ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 93, 4 6 9 - ~ 7 5 (1961)

Hydroxamic Acids: Relationship between Structure and Ability to Reactivate Phosphonate-lnhibited Acetylcholinesterase

GERALD GILBERT, 1 T I t E O D O R W A G N E R - J A U R E G G 2 AXD GEORGE M. S T E I N B E R G a

From the Research Directorate, United States Army Chemical Research and Development Laboratories, Army Chemical Center, Maryland ~

Received February 10, 1961

A series of hydroxamic acids have been examined for their ability to reactivate aeetylcholinesterase inhibited by isopropyl methylphosphonofluoridate. The active compounds fall into two structurally distinct groups, neither of which is closely related to the natural substrate of the enzyme.

Reactivation of the inhibited enzyme appears to 4nvolve a number of sites around the active center which absorb and orient the reactivators. Unless the specific structural requirements for this absorption are met, no reactivation occurs.

INTRODUCTION

During the past decade, there have been a large number of studies concerned with the elaboration of the gross structure, the active center, and the reactivity of the en- zyme acetylcholinesterase (ACHE). The subject has been extensively reviewed (1). These studies have been motivated by the major importance of this enzyme in the process of synaptie transmission and also by its vital role as one of the focal points of at tack by the family of highly toxic organo- phosphorus compounds which include toxic chemical agents and insecticides.

The most generally accepted view of the catalytic center of AChE is tha t of Wilson and Bergmann (2), who postulated that this region contains two important spatially separated elements or sites which they des- ignated as "anionic" (negatively charged)

1Present address: Applied Research Labora- tory, U. S. Steel Corporation, Monroeville, Penn- sylvania.

Present address: Siegfried Ltd., Zofingen, Swit- zerland.

To whom requests for reprints should be sent. 4The experimental results in this paper were

obtained several years ago, but its publication has been delayed for various reasons.

and "es terat ic ," whose respective functions are to bind the substrate and to cleave the ester linkage. The sites have been caleu- lated to be separated by a distance corre- sponding to tha t separating the quaternary and ester groups in its natural substrate, acetylcholine. The importance of the bind- ing site is emphasized by the ability of many simple quaternary ammonium com- pounds, which are themselves incapable of being hydrolyzed, to inhibit the esterolytic action of the enzyme. Also, the importance of this site is demonstrated by the higher rate of hydrolysis of esters containing suit- ably spaced quaternary ammonium groups over those of isosterie structure which lack the positive charge (3).

I t has been shown through the use of p32_ labeled compounds that inhibition of AChE from the electric eel and other sources by diisopropyl phosphorofluoridate (DFP) and isopropyl methylphosphonofluoridate (GB) results in a firm binding between the en- zyme and the phosphorus-eontaining group, persumably at the esteratic site (4), and also that upon reactivation there is a direct correlation between release of protein- bound phosphorus and return of enzymic

469

470 GILBERT, WAGNER-JAUREGG AND STEINBERG

ac t iv i ty (5). The pos tu la ted react ions are given in Eqs. (1) and (2).

R2P(O)F + E -> R~P(O)--E + F- (1)

R.~P(O)--E + reaetivator --> E + products (2)

where E = enzyme. I t would thus appear l ikely t h a t the re-

act ion be tween reac t iva to r s and the in- h ib i ted enzyme occurs a t the ac t ive center of the enzyme2 There fo re an inves t iga t ion of the re la t ionship be tween s t ruc ture and r eac t iva t ing ab i l i ty of a f ami ly of rapid reac tan t s for organophosphorus compounds should provide in fo rmat ion which will con- t r ibu te to the u l t i m a t e e luc ida t ion of the na tu re and pos i t ional re la t ionship of the chemica l groups present at this center.

In the present work, a series of hydrox- antic acids have been examined for thei r ab i l i ty to r eac t i va t e A C h E inhib i ted by G B 2

EXPERIMENTAL

A. REAGENTS

All reagents, with the exceptions noted below, were obtained commercially and were of the high- est purity available. The GB was obtained from the Chemical Research Division of these labora- tories in purity greater than 95%.

The electric eel cholinesterase was a highly purified preparation prepared by Dr. D. Nach- mansohn. It had an activity of about 30 g. acetyl- choline hydrolyzed/hr./ml, and contained approxi- mately 3 mg. protein/ml. This solution was lyophilized and dissolved in one-fifth of the origi- nal volmne of water, and this stock solution was kept in the frozen state when not in use. Its con-

Although this is a most likely explanation, one cannot exclude the possibility that return of ac- tivity results from a physical reorientation of the enzyme after removal of the blocking group; in this case the phosphoryl (or phosphonyl) group may be located at a position remote from the catalytic center. Alternatively, inhibition may have resulted in the disruption of this center and separation of its two sites. In any ease, reactiva- tion studies may be expected to yield information on the features of this particular area of the in- hibited enzyme.

s For a detailed bibliography of prior studies of the reactivation of phosphorylated AChE with hy- droxamic acids and other reagents, as well as of the reaction of these reagents with the organophos- phorus inhibitors, see Refs. (la, 6).

centration was computed by Dr. H. O. Michel of these laboratories on the basis of radioactive DFP binding to be approximately 2.8 × 10 -5 M (or equivalent).

B. HYDROXAMIC ACIDS

Reactions were run with the object of obtain- ing highly purified compounds rather than high yields. Hence the yields aetuaUy obtained are not meaningful and are not reported. The hydroxamic acids whose preparation is described below have not been reported previously.

3-Hy droxycar b amoyl- 1 -b enzylpyridinium Bromide (VI)

A solution of 8.55 g. benzyl bromide (0.05 mole) and 6.95 g. nieotinohydroxamic acid (0.05 mole) in 55 ml. of 80% ethanol was heated for 2V2 hr. on a steam bath. The product which precipitated upon addition of ether was recrystallized several times from ethanol, m.p. 100-105 ° dec.

Anal. Calcd. for C1,Ha3N~O=Br: C, 50.5; H, 4.2; N.E. (neutral equivalent), 309. Found: C, 50.1; H, 4.6; N.E., 320.

1-Hy droxycar b amoylme thylpyridinium Chloride (IX)

An equimolar quantity of 1-carbethoxymethyl- pyridinium chloride (7) was added to a solution of hydroxylamine in absolute ethanol. (This was prepared in situ by neutralization with sodium ethoxide to phenolphthalein end point of a solu- tion of hydroxylamine hydroehloride. Precipitated sodium chloride was removed prior to use.) After incubation for 4 hr. at room temperature, the pre- cipitated product was filtered and recrystallized from aqueous ethanol, m.p. 184-5 ° dec.

Anal. Caled. for CrHgO2N2CI: C, 44.6; H, 4.81; N.E., 188. Found: C, 44.2; H, 4.9; N.E., 189.

Hydroxycarbamoylmethyltrimethylam- monium Chloride (XI)

1. From Carbethoxymethyltrimethylammonium Chloride. Equimolar quantities of the quaternary ester and hydroxylamine were dissolved in abso- lute ethanol and stored overnight at room tem- perature. The solid which precipitated was reerys- tallized from 90% ethanol, m.p. 167-9 ° dec.

Anal. Calcd. for C~H18N20~Cl: N, 16.6; C1, 21.0; N.E., 168. Found: N, 16.4; C1, 21.1; N.E., 166.

2. From a-CMoroacetohydroxamic Acid. A so- Iution of 0.8 g. a-chloroacetohydroxamie acid and 0.5 g. trimethylamine in 10 ml. of absolute alcohol was stored at room temperature for 4 days. The

H Y D R O X A M I C ACIDS 471

product was precipitated with ether and recrystal- lized from 90% ethanol, m.p. 166.8 ° dee.

Anal. Calcd: C1, 21.0. Found: CI, 20.6.

2-Hydroxycarbamoylethyltrimethylam- monium Chloride (XII)

After several unsuccessful a t tempts to carry ou~ the reaction between the corresponding quaternary methyl ester and hydroxylamine under alkaline conditions, the reaction was successfully run in aqueous solution at pH 5.0, employing about four t imes the stoichiometric amount of hydroxylamine in the presence of the hydroehloride. After incu- bat ion at room tmperature for 3 days, the water was vacuum stripped and the residue recrystallized from ethanol containing a small amount of water, m.p. 175-7 ° dec.

Anal. Calcd. for C6tI~N~O~CI: C, 39.5; H, 8.3; N, 19.4; N.E., 182. Found: C, 39.6; It , 8.4; N, 19.0; N.E., 182.

8-Carbethoxypropyltrimethylammonium Chloride

A solution of ethyl -y-chlorobutyrate (5 g., 0.032 mole) and t r imethylamine (2.95 g., 0.05 mole) in 15 ml. ethanol was heated at 60°C. for 2 days in a sealed container. The solvent and excess amine were removed at reduced pressure, and the residual solid was recrystallized from ethanol-e thyl acetate (1.2 g., m.p. 95-7°).

Anal. Caled. for C~H~0NO~CI: C1, 16.8. Found: C1, 16.9.

3-Hydroxycarbamoylpropyltrimethylam- monium Chloride (XIII)

A solution of 3-carbethoxypropyltr imethylam- monimn chloride in methanol containing a twofold excess of hydroxylamine was stored overnight at room temperature, heated on a steam bath for 5 hr., and the reaction product precipitated by addi- tion of ethyl acetate; recrystallized from ethanol- ether, m.p. 185-6 ° dec.

Anal. Calcd. for CcH~¢N~02Cl: C, 42.7; H, 8.7; N.E., 197. Found: C, 42.9; H, 8.8; N.E., 198.

Ethyl N-Hydroxyoxamate (XV) Equimolar quantit ies of diethyl oxalate and

hydroxylamine were dissolved in ethanol and stored for several hours at 5-10°C. The residue which remained after the solvent was vacuum stripped was recrystallized from ether-ethanol , m.p. 85-7%

Anal. Calcd. for C4HTNO~: C, 36.1; tI, 5.3; N.E., 133. Found: C, 36.1; I-I, 5.2; N.E., 133.

Picolino-, nicotino-, and is0nicotinohydroxamie acids and their methiodides were prepared by Dr. R. E. Plapinger and Mr. O. O. Owens. Compounds

VII I and X were kindly supplied by the Hoff- mann-La Roche Company. Dr. B. E. Haekley, Jr., prepared oxalohydroxamic acid (XIV) .

The pK~ values of the acids were determined as the pH of half-neutral ization of the acid on po- tentiometric t i t rat ion with 0.1 N NaOH in aqueous solution.

C. Reactivation Method A weakly buffered s tandard solution of AChE

(2.8 × 10 -8 M) was prepared by the addit ion of 10 M. of stock AChE to 10 ml. of Veronal (0.02' M), KC1 (0.3 M) and gelatin (0.1%) adjusted to pH 7.4. A solution of inhibi ted enzyme was pre- pared by incubation for ½ - 1 ½ hr. of 2 ml. of the standard solution with 10 ~1. of 1.96 × 10 -~ M GB, enzymic activity having been reduced to 0-6% of the original. For reactivation study, 0.2 ml. each of inhibited enzyme solution and 0.02 M hydrox- amic acid were mixed and incubated at pH 7.4, 25°C. for 30 rain. The 30-rain. reactivation t ime was selected since it was convenient and experience showed tha t much longer t imes did not show sig- nificant differences.

The extent of inhibit ion of the untreated en- zyme was determined for each hydroxamic acid by an identical 30-min. incubation of hydroxamie acid with uninhibi ted enzyme.

For measurement of esteratic activity 0.10 mh of test solution was added to 2.9 mI. of acetyl- choline (7.3 × 10 ~ M) , KCI (0.3 M) , Veronal (0.0023 M) and gelatin (0.1%) at pH 7.4, 25°C., and standard sodium hydroxide solution was then added by microburet as required to mainta in con- s tant pH. The rate of addit ion of alkali is a meas- ure of AChE activity. 7 The extent of reactivation was then calculated from the expression :

[E] [EGR] X [ ~ - [EG]

% Reac t iva t ion = X 100 [ E l - [EG]

where, E G R = activity of react ivated enzyme; E : activity of enzyme (control) ; E R ---- activity of enzyme in the presence of react ivator; and EG = activity of enzyme after reaction with GB (inhibited enzyme).

An excess of GB was used in the preparation of inhibited enzyme, and this compound has been shown to react rapidly with hydroxamic acids (9) to yield the products of Lossen rearrangement: RNCO, R N I I C ( O ) N H R , R N H C ( O ) O N H C O R , and RNI-I2. I t seemed highly unlikely tha t the

7The method of Glick (8) was employed as adapted for microscale by J. H. Fleisher and It . O. Michel, Chemical Corps Medical Laboratories Research Report No. 112 (1952).

472 GILBERT, WAGNER-JAUREGG AND STEINBERG

products of this reaction would be inhibitory to the enzyme. Experimental verification for this as- sumption was obtained with two of the test com- pounds. A mixture containing 10 ml. of 0.02 M hydroxamic acid, 1 M. GB, and 1 mh water was adjusted to pH 7.4, allowed to stand for 3 days, and tested for inhibiting action, as described above. Activity was 90% of control value for the products of reaction of XI and 95% for I.

The values for reactivation and inhibition are given in Table I.

RESULTS AND DISCUSSION

Reactivation results and pK~ values for 15 hydroxamic acids are presented in Table I. These include ten compounds containing the quaternary nitrogen function, both ali- phatic and heterocyclic, in which the spac- ing between the positively charged group and the bydroxamic acid group ( - - C ( O ) - NHOH) spans the corresponding distance in acetylcholine and in V which is a well- known reactivator ; and also five compounds which contain no positive charge. Of these 15 compounds, only four, I, V, VI, and XIV show marked activity.

I t is noteworthy that the range in ob- served reactivating activity is very wide. This is particularly striking if one compares the reactivation reaction with the corre- sponding displacement reaction between hy- droxamic acids and GB, in which fluoride ion is displaced instead of enzyme. In this ease the variations in reaction velocities are less than threefold. One also observes a smooth curvilinear relationship between re- action rate and pK~ of hydroxamic acid with a maximum rate for the hydroxamic acid of pK~ = pH + 0.6 (10). No such re- lationship is evident in reactivation of the inhibited enzyme.

Before it could be concluded that the dif- ferences in behavior of hydroxamic acids toward the P-F and P-enzyme linkage are due to the steric and coulombic effects in the latter reaction, it was necessary to es- tablish that the products of the reactivation reaction were not inhibitory to the active enzyme. This possibility could be experi- mentally examined since the products of re- action between GB and hydroxamie acids should contain all of the components which result from the enzyme reactivation reac-

tion. As indicated in Experimental, the products of the reaction of GB with com- pounds I and XI even at much higher con- centrations than those attainable in the re- activation reaction produced little if any inhibition.

Thus, we may conclude that the differ- ences in reactivating ability among the hy- droxamic acids are primarily related to very specific differences in affinity for the enzyme at the specific site or sites involved in the reactivation reaction.

The data also indicate that the reactiva- tion reaction does not require a molecular configuration similar to acetyleholine, the natural substrate of ACHE. This is evi- denced by the similarity of XI, XII , and X I I I to acetylcholine and by the observa- tion that none of these substances is an ef- fective reactivator.

(CHa) aN CH2CH2--C--NHOttJ

"- O ÷

L(CHa)aNCH2C}I2CH2__~__NI_IOI_II C1- XIII

c u " /I c1

aeetylcholine chloride

The distances between the quaternary group and the hydroxamie acid function in XI. XII , and X I I I also cover the range in whicl~ V must fall. The fact that V is a very effec- tive reactivator cannot therefore be ex- plained on the basis of an optimmn distance between groups. Furthermore, the reactivat- ing ability is not related to the size of the hy- droxamic acid molecule since VI, in which the mel, hyl group of V is replaced by the bulkier benzyl group, is still a very effective reactivator.

O LI CNHOH

,I !

/ I - CH3

V

HYDROXAMIC ACIDS

TABLE I

REAC~iVATION OF ISOPROPYL MET~IYLPHOSPHONYLATED EEL ACETYLCHOLINESTERASE BY H'ZDROX•MIC ACIDS, 25°C., pH 7.4

473

Compound pKa Reactivation a Inhlbition a

% % J ~

~N)~CONHOH

Pieolinohydroxamie acid

II

\ N y

Nieotinohydroxamic acid

I l l

Isonicotinohydroxamic

IV

CONHOH

' ~ N / /

acid

r I - CH~

2-Hydroxyearbamoyl- 1-met hylpyridinium iodide

CONHOH

I I - CH,

3-Hydroxyearbamoyl- 1-methylpyridinium iodide

VI (/ I--CONSOH "%I~ j Br-

I / ~ x , CH~-- ~ _ ~

3-Hydroxyearbamoyl- 1-benzylpyridinium bromide

CONHOH E

~I~//" I - I

CH3

4-Hydroxyearbamoyl-l-met hylpyridinium iodide

8.7 51 0

8.3 4 (1 hr.) 0

7.8 0 0

5.5 0 0

6.5 40 0

6.4 28 20

6.3 0 0

474 GILBERT, W A G N E R - J A U R E G G AND S T E I N B E R G

TABLE I--Continued

Compound pKa Reactivation a Inhibition a

% %

V I I I ~ C O N H O H

CI-I~

1 , 6 - D i m e t h y l - 3 - h y d r o x y c a r b a m o y l p y r i d i n i u m iodide 6.0 0 0

ix /\11

I C I t 2 C O N H O H

CI-

1 -Hydroxyearbamoylmethy lpyr id in ium chloride 7.2 2 0

x Q\IIoH ooN o N I - c H3

1-Me~hyl-3-hydroxycarbamoylmethylpyr id in ium iodide 8.4 0 4

X I + (CH3)~NCH2CONHOH 7.0 3 14

C1-

I t yd roxyea rba lnoy lme thy l t r ime thy lammonium chloride

X l I + (CH3)sNCH2CH2CONHOH 8.4 0 0

C1-

2 -Hydroxyca rbamoyle thy l t r ime thy lammonium chloride

X I I I + (CH~) 3N CIt 2Ctt 2CH 2CON HO H 8.9 0 0

C1-

3 -Hydroxyca rbamoylp ropy l t r ime thy lammonium chloride

X I V CONHOH 6.9 (pK1) 17 0 i CONHOH

Oxalohydroxamie acid

XV CONHOH I COOC2H5 6.9 0 0

E t h y l N-hydroxyoxamate

t = 30 m i n .

HYDROXAMIC ACIDS 475

Among the four compounds having the greatest react ivat ion rates there is no single steric or electronic configuration tha t is consistent with the binding requirements for a single site on the enzyme. However, if one will consider the possibility tha t two different binding sites are available each of which is properly positioned to assist in the a t t a ck of the hydroxamic acid moiety on the phosphonyl group, a consistent picture .can be developed. Thus, one site m a y be de- fined which will bind the moiety X V I and .a second which will bind X V I I ; compounds

O

~NHOH

H I R

XVI XVII

V and VI would fall into the first category, and I and X I V (in its tautomeric hydroxi- mic form) into the second.

We would like to suggest further tha t the number of binding sites need not be limited to two. In fact, there may be a greater var ie ty of sites grouped around the center of chemical reaction. Recent studies have in- dieated tha t most proteins are highly ori- eared structures even when present in solE- tion and tha t maior portions exist in a helical arrangement. See, for instance, Dory, e t al. (11).

Because of the diversity in size, charge, polarity, and chemical affinity of the chem- ical groups at tached to the helical poly- amide chain, we may visualize tha t the re- action site is surrounded in a three-dimen- sional a r ray by binding groups of a multiplici ty of types. Whether or not these groups will function in orientation will de- pend upon the substrate. Those substrates which do not satisfy the steric and polar re- quirements for a good fit will be excluded. However, since there are a nmnber of diree- tions from which the reaction site may be approached, a substance which fits the structural requirements for any one of these

and in addition contains the chemically active group may be expected to exhibit re- activity. Thus, the substrates for an en- zymic reaction, or react ivat ion of an en- zyme, may be expected to fall into c l a s se s or families. This assumption appears to be verified by the results of this investigation.

ACKNOWLEDGMENTS

The authors are indebted to Drs. H. O. Michel and B. J. Jandorf for valuable comments; to Dr. R. E. Plaloinger, Mr. O. O. Owens, and Dr. B. E. Haekley, Jr., and the Hoffmann-La Roche Com- pany for supplying chemical samples; and to Messrs. J. Snyder and V. Vely for certain of the reactivation data.

REFERENCES

1. (a) DAVIES, D., AND GREE~', A. L., Advances in Enzymol. 20, 283 (1958); (b) ALDRIDGE, W. N., Ann. Repts. on Prog. Chem. (Chem. Soe. London) 53, 294 (1956); (e) WACNER- JAUREGG, T., Arzneimittel-Forsch. 4, 527 (1954); (d) BALLS, A. K., AND JANSEN, E. F., Advances in Enzymol. 13, 333 (1952); (e) NACI-IMANSOJ-IN, D., AND WILSON, I. B., Ad- vances in Enzymol. 12, 259 (1951).

2. WILSON, I. B., AND BERGMANN, ]~., J. Biol. Chem. 186, 683 (1950).

3. ADAMS, D. }I., AND WttITTAXER, V. P., Biochim. et Biophys. Aeta 49 543 (1950).

4. BOURSNELL, J. C., AND WEBB, E. C., Nature 164, 875 (1949); MICHEL, I-I. 0., AND KROP, S., J. Biol. Chem. 190, 119 (1951) ; MICHEL, tI. O., Federation Proe. 11, 259 (1952); JAnSEn,, E. F., JANG, R., Ah'D BALLS, A. I~., J. Biol. Chem. 196, 247 (1952).

5. JANDORF, B. J., CROV~'ELL, E. A., AND LEVIN, A. P., Federation Proe. 14, 231 (1955).

6. GREEN, A. L., AND SMITH, H. J., Biochem Y. 68, 28 (1958); GREEN, A. L., AND S.~ITH, It. J., Biochem. J. 68, 32 (1958); WA~NER-JAUREGG, T., Arzneimittel-Forseh. 6, 194 (1956).

7. MELNIKOV, N. N., SUKtIAREVA, M. D., AND ARI~IPOW, O. P., Zhur. Pri/dad. Khim. 20, 642 (1947) ; C. A. 43, 6977b (1949).

8. GLICK, D., J. Gen. Physiol. 21, 289 (1938). 9. I-IAcKLEY, B. E., PLAPINGER, R., STOLBERG, M.,

AND WAGNER-JAUREGG, T., J. Am. Chem. Soc. 77, 3651 (1955).

10. SWIDLER, R., PLAPINGER, R. E., AND STEINBERG, G. M., J. Am. Chem. Soe. 81, 3271 (1959).

II. DOTY, P., IIOLTZER, A. M., BRADBURY, J. H., AND BLOUT, E. R., J. Am. Chem. Sac., 76, 4493 (1954).