring for 5 hr. After it had cooled to room temperature, the black-brown slurry was filtered and the filtrate carefully distilled under vacuum. After a small forerun the product distilled at 59-60°C at 30 mm to give 3.2 g (71%) yield; NMR (‘H) 6 4.11 m and 6 3.38 m (2 CHZ), 6 1.29 s and 6 0.80 s (2 CHa) ; (“‘P) 6 - 112.1 d IJpr = 1176Hz; and (lgF) 6 65.4 d. Preparation of 2-jluorod-oxo-5,&dimethyl1,3,2-dioxaphosphorinane. (B). The phosphorofluoridite prepared above was oxidized with Nz04 in CCL solution by a previously described method (10). Evaporation of the
465
solvent left a solid which was purified by sublimination at 50°C and 0.1 mm in 76% yield; NMR (rH) 6 4.00 and 6 3.29 m (2 CH*) 6 1.17 s and 6 0.70 s (2 CHI); (“1P) 6 17.0 d lJpr = 1008 Hz; and (lgF) 6 87.0 d. Preparation of 2-jtuoro-Q-methyl-l ,S,W-dioxaphosphorinane. This compound was prepared in 709ib yield (bp 50°C at 30 mm), NMR (lH) 6 4.54 m, 6 3.94 m and 6 1.89 m (5 ring CH), 6 1.36 d (CH3) 3Jnn = 7 Hz; (“rP> 6 118.3 d, lJpr = 1170 Hz; and (lgF) 6 62.3 d. Preparation of 2-Jluoro-2-oxo-.&methylI,3,2-dioxaphosphorinane (C). Oxidation of the preceding compound with N204 produced a 73% yield of C. The product was distilled under reduced pressure, bp lOO2°C at 0.1 mm; NMR (*H) 6 4.55 m and 6 1.94 m (5 ring CH), 6 1.37 dd (CHs) 4JpH = 2.5 Hz Ajax = 6.7 Hz; (“‘P) 6 16.1 d, rJpr = 1005 Hz; and (lgF) 6 86.1 d. Preparation of 2-$uoro-&,6a-dimethyl1,3,2-dioxaphosphorinane. The meso phosphorofluoridite was prepared in 49% yield (bp 60°C at 35 mm); NMR (‘H) 6 4.49 m and 6 1.55 m (5 ring CH), and 6 1.18 d (CW,), 3Jnn = 6 Hz; (“‘P) 6 - 116.2 ci, lJpF = 1165 Hz; and (lgF) 6 73.4 d Preparation of 2-$uoro-2-oxo-.&,6a-dzmethyl-l ,3,2-dioxaphosphorinane (D) . Dinitrogen tetroxide oxidation of the preceding compound gave a 65Yo yield of D (bp lOl-102°C at 0.1 mm); NMR (‘H) 6 4.54 m and 6 1.73 m (5 ring CH), 6 1.29 dd (2 CH3) 4Jpn = 3 Hz, 3Jrrn = 6 Hz; (“‘P) 6 16.0 d, ‘JPF = 1002 Hz ; and (lgF) 6 85.4 d. Preparation of 2-Jluoro-&G/3-dimethyt1,3,2-dioxaphosphorinane. This d, t phosphorafluoridite was isolated in 707. yield (bp 46°C at 35 mm); NMR (lH) 6 4.46 m and 6 1.83 m (4 ring CH), 6 1.41 d (CH3) 3Jnn = 7.5 Hz and 6 1.22 d (CBS) 3Jnn = 7.0 Hz; (“‘P) 6 - 118.6 d, ‘Jpr = 1185 Hz ; and (lgF) 6 43.2. Preparation of 2-jluoro-2-oxo-&,6&dimethyl-l ,3,2-dioxaphosphorinane (E). The above compound was isolated from the N204 oxidation reaction in 80% yield (bp
466
HART
.“01
107-108°C at 0.5 mm) ; KRIR (‘H) 6 4.63 nz and 6 1.78 m (4 ring CH), 6 1.32 dd (2 CH,) ?JPn = 3 Hz and 3JBn = 5.4 Hz; (“‘I’) 6 16.1 (1, ~JPF = 1001 Hz; and (‘“E’) 6 72.7 cl. Dioxaphosphorinane derivatives w-erc purified as described in the Methods scction. All other solvents and reagents were A. Ii. grade or Laboratory grade. The enzyme used was bovine erythrocyte acetylcholincsterase (EC 3.1.1.7) obtained from Stcrwin Chemicals. The chromogenic substrate XWLYp-nitrophenyl acetate (Eastman). Reactions were followed by monitoring the change in absorbance at 402 nm dw to relcnw of p-nitrophenolate ion, using an Acta III recording spectrophotometer, at pH ‘7 and 25°C. The K, obtained for the interaction of this substrate with the cnzyme was 2.04 mM. Two criteria mere used for detecting impurities in the inhibitors. The three derivatives which are liquid at room tempernture (compounds C, D, I!: of Table 1) were examined by ascending chromatography using Gelman silicic acid paper with loo/;, (by volume) water in acetone as solvent’. After development, the chromatograms were viswlizcd by exposure to iodine vapor
”
Ill,.
for several minutes. Compounds C and D appeared to be pure by this criterion, whereas compound E was impure. A further purity test was c*:vricld out in order to check for the presence of chloro dioxsphosphorinanes, jvhich might not bc distinguishable from the fluoro d~~rivatiws cthromatogr:lphicall~-. Ashani et nl. (:;) have noted that) the chloro derivativrb may be prtwnt as :L contaminant! in the parent fluoro compound (A). The procc~tlurr used in the prwcnt study was an udaptst,ion of the method described by Ash:mi ~1. (3) : An appropriate volume of c~tli:uiolic inhibitor solution was added to an at least 10X greater volume of 0.1 ill phosphate buffer (pH 7) in ;l volunwtric~ flask and allowed to stand at room tcmptwture for about 30 min. The (~th:tnolic~ xubstrate solution \Y:M thw added and thcs tystem m:id(a up to final volume n-it,h buffer. The chloro tlcrivatiw h:ls :i half-life of -I min in 0.1 N phosphate buffer at pfI 7, I\-hereas the fluoro dcrivutive has a (it’ :lIwllt 500 min under the same wnditions !S). The 30-min c~xposuretc) buft’c,r wrvw, t h~~refow, tc) rcmow~ trnws of th(s (,orlt:rltlirlating hiphlj. wtiw ahloro dcriv:~tivc~ \vithollt ct
ti
TABLE Phosphorylation -___
1
Rate, Dissociation, and Bimolecular Reaction Acetylcholinesterase by 1,3,2,-Dioxaphosphorinane
Constants for the Inhibition 2-Oxcide Derivatives’”
of
-0
4 $‘”
F’
R, ‘0
R,
3 R2
Compound
122
R1
R3
124
Kd (rniu)
kd (mix’)
10-z ;< R, (N-’
---~-
-
Ab B c 1) E
H H H CH, C&
H H
(es) (eq)
CJL CH, CHS
(es) (es) (ax)
H
H
CHs H H H
CHI H H H
0.54 1.14 0.151 0.712 0.990
--..-~I_~---.-~~--.-.--~-.. (f0.41) (f0.022) (hO.036) (~~0.045)
Q In the presence of p-nitrophenyl acetate at 25°C in 0.1 M phosphate shown in parentheses. The data shown for each compound were obtained the type described in the text.
bFrom (3).
0.43 1.11 1.19 0.84 0.280
(fO.45) (&0.32) (50.086) (f0.034)
mirr--I) -~
1.27 1.03 0.128 0.849 3.54
~--
(ztO.30) (ZtO.038) (f0.094) (~kO.46)
buffer (pH 7). iStandard errors from double-reciprocal plots, of
‘&FLUORO,
significantly affecting the concentrations of the fluoro derivative. In those cases where the rate of inhibition of the enzyme was appreciably decreased by the buffer-exposure procedure, it was concluded that contaminating amounts of the chloro derivative had been present in the original material. This decrease in potency after buffer-exposure was observed with compounds B, C, and D, which were, therefore, each treated as follows: about 0.25 ml or grams of impure inhibitor was dissolved in approximately 50 ml of 0.2 M sodium phosphate buffer and agitated for about 30 min at room temperature. The inhibitor was then extracted from the aqueous buffer using three lo-ml portions of chloroform. The chloroform extracts were then bulked and the chloroform evaporated under reduced pressure over a waterbath at 50-60°C. Finally, where impurities were detectable chromatographically (compound E), the preparative chromatographic separation and elution technique, followed by evaporation of the solvent, was used as described earlier (6, 7). This was followed by the buffer-exposure, extraction, and evaporation procedure outlined above. RESULTS
AND
467
2-0X0-1,3,%DIOXAPHOSPHORINANES
DISCUSSION
Before carrying out the detailed kinet.ic analysis for the determination of kz and Kd for the interaction of acetylcholinesterase with the various inhibitors, the effectiveness of the buffer-exposure procedure for removing contaminants was examined. Figure 1 shows a semilogarithmic plot for inhibition of the enzyme by unpurified compound D. The plot was constructed by drawing tangents to the recording spectrophotometer trace and then plotting the log of these tangent values against time (see (4-7) for detailed explanation of plotting procedures and kinetic derivations). The low concentration of inhibitor used (0.0102 mM) allowed the contaminating potent impurity to be depleted during the course of the reaction, resulting in the biphasic
I 1
I 2 1o-2 x time.
I 3
I 1,
I 5
seconds
FIG. 1. Semilogarithmic plot showing biphasic profile obtained using a relatively Eow concentratzon (0.0102 mM) oj unpur$ed compound D (see Table 1) to inhibit acetylcholine-sterase (10 pm ml+) in the presence oj substrate (0.2006 mM p-nitrophenyl acetate). 0.1 M phosphate buffer in 2.1% aqueous ethanol; pH 7; 25°C.
plot shown. The plot should have been linear if the inhibitor were pure since, under the conditions used, the apparent inhibitor/active-site molar concentration ratio was about 218, which is fully adequate to satisfy the requirements for pseudo first-order conditions. Figure 2 shows the profile obtained after the inhibitor had been subjected to the buffer-exposure procedure described above. Concentrations and conditions were identical in the experiments upon which Figs. 1 and 2 were based, except for the bufferexposure procedure used in the latter case. The linearity of the plot in Fig. 2 indicates that the buffer-exposure method is a satisfactory approach to removing the contaminant. Proceeding to the detailed kinetic analysis: Ashani et al. (3) have made the interesting observation that compounds with structure A give phosphorylated acetylcholinesterase (eel) that spontaneously regains activity in aqueous solution more rapidly than is usual for phosphorylated cholinesterases. They found the half-life for aqueous hydrolysis of the 2-enzymo-2fluoro-2-oxo-1,3,2dioxaphosphorinane to be about 12 min. Such compounds, which give
-k&3
HART
.“”
*’
a low kz in combination with a relatively high ka (for the dephosphorylation step in which EP 2 E + P), may be expected to show the type of “pseudo steady-state” behavior illustrated by the plots for the inhibition of the enzyme by compound C as shown in Fig. 3. The initial, linear portions of the In V versus t plots (i.e., at times ~500 set in Fig. 3) were used to estimate the first-order rate constants from which the kz and Kd values were determined using a double-reciprocal inhibition plot of the type described previously (4-7). The double-reciprocal plot was satisfactorily linear (correlation coefficient 0.94) suggesting that the use of the initial portions of the biphasic In V versus t plots is a valid approach. Turning to compounds B, D, and E, it is of interest to note that these inhibitors give linear semilogarithmic plots of reaction velocity versus time. Plots were obtained that were linear over more than four halflives, there being no apparent change in kinetic order-unlike the result obtained with compound C. This suggests that enzymo-B, -D, or -E does not show the relatively rapid kB step which occurs with enzymo-C or -A.
1
2 1O-2
3 x
time,
4
5
seconds
Fro. 2. Semilogarithmic plot constructed from a reaction projile obtained using pura$k.d Compound D (see Table 1) to inhibit acetylcholinesterase in the presence of substrate. The plot is sensibly linear, the least-squares line shown has a correlation coeficient of 0.979. Calculated concentrations and reaction conditions were identical to those described in Fig. 1.
FIG. pound in the acetate). 2.51; 0.750; aqueous
3. Semilogarithmic plots obtainedWusing comC to inhibit acetylcholinesterase (10 pm ml-‘) presence of substrate (0.2 mM p-nitrophenyl Inhibitor concentrations (mM): 0, 6.02; X, 8, 1.72; q , 1.26; A, 1.00; D, 0.831; A, Q, 0.664. (0.1 M phosphate bufler in 2.10/, ethanol; pH 7; 26°C).
The kinetic and dissociation constants for inhibition of the enzyme by dioxaphosphorinane derivatives and the parent compound are shown in Table 1. The kz values are all very low in comparison to those usually obtained using organophosphorus esters. In addition, the differences between the Kd values are generally not large enough to be statistically significant. Nevertheless, although there is no systematic pattern, it is of interest to note that methyl substitution affects both k2 and Kd, as does geometric isomerism. Thus the introduction of an equatorial methyl group at position 4 (compare compounds C and A) produces a X6-fold decrease in k, and a 2.S-fold increase in Kd, the combined effect being a 9.9-fold decrease in ki. A second equatorial methyl group (i.e., at position 6: compound D) starts a trend toward increasing potency (comparing compounds D and C). In this case there is a 4.7-fold increase in
2-FLUORO,
469
2-0X0-1,3,2-DIOXAPHOSPHORINANES
kz and a 1.4-fold decrease in Kd, which combine to give a 6.6-fold increase in ki. The replacement of the equatorial methyl group at position 4 by an axial methyl group (compare compounds E and D) produces a further l.Cfold increase in k2, coupled with a 3-fold decrease in Kd, resulting in a 4.2-fold increase in Ici. The gem-dimethyl compound (B), (on the other hand) while giving the highest kz, shows poor affinity (high Kd) and is of intermediate overall potency, as indicated by its ki. Of interest in these compounds is the stereochemistry at phosphorus. Systems of this type which possess an electronegative phosphorus substitutent (fluorine in the present case), tend to occupy the axial position in a chair conformer (11) and this tendency has been rationalized recently on electronic grounds (12) :
very likely occurs as shown in Eq. [3] since this stereochemical course has been verified for-the
c31 chloro derivative methanol (15) :
of C in the presence of
c41 By this process, enzyme phosphorylation would produce a phosphorus stereochemistry in EPn and EPo which is isomeric with that in EPA, EPz, and EPz, owing to the conformational mobility of the phosphate ester rings in these cases along with the proclivity of alkoxy groups to be axial (11, 12):
I31 X-Ray diffraction experiments on a single crystal of B reveal a chair conformer with an axial fluorine, which also strongly suggests that the dominant conformer in solution resembles 1 rather than 2 (13). That compounds A-E all have axial fluorines (14) is indicated by the similar 31P-1gF coupling constants (ca. 1000 Hz) observed for B-E. Other NMR active substituents in related systems have demonstrated that these directly bonded coupling constants depend markedly on whether they are axially or equatorially oriented. It thus can be reasonably concluded that at least in the case of C and D which are conformationally locked by one and two equatorial methyl groups respectively, the EPX complex (Eq. [l]) probably contained the phosphorylating agent in the conformation represented by 1 shown in Eq. [a]. If this is the case, phosphorylation of the :nzyme’s hydroxyl function by C and D
Steric factors near the phosphorylation site within the enzyme, however, could favor the less sterically encumbered phosphorus stereochemistry represented by EPn in all cases. ACKNOWLEDQMENTS
We wish to thank Betty Wolfson for excellent technical assistance. This work WM supported in part by a grant from the National Cancer Institute (J. G. V.) and by Grant DAHCOQ 74G0237 from the United States Army Rssesrch Office (R. D. 0.). RBFERENCES
1. F.
H. Westheimer, Pseudo-rotation in the hydrolysis of phosphate esters, Ace. Chem. Res. 1, 70 (1968).
470
HART
2. 1. Ugi and F. Ramsey, Stereochemistry of five coordinate phosphorus, Chem. Brd. 8, 198 (1972). 3. Y. Ashani, S. L. Snyder, and I. B. Wilson, Inhibition of cholinesterase by 1,3,2-dioxaphosphorinane a-oxide derivatives, Bioch~mislry 11, 3518 (1972). 4. G. J. Hart and R. D. O’Brien, Recording spectrophotometric method for determination of dissociation and phosphorylation constants of the inhibition of acetylcholinesterase by organophosphates in the presence of substrate, Biochemistry 12, 2940 (1973). 5. G. J. Hart and R. D. O’Brien, Stopped-flow studies of the inhibition of acetylcholinesterase by organophosphates in the presence of substrate, P&c. Biochem. Physiol. 4, 239 (1974). fi. G. J. Hart, The determination of dissociation and phosphorylation con&ants for the inhibition of cholinesterases by organophosphorus esters in the presence of substrate, Ph.D. Thesis, Cornell University, 1974. 7. G. J. Hart and R. D. O’Brien, Dissociation and phosphorylation constants for the inhibition of acetylcholinesterase by a series of novel O-ethyl 0-phenyl S-n-propyl phosphorothioates, Pestic. Biochem. Physiol. 6, St5 (1976). 8. D. W. White, R. D. Bertrand, G. K. McEwen, and J. G. Verkade, Structural implication of nuclear magnetic resonance studies on l-lt-lphospha-2,6,-dioxacyclohexanes, J. Amer. Chem. Soe. 92, 7125 (1970).
ET AL.
9. R. Schmutzler, Synthese and Koordinationschimie der Fluorophosphite, Chcm. Ber. 96, 2435 (1963). 10. J. R. Cox, Jr. and F. H. Westheimer, The oxidation of trisubstituted phosphites by dinitrogen tetroxide, J. Amrr. Chcm. SW. SO, 5441 (1938). 11. J. G. Verkade, Interplay of st,eric and electronic influences in the chemistry of monocyclic and bicyclic phosphorus rstrrs, Plenary Lecture, 5th International Conference on Organic Phosphorus Chemistr~~. Gdansk, Sept., 1974; Phosphorus, to appear. 12. R. F. Hudson and .J. G. Verkade. The conformation and reactivity of 1,3,2dioxaphosphorinanes, Tdrahactron, Mt. 323 I (1075) and references therein. 13. D. 8. Mlbrath, J. Springer, J. C. (‘lardy, and J. Cr. Verkade. Crystal and molecular structure of 4,6-dimethyl-2-fluoro-2-r~xo-l,3,2-dioxaphosphorinane, to appear. 14 In t,he case of E, a “twist-boat” conformation is not unlikely in order to relieve 1-r: diaxial interaction of fluorine wit,h :I methyl substituent. Such a conformation st,ill permits the fluorine to be pseudo axial, howcvf~r. 15. W. Stec and M. 41. Mikolajcq.k, Stereochemistry of organophosphorus cyclic (‘( mpounds. II. Stereospecific synthesis of’ c+s arltl /runs 2hell,geno-2-oso-4-mdhyl-I ,:1,P-tiioxaphosphormans and their chemic*al tr:rrl~ir)l,rnations. 7’elruhdron 29, 539 (19733).
BIOCHEMISTRY
AND
Dissociation
(1976)
and Phosphorylation Constants for the of Acetylcholinesterase by 2-Fluoro, 2-0x0-1,3,2-dioxaphosphorinanes J. HART AND RICHARD
GEOFFREY Section
6, 464470
PHYSIOWGY
of Neurobiology
and Behavior,
D. S. MILBRATH Department
oJ Chemistry,
Iowa
Cornell
Inhibition
D. O’BRIEN
University, Ithaca,
New
York
14863
AND J. G. VERKADE State
Unive+sity,
Ames,
Iowa
60010
Received September 24, 1975; accepted January 28, 1976 Phosphorylation, dissociation and bimolecular reaction constants were determined for a number of fluoro 1,3,2-dioxaphosphorinane a-oxide derivatives in the presence of substrate. The data are discussed in terms of the steric effects of dioxaphosphorinane ring substituents on the kinetic and dissociation constants for the inhibition reaction.
Interpretation of studies on the structureactivity relationships of organophosphorus esters may be hampered by the conformational flexibility of substituents. This may present a major obstacle to deducing the topography of the active zone of the enzyme by extrapolating from the known structures and conformations of the inhibitors. ln the majority of enzymological studies using organophosphorus esters, the pentavalent phosphorus atom has carried substituents which are not sterically constrained in ring structures. However, the work of Westheimer (1) and others [see (2) for review], on organic reaction mechanisms involving phosphorus ring systems, has shown that such structures may provide a very useful basis for stereochemical interpretations. In view of this evidence we decided to undertake a study using a suitable ring structure to provide a fairly rigid framework upon which various substituents could be located with some certainty as to their spatial orientation. We chose a six-membered, unstrained dioxa464 Copyright All rights
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
phosphorinane ring system which provides a number of carbon positions at which to introduce substituents of known orientation. The dioxaphosphorinane compounds were :
Ashani et al. (3) have reported on compound A. In the present paper we report our findings using methyl and dimethyl derivatives (B-E) of the parent compound, A. The most generally useful kinetic scheme for describing the progressive inhibit,ion of acetylcholinesterase by organophosphorus esters is : E+P&
k-l
EPXEEP
+ X
[l-J
2-FLUORO,
2-0X0-1,3,2-DIOXAPHOSPHORINANES
where E represents the enzyme, PX the organophosphorus ester with its leaving group X, EPX the reversible enzyme/inhibitor complex, and EP the covalently inhibited enzyme. The dissociation constant, and the phosphorylation Kd (= Wh), constant, kz, were determined for each of the dioxaphosphorinane derivatives in the presence of substrate, using the methods described previously (4-7). EXPERIMENTAL
Materials
and Methods
Because the preparations of compounds B-E are similar, the procedure will be outlined in detail only for B. All NMR spectra were run in CDCII. The 3rP and lgF spectra were run with protons decoupled on a Bruker HX-90 spectrometer operating in the FT mode using 850/, HsP04 and CFCla as external standards, respectively. Preparation of 2-chloro-1,3,2-phosphorinanes. These compounds were prepared from PCl, and the appropriate diols as has been previously described (8). Preparation of W-Jluoro-6,6-dimethyl-1,3,2dioxaphosphorinane. The procedure used is a modification of Schmutzler’s method (9). Antimony trifluoride, 8.6 g (0.04 mol), was added to 2-chloro-5,5-dimethyl-1,3,2-dioxaphosphorinane, 5.0 g (0.032 mol), in a 50-ml round bottom flask under a positive pressure of nitrogen. The heterogeneous mixture was heated to 50°C with stirring for 5 hr. After it had cooled to room temperature, the black-brown slurry was filtered and the filtrate carefully distilled under vacuum. After a small forerun the product distilled at 59-60°C at 30 mm to give 3.2 g (71%) yield; NMR (‘H) 6 4.11 m and 6 3.38 m (2 CHZ), 6 1.29 s and 6 0.80 s (2 CHa) ; (“‘P) 6 - 112.1 d IJpr = 1176Hz; and (lgF) 6 65.4 d. Preparation of 2-jluorod-oxo-5,&dimethyl1,3,2-dioxaphosphorinane. (B). The phosphorofluoridite prepared above was oxidized with Nz04 in CCL solution by a previously described method (10). Evaporation of the
465
solvent left a solid which was purified by sublimination at 50°C and 0.1 mm in 76% yield; NMR (rH) 6 4.00 and 6 3.29 m (2 CH*) 6 1.17 s and 6 0.70 s (2 CHI); (“1P) 6 17.0 d lJpr = 1008 Hz; and (lgF) 6 87.0 d. Preparation of 2-jtuoro-Q-methyl-l ,S,W-dioxaphosphorinane. This compound was prepared in 709ib yield (bp 50°C at 30 mm), NMR (lH) 6 4.54 m, 6 3.94 m and 6 1.89 m (5 ring CH), 6 1.36 d (CH3) 3Jnn = 7 Hz; (“rP> 6 118.3 d, lJpr = 1170 Hz; and (lgF) 6 62.3 d. Preparation of 2-Jluoro-2-oxo-.&methylI,3,2-dioxaphosphorinane (C). Oxidation of the preceding compound with N204 produced a 73% yield of C. The product was distilled under reduced pressure, bp lOO2°C at 0.1 mm; NMR (*H) 6 4.55 m and 6 1.94 m (5 ring CH), 6 1.37 dd (CHs) 4JpH = 2.5 Hz Ajax = 6.7 Hz; (“‘P) 6 16.1 d, rJpr = 1005 Hz; and (lgF) 6 86.1 d. Preparation of 2-$uoro-&,6a-dimethyl1,3,2-dioxaphosphorinane. The meso phosphorofluoridite was prepared in 49% yield (bp 60°C at 35 mm); NMR (‘H) 6 4.49 m and 6 1.55 m (5 ring CH), and 6 1.18 d (CW,), 3Jnn = 6 Hz; (“‘P) 6 - 116.2 ci, lJpF = 1165 Hz; and (lgF) 6 73.4 d Preparation of 2-$uoro-2-oxo-.&,6a-dzmethyl-l ,3,2-dioxaphosphorinane (D) . Dinitrogen tetroxide oxidation of the preceding compound gave a 65Yo yield of D (bp lOl-102°C at 0.1 mm); NMR (‘H) 6 4.54 m and 6 1.73 m (5 ring CH), 6 1.29 dd (2 CH3) 4Jpn = 3 Hz, 3Jrrn = 6 Hz; (“‘P) 6 16.0 d, ‘JPF = 1002 Hz ; and (lgF) 6 85.4 d. Preparation of 2-Jluoro-&G/3-dimethyt1,3,2-dioxaphosphorinane. This d, t phosphorafluoridite was isolated in 707. yield (bp 46°C at 35 mm); NMR (lH) 6 4.46 m and 6 1.83 m (4 ring CH), 6 1.41 d (CH3) 3Jnn = 7.5 Hz and 6 1.22 d (CBS) 3Jnn = 7.0 Hz; (“‘P) 6 - 118.6 d, ‘Jpr = 1185 Hz ; and (lgF) 6 43.2. Preparation of 2-jluoro-2-oxo-&,6&dimethyl-l ,3,2-dioxaphosphorinane (E). The above compound was isolated from the N204 oxidation reaction in 80% yield (bp
466
HART
.“01
107-108°C at 0.5 mm) ; KRIR (‘H) 6 4.63 nz and 6 1.78 m (4 ring CH), 6 1.32 dd (2 CH,) ?JPn = 3 Hz and 3JBn = 5.4 Hz; (“‘I’) 6 16.1 (1, ~JPF = 1001 Hz; and (‘“E’) 6 72.7 cl. Dioxaphosphorinane derivatives w-erc purified as described in the Methods scction. All other solvents and reagents were A. Ii. grade or Laboratory grade. The enzyme used was bovine erythrocyte acetylcholincsterase (EC 3.1.1.7) obtained from Stcrwin Chemicals. The chromogenic substrate XWLYp-nitrophenyl acetate (Eastman). Reactions were followed by monitoring the change in absorbance at 402 nm dw to relcnw of p-nitrophenolate ion, using an Acta III recording spectrophotometer, at pH ‘7 and 25°C. The K, obtained for the interaction of this substrate with the cnzyme was 2.04 mM. Two criteria mere used for detecting impurities in the inhibitors. The three derivatives which are liquid at room tempernture (compounds C, D, I!: of Table 1) were examined by ascending chromatography using Gelman silicic acid paper with loo/;, (by volume) water in acetone as solvent’. After development, the chromatograms were viswlizcd by exposure to iodine vapor
”
Ill,.
for several minutes. Compounds C and D appeared to be pure by this criterion, whereas compound E was impure. A further purity test was c*:vricld out in order to check for the presence of chloro dioxsphosphorinanes, jvhich might not bc distinguishable from the fluoro d~~rivatiws cthromatogr:lphicall~-. Ashani et nl. (:;) have noted that) the chloro derivativrb may be prtwnt as :L contaminant! in the parent fluoro compound (A). The procc~tlurr used in the prwcnt study was an udaptst,ion of the method described by Ash:mi ~1. (3) : An appropriate volume of c~tli:uiolic inhibitor solution was added to an at least 10X greater volume of 0.1 ill phosphate buffer (pH 7) in ;l volunwtric~ flask and allowed to stand at room tcmptwture for about 30 min. The (~th:tnolic~ xubstrate solution \Y:M thw added and thcs tystem m:id(a up to final volume n-it,h buffer. The chloro tlcrivatiw h:ls :i half-life of -I min in 0.1 N phosphate buffer at pfI 7, I\-hereas the fluoro dcrivutive has a (it’ :lIwllt 500 min under the same wnditions !S). The 30-min c~xposuretc) buft’c,r wrvw, t h~~refow, tc) rcmow~ trnws of th(s (,orlt:rltlirlating hiphlj. wtiw ahloro dcriv:~tivc~ \vithollt ct
ti
TABLE Phosphorylation -___
1
Rate, Dissociation, and Bimolecular Reaction Acetylcholinesterase by 1,3,2,-Dioxaphosphorinane
Constants for the Inhibition 2-Oxcide Derivatives’”
of
-0
4 $‘”
F’
R, ‘0
R,
3 R2
Compound
122
R1
R3
124
Kd (rniu)
kd (mix’)
10-z ;< R, (N-’
---~-
-
Ab B c 1) E
H H H CH, C&
H H
(es) (eq)
CJL CH, CHS
(es) (es) (ax)
H
H
CHs H H H
CHI H H H
0.54 1.14 0.151 0.712 0.990
--..-~I_~---.-~~--.-.--~-.. (f0.41) (f0.022) (hO.036) (~~0.045)
Q In the presence of p-nitrophenyl acetate at 25°C in 0.1 M phosphate shown in parentheses. The data shown for each compound were obtained the type described in the text.
bFrom (3).
0.43 1.11 1.19 0.84 0.280
(fO.45) (&0.32) (50.086) (f0.034)
mirr--I) -~
1.27 1.03 0.128 0.849 3.54
~--
(ztO.30) (ZtO.038) (f0.094) (~kO.46)
buffer (pH 7). iStandard errors from double-reciprocal plots, of
‘&FLUORO,
significantly affecting the concentrations of the fluoro derivative. In those cases where the rate of inhibition of the enzyme was appreciably decreased by the buffer-exposure procedure, it was concluded that contaminating amounts of the chloro derivative had been present in the original material. This decrease in potency after buffer-exposure was observed with compounds B, C, and D, which were, therefore, each treated as follows: about 0.25 ml or grams of impure inhibitor was dissolved in approximately 50 ml of 0.2 M sodium phosphate buffer and agitated for about 30 min at room temperature. The inhibitor was then extracted from the aqueous buffer using three lo-ml portions of chloroform. The chloroform extracts were then bulked and the chloroform evaporated under reduced pressure over a waterbath at 50-60°C. Finally, where impurities were detectable chromatographically (compound E), the preparative chromatographic separation and elution technique, followed by evaporation of the solvent, was used as described earlier (6, 7). This was followed by the buffer-exposure, extraction, and evaporation procedure outlined above. RESULTS
AND
467
2-0X0-1,3,%DIOXAPHOSPHORINANES
DISCUSSION
Before carrying out the detailed kinet.ic analysis for the determination of kz and Kd for the interaction of acetylcholinesterase with the various inhibitors, the effectiveness of the buffer-exposure procedure for removing contaminants was examined. Figure 1 shows a semilogarithmic plot for inhibition of the enzyme by unpurified compound D. The plot was constructed by drawing tangents to the recording spectrophotometer trace and then plotting the log of these tangent values against time (see (4-7) for detailed explanation of plotting procedures and kinetic derivations). The low concentration of inhibitor used (0.0102 mM) allowed the contaminating potent impurity to be depleted during the course of the reaction, resulting in the biphasic
I 1
I 2 1o-2 x time.
I 3
I 1,
I 5
seconds
FIG. 1. Semilogarithmic plot showing biphasic profile obtained using a relatively Eow concentratzon (0.0102 mM) oj unpur$ed compound D (see Table 1) to inhibit acetylcholine-sterase (10 pm ml+) in the presence oj substrate (0.2006 mM p-nitrophenyl acetate). 0.1 M phosphate buffer in 2.1% aqueous ethanol; pH 7; 25°C.
plot shown. The plot should have been linear if the inhibitor were pure since, under the conditions used, the apparent inhibitor/active-site molar concentration ratio was about 218, which is fully adequate to satisfy the requirements for pseudo first-order conditions. Figure 2 shows the profile obtained after the inhibitor had been subjected to the buffer-exposure procedure described above. Concentrations and conditions were identical in the experiments upon which Figs. 1 and 2 were based, except for the bufferexposure procedure used in the latter case. The linearity of the plot in Fig. 2 indicates that the buffer-exposure method is a satisfactory approach to removing the contaminant. Proceeding to the detailed kinetic analysis: Ashani et al. (3) have made the interesting observation that compounds with structure A give phosphorylated acetylcholinesterase (eel) that spontaneously regains activity in aqueous solution more rapidly than is usual for phosphorylated cholinesterases. They found the half-life for aqueous hydrolysis of the 2-enzymo-2fluoro-2-oxo-1,3,2dioxaphosphorinane to be about 12 min. Such compounds, which give
-k&3
HART
.“”
*’
a low kz in combination with a relatively high ka (for the dephosphorylation step in which EP 2 E + P), may be expected to show the type of “pseudo steady-state” behavior illustrated by the plots for the inhibition of the enzyme by compound C as shown in Fig. 3. The initial, linear portions of the In V versus t plots (i.e., at times ~500 set in Fig. 3) were used to estimate the first-order rate constants from which the kz and Kd values were determined using a double-reciprocal inhibition plot of the type described previously (4-7). The double-reciprocal plot was satisfactorily linear (correlation coefficient 0.94) suggesting that the use of the initial portions of the biphasic In V versus t plots is a valid approach. Turning to compounds B, D, and E, it is of interest to note that these inhibitors give linear semilogarithmic plots of reaction velocity versus time. Plots were obtained that were linear over more than four halflives, there being no apparent change in kinetic order-unlike the result obtained with compound C. This suggests that enzymo-B, -D, or -E does not show the relatively rapid kB step which occurs with enzymo-C or -A.
1
2 1O-2
3 x
time,
4
5
seconds
Fro. 2. Semilogarithmic plot constructed from a reaction projile obtained using pura$k.d Compound D (see Table 1) to inhibit acetylcholinesterase in the presence of substrate. The plot is sensibly linear, the least-squares line shown has a correlation coeficient of 0.979. Calculated concentrations and reaction conditions were identical to those described in Fig. 1.
FIG. pound in the acetate). 2.51; 0.750; aqueous
3. Semilogarithmic plots obtainedWusing comC to inhibit acetylcholinesterase (10 pm ml-‘) presence of substrate (0.2 mM p-nitrophenyl Inhibitor concentrations (mM): 0, 6.02; X, 8, 1.72; q , 1.26; A, 1.00; D, 0.831; A, Q, 0.664. (0.1 M phosphate bufler in 2.10/, ethanol; pH 7; 26°C).
The kinetic and dissociation constants for inhibition of the enzyme by dioxaphosphorinane derivatives and the parent compound are shown in Table 1. The kz values are all very low in comparison to those usually obtained using organophosphorus esters. In addition, the differences between the Kd values are generally not large enough to be statistically significant. Nevertheless, although there is no systematic pattern, it is of interest to note that methyl substitution affects both k2 and Kd, as does geometric isomerism. Thus the introduction of an equatorial methyl group at position 4 (compare compounds C and A) produces a X6-fold decrease in k, and a 2.S-fold increase in Kd, the combined effect being a 9.9-fold decrease in ki. A second equatorial methyl group (i.e., at position 6: compound D) starts a trend toward increasing potency (comparing compounds D and C). In this case there is a 4.7-fold increase in
2-FLUORO,
469
2-0X0-1,3,2-DIOXAPHOSPHORINANES
kz and a 1.4-fold decrease in Kd, which combine to give a 6.6-fold increase in ki. The replacement of the equatorial methyl group at position 4 by an axial methyl group (compare compounds E and D) produces a further l.Cfold increase in k2, coupled with a 3-fold decrease in Kd, resulting in a 4.2-fold increase in Ici. The gem-dimethyl compound (B), (on the other hand) while giving the highest kz, shows poor affinity (high Kd) and is of intermediate overall potency, as indicated by its ki. Of interest in these compounds is the stereochemistry at phosphorus. Systems of this type which possess an electronegative phosphorus substitutent (fluorine in the present case), tend to occupy the axial position in a chair conformer (11) and this tendency has been rationalized recently on electronic grounds (12) :
very likely occurs as shown in Eq. [3] since this stereochemical course has been verified for-the
c31 chloro derivative methanol (15) :
of C in the presence of
c41 By this process, enzyme phosphorylation would produce a phosphorus stereochemistry in EPn and EPo which is isomeric with that in EPA, EPz, and EPz, owing to the conformational mobility of the phosphate ester rings in these cases along with the proclivity of alkoxy groups to be axial (11, 12):
I31 X-Ray diffraction experiments on a single crystal of B reveal a chair conformer with an axial fluorine, which also strongly suggests that the dominant conformer in solution resembles 1 rather than 2 (13). That compounds A-E all have axial fluorines (14) is indicated by the similar 31P-1gF coupling constants (ca. 1000 Hz) observed for B-E. Other NMR active substituents in related systems have demonstrated that these directly bonded coupling constants depend markedly on whether they are axially or equatorially oriented. It thus can be reasonably concluded that at least in the case of C and D which are conformationally locked by one and two equatorial methyl groups respectively, the EPX complex (Eq. [l]) probably contained the phosphorylating agent in the conformation represented by 1 shown in Eq. [a]. If this is the case, phosphorylation of the :nzyme’s hydroxyl function by C and D
Steric factors near the phosphorylation site within the enzyme, however, could favor the less sterically encumbered phosphorus stereochemistry represented by EPn in all cases. ACKNOWLEDQMENTS
We wish to thank Betty Wolfson for excellent technical assistance. This work WM supported in part by a grant from the National Cancer Institute (J. G. V.) and by Grant DAHCOQ 74G0237 from the United States Army Rssesrch Office (R. D. 0.). RBFERENCES
1. F.
H. Westheimer, Pseudo-rotation in the hydrolysis of phosphate esters, Ace. Chem. Res. 1, 70 (1968).
470
HART
2. 1. Ugi and F. Ramsey, Stereochemistry of five coordinate phosphorus, Chem. Brd. 8, 198 (1972). 3. Y. Ashani, S. L. Snyder, and I. B. Wilson, Inhibition of cholinesterase by 1,3,2-dioxaphosphorinane a-oxide derivatives, Bioch~mislry 11, 3518 (1972). 4. G. J. Hart and R. D. O’Brien, Recording spectrophotometric method for determination of dissociation and phosphorylation constants of the inhibition of acetylcholinesterase by organophosphates in the presence of substrate, Biochemistry 12, 2940 (1973). 5. G. J. Hart and R. D. O’Brien, Stopped-flow studies of the inhibition of acetylcholinesterase by organophosphates in the presence of substrate, P&c. Biochem. Physiol. 4, 239 (1974). fi. G. J. Hart, The determination of dissociation and phosphorylation con&ants for the inhibition of cholinesterases by organophosphorus esters in the presence of substrate, Ph.D. Thesis, Cornell University, 1974. 7. G. J. Hart and R. D. O’Brien, Dissociation and phosphorylation constants for the inhibition of acetylcholinesterase by a series of novel O-ethyl 0-phenyl S-n-propyl phosphorothioates, Pestic. Biochem. Physiol. 6, St5 (1976). 8. D. W. White, R. D. Bertrand, G. K. McEwen, and J. G. Verkade, Structural implication of nuclear magnetic resonance studies on l-lt-lphospha-2,6,-dioxacyclohexanes, J. Amer. Chem. Soe. 92, 7125 (1970).
ET AL.
9. R. Schmutzler, Synthese and Koordinationschimie der Fluorophosphite, Chcm. Ber. 96, 2435 (1963). 10. J. R. Cox, Jr. and F. H. Westheimer, The oxidation of trisubstituted phosphites by dinitrogen tetroxide, J. Amrr. Chcm. SW. SO, 5441 (1938). 11. J. G. Verkade, Interplay of st,eric and electronic influences in the chemistry of monocyclic and bicyclic phosphorus rstrrs, Plenary Lecture, 5th International Conference on Organic Phosphorus Chemistr~~. Gdansk, Sept., 1974; Phosphorus, to appear. 12. R. F. Hudson and .J. G. Verkade. The conformation and reactivity of 1,3,2dioxaphosphorinanes, Tdrahactron, Mt. 323 I (1075) and references therein. 13. D. 8. Mlbrath, J. Springer, J. C. (‘lardy, and J. Cr. Verkade. Crystal and molecular structure of 4,6-dimethyl-2-fluoro-2-r~xo-l,3,2-dioxaphosphorinane, to appear. 14 In t,he case of E, a “twist-boat” conformation is not unlikely in order to relieve 1-r: diaxial interaction of fluorine wit,h :I methyl substituent. Such a conformation st,ill permits the fluorine to be pseudo axial, howcvf~r. 15. W. Stec and M. 41. Mikolajcq.k, Stereochemistry of organophosphorus cyclic (‘( mpounds. II. Stereospecific synthesis of’ c+s arltl /runs 2hell,geno-2-oso-4-mdhyl-I ,:1,P-tiioxaphosphormans and their chemic*al tr:rrl~ir)l,rnations. 7’elruhdron 29, 539 (19733).