Further observations on substrate-derived chloromethyl ketones that inactivate trypsin

ARCHIVES

OF

BIOCHEMISTRY

Further

ANI)

BIOPHYSICS

Observations

on

Ketones ELLIOTT Biology

Department, Received

298-305

139,

Substrate-Derived

that SHAW

(1970)

Inactivate Ah-D

Chloromethyl

Trypsin’

GEORGE

Brookhaven

.Vct/ional

Laborator!y,

Fcbrimry

12, 1970;

accepted

GIlOVER [‘plan,

April

A7ew York

11978

22, 1970

The inactivation of trypsin by TICK was csamined with respect to dependence on the concentration of the inhibit,or. Sat,ur:ttion kinetics were observed as espected of an active-site directed reagent.. The int,ermediat,e complex has a dissociation const,ant of 2.1 X 1OP RI and forms inact,ive enzyme with ky = 0.16 Ininat. pH 7.0,25’. An improved synt,hesis of TLCK is described which also facilitated the preparation of J)-TLCK and of LCK (the chloromcthyl ketone derived from Il\-sine). The former is essentially inact,ive. LCK alkylates ~rypsin at, N-3 of a histidine residue. Alt,hough it has less allinity for trgpsin than TLCK, LCK forms a complex which proceeds to alkylated enzyme more rapidly, i.e., k? = 0.50 min-1. The inactivat.ion of trypsin by TLCK was examined in the region of pI1 1.0-4.4 at 37”. Under these conditions considerable denaturation was occturing. However, alkylation took place only vvit,h active enzyme, esclttsively at, N-3 of a histidine residue. Attempts made to synthesize chloromethyl ketones derived from N*-substituted ornithine and arginine encountered difficulty chiefly due to cyclizat ion. However, preparations containing a low content of the chloromethyl ket.one from NW-p-nitrobenzylosycarbonylarginine (p-NOT-ZACK) were obtained; this agent is an rstremelv rapid inact,ivntor of trypsin.

The identification of an active center hi&dine in trypsin at, His-46 by specific chemical modification u-as described earlier (1, 2). The reagent used, TICK, t,he chloromethyl ketone derived from Ka-to&Llysine, was active site-directed in it,s design, t,hat is, it was expect’ed to alkylate trypsin as the result of a substrate-like complex formation with the enzyme which localized the covalent modificat)ion at t’he active center. This paper describes the results of studies to establish whether evidence for the formation of an intermediate complex could be obt,ained in the inactivation of trypsin by TJLX. In addition, the importance of the stereochemistry of the reagent was examined as well as the effect, of pH unfavorable for histidine alkylation.

Some at,tempts were made to explore the effect of structure on the nature of the inhibitory action, particularly in view of the variety of trypsin-like enzymes found in nature and t,he extension of their specificities to include action at arginine and ornithyl bonds to a greater or lesser degree t’han at lysyl residues. MBTISl:I.4I,S

ANI)

MIGTHOJ>S

Uovine trypsin (Worthington two times tryst alized, lyophilized) was purified by chromat,ography (3) and the single chain form (&trypsin) was tised. l:at,c assays were carried ottt, with benzoylargininr: p-nitroanilide (BAPA) (4) or with benzoylargininc ethyl ester (5). Trypsin soltttions \vere titrated with p - nitrophenyl p’ gSrlanidinobenzoat(, (p-NPGU) as described (6). Performic acid oxid+ t,ions of alkylatcd t,rypsin dcrivai ivcs were carried out, by the literattuc method (7). Infrared spectra wore observed vvith :I I’erkinElmer 237B spectrophotometer and NMIL spectra were measured with a Varian T-60 spectrometer.

’ l
SITE:-SPECIFIC

ALKYLATION

~licroat~:d~-ses \vere carried out, hy Alfred Bernhardt,, Rlttlheim, Germany; the Applied Science Ikp:nhent, Brookh itven National Lnltorntor?-; and Schwarzkopf Microanalytical I,nhorat.ory, \Voodsidc. Xe\\r \-ork. t) - Lysittc, dihenzyloIu?-c:trt~ott~l Iysine, Ne I)c~tlzSlos~cnrb~)nyl lysine, and N6-henzylosycartwttyl ornithincwere plrrchnsed from Cycle Chemic:li. ,411 other reagents wpre of lhe besl :ivailak)lc c~omntr~rcial grade.

c!r the Iuactivatiou by I,;-TLC’K

liitwtics

0.f Trypsirz

Stock so111l ions were prepared of p-t.rypsitt (J.-l tttg/ml, 1V $1) and of LTLCK, 5 ml!, both in lW3 s HCl. The ittac*l ivalion rate at ii given inhibit 01 ~orlc:rn~ ratiott was observed by removal of aliclitots from :L reacl ion mistltre, rapidly lowering ihc pII to stop t,he inactivation (quenching), and s~iltsrqlwn(ly tlelf~rtnittit~g residtial tr,vptir activit) itt :I rate assay. This was pnrticttlarly important at highrr

itihit~ilor

cwncetitrstions

which

providrtl

rapid in:tctiv:ttioti rclalivc to t.he ditration of rate ;w:t~-S; wttsec~uct~~ly rapid sampling and qltcnchitrg was reqliired. ‘l’hc rract iott tnistruw xvcrc cotnposcd of stock ;?-lrypsilt solrction (4 ml), 0.10 M Tris bluffer, p1-I 7.0. 0.05 M it1 c:nlrium ion (10 ml), stock inhibitor ,SIIIIII iott (rtlt to -1 ml), and water as needed to at tnin :i final vollitne of 20 ml. A timer \vas started at ~IIIC~. For qlrcttching, 1.0.ml aliquols were removed :1t1tl atldrtl to 0.10 N HCI (0.5 ml). assays \vere farrirtl alit sl~ectrophot~omc~ric:~ll?; on 1.0 ml of clltctttcnltrd sample attd 3.0 ml of ISAPA in 0.10 M ‘I‘ris. 1111 8.2.

OF

intermediates lvere obt.ained had an infrared

The

299

TRYPSIN

comparahlc to those of the J,-isOttler at each step and the final prodttct spwtrrun identical to L-TI,CK.

Chloromethyl Ketone Delived L:ysine (LCK)

from

The dihydrochloride of this derivat,ive is readily crystalliznblc and is insoluble in absolltte alcohol, properties which facilitate its isola1ion. Conseqttently it coldd br prepared from cu,e-dihenzylosycarhonyl-lysine without. char:lctc~rizat,iort of the intertnediates. The hlocketl lysitw (I .2 g) in trenzcnr (20 ml) was dried by dist iilation of a few milliliters of benzene, after which the solution !vas broright t,o 0’ and treated with phosphorolts pentachloride (0.75 g) with stirring. After lj mitt, e1hereal diazomcthane was added in excess (pcrsislent yellow color), and Ihe sol~ttion \vas left at 0” overnight. The rc:tc(ion rnist.~~rr \V:LS PStracted with aqttco~~s sodiltm ttic:wlwtrate rcpeatedly and dried over anhydrolts tnagncsittm sttlfa(e. The ethereal tliazokcttrttc sol~ttiotl was I hen I rest ed with :I stream of anhydrotts hytlrogcn chloride in art ice t)at,h for 2 hr and 1 aken 10 dr>-ness. The rcsidtte was dcblockcd {vii h t riflrtoroacetic acid, 8 ml, and converted to the hydrochloride as drscribetl above for TLCK. Aft,er ~‘1 het eslraction of benzyl friflrloro:tcet:lt,e, the 1,(X was readily crystallized on addition of ahsol~tte alcohol or by sollltion in 957; ethanol, conct~nirat.ion, and seeding to provide 2% tng, mp l(ji-173”. 33’;; overall yield. An analytical sample \VRS (,I)tained from 95C,;, alcohol and ether, mp 170-172”. Anal. calcd for C,II,,X,Cl,O: C, 313.11; II, (;.$I; Cl. 12.28; r\‘, 11.03

ii-Benzlllo~ycaI,bo,lyl-ol-tosylornithillyl Nf-Bcnzylos?;carbonyl-L-TLCK (2.5 g) (I) was tw:LCed in t.rifi~loroacctic acid (10 ml) al, 90” for 20 nlitt. ‘l% triflltoroacet ic acid was removed ltndet rrd~ifcd prcssttre in 11. rotary evaporator. The rwidrlct was dissolved in excess ethnnolic hydrogen c.llloritlc and taken to a thick syrrtp once more urlder redriwtl prcssrtrr. To remove Ixnzyl t,riIlru)ro;wetatc~. the oily protlttrt was stirred with several port ions of anhydrotts et,hrr (50 ml) which \wre tlisc~:trtlrtl. At this point, the TLCK could be crysfallizc~d from :I concentrated sollttion in ;tl~sol~ttc c~11:rnul irt its ch:tr:l~tcristic:lliy slow fxshion in 70’ c, yield. Thr prodrwt was identical t 0 1ha1 dwfribed earlier (1). The improved yield \\ as parlicrtlnrly appreciated iti syntheses of TI,CK-3H which started with t.-lysitte-311. I )-TTX’K was prepared from wlysine; the Nf-hctrz~los~~c~:Irl)onyl derivative \vas prepared 1)~~ \Vny of the copper salt (S), converted to Ne-benzy~~xyc:trbonyl~TLCK by the pllhlished method (l), :tti(l tlt~t~lockecl as described above. Crystalline

chloride I:ea.ct.iott of phosphorous pent,achloride with 6-t)enz~lvsyc:~r~o~~yl-a-tosylornit,hine (!I), carried out as Iwed by Synge (10) to prepare a-&dibenzylosvcarbonylorrlithili~l chloride, gave ‘317; of the desired prodrlct. Infrared spectr\tm (Kljr) : 1790 1097, 1535 ctn-‘, mp X2-84”. Anal. calcd for CZoHZgN~6)&XX: C, 5J.iG; II, 5.Z; N. 6.37; s, 7.32 Fo~ultl: C, 54.86; H, 5.13; N, (i.7-2; 9, 7.a(i

6-L3e7lxylol:ycarbonyl-cu-t~sylorr2ithinylrliazorrletllane The acid chloride, A.0 g (9.1 mmoles), was 211s. pcnded in ether, cooled to O”, and treated with excess dinzumrthane in et her (persister1 t yellt)w color) for 2 hr. The product \vas obtained in 75:; yield by filtration of the reaction mixture. 1~ frared spectrum (KUr) : 2107, 1685, 1630, 1530 cnr’, mp 42-Q”.

300

SHAW

Anal. calcd for C2111?~Y405S: C, 56.78; II, N, 12.60; S, 7.21 Found: C, 56.32; H, 5.72; N, 11.07; S, 5.29

AN11 5.44;

&Benxyloxycarbonyl-cr-tosylorlzitkinylchlo~omethane A suspension of 0.5 g (1.1 mmoles) of diazoketone in 20 ml 4 N HCl \vas st.irred at 0” for 2.5 hr and at 25” for 14 hr. The product, was filtered off and dried by dissolving in ethylacetate, stirring with anhydrous sodium sulfate, filtering, and evapnriition of the ethyl acetate. The residue was recrysi.allized from ethyl acetate-hexane yielding 0.32 g (71%) of chloromcthylketone. Infrared spectrum (Nlljol): 1740, 1695, 1550 cm-l. NMf1. spectrum in trifluoroacet,ic acid (8, ppm): 1.75 (m, 4H), 2.39 (s, 3H), 3.15 (m, 2H), 4.13 (s, 2H), 4.20 (m, lH), 5.30 (s, 2H), 7.27 (s, 5II), 7.27 and 767 (d, 2H).

Attempted PreparatioTl of ff-(p-nitroberczyloxycarbonyl)arginylchloromethane hydrochloride (p-NOZ-ZACK) ol-(p-Pu’itrohenz~loxy~~~rbonyl)argin~~l chloride hydrochloride, prepared from 5 mmolcs of acid according to Gish and Carpenter (II), was suspended in ether and treated with an excess of

dixzomethane (persistent

?-ellow color) at 0”.

After stirring for 2 hr at 0” t,he solution was filtered. The prodllct, l.A3 g, was rcslqended in 120 ml of ether and the mixt,lwe was snturat,ed with hydrogen chloride gas at 25”. After 16 hr at 25”, t,he light yellow-colored solid was filtered yielding 1.5 g of material, mp 10441Oi” (71y0 yield). Infrared spectrum (KBr) : 1735, 1695, 16i5, 1520 cnl-I. NMR spectrum in trifluoroacetic acid (6, ppm): 2.0

(m, 4H), 3.48 (m, 2H), 3.92 (s, 2H), 4.40 (m, III), 5.30 (s, 2B), 7.57 and 8.57 (d, 27-I). Titration

of p-NOa-ZACK Preparations with Trypsin

A st.ock solut.ion of 10e4 hi @-trypsin (det,ermined by titration wit,h p-SPGB) was made up in lob3 M HCI. The pmNO,-ZACK preparation was made 11p at a concentration of 21.1 mg per 10 ml of lop3 31 HCl. Reaction mixtures consisted of 0.1 ml of standard trypsin sohltion, 1.0 ml of 0.1 14 Tris, pH 7.0, 0.02 M in Ca2+ and gradecl amounts of p-NO,-ZACK solution plus water to give a final volume of 2 ml. Trypsin was thlls at 5 X 10L6 M. Concentrations of p-i%Oa-ZACK preparations of 8.4, 12.7, lG.9, 21.1, and 42.2 X 10m5 g/2 ml were employed. The inhibition was followed by qwnching 0.2.ml aliquots with 0.1 ml of 0.2 N HCl at appropriate times and assaying ‘arnidase activity with 1.7 ml of lop3 hc BAPA in 0.1 M Tris, pH 8.2.

GJ,O\‘RI:

Inhibition OS Trypsin by p-NOz-Zi4CK Determine the Xite of Inactivatiott

To

The p-trypsin 1.0 ml ZACK

inhibition mixture consist,ed of 5 mg of and 4 ml of 0.25 M Tris, pH 7.0, to which of water containing 4.22 mg of a p-NC),preparat,ion was added (the ttctrral concentration of p-N&ZACK was 3.4 X 10-” 11). The inhibition was followed by assaying 10-J aliyuots for cstcrase activity. All activit.y was gone within 30 min. The mixt,lwe was dialyzed against several

changes of deionized wnt,er and lyophilized. RESULTS

,4?J11

I)ISCUSSI(.)K

Chemical Studies The exploration of structure-inhibitory relationships among active site-directed reagents for trypsin is of interest to ext#end our knowledge of this method of selective chemical modification of enzymes. In addit,ion, the abundance of trypsin-like enzymes in nature increases the probability of obt.aining useful information about physiologically im portant processes from this type of specific inhibitor. Unfortunately, t,he chemistry of the basic amino acids raises difficulties in synt’hetic work leading to chloromethyl ketone derivatives. In the initial synthesis of TLCK, remowl of t,he benzyloxycarbonyl group, the final st’ep, was t,roublesome, but a low yield (about 20%) was obtainable by acid hydrolysis (1). Catalytic reduction with palladized charcoal resulted in dehalogenation. The use of trifluoroacetic acid for general removal of the benzyloxycarbonyl group was demonst,rated by Weygand and Steglich (12) and, as described above, has greatly improved the synthesis of TLCK. The synthesis of the chloromethylket.one drrived from lysine iiself (LCK) also becnmc feasible. Attempts to prepare tosylornithine chloromethy ket,one, however, were not successful because of competing side reactions leading t,o the formation of cyclic by-products. Thus, although N6-benzyloxycarbonyl-N*tosylornithinyl diazomethyl ketone (I) n-as readily obtained, attempted conversion to the chloromethpl ketone with hydrogc>n chloride in ether (1) did not lead to the chloromethyl ketone (IV) as judged by the

:tbscnw of :I singlet lvith t\vo hydrogen:: abollt 4.1 ppm in t,hc N.\IR spectrum.

I

II

Hz’%“eH c-c-c-c-c-c-c1 AH

at

m

0 II “2 2

=

benryloxycorbonyl

Ts

=

p-toluenesulfonyl

fiti

i

ts u

In addition, the infrared spectrum hsd :t singlr cnrbonyl absorption at 1705 cn1-l thle to t,he carbobenzox; group, but no ;rmidc -II :tbsorpt,ion bet,neen 1500 and 0 II I:j,j() cm-1 indic:kive of :L (l--NH-grouping. These facts, together with consistent PL\IR nbsorptions, point to u cyclic structure III arising by dehydmtion of II for this product. Removal of the blocking group bx refluxing in hot t~rifluoroacetic acid led to :t product which had lost the benzyloxycarbonyl group and st,ill n-ns not, n chloromethyl ketone. Thr KA1IL spectrum was consistent with III \\-ith no benzyloxycnrbonyl group. The protected chloromethyl ketone (IV) could be obkned by treatment of the diazokctonr \vith aqueous hydrochloric acid :lccording to Miescher and IGgi (13). The KALR and infrared spectra \vere in accord \\-ith those of the corresponding lysine derix-atiw. This mxt~erial \V:IS difficult to recrystallize, since it vxs unstable in sol77tion. l)c~blocking of t,he chloromethyl ketone in boiling trifluoroncetic acid gave the same product obt,:Cned from deblocking of III.”

In the at,tempted synthesis of NI arginine chloromethyl ketone derivative, use JK~S made of the laoa-n formation of the acid chloride of a-p-nitrobcnzyloxycnrbonglarginine (11 ), which avoids blocking t)he guanidino group, \vith the expeckation of synthesizing the chloromet.hyI ketone derived from So-p-~~it~lobenz~lox~carbonyl~rginine (VII). Reaction of the acid chloride ~1sa suspension I\-ith excess dinzomet,hane in ether, folloned by treatment. I\-ith ;mhydrous hydrogen chloride yielded :I product :ulalyzing for the expected composition (by C, H, and X analysis). Ho~vea~r, hhe elemental analysis changed wit,h time and M chlorine :malysis after several months indicated only about 50 “; of the expect,ed value. Repetition of the reaction yielded the Fame product. 0

Hz Hz Hz H :: 4 y-c-c-c-c-c-cl / NH NH

HN 0’

04 / NH2

NH2

I

I H2N=C+

r4-;=wi, El

xc\

OCH*

/ -cF

LH,

-

\

NO,

NO?

The X\TllR spectrum was quite definitive. All the expeckd resonances n-ere present, \\-ith :L sharp singlet at’ 3.92 ppm for the methylene int’roduced by diazomethane. This resonance occurs 0.2 ppm too high to be a chloromethyl ket.one. The best struct,ure is one annlogous to Y, namely VIII, substitut.ion of nikogen for chlorine on the ketone methyl group being consist’ent with the obwri-4 chcmicul shift difference. Al-

‘NHTs

302

SHAW

AND

though the principal product is VIII, some p-NO,-ZACK (VII) is present since the material inhibits trypsin irreversibly. The concentration of p-N02-ZACK in the product was determined by reaction with a small excess of a standard trypsin solution. The inhibitor is very reactive; consequently, reasonabIe end points indicating exhaustion of the inhibitor were obtainable (Fig. 1). The amount of trypsin inhibited was considered to be equal to the amount of p-NOZ-ZACK in the reaction mixture. From Fig. 1, values of 1.5, 1.5, 1.9, and 2% p-1V02-ZACK were obtained giving an average value of 1.7% for the p-NOzZACK content of t’he preparation. The inhibition itself did not prove t,he presence of p-N02-ZACK (VII). However, like TLCK inhibition, p-N02-ZACK inhibition resulted in bhe loss of a hist’idine residue (0.7 residue per mole of trypsin), and

i

J MINUTES

FIG. 1. Reaction of p-NO%-ZACK preparations with standard trypsin solution for titration. The curves from top to bottom represent increasing amounts of p-NO*-ZACK as described under Titration of p-NO?-ZACK preparations with trypsin.

GLOVEI:

performic acid oxidation of the inhibited enzyme produced 0.81 residue of 3-carboxymethyl histidine. Kinetic Xtudies To establish whether, in the inactivation of trypsin by TLCK, an intermediate complex was formed as expected of a subst,ratelike alkylating agent,, the concentration dependence of t,he inact,ivation \vas studied essentially I+,+

1.E 3 I complex

alkylated

enzyme

as described by Kit’z and Wilson (15). In their work, it was established that the inactivat,ion of acetylcholinesterase by esters of methanesulfonic acid proceed by \vay of complex formation, if the structure of the alcohol component. provides affinity to t.he cationic binding site of the enzyme. It was pointed out that, in the range of inhibitor concentrations adequate to provide apparent first-order inactivation of the enzyme, the dependence of this inactivation constant on inhibitor concentration permitted a graphic analysis for evaluation of t,he importance of prior complex formation in the inactivation process. Certain methanesulfonates, in fact, provided evidence for saturation kinetics, and their affinity to acet’ylcholinesterase was measured (15). This kinetic analysis is of general value in chemical modification studies to establish whet,her a given reagent is acting in a substrate-like manner. The inact,ivation of fl-trypsin at pH 7.0, 25”, by TLCK at a number of inhibit,or concentrations was observed to obey first,order kinetics through a major portion of the inact,ivation process (Fig. 2). A reciprocal plot of the apparent first-order inactivation rate constants so obtained vs inhibitor concentration (Fig. 3) gave a positive intercept on t.he abscissa indicating sat.uration of trypsin by TLCK with a limiting rate of inactivation, kz, of 0.16 min-I. From the slope = K,/kz (15), a K, of 2.1 X lo-” M \\-as calculat,ed. The limiting value of inactivation indicates a half-life for trypsin of 4.4 min at saturation. The most rapid actual

SITE-SPECIFIC

AI,KYLATION

OF

36

5

IO

15 20

25

30

35

40

45

50

55

so

65

303

TRYPSIN

t

70

MINUTES

FIG. 2. Inactivation of bovine trypsin (3 X 10V5 M) hy various concentrations of TLCK at pH 7.0, 25”, in 0.05 M Tris buffer, 0.02 M in calcirun ion; loss of amidase activity measlwed.

experiment~al observat,ion, 6.3 min, was made with mM TLCK. As expected, D-TLCK was not very effective. Under the conditions described above (as in Fig. 2) mM D-TLCK produced a loss of activity of 2ci%l in 2 hr, about 50 times slower than the L-isomer at the same concentration. It could not, be excluded that the observed low Mivity might be due t,o the presence of residual L-isomer in the preparation. The in&ivation of trypsin by LCK at a series of inhibitor concentrations also gave apparent first-order decay curves comparable to those observed with TICK (Fig. 2). From the reciprocal plot of such dat,a (Fig. 3), a k2 of 0.50 min-l and R, = 1.3 X 1O-3 nf \vere determined. The results \vit,h TLCK and LCX offer an interesting comparison. 130th alkylatf~ hist,idinc at S-3. Although LCK is less avidly complexed to trypsin by an order of magnitude, the complex breaks down to alkyl enzyme several times more rapidly (0.50 mu-’ vs 0.16 mire-I) than in the case with TICT<. That, is, the degree of aflinit~ is not necessarily correlated w&h rapidity of inwtivat,ion. Because of the essential role

0

4

8 I / [TLCK]

12 x 10’

16

20

M-I

PIG. 3. Saturation kinetics in inactivation of bovine trypsin by TLCK. The k,,,,,, of inac t ivnt ion at, various TLCK concentrations that providr first-order inactivation kinetics (Fig. 2) replotted in double-reciprocal fashion to obtain kinetic propert,ies of intermediate trypsin-TLCK wmpies.

of the intermediate complex, however, the observed rate of inactivatjion is very much dependent, on inhibitor concentration. A comparison of the two reagents at, 5 X lo-“’ M reveals TLCK about six times more rapid than LCK; at Fi X lo-” M they are about equally effective; at lo-” n1, on the other hand, TCK is about’ twice as rapid as TICK. l’resumably His-46 ~vns alkylated (3. An attempt’ \\-a~ made to determine whet.her the action of TICK on t,rypsin might’ be directed to an amino acid residue ot,her t’han histidine by incubation at low pH values. At 37” and pH 4.4, t’hr loss of activity of purified trypsin (1OP M) in the presence of 14C-TT,CT< (50 X lo-” M)

304

24

SHAW

ANI)

-

4

t

4

k zo/-

I

i

,E 'T fE t IZ-

8i

OOb I/[LCK]

FIG. bovine

x IO3

M-1

-2. Saturation kinetics in t,rypsin by LCK. Conditions

innctiwltion as in Fig.

of 2.

reached 85% in 7 hr. However, chromatography of the product, provided 56% as unlabeled protein in an elution position characteristic of denatured trypsin (3); the remainder of the product \vas accounted for as a histidine-alkylated product by stoichiometric incorporation of radioactivity, loss of histidine content (l), and formation of 3-carboxymethyl histidine on performic acid oxidation (7). These results show that the specificity of TLCK for the alkylation of histidine in trypsin is maintained under rather extreme conditJions since the amount of unprotonated His-45 can be expected to be very small at pH 4.4. So new site of alkylation was uncovered; all of the reagent incorporated was accounted for attached t’o histidine. Although efforts to extend the chloromethyl ketone series to ornithine and arginine derivatives met with chemical difficulties, a preparation containing p-NOZ-ZACK (VII) \vas obtained. The speed with which

GLl)VEII

this material reacts with trypsin made it possible to obtain reasonable estimates even of t,he low content present in its mixture with a cyclic by-product (VIII). It is evident that t.his agent is extremely effective. I:rom the data in Fig. 1 it was est,imated that, p-X02-ZACK is at, least, two orders of magnit,ude more rapid than TLCK in the inactivation of trypsin at pH 7 and t)hus merits further attention. This increased effecGveness may be due, in part), t,o the difference of side chains and not merely to advantage of arginine over lysine. As shown in the case of subt,ilisin, a change from tosyl to benzyloxycarbonyl in the chloromethyl ketone derived from phenylalanine result,ed in the conversion of an inert, compound to an effect’ive enzyme inxctivator (16). It is hoped that future \vork \vill permit a more systemat,ic exploratjion of the relation of structure to inhibitory activity in t,hese active-site directed agents for trypsin, since the present observations indicate t,hat the scope of their effectiveness is considerably greater than i&ally anticipated. After the preparation of this manuscript, the blocked chloromethylketone, IV, was finally successfully deblocked in trifluoroacetic acid at 35”. The reaction could be followed because the n’l\lR absorption for benzyltrifluoroacetate occurs at lower field than that of t,he benzyloxycarbonyl group. The crude product, had the odor characterist,ic of TTXK. However, the reagent is apparently unstable in water; this fact’ w-as confirmed by dissolving freshly deblocked material in D20 and observing that the absorption at 4.1 ppm, characteristic of a chloromethyl ket,one, disappeared. The same absorption for TLCK remains unchanged in D,O. This instability probably accounts for the failure to observe an inhibitory action on trypsin even in large amounts.

1.

sH.\\Y,

I:.,

~I.\llE:s-(:UI.\,

Bioche?)lis/~y 2.

Sr1aw,

E.,

Bioph,~/s. 3.

SCHROEDER,

hr.,

,\XD

COIIEK,

w.,

4, 2219 (1965). .\SI)

SIwINGHoIm,

Res. Conm?m. I).,

ANI)

SHA\V,

s.,

l~iochnn.

27, 301 (1987). K;., J. Biol.

Chenr.

243, 2943 (1968). 4.

ERL.~NGER,

W., Arch.

B.

F.,

&liO\\

Biochem.

SKY.

Hiophys.

xc’.,

.\NU

&HEX,

96, 271 (1961).

SITI<:-SPECIFIC

T.LKEN.IL\. I’., Gio16, 570 (1955). T., JR., .\h-n ~FI.\\\-, I%:., ~~iod/w,~~. Biophys. Rex. Commun. 29, 508 (1967). i. PETRA, P. H., COHEX. W., AIVI~ SK\\\-, I<;., I3iochm. Bioph.ys. Iles. (“ottlv/~trr 21, 612 (1965). 8. NEV~ERGEN, A., .\ICI) ?~.\SGEIC, P., Lliochem. J. 37, 515 (1943). 9. Id, (:. Ii., %!H~.\rrEr~, B., .uw ~~LJNG, Lf., .f. A77,er. (‘hem. sot. 82, 201m (l!)GO). 10. SYNGE, 1:. I,. 11.. Hioc,h.et,t..J. 42, 99 (1948). 5.

~HWEILT,

chim. 6. CHaSE,

G.

Biophys.

W.,

mn

AI,KYI,ATION

dcln

OF

Tl:YPSIK

305