ftD-R192 561 RCETYLCHOLINESTERRSE AND RCETYLCHOLINE RECEPTOR(U) 1/19RIIEIS UNIV UALTHRM HR DEPT OF CHEMISTRY S G COHEN21 JAN 06 DAM17-93-C-325i

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Annual Report

Saul G. Cohen, Ph.D.

January 21, 1986

Supported by

U. S. ARMY MEDICAL RESEARCH AND DEVELOPMENT COMMCANDFort Detrick, Frederick, Maryland 21701-5012

Contract No. DAMD17-83-C-3251 1DTICBrandeis University

Department of ChemistryWaltham, Massachusetts 02254

Approved for public release; distribution unlimited

The findings in this report are not to be construedas an official Department of the Army position

unless so designated by other authorized documents.

a, I

SIECURITY CLASSIMATION OF TkiS PAGE

REPORT DOCUMENTATION PAGE O O nI

Ia. REPORT SECuRITY CLA.SSIICATION lb RESTRICTIVE MARKIN4GSUnclassified_________________________

Ia. SECUITY CLASSIFICATION AUT4HORITY 3 OISTRIBU TION/ AVAMASIUTY OF REPORTApproved for public release; distribution

2b. OEO.ASSIPIA?*tiONIO*NGRAOIN S"O4E l unlimited

4. PERFOINW ORGANIZAT"O REPORT NUMBERIII(S) S MOITORING OOGANIZATION REPOfT NUMNER()

ft. 01A OF PERfORMIN ORGANdIZATION 6b. 1144a SYMBOL 78. NAME Of MONTORIG 0RGANUZTION

Brandeis University O ~at

)k A00NESS (Our. Raft and 2W Cod) 7b. AOORIESS (Oiy. Semee. ad 20 C.*)

Waltham, Massachusetts 02254

I&. NAME 0OF UNORINGSPONSORING ft. OFFICE SYMOOL I PSOCUREMENT INSTRumENT iIENTIFCAT1ON NUMBER1OWAIZTI010U.S. Army Medicalj(f&vat AM1-3C35

Ressrch DAN 17-83-c-t 3251aDL AMSS .Rft. W A Cod) 1 SOURCE OF PLACING NUMBERS

Fort Detrick ELNT"

Frederick, Maryland 21701-5012 6LI112AO 00 3Ml- [o.

11. TT *Aha SKaWu amucsesACETYLCUOLINESTERASE AND ACETYLCHlOLINE RECEPTOR

12. PERSONAL AUTHORMSCohen, S.G.

I is. TYPE OF xROfrT 113b. TOM COVERED I1 DT Of REPORT IVW I @W D 60 I PAGE COUT

Annual JU PUMd..ITOJ.~S14 1986 JAMUL.2"I 316. SUPPLMEN01RTARY NOTATIOIN

17 COSAri COoES Ia. RSBEC TEim"Scot on' mme dI meggo, ww eatfp IVho f

FIEILD GROUP SU4FOUP Acetylcholinesterase.Active site.Substrates.Inhibitors.

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we are studying the properties of the active site, and the regionperipheral to it, of acetylcholinesterase (AcChB), the enzyme whichhydrolyzes and thus terminates the action of the important neurotrans-mitter acetylcholine (AcCh). We have studied the response of the enzymeto synthesized substrates and inhibitors of varied structure for indica-tions about the parts of the enzyme that are complementary to thesesubstrates. From this information radioactive active-site-directed irre-versible inhibitors have been designed for use in labeling amino acids inthe active site. In this work AcChl was isolated from In1ked n~jI2J~An.Novel uncharged reversible inhibitors were also prepared; they may havepotential application. Compounds were also prepared for study of theiraction on the AcCh receptor by Professor J.B. Cohen, Department of Anatomyand Neurobiology, Washington University School of Medicine. St. Louis. .

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3

SUM0ARY

Appropriate literature and work in our laboratory are

reviewed, which have led to our proposal that the part of theactive site of AcChZ at which the cationic p-substituent ofAcCh binds is not anionic, as is qenerally accepted, but isuncharqed and complementary to the spherical shape of thesubstituent. Since there are anionic charges peripheral to theactive site with which cationic reagents, intended to label anamino acid in the active site, may react, we propose to useuncharged active-site-directed labeling reagents. 1-Bromo-pinacolone, DrCHCOC(CH,)3 (BrPin) has been selected as thefirst such reagent for detailed study.

AcChZ was isolated from T D9j A, and its inacti-vation by 3 H-DFP and 5rPin has been studied. The procedure forsynthesis of BrPin on a 60 poole scale, from (i) reaction ofCM1 COI and (C) 3 COqCl to form pinacolone, CW1 COC(CH )3, (ii)followed by bromination is being worked out, for application tosynthesis of BrCa IICOC(C, )3 (1 'C-BrPin) .

Properties of the binding site for the p-substituent havebeen examined further by study of reversible inhibitorscontaininq(C),Si-, (CII) 2 N(0")-, and COIS(O)- groups. Thesite binds (C.) 35i- and OI3S(O,)- groups as well as it does(CH,),C-, but the N' (0-)- group decreases binding. Thelatter substituent, as well as CHiS(O3)-, CN3 S(O)- and CI3S-decrease substrate reactivity.

Nono- and di-substituted benzene derivatives bindreversibly to AcChI. Binding is increased by electronwithdrawinq and (CI,)2 N- substituents, and is described by theHammett relationship, with a value of p of 2. An aryl bindingsite is proposed adjacent to the trimethyl and esteratic sites.Strong synerqism is found in effects of meta-tert-butyl andmeta-trimethylammonio groups with phenolic hydroxyl group.

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FOREWORD

Citations of commercial organizations and trade names in thisreport do not constitute an official Department of the Armyendorsement or approval of the products or services of theseorganizations.

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5

Table of Contents

Page

Sumary 3

Foreword 4

ReportStatement of the Problem Under Study 7Background and Review of Appropriate Literature 7Rationale for the Current Study 10Experimental Methods 11Results

A. Inactivation of AcChZ 11B. Synthesis of BrPin, 60 plole Scale 13C. Sulfonyl, Sulfonate and Related

Compounds 15D. Reversible Inhibition by Derivatives

of Bensene and Phenols 16Discussion and Conclusions 18Table I. Hydrolysis of X-CH2 CH2 OCOCH3 by

Acetylcholinesterase (pH 7.8, 25"C, 0.18 N NaC1)and by Hydroxide 20

Table II. Reversible Inhibition of Hydrolysis byAcetylcholinesterase of Acetylcholine and3,3-Dimethylbutyl Acetate, in 0.18 N NaCi, pH 7.8,at 25"C 21

Table III. Inactivation by Sigma AcChE ny MEN 3H-DFP 22Table IV. Reversible Inhibition of Hydrolysis ofAcetylcholine by Acetylcholinesterase, pH 7.8,256C, 0.18 N NaCl 23

Table V. Competitive and Uncompetitive Inhibitionby Benzene and Substituted Benzenes of Hydrolysisof Acetylcholine by Acetylcholinesterase,in 0.18 N NaCl, pH 7.8, 25"C 24

Table VI. Reversible Inhibition by Phenols 26Figure 1. Normalized Reactivity of X-CHiCHOCOCH,,

log(k2 .)/k ),, and Apparent Molal Volume (V;,) 27Figure 2. Normalizedd Reactivity of X-CHCH2OCOCH3 ,

loq(k a,,/k,),, and Molar Refractivity (MR) 28References 29

Distribution List 33

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(1) Stat^ nt of the Problem Mnder Study

We are studying the properties of the active site, and theregion peripheral to it, of acetylcholinesterase (AcChE), theenzyme which hydrolyzes and thus terminates the action of theimportant neurotransmitter acetylcholine (AcCh). We havestudied the response of the enzyme to synthesized substratesand inhibitors of varied structure for indications about theparts of the enzyme that are complementary to these substrates.From this information, radioactive active-site-directed irre-versible inhibitors were designed for use in labeling aminoacids in the active site. In this work AcChE was isolated fromTormedn iana. Novel uncharged reversible inhibitors werealso prepared; such compounds may have permeation propertybetter than that of cationic inhibitors, and potential use asmedicinals.

(2) Maokqround and Review of Appropriate Literature

The structural features involved in the interactions ofAcCh, (CH 3),N*CH2 CHOCOCH3 , with AcChE are the three N-methylgroups, the positive charge, and the ester groups. The part ofthe active site of AcChE at which the trimethylammonium groupbinds has generally been depicted as anionic (1-3), with itsnegative charge increasing enzymic activity by attracting,binding, and orienting cationic substrates (4). It has beennoted that successive methylation of alkylamonium ions,starting with methylamine and ethanolamine, improves binding,and trimethylation of substrates starting with p-aminoethylacetate increases both binding and reactivity, and the methylgroups contribute more to binding than does coulombic attrac-tion (4-6). Thus, it has been proposed that less than 10% ofthe binding of tetraalkylammonium ions is due to their chargeand there is no negative charge in the "anionic" binding site(7). However, this calculation did not take into accountdecrease in solvation of ammonium ions with increasing methyl-ation (5), and the presence of a negative charge in the"anionic site" remained widely accepted (8).

Addition of methyl substituents in uncharged analogues ofthe O-ammonioethyl acetate, from ethyl propionate to 0,0-di-methylbutyl acetate, (CH 3)3CCH2 CH2 OCOCH3 (DMBAc), the carbonanalogue of AcCh, also led to progressively increasing reactiv-ity toward AcChE (1,9). Thus the rate constant for reaction ofDMEAc with AcChE is very high, 2 x 106 M-'sec-1 (10), thoughsomewhat lower than that for AcCh.

The p-amonio substituents increase the intrinsichydrolytic reactivity of such esters toward hydrolysis, by

8

hydroxide, as compared with the uncharged, carbon analogues.We have deemed it appropriate to apply normalization factors tothe enzymic hydrolysis rates, as has been done in studies ofchymotrypsin, which also reacts via an acyl-serine intermediate(11). This was done for a series of 14 #-substituted ethylacetates, X-CH2 CH2OCOCH 3 , with X = the four ammonio groups,H3N to (CH3 )3N4 , the analogous carbon substituents, and, CH30,HO, Cl, Br, N-C and H, Table I (12). The normalization factorswere the ratios of rate constants of base catalyzed hydrolysisof AcCh to each of the substrates. We then found that thenormalized enzymic reactivities [log(k2 (,)/K,), where k(,) isthe normalized acylation rate constant and K is the bindingconstant] for these compounds were proportional to the apparentmolal volumes (V;5 ) of the p-substituents, X, eq. 1, Figure 1.

log(k2 (n)/K.) = a V25 + C (1)

The enzymic reactivity of these acetate esters of widely variedstructure was accounted for by two factors (i) their intrinsichydroxide catalyzed reactivity and (ii) the volume of thefi-substituent and thus its fit into the active site, and theresulting effect on placing the ester group at the esteraticsite. K for AcCh and DMBAc are similar, indicating nosubstantlal coulombic effect, and the effects on binding in theother cationic and uncharged parts of substrates were evensmaller, Table I.

The effect of the positive charge in AcCh is to increasethe intrinsic reactivity over that of DMBAc by a factor ofabout 25, and the normalized reactivities are quite similar.No specific effect of anionic charge on the hydrolysis rate andthus no evidence for an "anionic" site is observed. On thisbasis we propose that the part of the active site at which thetrimethylammonio group of AcCh and the P-substituent of otheracetate substrates bind is not anionic, and is more accuratelyconsidered trimethyl, complementary to this character of theP-substituent of AcCh rather than to its positive charge.

Others have noted a relation between enzymic reactivityand the hydrobicity (w) of the P-substituents of alkyl esters,II, IV, VI and VIII (10). Hydrophobicity is a parameterderived from 2-octanol/water distribution coefficients; it isrelated to capacity for hydrophobic interactions and used indrug design (13). For these hydrocarbon substituents, valuesof * are proportional to volume, but use of this parameter doesnot allow the non-polar and the water-soluble ammonio deriva-tives to be correlated on the same scale. The correlation withvolume, Figure 1, does allow the reactivity of both classes ofsubstrates to be so correlated.

We then examined the effect of positive charge on bindingof reversible inhibitors, Table II (14). The enzyme has iso-electric point of about pH 5 (15,16) and thus excess negative

9

charge on its surface at pH 7-8. Cationic reversible inhibi-tors structurally related to AcCh do bind better than theiruncharged analogues by small factors, Table II, correspondingto about 1 kcal/mol of binding energy, much less than would becaused by interaction of (CH3 )3N+ - with a "contact" anionic 0-that is implied by a specific anionic site (14). Also, ionicstrength effects on binding and on hydrolysis have been inter-preted in terms of anionic charges on the enzyme surface, peri-pheral to the active site (16).

Each of the inhibitors which we studied, structurallyrelated to AcCh, whether neutral or cationic, showed essential-ly the same binding constant when retarding hydrolysis of AcChand its uncharged analogue, DMBAc. This indicated that the6-trimethylammonio and p-tert-butyl groups of the two sub-strates and of the related inhibitors bound at the same subsite(14). The substrate study had indicated that this subsite isuncharged trimethyl, and the nature of this site could then beexplored more specifically with uncharged reagents (12).Indeed, arylaziridinium reagents, intended to alkylate the"anionic" site and prevent substrate access, completelyinhibited hydrolysis only of cationic, but not of neutralesters (18,19). We took this to indicate, not that there areseparate anionic and neutral subsites, but that cationicirreversible inhibitors may react with peripheral anionicgroups, increasing positive charge and repelling cationicsubstrates and inhibitors; they modify the active site domainbut allow uncharged substrates to bind at the one trimethylsite and react, albeit at reduced rate.

This view was borne out in studies with 1-bromopinacolone,BrCH2 COC(CH3 )3 (BrPin) (20). This uncharged reagent inhibitedAcChE irreversibly with the same rate for hydrolysis of avariety of both cationic and neutral substrates, and itsinhibiting reaction was retarded by reversible quaternary andtrimethylammonio inhibitors to an extent appropriate to theirbinding constants. These results indicate that both classes ofcompounds bind at a single trimethyl site (20). BrPin is beingused to label the active site.

While we use the term trimethyl for the binding site forO-substituents of substrates and inhibitors related to them,this site also accommodates other groups. As described in theprevious Annual Report (21), trichloroethanol (Cl3CCH2OH) bindssimilarly to its carbon analogue, neopentyl alcohol, andchloral binds better than its carbon analogue, pivalaldehyde.In the latter case the effect of the chlorine in rendering thecarbonyl electrophilic may be important.

In an extension of the study of the relation of enzymicreactivity to the volume of the p-substituent of B-X-ethylacetate substrates, we substituted refraction volumes (MR) (22-25) for apparent molal volume (V;,) in eq. 1, allowing us to

10

examine a broader range of substrates containing more hydro-philic and larger p-substituents, Figure 2 (26). The largersilyl compound, (CH3 )3 SiCH2 CH2 OCOCH3 , was accommodated in theactive site, binding slightly better and with slightly greaterkc,, than the analogous carbon compound and, possibly, withslightly greater normalized reactivity than AcCh. Essentially,normalized reactivity levels off at the volume of the naturalsubstrate. On the other hand, hydrophilic p-substituents,methylsulfonyl (CH3 SO2 _-), dimethylamine oxide [(CH3 )2WN (0)-]and methylsulfoxy (CH, SO-) led to substantially lower reacti-vities than were consistent with their MR values. Howevertheir reactivities, like those of the reactive (CH 3)WN - and(CH3 )2 S'- compounds, were substantially higher than was consis-tent with hydrophobicity (r) (13,22,27-29), and reactivities ofthe cationic compounds correlated with MR. Overall, reactivitycorrelates with volume (V0 or MR) better than with hydrophobi-city.

It is noteworthy that the hydrophilic surface of the6-CH3 SO2- substrate did not decrease its binding as comparedwith that of the analogous (CH3) 3 C- compound (26). The effectwas on kct.1, indicating that desolvation, removal of water, is,paradoxically, an important aspect of enzymic hydrolytic react-ivity (30).

In the last Annual Report (21) we described results onother novel irreversible and reversible inhibitors; theseresults are summarized briefly here. Chloromethyl pivalate((CH 3 )3 CCO2CH2C1] and chloromethyl acetate (CH3COCH2Cl) mayalkylate a nucleophile in the active site; then, if the boundester group hydrolyzes, the hydroxymethyl substituent may reactfurther to alkylate again and possibly form a cross-link. Thetwo compounds appear to have similar inactivating effective-ness. Trimethylammoniomethyl acetate (CM3 CO2 CH2 N (CH3 )3] is acationic analogue which may behave similarly. It may behydrolyzed, as a lower homologue of choline, and may eject thepotential alkylating group. It seems to be a less effectiveinactivator than the chloromethyl compounds.

Methyl methanesulfonate and methyl benzenesulfonateapparently act as methylating agents. The benzene derivativeis of interest in comparison to styrene oxide, which mayintroduce an aryl group as it inactivates irreversibly, andalso in comparison to a series of substituted-benzene reversi-ble inhibitors, which act largely competitively and may bedirected against the active site. These compounds will bediscussed in the results section of this report.

(3) Rationale Used in the Current Study

(i) BrPin has properties of an active-site-directed

11

inactivator, since it is equally effective against substratesof widely varied structure and its own action is retarded bysubstrate-related reversible inhibitors (20). This year 14 C-labeled BrPin [BrCH21 4 COC(CH3 )3 ] has been examined as a radio-active label for the active site of AcChE. The enzyme wasisolated from Toredo, or eel enzyme was purchased from Sigmaand purified.

(ii) Properties of the subsite at which the fi-substi-tuents of P-X-CH2CH2OCOCH3 substrates bind have been exploredby study of the reactivity of such substrates (16). In thecurrent year they have been studied further with structurallyrelated reversible and irreversible inhibitors.

(iii) Simple benzene derivatives have been found toretard and accelerate hydrolysis by AcChE in a manner compara-ble to those of common cationic and our newly studied unchargedaliphatic inhibitors. Hence a more detailed study of aromaticinhibitors was performed this year.

(4) Experimental Methods.

Isolation of AcChE from frozen electric organ of Torpedon was described in the revious Annual Report (21).Literature procedures were essentially followed for enzymeisolation (16,31), for its purification by affinity chromato-graphy (32,33), for the Ellman assay (34), for enzyme charac-terization by gel electrophoresis (35,36), and examination ofgels labeled with [3H]diisopropylfluorophosphate (3H-DFP) [NewEngland Nuclear (NEN) Boston, Massachusetts] by exposure toX-ray film (37). pH stat kinetic studies were carried out asdescribed previously (12,14), the value of kca of 3.3 x 105min-' for Torpedo caiori was used (38).

Analysis for BrPin when synthesized on micromolar scalewas carried out by gas-liquid chromatography (glc) on 10% SE 30chromosorb WHP 100/120, 110"C, Perkin-Elmer 990 gas chromato-graph.

(5) Results

A. Inactivation of AcChE

Acetylcholinesterase, about 10 mg, was isolated fromelectric organ of Torpedo, purified as described in the previ-ous Annual Report (21), and stored in pH 8 buffer at -70"C. Ithydrolyzes AcCh with K, of 0.06 mM, compared with 0.33 mM forSigma eel enzyme. 1-Trimethylammonio-4-pentanone (TAP) has Ki

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12

of 0.05 mM as compared with 0.02 mM for the eel enzyme. BrPin,has Ki of 0.2 mM, the same as its K1 for eel enzyme.

Gel electrophoresis at high loading, >11 pg of protein,showed small bands at 133,000 D, 107,000 D, and 95,000 D, and amajor band at 70,000 D comprising about 75% of the total.Treatment with 1.3 AM 3H-DFP for 1 hr and autoradiographyshowed tritium incorporation at 70,000 D and 133,000 D, thelatter band apparently consisting of a dimer. Pretreatmentwith 1 mM carbamyl choline for 1 hr prevented incorporation of3H-DFP.

Inactivation by 3H-DFP was studied along with that byother inactivators, BrPin, methyl sulfonates, and styreneoxide, for information as to their mode of action, while thesynthesis of 14C-BrPin was being worked out. The course ofinactivation of Sigma AcChE by 3H-DFP was followed at 0 and25"C for guidance in study of the purer but less readilyavailable T enzyme, Table III. Since the 3H-DFP wasobtained in propylene glycol, the stability of DFP in thehydroxylic solvent and thus its true concentration was investi-gated. The correct concentration would be important in label-ing and counting experiments if the radioactive reagent wasdiluted with unlabeled DFP. A solution of unlabeled DFP inpropylene glycol was obtained from NEN for use in model experi-ments and found to have no inhibiting activity. We turned toneat DFP from Sigma and made our own dilutions. Concentrationsand times for half-inacitvation (t1 /2 ) of Sigma eel AcChE forthis DFP were: 48 MM, 2 min; 10 pM, 4 min; 2 AM, 18 min; 1 pM38 min; and 0.5 MM, 78 min. The value of t1 ,2 for a nominalconcentration of 2 AM 3H-DFP was 30 min, corresponding to aneffective concentration of 1.4 AM, 30% less than that indi-cated. This was confirmed by inactivation of Torpedo AcChE by3 H-DFP and 3:1 DFP:3H-DFP, with 3H-DFP content based on theeffective concentration, 70% of nominal. Observed counts afterdialysis were in the ration of 3.9:1.

Sigma AcChE was inactivated by 7 pM and 1.4 MM 3 H-DFP for1 hr and subjected to gel electrophoresis and autoradiography.The gels were streaky, with many bands and much low molecularweight material. The 7 pM 3H-DFP sample showed autoradiographbands at 57,000, 53,000, 34,000 and 31,000 D; the 1.4 pM samplegave labeled bands only at 57,000 and 53,000 D.

Sigma AcChE samples that (i) were 98% inactivated by 20 hrtreatment with 3.8 mM BrPin or (ii) were not pretreated withBrPin were treated with 51 MM 5:1 DFP:3H-DFP, containing 6.3 AM3H-DFP. Electrophoresis and autoradiography showed no 3H-DFPin the BrPin-pretreated AcChE, and incorporation of 3H-DFP inuntreated enzyme at 55,000 and 50,000 D.

Sigma AcChE was first exposed to (i) complete inactivationby treatment with 3.7 mM BrPin for 36 hr; (ii) 48% inactivation

13

by 2.5 mM BrPin treatment for 3.5 hr; and (iii) no preinactiva-tion by BrPin. Then incorporation of 3H-DFP into AcChE by 2:1DFP:3H-DFP, 46 pM containing 12 UM 3H-DFP, for 36 hr wasexamined. After dialysis, samples showed (i) 64,000 cpm, (ii)226,000 cpm and (iii) 355,000 cpm. If 64,000 cpm is due tonon-specific reaction of impure Sigma AcChE and incompletedialysis, subtraction leads to 162,000 cpm for (ii), 291,000cpm for (iii), giving a ratio of 0.56, consistent with 52%residual activity.

In a parallel experiment, Torpedo AcChE was subjected to(i) complete inactivation by treatment with 1.5 mM BrPin for 16hr, (ii) 53% inactivation by treatment with 0.88 mM BrPin for60 min, and (iii) no preinactivation by BrPin. Then incorpo-ration of 3H-DFP into T AcChE by 2:1 DFP:3H-DFP, 49 pM,containing 13 pM 3H-DFP, for 36 hr was examined. Afterdialysis, samples showed (i) 16,5000 cpm, (ii) 58,000 cpm and(iii) 204,000 cpm. Torpedo AcChE was more rapidly inactivatedby BrPin than was Sigma AcChE. The time course of inactivationby 49 pM DFP indicated that in (ii) 30 min would be requiredfor the remaining 47% inactivation by DFP. In this period theBrPin would contribute about 35% of the remaining inactivation,about 16% of total inactivation, leading to about 69% totalinactivation due to BrPin and about 31% due to 3H-DFP. Countsin (ii) are 29% of the counts in (iii), consistent with thiscalculation. However, if the counts in (i) are subtracted,42,000 cpm are left in (ii), 22% of the 188,000 cpm left in(iii), a poorer but not unsatisfactory correspondence in viewof the difficulty arising from comparable rates of inactivationdue to BrPin and 3H-DFP in this case. The total number ofcounts calculated for 6.15 x 10-11 moles of Torpedo AcChE usedin these. experiments, 100% inactivated by 3H-DFP, was 183,000cpm, to be compared with 204,000 cpm observed, and 188,000 cpmcorrected, and in the enzyme inactivated about 69% by BrPin,57,000 cpm calculated, 58,500 cpm observed, and 42,000 cpmcorrected. Our isolated TorSedg enzyme appears to be ratherpure, but very easily inactivated by BrPin. Pretreatment withBrPin or with other complete or partial inactivators leads toappropriate exclusion of 3H-DFP. This indicates that partialinactivation does not lead to enzyme that is modified peri-pherally to yield decreased activity and slower 3H-DFP incorpo-ration. Instead, these results are consistent with enzyme thatin part totally inactivated and in part totally active. Thismay be evidence for the specificity of action of BrPin.

B. Synthesis of BrPin, 60 pmole Scale

Many experiments were carried out on the micromole scalesynthesis of BrPin that would be required for synthesis of theradioactive material. Preliminary large-scale experiments werecarried out on 500 pmole of CH3 COCl and 400 pmole of 2 M(CH3 )3CMgCl in 200 pL of tetrahydrofuran (THF), to form

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14

pinacolone. A substantial by-product was detected, which wasidentified as tert-butyl acetate by its retention time andconversion by 6 M HCl to tert-butyl alcohol and tert-butylchloride. It is formed from CH3 COCl and (CH3 )3 COH, the latterbeing formed from the Grignard reagent and 02 or peroxidepresent in THF solvent. Air or peroxide and moisture must berigorously excluded in the small-scale synthesis by thisprocedure.

Other possible syntheses of pinacolone were examined:treatment of (CH 3 )3CMgCl with ethyl and methyl acetate, or withCdCl2 prior to treatment with CH3 COC1, failed to form pina-colone in good yield. Then it was found that CH3COCI and(CH3)3CMgCl, 60 pmole, in the presence of about 0.5 mg Cu2 Cl2catalyst, led reproducibly to pinacolone in >80% yield (39).However, Cu was found to inactivate AcChE instantaneously.Complexing with ethylenediaminetetraacetic acid (EDTA) retardedbut did not stop this inactivation; Cu , formed from Cu byair oxidation or in the subsequent reaction with Br2 , will haveto be removed completely from synthesized "4C-BrPin.

CH3 14COCd obtained from NEN in a sealed ampoule, wastransferred by pipette, with rinsing, to a Reacti-vial, whereit was treated with the Grignard reagent. Unlabeled CH3COC1 inTHF in the standard sealed ampoule, obtained from NEN for usein a model experiment, failed to yield the desired product. Wedecided to forego the NEN dilution with solvent and make ourown dilutions.

In a series of brominations of pinacolone on 20-400 #molescale it was difficult to achieve reproducibility. Room lighthad no effect. Added acetic acid and AlCl3 had no effect.Aqueous HCl prevented bromination. It was determined thatsmall amounts of water led to low or no yields of BrPin. Afterwashing to remove copper and magnesium salts present in thepinacolone synthesis, it proved difficult to dry THF, thesolvent in which (CH3 )3 CMgCl was obtained; this problem led toirreproducible results. The Grignard reagent was then obtainedin ether and the bromination proceeded well. Bromination withequivalent quantities of bromine and pinacolone leaves residualbromine, which inactivates AcChE instantaneously and must bewashed out. After the period covered by this report it wasfound that the mixture formed in the reaction of CHCOC1 withthe Grignard reagent could be brominated directly withoutwashing. Residual bromine, Cu and other salts can be washedout readily after the bromination. Successful micromolarsynthesis of BrPin, suitable for inactivation and labelingAcChE, was achieved. The use of CH3 1'COCl was worked out afterthe period covered by this report.

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15

C. Sulfonyl, Sulfonate and Related Compounds

In further study of the effects of charge, volume andsurface properties on binding at the trimethyl site, supple-mentinq our work on substrates (26), a series of relatedreversible inhibitors of AcChE was tested (40). The resultsare summarized in Table IV and as follows:

Sterically similar alcohols with tetra-substituteduncharged p-groups, (CH3 )3SiCHCH OH (I), (CH3 )3CCHaCH2OH (IA)and CHS(02 )CH CHOH (VII) bind similarly, K, - 3-9 mn, and thebinding of each is similar to that of the corresponding acetatesubstrate. Cationic analogues, (CH3 )3 NCHCH2OH (IB) and(CH3)S C CH 2 OH (II), bind similarly, Ki - 0.4 mM, have N,values similar to those of their acetate substrates, and bindmore strongly than the uncharged alcohols by about 1.5 kcal/mol.In comparisons of VII with CH3 SO2 CH3 , II with (CH )3S*, and IBwith (CH3 )W,, hydroxyethyl leads to more favorable bindingthan methyl by about 0.8 kcal/mol, despite lower hydrophobi-city. Two hydrophobic methyl groups (comparison of IA withn-butanol) and two hydrophilic sulfone 0 atoms, (comparison ofVII with 2-methylthioethanol) increase binding similarly, by1.0 kcal/mol. Conversion of (CH3 ) 3 S to (CH3 ) 3 S O alsoimproves binding. However, (CH3 N*O- does not bind to AcChE,and conversion of 1-dimethylamumonio-4-pentanone and 2-dimethyl-aumonioethyl acetate to their N-oxides, changes of IFH to" 1W (0) decrease binding by 1.5 kcal/mol. Although the -COCH3group in esters with strongly binding P-substituents makes es-sentially no contribution to binding over that of the alcohols,in esters with weakly bound O-substituents, (CH3 ) 2 N* (O")-,CH3NWH,-, CH3 S(O)-, CH3 CH -, and CH3 S-, binding is dominated bythe ester -COCH3 group, with values of K about 16 mM.

Inhibitors containing the methanesulfonyl group (CHA SO2 -),a polar analogue of the tert-butyl group, are being studied.The effectiveness of methanesulfonyl chloride (MSCl) is similarto that of methanesulfonyl fluoride (CH3SO2 F; MSF). Thesecompounds introduce methanesulfonyl groups. Irreversibleinhibition by MSF is not retarded efficiently by TAP. It isaccelerated by small quaternary a-monium compounds (41).Irreversible inhibition by methyl methanesulfonate (CH3 SO2 0 CH3 ;MKS), a methylating agent, is not retarded efficiently by(CH3)4 . Hydrolysis of methylsulfonylethyl acetate (CH3 SO2 -CH2CH2OCOCH3 ; MSAc) by AcChE is retarded efficiently by TAP.The small CH3SO2- inhibitors may not utilize the trimethylsubsite and thus their action may not be retarded, and may beaccelerated, by small cations. It is difficult to account forthe inefficiency in retardation by TAP of inhibition by methane-sulfonyl compounds.

Methyl benzenesulfonate (C. H5 SO2 OCH3 ; MBS), a methylatingagent, at 3 mM inactivated AcChE 70% in 1 hr and 90% in 2 hr;

this inactivation wasn not retarded by 29 mH (CHI ),NW Benzenederivatives related to 113 are reversible inhibitors with sub-stantial competitive activity. It appears that the active sitecan accommodate the (Cli),r at the trimethyl subsite, and thebenzenesulfonyl group at another site.

While simple tert-butyl and trichioromethyl compounds weremoderately effective reversible inhibitors, tert-butyl isothio-cyanate and trichioromethylethylene oxide were not effectiveirreversible inhibitors, unlike the related compounds BrPin andstyrene oxide.

Although methylsulfonylethanol and its acetate bound toAcChE similarly to the analogous tert-butyl compounds, 1,3-bis-methylsulfonyipropane (CN3SOCH2CI2CII2SO CH,), which is solubleonly to about 1 Ml, did not affect reactivity of AcChE. itseemed possible that the sulfone group might bind at both thetrimethyl and acetyl (esteratic) subsites, but this does notappear to occur. Similarly the bis-methylsulfinyl compoundCH3S(O) CR2 CHCHS(O)C2I is a weak inhibitor, at 10 mul decreas-ing activity by about 30%. 2-Methylsulfinylethanol will beexamined.

D. Reversible Inhibition by Derivatives of Benzene andPhenols

An aspect of a study of inhibition of AcChE by benzene andsome mono- and di-substituted derivatives was completed. Inthis study effects of substituents are satisfactorily inter-preted in terms of the parameters of the Ha minett equation (42),in which a-values are measures of the electron withdrawing(positive values) and electron donating (negative values) powerof the substituents, and p is a measure of the sensitivity ofthe interaction to the electronic effect, positive valuesarising when effects are increased by electron withdrawal.Results are su mma rized in Table V and as follows:

Benzene and 14 mono- and di-substituted derivatives, andstyrene oxide, pyridine and 4-tert-butylpyridine were examinedas inhibitors for hydrolysis of AcCh by AcChE. Linear rela-tions between binding, log 1/K1 , and summed a-values wereobserved as follows: (i) In competitive inhibition, binding ofCH,-Y, where Y - H N (IX), H (I), CH A COH (II), CH.CO (III),and 02H (IV), and binding of di-substituted compounds para-02N,KHCOCH, (XI), meta-02 N,NH2 (XII), and para-K2t4,COCH, (XVI)led to # of 1.85 ± 0.09, correlation coefficient (r) of 0.99,with K, values from 46 mMl for IX to 0.63 mH for IV, and 0.46 Mfor XI. Inclusion of meta-02H,NHCOCH, (X) gave K, of 0.26 .14,and para-0 2 N,NH2 (XIII) led to p of 2.03 ± 0.15 and r of 0.98.(ii) The di- and trialkylamino compounds with substituents(CH3 )2 N (VIII), (CII,)3N* (VI), ueta-(C14,),N0 2O (XIV), para-(CHN,N0 2 (XV), and para-(CH.),N,COC4, (XVII) bound with

17

higher values of p, 2.36 ± 0.16, and r, 0.99; K, values rangedfrom 11 mN for VIII to 0.091 mM for XV and 0.077 mM for VI.The cationic charge in VI had no effect beyond that of itsa-value. The fact that binding was increased by electronwithdrawal is attributed to charge-transfer interaction. CH3groups of (CH 3 ) N increase competitive binding by interactionin the trimethyl site. The effect is consistent with that ofCH. groups in the pyridine, tert-butylpyridine pair. TheCM3 CO- substituont leads to enhanced noncompetitive binding.The substituent pairs mete- and parar-(CH3 )2N,NO and para-HAN,NO2 are mutually reinforcing; para-(CH),N,COCH, interac-tion is small. Styrene oxide inhibits irreversibly.

There is evidence that protein tertiary anu quaternarystructure is stabilized by aromatic-aromatic interactions ofamino acid side chains, contributing 1 to 2 kcal/mol perinteraction (44). The effects found in this study indicatethat added aromatics may compete with these stabilizingactions, affecting structure and, in the case of AcChE, deceas-ing reactivity. This ay be a general phenomenon that is foundwith other enzyme which, like AcChE, have no apparent involve-ment of aromatic groups in their normal substrate interactions.In the present case the added aromatics act as electronacceptors, with enzyme groups as donors; in others the reversemay be true.

This study is now being extended to derivatives of phenol.Some data are summarized in Table VI.

In phenol the hydroxyl group led to binding weaker thanbenzene, and similar to that of aniline, consistent with thehydroxyl a-value. Effects of the tert-butyl group are remarka-ble. In the met& position (III), it increased competitivebinding over that of phenol (I) by 4.5 kcal/sol and noncompeti-tive binding by 3.8 kcal/mol, far greater than its effects onpyridine (Table V). However, when tert-butyl was in the paraposition (IV) no inhibition was observed up to 0.65 mM, a limitset by its low solubility. In 4-tert-butylcatechol (VII) 25%of the binding energy of the 3-tert-butylphenol was lost inboth binding modes, the para-hydroxyl causing this decrease.Binding of tert-butyl in the trimethyl site led to a specificinteraction of the mete-hydroxyl, but not of the para.However, in the nitrophenols different effects were observed.In the meta compound, the hydroxyl decreases binding, consis-tent with its positive a, thus not affecting the nitrophenylbinding per se. But in the para position it removes allcompetitive binding and retains the noncompetitive bindingcharacteristic of nitrobenzene. Those are remarkable effectsand will be studied further.

The effect of the meta-tert-butyl in III indicates that itfits into the trimethyl site, and that that the benzene ringitself does not utilize this part of the site. This is

18

consistent with the observation that tetramethylammonium iondoes not retard inactivation by methyl benzenesulfonate.However, the benzene ring is in the active site and thebenzenesulfonate remains an interesting labeling compound. Thenon-synergistic action of 02 N and HO in V supports the viewthat ON makes no specific steric interaction in the activesite and acts only by electron withdrawal, as indicated by the

relation.

The strong binding of meta-tert-butylphenol (III) leads toan interesting application of the p-u relation determined inthe study of benzene derivatives (Table V). meta-Trimethyl-ammoniophenol, the cationic analogue of III, was reported (45)to bind strongly, with Kt of 0.00031 mM and AG of -8.8 kcal/mol;III, with K of 0.024 mM, has AG of -6.24 kcal/mol. Thedifference in the a-values of (CH3 )3 C- and (CH,)3N-, 0.94(Table V) and application of the Hammett relationship,log K - log K, - pav, where log K, refers to meta-tert-butyl-phenol and has the value of 4.62, and p has the value of 2.36,for polymethylamino compounds, above, leads to the value of logK for meta-trimtheylamnoniophenol, 6.84. This corresponds toaG - -9.2 kcal/mol for the binding energy of the cationiccompound, the same, within the error of such measurements(Table V), as the value reported in 1958! (45). It will beinteresting to check this measurement. This is an importantcompound, which we will prepare. Our study of aromaticinhibitors leads to a reliable correlation of inhibitory powerto structure which has predictive value for the effects ofhitherto unstudied compounds.

(6) Discussion and Conclusions

We were able to isolate and purify AcChE from Irdonobiina, and to purify commercially available eel AcChE. Wecan now prepare 1'C-BrPin on a 60 pmole scale, and we willstudy the incorporation of this labeled inactivator into theenzyme, and the effects of other active-site-directed reversi-ble and irreversible inhibitors on this incorporation. We willundertake to identify the group or groups labeled by thiscompound.

We studied the effects of novel irreversible alkylatinginhibitors -- BrPin, methyl methanesulfonate, methyl benzene-sulfonate and styrene oxide -- on subsequent incorporation ofthe inhibitor 3H-DFP, which acylates the active site serine.Partial inactivation of Sigma eel AcChE by BrPin leads to aproportional decrease in subsequent incorporation of IH-DFP. Asimilar result is obtained with Tggd AcChE, but this issomewhat more difficult to assess quantitatively because of thevery rapid inactivation of this enzyme by BrPin. The partiallyinactivated material appears to comprise fully inactivated and

19

uninactivated material, supporting the specificity of action ofthis inhibitor. This study will be extended to the sulfonate-and benzene-related inhibitors.

We explored the active site of AcChE by studying thereactivity of p-X-CHCH2OCOCH3 substrates, and reversible andirreversible inhibitors related to these substrates. We havetermed the subsite at which the 8-X substituent binds trimethylrather than anionic because of the relative reactivity andbinding of analogous compounds in which X is (CH3 )3C- and(CH3 )3N -. We found that CH3 S(02 )CH2 CH2 - compounds bind aswell as analogous (CH3 )3CCH2 CH2 - compounds, indicating thattrimethyl may also be too specific a name. Small CH3 SO2-compounds, CH3SO2 Cl and CH3SO OCH3, inactivate AcChE as theyacylate and alkylate, respectively. These processes are notretarded by (CH3 ).N ion, indicating that they do not utilizethe trimethyl subsite, unlike the corresponding substrate,CH3 S CH2 CH2 OCOCH 3 .

Simple mono- and di-substituted benzene compounds,containing as substituents CH3CONH-, CH3 CO-, O N-, H2 N-,(CH3 ),N-, and (CH3)3N+ -, act as reversible inhibitors forAcChE, in which inhibiting efficiency increases with electronattraction by the substitUent as indicated by correlations witha-values. Ki values fall from about 50 to 0.1 mM. Binding isalso increased by (CH3)2N- amd (CH3 )3C- substituents, indicat-ing that these occupy the trimethyl site and that the benzenering itself does not. The meta-(CH3 )3C- and (CH3 )3N*- groupsgreatly increase the binding of phenol, from K of 50 mM to0.024 mM and 0.0003 mM, respectively, as the substituents bindin the trimethyl site. The difference between the neutral andcharged substituents can be calculated accurately from theira-values and p obtained in the study of benzene derivatives.

Methyl benzenesulfonate is an effective methylatinginactivating agent, and it is not retarded by (CH3 ),N* ion,supporting the view that the benzene ring does not occupy thetrimethyl site. Styrene oxide is also an alkylating inacti-vator, and effects of these aromatic compounds on subsequentintroduction of 3H-DFP will be examined. Effects of (CH 3 ))C-,(CH3 ) 2 N- and O2N- substituents on inactivation by methylbenzenesulfonate and styrene oxide may be examined andappropriate e'C-labeled materials prepared for study on AcChE.

.. ... .. ~ ~~ . . ... . ', .. -

20

oN I* C - 9-4

N? InIIn4 NO 0 OOO o 4

r4M N 10 atO~ UiiN NM f N m e-4 m

Nr-

6 N P-4 N4 N4F

jfd NJ4" M NN NO O

inN

111.:

21

Table II

Reversible Inhibition of Hydrolysis by Acetylcholiriesteraseof Acetylcholine and 3,3-Dimethylbutyl Acetate,

in 0.18 N NaCi, pH 7.8, at 25*C

Inhibitor K

No. Compound Concen- Acetyl- DMBActration choline(UN) (UK) (mm )

Ib (CH3 )3*NCH2 HCH2 COCH3I- 0.1-0.3 0.022 0.024

J1 b (C3)3C H H H OH l 0.1-1 0.16 0.14

Jjb (CH3) 3CSO% 04 COCK3 1-3 0.40 0.33

IVb (CH ) 3 C.NH3Cl- 0.1-2 0.43 0.44

Vb (CH3 )2.NHCH2cHCHCOCH.Cl- 1-5 0.77 0.71

VIC (CH,)3-NCMHIOHC1- 0.1-2 1.0 0.63

VIIC (CH3 ) 3 -NH 3 C- 0.4-2 1.5 1.0

VIlIb (C33OHCOH 0.4-3 1.6 1.0

I (01, ) 2Cr NH2CICH2 COHC 1-5 2.0 1.5

Xb (C1, ) .CIIOCH, CH2 COCH, 2-9 4.8 3.1

XIC (CH3 ) 3CCH2 CH0H 2-16 7.5 5.7

t 25%.

bShowed competitive inhibition.

Mixed or noncompetitive inhibition.

22Table III

Inactivation Of Sigma AcchE by NWl 3H-DFP

3H-DFP T Time Inacti-vation

(ON) CC) (am) M%

1 0 10 20

10 0 60 55

10 25 10 55

10 25 30 88

10 25 60 94

48 25 5 75

48 25 15 84

48 25 30 97

23

Table IV

Reversible Inhibition of Hydrolysis of Acetylcholine byAcetylcholinesterase, pH 7.8, 25"C, 0.18 M NaCi

No. Ccapound K1 (cow) K (nonc)a !b

(uM) (') (M) (cc)

I (C) 3 SidC C" OH 3.3 3.5 25.0

IA (C%) 3 CC2 C2 OH 7 . 5d 19 d 5.3 19.6

IB (C%) 3 N 2 CH2 OHCI 0.40 7.60 0.33 17.2

II (C) 2 S CH2 CH2 OHI - 0.4 13 0.33 16.4

III (C3)3 S* I 2.0 7.2

IV (CH3 )3 S+or 1.3

V (CH 3 wo- >>200 18

VI (CH3 ) O (0-) CH C0CH3 14 18 16.4

VII CH3S(O 2 )O C CH20H 8.7 100' 6.2 13.9

VIIA CH3 S (O2 ) CCH 2OCOCH3 6.4 11 6.2

VIII (C ) 2 S0z 28 260

IX (cH3) 2 So 25 16 14.1

X CH3 SC2CH2 0H 40 15 13.3

XI C3 CH2 COaiCHOH 47 13 10.3

a ±20%; corn - competitive; nonc - noncompetitive.

b yapp) of corresponding acetate, Ref. 26.

c Refraction volume of p-substituent, (CH3 )3 Si-, (C 3 ) 3 C-, (CH3 ) 3 N-,

(CHi) 2 S+ , (C)) 2WN(O-)-, CiS(O2 )-, C 3S(O)-, C 3 S-, CCH2, Ref. 12.d Ref. 14.

- D. Bell, unpublished result.

f This value has high uncertainty.

24

I %rc4 'lO l0 ILh vA r- a)? I AN h11;7 A1

r-4 m

00 0D '0I 80 '0 LO(

C! i

Co 0 in C; C; OM m .- I

In~I IniI -NA A4' M4 M m~

AA r4

Ul , p * * -p - re I I I Ip

000 00 00

0

10

*1

25

Table V (Cont.)

In hydrolysis of 3,3-dimthylbutyl acetate.

b Data from KrU)ka (Ref. 43).

c Studied at pH 8.5.

d < _0. 1 )al/Mol.

± _ 0.2 kcal/mol.

± 0.15 kcal/n l.

± ± 0.3 kcal/mol.

h ± 0.4 kcal/mol.

26

Table VI

Reversible Inhibition by Phenols

No. Compound K,(COM) Ki(nonc)

(MM) (MM)

I CS H5 OH 50 110

II 3-CH3 C6 H4 OH 10 40

III 3-(CH3 )3 CC6 H4 OH 0.024 0.17

IV 4- (CH 3 ) 3 CCr H4 OH

V 3-02 NCH 4 OH 0.92 1.7

VI 4-02 NC, H4 OH

VII 1, 2-di (HO) -4- (CH 3) 3 CC6 H3 0.35 1.8

Lwv= -

27

Figure 1. Normalized Reactivity of XCH 2C 2 OCOCH 3 '

log ( /K and Apparent !4olal Volume (V25)

(CH,3Nt@

(CH5)3C-

7.0 (CH3)aCH-

(CH4NH- 0

CH3CH2-log k2(n)

H0Ks 6.0 H5

CI-.

0 CH3 N-

5.0 + C3 N,N-N/40o

40 sop

Vj(R-

28

U 0 0

C4 0 N -= 41

+z 0to (A a

0 ~4)

C4 N 4J0

+to

C')4 .'

N r.

o 0o

0

N N

C.t)I 00'4 fO

29

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45. "Acetylcholinesterase Studies on Molecular Complementari-ness", Wilson, I. B., and Quan, C. (1958) Arch. Biochem.Biphs 7,11-143.

33

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