haloenol lactones

9
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 258, No. 24, Issue of December 25, PP. 15046-15053.1983 Printed in U.S.A. Haloenol Lactones POTENT ENZYME-ACTIVATED IRREVERSIBLE INHIBITORS FOR a-CHYMOTRYPSIN* (Receivedfor publication, May 2, 1983) Scott B. Daniels, Edward Cooney, Michael J. Sofia$, Prasun K. Chakravarty, and John A. Katzenellenbogeng From the School of Chemical Sciences, University of Illinois, Urbana, Illinois 61801 Haloenol lactones can act as enzyme-activated irre- The basic concept behind enzyme-activated irreversible versible inhibitors for a-chymotrypsin: acyl transfer inhibitors is thatan enzyme, by performing its catalytic to the active site serine releases a halomethyl ketone function upon a substrate analog with latent reactivity, re- that remains tethered in the active site during the veals within its active site a reactive species which then lifetime of the acyl enzymes Poised to alkylate an ac- proceeds to inactivate the enzyme by covalent attachment to cessible nucleophilicresidue. TO investigate the struc- essential residues or tightly bound cofactors. most exam- tion with lactones, we prepared a series Of isomerization, an oxidation, or an elimination as the essential nine laCtonesl differing in ring size @-memberedval- activating reaction. There are only a few examples in which erolactones and butyrolactones) and in an enzyme-catalyzed hydrolysis has been the event that acti- the nature of the aromatic substituent (phenyl and a- vated the suicide substrate (5-12). naphthyl), and the halogen (bromine and iodine). The inactivating behavior of these lactones is characterized Recently, we have described several synthetic approaches by a binding (Ki) and three rate constants, to two haloenol lactone systems: 5-halomethylene tetrahydro- for inactivation ( kz), catalytic hydrolysis (kc), and furan-2-ones and 6-halomethylene tetrahYdroPYran-2-ones spontaneous hydrolysis (k). The six-membered (13). These lactones were designed to act as suicide substrates erolactones were much more potent inactivators than for serineproteases, as illustrated in Scheme 1. Acyl transfer the butyrolactones, having both higher affinity and ofthe lactone carbonyl to theserine hydroxyl (a * b) would more rapid inactivation; the a-naphthyl-substituted release the haloenolate, which upon protonation and ketoni- lactones were also more effective, but the nature of the zation would reveal a halomethyl ketone. Because of the cyclic halogen had relatively little effect. The spontaneous nature of the lactone, this powerful alkylating agent would be rate of hydrolysis of all of these lactones is low. The tethered to the active site serine, so that it would be available turnovers per inactivation of these lactones vary from for reaction with suitably-positioned nucleophilic residues 91-1.7, with some of the a-naphthyl-substituted lac- near the active site (b 3 e). Covalent attachment to these tones approaching ideal behavior (stoichiometric in- residues should impair the catalytic activity of the enzyme activation). These studies indicate that several hal- (c) even after &acylation (d). oenol lactones are effective enzyme-activated irreVerS- We also have presented a preliminary study of the inacti- ible inhibitors of chymotrypsin, and that their Potency vation of chymotrypsin by two of these agents in which we and efficiency depends markedly Won certain strut- documented that the inhibition fulfilled the criteria of enzyme tural features of the lactone system. inactivation (14). In this report, we describe the preparation of an extended series of aryl-substituted halomethylene tet- rahydrofuranones and tetrahydropyranones and thedevelop- The development of effective inhibitors for enzymes has ment of a comprehensive kinetic analysis of their inactivation provided a continuing challenge to biochemists and medicinal of chymotrypsin. We have found that the effectiveness of chemists (1). While effective inhibition of enzymatic activity these compounds as suicide substrates depends strongly on can often be achieved with a reversibly binding inhibitor or the ring size of the lactone and to a lesser extent on the nature with a reactive substrate analog, there is currently much of the aryl group and halogen. The most effective inactivators interest in the development of enzyme-activated irreversible show properties that approach ideal behavior (ie. stoichio- inhibitors (2-4). These suicide substrates or kcat inhibitors metric inactivation). hold the potential of affording inhibition that is sufficiently selective for use in in vivo situations. turd determinants for chymotrypsin suicide inactiva- ples thus far described, the activation event has involved an *This work was supported by Research Grant PHs 5 R01 AM 27526 from the National Institutes of Health. High field NMR spectra were obtained using spectrometers in the National Science Founda- tion Regional Instrumentation Facility (NSF CHE 79-16100) and high resolution mass spectra were obtained in the Mass Spectrometry Laboratory, supported in part by Grant GM 27029 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked ''advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ Supported by a fellowship from the University of Illinois and by a National Institutes of Health Traineeship. f To whom request for reprints should be addressed. SCHEME 1 15046 by guest on February 7, 2018 http://www.jbc.org/ Downloaded from

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Page 1: Haloenol Lactones

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 258, No. 24, Issue of December 25, PP. 15046-15053.1983 Printed in U.S.A.

Haloenol Lactones POTENT ENZYME-ACTIVATED IRREVERSIBLE INHIBITORS FOR a-CHYMOTRYPSIN*

(Received for publication, May 2, 1983)

Scott B. Daniels, Edward Cooney, Michael J. Sofia$, Prasun K. Chakravarty, and John A. Katzenellenbogeng From the School of Chemical Sciences, University of Illinois, Urbana, Illinois 61801

Haloenol lactones can act as enzyme-activated irre- The basic concept behind enzyme-activated irreversible versible inhibitors for a-chymotrypsin: acyl transfer inhibitors is that an enzyme, by performing its catalytic to the active site serine releases a halomethyl ketone function upon a substrate analog with latent reactivity, re- that remains tethered in the active site during the veals within its active site a reactive species which then lifetime of the acyl enzymes Poised to alkylate an ac- proceeds to inactivate the enzyme by covalent attachment to cessible nucleophilic residue. TO investigate the struc- essential residues or tightly bound cofactors. most exam-

tion with lactones, we prepared a series Of isomerization, an oxidation, or an elimination as the essential nine laCtonesl differing in ring size @-membered val- activating reaction. There are only a few examples in which erolactones and butyrolactones) and in an enzyme-catalyzed hydrolysis has been the event that acti- the nature of the aromatic substituent (phenyl and a- vated the suicide substrate (5-12). naphthyl), and the halogen (bromine and iodine). The inactivating behavior of these lactones is characterized Recently, we have described several synthetic approaches by a binding (Ki) and three rate constants, to two haloenol lactone systems: 5-halomethylene tetrahydro- for inactivation ( kz), catalytic hydrolysis (kc), and furan-2-ones and 6-halomethylene tetrahYdroPYran-2-ones spontaneous hydrolysis (k) . The six-membered (13). These lactones were designed to act as suicide substrates erolactones were much more potent inactivators than for serine proteases, as illustrated in Scheme 1. Acyl transfer the butyrolactones, having both higher affinity and ofthe lactone carbonyl to the serine hydroxyl (a * b ) would more rapid inactivation; the a-naphthyl-substituted release the haloenolate, which upon protonation and ketoni- lactones were also more effective, but the nature of the zation would reveal a halomethyl ketone. Because of the cyclic halogen had relatively little effect. The spontaneous nature of the lactone, this powerful alkylating agent would be rate of hydrolysis of all of these lactones is low. The tethered to the active site serine, so that it would be available turnovers per inactivation of these lactones vary from for reaction with suitably-positioned nucleophilic residues 91-1.7, with some of the a-naphthyl-substituted lac- near the active site ( b 3 e). Covalent attachment to these tones approaching ideal behavior (stoichiometric in- residues should impair the catalytic activity of the enzyme activation). These studies indicate that several hal- (c) even after &acylation (d ) . oenol lactones are effective enzyme-activated irreVerS- We also have presented a preliminary study of the inacti- ible inhibitors of chymotrypsin, and that their Potency vation of chymotrypsin by two of these agents in which we and efficiency depends markedly Won certain strut- documented that the inhibition fulfilled the criteria of enzyme tural features of the lactone system. inactivation (14). In this report, we describe the preparation

of an extended series of aryl-substituted halomethylene tet- rahydrofuranones and tetrahydropyranones and the develop-

The development of effective inhibitors for enzymes has ment of a comprehensive kinetic analysis of their inactivation provided a continuing challenge to biochemists and medicinal of chymotrypsin. We have found that the effectiveness of chemists (1). While effective inhibition of enzymatic activity these compounds as suicide substrates depends strongly on can often be achieved with a reversibly binding inhibitor or the ring size of the lactone and to a lesser extent on the nature with a reactive substrate analog, there is currently much of the aryl group and halogen. The most effective inactivators interest in the development of enzyme-activated irreversible show properties that approach ideal behavior (ie. stoichio- inhibitors (2-4). These suicide substrates or kcat inhibitors metric inactivation). hold the potential of affording inhibition that is sufficiently selective for use in in vivo situations.

tu rd determinants for chymotrypsin suicide inactiva- ples thus far described, the activation event has involved an

*This work was supported by Research Grant PHs 5 R01 AM 27526 from the National Institutes of Health. High field NMR spectra were obtained using spectrometers in the National Science Founda- tion Regional Instrumentation Facility (NSF CHE 79-16100) and high resolution mass spectra were obtained in the Mass Spectrometry Laboratory, supported in part by Grant GM 27029 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked ''advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Supported by a fellowship from the University of Illinois and by a National Institutes of Health Traineeship.

f To whom request for reprints should be addressed. SCHEME 1

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Chymotrypsin Enzyme-activated Irreversible Inactivators 15047

EXPERIMENTAL PROCEDURES’

RESULTS

Synthesis of Chymotrypsin Suicide Substrates The structures, compound numbers, and abbreviations for

the lactones we have studied (1-9) are shown in Scheme 2. The preparation of lactones 1-3 has been described before (13, 14). In all cases, the final step involves halolactonization of a suitable acetylenic acid precursor; thus, the halometh- ylene units all have the E stereochemistry.

The preparation of the 3-aryl tetrahydrofuran-2-ones (2- 5) is illustrated in Scheme 3. The precursor acetylenic acids (12 and 13) for these lactones were prepared by alkylation of the dilithium salt of the 2-arylacetic acids (10 and 11) with 1-chloro- or 1-bromo-2-propyne; bromo- and iodolacton- ization with the corresponding N-halosuccinimides in meth- ylene chloride-potassium bicarbonate was rapid.

Synthesis of the homologous tetrahydropyran-2-ones 6-9 is shown in Scheme 4. The dianion of the 2-arylacetic acid was alkylated with 1-bromo-3-butyne. Unlike the synthesis of the tetrahydrofuranones, the cyclization of 14 and 15 to the tetrahydropyranones was very slow. Bromolactonization pro- duced lactones 6 and 8 in only moderate yield. Small amounts of the 2-isomer were produced during the extended reaction period, but these were removed by recrystallization. Iodolac- tonization with N-iodosuccinimide was also slow; however, lactonization with iodine in acetonitrile-potassium bicarbon- ate was more successful.

Analysis of Inactivation Kinetics Initial Rates of Inactiuation-The interaction of a suicide

substrate with a protease can be considered to proceed ac- cording to the kinetic scheme below (Equation l), where E is the enzyme, I the suicide inactivator, E . I the Michaelis complex, E - I the acyl enzyme, and ET the inactivated enzyme with the inhibitor covalently attached (-), Since the progress of the reaction is monitored by the decrease in total active enzyme concentration (accumulation of E f ) , E . I and E - I are not observed independently. Thus, this expression can be simplified using the enzyme-inhibitor complex E*I to represent the kinetic composite of E . I and E - I (Equation 2). This

simplified kinetic scheme conforms to that used to describe an active site-directed irreversible inhibitor and can be ana- lyzed by the method of Kitz and Wilson (15) to give the integrated rate expression,

13)

where t is the enzyme activity ( 6 = E, - E?). Therefore, a plot of the natural log of the enzyme activity uersus time should give a straight line with a slope -k&. In an earlier study (14) we demonstrated that the initial rate of inactivation

Portions of this paper (including “Experimental Procedures,” “Appendix,” and additional references) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of standard

of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. magnifying glass. Full size photocopies are available from the Journal

Request Document No. 83-1200, cite the authors, and include a check or money order for $4.00 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

Ph Ar Ar

Ph 5 Br Me (I-) Ar Hol Ar Hai

Ph5Br (2) Ph6Br (6) P h 5 I (2) P h 6 I (2)

Ph = phenyl Np5Br (2) Np6Br Np = I-naphthyl N p 5 I (5) N p 6 I (2)

SCHEME 2

SCHEME 3

(Ar=Ph) I t fnr=aNp) ‘,5 (ArzaNpI

SCHEME 4

of chymotrypsin by these lactones follows first order kinetics. With the less efficient and less reactive lactones (1-5), the

initial inactivation rates could be monitored by exposing a relatively concentrated (2 p ~ ) solution of chymotrypsin to various concentrations of inactivator. The inactivation is effectively stopped by periodic removal of aliquots which are then diluted into an assay mixture containing the chromo- genic chymotrypsin substrate benzoyl tyrosine ethyl ester. Suitable corrections are needed to account for competitive inhibition by unhydrolyzed lactone (see “Experimental Pro- cedures”) and for changes in a pure enzyme control. This analysis of initial rates of inactivation under steady state conditions assumes that at most only a small fraction of inhibitor is being consumed or undergoing spontaneous or enzymic hydrolysis during the observation period. This was true in the case of the inhibitors analyzed by this method but not for the more efficient and reactive lactones (see below) which were studied by kinetic analysis of a progress curve.

Determination of Inactivation Constants Ki and k,-Accord- ing to Equation 4, which is obtained by rearrangement of the definition of kobs, a plot of

I o 1.7 K %, kz k z - = - + - (4)

Io/kobs uersw I , should be linear. The values of the inactivation constants can then be evaluated from the slope (l/kn) and x intercept (-Kt) of this plot. Such plots for lactones 1-5 are shown in Fig. 1 and the kinetic constants determined by this method (Method A) are given in Table I.

The inactivation of chymotrypsin by the more efficient and reactive lactone suicide substrates (6-9) cannot be studied by the measurement of initial inactivation rates under steady state conditions because inactivation is too rapid and inhibitor concentrations change significantly during the observation interval. To analyze the kinetics of chymotrypsin inactivation with these lactones, we followed the progress curve of N- acetyltryptophan p-nitrophenyl ester (hydrolysis run in a competitive fashion. This approach has the advantage that

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15048 Chymotrypsin Enzyme-activc

the rate of inactivation can be slowed by raising the concen- tration of the competing substrate.

An example of such a series of progress curves with lactone 9 and N-acetyltryptophan p-nitrophenyl ester is shown in Fig. 2A. From the leveling of the slope, it is evident that by 1 min inactivation by this lactone is complete. The constants K, and k2 can be obtained by analysis of the changing slopes of the progress curves with time, according to the approach described by Main (16).

Main has derived an expression (Equation 5) to describe the time-dependent decrease in the rate of substrate hydrol- ysis that occurs when an enzyme is exposed simultaneously to a substrate and an active site-directed inactivator

" -100 0 100 200 300 400 500

I, (pM) FIG. 1. Determination of Ki and kz for lactones 1-5. Chy-

motrypsin was incubated with inhibitor at pH 7.2 and 25 "C and remaining activity was determined in aliquots removed at various times. A semilogarithmic! plot of the percentage of enzyme activity remaining versus time (not shown) gives a straight line with a slope of -kobs. A plot of the initial inhibitor concentrations versus Io/&bs

gives a straight line with a slope of l /kz and an z intercept of -K, for each of the lactones 1-5.

1.01 1

zted Irreversible Inactivators

The assumption has been made that the substrate and inhib- itor concentrations are not changing during the assay and can be represented by the initial concentrations So and Io, respec- tively. K, is the Michaelis constant for the substrate (N- acetyltryptophan p-nitrophenyl ester), and V, and V are the velocity of substrate hydrolysis at initial time and time t, respectively. According to Equation 5, a plot of In( V/Vo) uersus time will give a straight line, with a slope of -p (see "Experimental Procedures"). An example of such a plot with lactone 9 is shown in Fig. 2B. Equation 6, which is obtained by a rearrangement of the definition of p, indicates that a plot of I , /p uersus I, will give a straight line with a slope of l /k2 and an x intercept of -Ki.(l + SJK,,,). Since So is known and K,,, for N-acetyltryptophan p-nitrophenyl ester has been determined to be 31 p ~ , one can also obtain the value of Ki. Plots for the four lactones 6-9 are shown in Fig. 3.

TABLE I Kinetic constants for haloenol lactone inactivators and chymotrypsin

The inactivation behavior of these haloenol lactones is character- ized by the binding constant (K i ) , and the three rate constants, for inactivation ( k2) , catalytic hydrolysis (kc), and spontaneous hydroly- sis (kh) (see Scheme 5). The term, ( k + 4 ) / k 2 , is a partition ratio and represents the average number of enzyme turnovers per inacti- vation event. The ratio, k, /K; , is the bimolecular rate constant for the inactivation and represents the inactivation potency of the inhib- itor. Values for Ki and kz were determined by analysis of initial inactivation rates (Method A) for lactones 1-6 and inactivation progress curves (Method B) for lactones 6-9 (see text). All kinetic measurements were performed in pH 7.2 DhosDhate buffer at 25 'C.

Kt k z k / ('+ b / K ;

p~ min" min" min" h P s - 1

kho M / k *

Ph5BrMe 1 98.3 0.0368 3.31 0.00185 91 6.23 Ph5Br 2 50.0 0.0695 5.84 0.00398 85 23.2 Ph5I 3 42.2 0.0512 1.31 0.00926 38 20.0 Np5Br 4 17.3 0.0355 0.355 0.00538 11 34.2

Np5I 6 11.8 0.0247 0.126 0.00953 6.0 34.8

Ph6Br 6 1.94 8.59 223 0.00162 27 73,000 Ph6I 7 1.05 8.17 212 0.00252 27 130,000 Np6Br 8 0.636 7.69 23.1 0.00098 4.0 202,000 NpGI 9 0.339 5.94 4.16 0.00128 1.7 292,000

"Values in parentheses were determined by high performance liquid chromatographic analysis of rate of loss of lactone. Other values for kh were determined by UV and for kc by kinetic fitting of the ultimate activity data (see text).

(0.274) (0.0057)

(0.163) (0.013)

TIME (sec) TIME (sec1

FIG. 2. Inhibition in the presence of substrate. Chymotrypsin was added to a solution of inhibitor containing a final N-acetyltryptophan p-nitrophenyl ester concentration of 200 p~ at pH 7.2 and 25 "C, and the hydrolysis of the substrate was followed by UV at 400 nm. As the enzyme is inhibited the rate of substrate hydrolysis decreases, producing an inhibition progress curve. A series of these curves is shown in A for different concentrations of Np6I (lactone 9). The first derivative of the progress curve was obtained directly from a computer-assisted spectrometer and indicates the velocity of substrate hydrolysis as a function of time. A semilogarithmic plot of the ratio of the velocity to the initial velocity of the substrate hydrolysis versus time gives a straight line with a slope of -p for each inhibitor concentration ( B ) .

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Chymotrypsin Enzyme-activated Irreversibte Inactivators 15049

Stoichiometry of Inactivation, Spontaneous and Catalytic ~ y d ~ ~ y s ~ , and ~ l t ~ ~ a t e A c t ~ v i ~ Assa~-Th~retically, a sin- gle equivalent of an ideally behaved suicide substrate would be sufficient to inactivate an enzyme. Thus, a plot of the fraction of enzyme activity (EIE,) that ultimately (at “infi- nite” time) survives exposure to different ratios of an ideal suicide substrate ( Io/Eo) would be a straight line intersecting the x axis at Io/Eo = 1 (see Scheme 5 and Fig. 4, curve kh = kc = 0).

The efficiency of a suicide substrate can be compromised by premature dissociation of the reactive intermediate from the active site, prior to covalent attachment (inactivation), to

-20 -10 0 IO 2 0 3 0

I, @ M I FIG. 3. Determination of Kt and Fz2 for lactones 6-9. A plot

of the initial inhibitor concentration (I,) versus I J p (the p values are obtained from plots similar to those shown in Fig. 2) gives a straight line with a slope of l / k 2 and an x intercept of -Ki ( 1 + SJK,), where So and K , are the initial concentration and Michaelis constant for the substrate.

I .o I , Ph5 I

0 W \ W

IO‘EO

FIG. 4. Ultimate activity assay. Chymotrypsin was incubated with different concentrations of PhBI (lactone 3) at pH 7.2 and 25 “C. After all the inhibitor had been consumed (>15 h), the remaining enzyme activity was determined. The figure shows a plot of the initial inhibitor to enzyme ratio (Z,,/E,,) uersw the fraction of enzyme activity remaining (E/E,). The spontaneous hydrolysis of the inhibitor gives an outward curve to the plot. In theory (see “Appendix”), if there were no spontaneous hydrolysis ( k h = 0), the plot would be a straight line with an intercept on the Zo/E,, axis which indicates the turnovers per inactivation ( ( k 2 + kc)/k2) . An ideal inhibitor would have no spontaneous or catalytic hydrolysis (kh = kc = 0) and have an intercept of 1. The curve fitting method is explained in the “Appendix.”

produce the hydrolyzed inactivator I‘. This enzyme-catalyzed hydrolysis can be described by a catalytic rate constant kc (Scheme 5). The effect that the catalytic activity of the enzyme on the suicide substrate has on the plot of E/Eo versus Io/Eo is to reduce the slope of the line so that it extends to an intercept on the IJE0 axis of (k, + kz)/kz (see Fig. 4, curve kh = 0). This ratio is termed the partition ratio, and it represents the average number of enzyme “turnovers per inactivation.”

The efficiency of a suicide substrate can also be compro- mised by the spontaneous degradation (hydrolysis) of the suicide substrate. While this may not be a sensible chemical alternative in the case of suicide substrates whose activation is based on isomerization, elimination, or oxidation, it is a real concern with the lactones that are the subject of this study, because they are moderately activated toward sponta- neous hydrolysis. The effect that spontaneous hydrolysis (characterized by the rate constant kh; see Scheme 5) has on the ultimate activity curves is to cause a decrease in slope and a progressive outward curvature of the line (Fig. 4).

The data in Fig. 4 are, in fact, from an ultimate inactivation assay on lactone 3. The data points were fitted according to the method described in the “Appendix.” Plots for the ulti- mate inactivation of chymotrypsin by all of the nine lactones are shown in Fig. 5. It is apparent that the efficiency of chymotrypsin inactivation by the lactones varies dramatically (see discussion^).

It is possible to derive a simple analytical expression that describes the ultimate activity curves (E/E, versus I,,/E0), and if K: and kp are known, to determine kc and kh by a least squares curve fitting routine (see “Appendix”); however, be- cause kc and kh are not uncorrelated variables in the curve fitting process, estimates of their values can be quite inaccu- rate. Therefore, we have used a more direct method for determining kh and kc.

Since there is a small, but significant change in the ultra- violet absorbance upon hydrolysis of the enol lactones, this change can be monitored with time to determine the hydrol- ysis rate constants directly. These kh values are summarized in Table I. In conducting this assay, we have noted that the

1.0

0.8

0.6 0 w .

w 0.4

0.2

‘0 20 40 60 80 100 0 10 20 30 40 50 60

1o’Eo 10) Eo

Fro. 5. Ultimate activity assays of lactones 1-9. Chymotryp- sin was incubated with different concentrations of i n h i b i ~ r at pH 7.2 and 25 “C. After all of the inhibitor had been consumed (>15 h), the remaining enzyme activity was determined. A shows the plot of the initial inhibitor to enzyme ratio versm the fraction of enzyme activity remaining for the hutyrolactones 1-6. B shows the same plot for the valerolactones 6-9. Data from these plots can be fitted to a function (see “Appendix”) to obtain the values for the turnovers per inactivation, (kz + k,)/k2, and kc (if the value of k2 is known).

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15050 Chymotrypsin Enzyme-activated Irreversible Inactivators

FIG. 6. Determination of kh and k, by HPLC analysis. A typical chromat- ogram (for details see “Experimental Procedures”) of a partially hydrolyzed sample of Np5Br is shown in A . Unhy- drolyzed lactone elutes at approximately 4 min, the internal standard at about 7.5 min, and hydrolysis products at 10.2 and 14.7 min. The rate of lactone hydrolysis was determined by electronic integration of the lactone peak relative to that of the internal standard. Rates of spontaneous and chymotrypsin-catalyzed hydrolysis of Np5Br at pH 7.2, 25 “C, are shown in B. 0, no enzyme added A, [a-chymo- trypsin] = 4 nM.

l r

.L

A

_h I I I I I I I ( I

4 8 I2 16 0

hydrolysis is catalyzed by Tris buffer; therefore, all the hy- drolysis rate measurements, as well as all of the inactivation assays described previously, were conducted in phosphate buffers, which have no effect on the hydrolysis rates.

Since the hydrolysis of the haloenol lactones can be a complicated multistep process (it proceeds first to the halo- keto acid, which can then go to the hydroxyketo acid or possibly cyclize to the ketolactone), we felt that it was nec- essary to confirm our estimates of the hydrolysis rate constant using a more specific method. Therefore, in certain cases, we followed the decrease in haloenol lactone concentration with time by HPLC’ analysis using an internal standard.

An HPLC trace illustrating the hydrolysis of lactone 4 is shown in Fig. 6A. Under the normal phase conditions, the lactone is the first to elute (3.5 min), followed by the internal standard 4,4’-dimethoxybenzophenone (7 min). Hydrolysis products assigned as the bromoketoacid (10.2 min) and the hydroxyketoacid (14.7 min), by comparison of HPLC reten- tion times with authentic samples, elute at later times. The rate of lactone hydrolysis was determined by electronic inte- gration of the peak for the lactone relative to the internal standard peak. An example of such a hydrolysis rate assay is shown in Fig. 6B, where the rate of spontaneous hydrolysis of lactone 4 can be determined from the slope of the lower line (solid circles). In those cases where we have determined k h by HPLC, we have obtained values that are in reasonable agreement with those determined by the UV method (see Table I; values determined by HPLC are in parentheses).

The catalytic rate constant kc of a suicide substrate is usually determined by measuring the initial rate of substrate consumption or product formation. This can be difficult if inactivation is rapid. Moreover, we have not been able to make reliable determinations of kc by following the loss of substrate by UV, because of unpredictable transients in ab- sorbance that occur when enzyme is added to the suicide substrate.

As described above, we have been able to measure the loss of substrate ( i e . lactone) by HPLC analysis, and by this to determine kc. An example of such a determination is shown in Fig. 6B, where initially the rate of lactone consumption in the presence of enzyme is more rapid, having an inactivation, a catalyzed and a spontaneous component; as the enzyme becomes inactivated, the rate of loss of lactone returns to the rate due to spontaneous hydrolysis alone. The molar quantity

* The abbreviation used is: HPLC, high performance liquid chro- matography.

TIME (mml TIME ( m l n l

of lactone consumed (above that due to spontaneous hydrol- ysis) relative to the molar quantity of enzyme added to the incubation gives directly the ratio ( k c + kz ) /kB , and since k2 is known, kc can be evaluated. Values of kc determined in this manner are shown in Table I (in parentheses).

We have also been able to determine kc indirectly, using the data from the ultimate activity assay: since the values for K,, kz, and kh are known, kc can be determined accurately by kinetic modeling (see “Appendix”). The values of kc deter- mined by this method are shown in Table I.

DISCUSSION

The use of haloenol lactones as potential enzyme-activated irreversible inhibitors for serine proteases was first proposed by Rando in 1974 (17). Aside from our preliminary account (14), there have been no reports on the use of haloenol lactones as suicide substrates since Rando’s proposal, al- though the use of nitrosolactams (5 , 6), 6-chloro-2-pyrones (7) , and chloromethyl coumarins (8-12) as inactivators of chymotrypsin has been described.

In our preliminary account (14), we sought to establish definitively that two aryl-substituted haloenol lactones inac- tivated chymotrypsin by an enzyme-mediated process. This involved a series of control studies using other model com- pounds to establish that inactivation depended upon interac- tion of the lactone with the active site of chymotrypsin, that it required a cyclic (lactone) type of ester so that the reactive haloketone would be tethered in the active site during the lifetime of the acyl enzyme, and that the halogen-alkylating agent was required. In the present account, we have expanded upon these studies in two ways. We have investigated nine aryl-substituted haloenol lactones as suicide substrates of chymotrypsin. These display a graded series of structural changes: 5- and 6-membered ring lactones, phenyl and a- naphthyl aryl substituents, and bromine or iodine as halogens. We have also endeavored t G make a more comprehensive analysis of the kinetic features of the inactivation process, which we can now characterize in terms of a binding param- eter K,, and an inactivation rate constant $, these together determining the inactivation potency of the inactivator (b/ K,). We have also measured the catalytic rate constant kc as well as the rate constant for spontaneous hydrolysis k h . Thus, we can also determine the partition ratio ( k c + kZ)/k2, termed “turnovers per inactivation,” as well as the overall efficiency of the inactivation process. There are many interesting ways in which the kinetic constants reflect the systematic alteration in structure of these haloenol lactone substrates.

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Chymotrypsin Enzyme-activated Irreversible Inactivators 15051

Kinetic Analysis

Binding Constant (Ki)-The affinity of suicide substrate interaction with chymotrypsin can be characterized by the binding constant K,. The value of this constant varies some 300-fold, the highest affinity compound (9) being among the very highest affinity compounds for the active site of chymo- trypsin (18). The structural parameter that has the major effect on Ki is the lactone ring size, with the 6-membered valerolactones binding with 20- to 40-fold higher affinity than the butyrolactones. The higher affinity of the larger ring lactones must reflect, simply, a better "fit" to the active site, as they are not much more hydrophobic. Next to the effect of ring size, the higher affinity of the a-naphthyl versus the phenyl-substituted lactones (3-4-fold) and the very minor effect of iodine versus bromine (up to 2-fold) are relatively insignificant and may represent the general preference of chymotrypsin for lipophilic substances.

Inactivation Rate Constant (kd-The rate constant for the inactivation of chymotrypsin by the haloenol lactones de- pends almost exclusively on the ring size of the lactones, the 6-membered ones having 100-200-fold higher activity. This may be a reflection of the fact that in the acyl enzyme, the chain bearing the alkylating group derived from the 6-mem- bered ring lactone is longer and may have better access to the site of alkylation than the shorter chain derived from the butyrolactones. The effect of the aryl substituent (about 2- fold) or halogen (essentially negligible) are, by contrast, al- most insignificant.

The fact that the inactivation rate constant does not depend upon the nature of the halogen i s surprising, since the iodo- methylketone should be a more reactive alkylating agent than the bromomethylketone. Still, it is not certain that alkylation is a rate-limiting step, nor that the iodomethylketone, because of its greater steric demands, is disposed as favorably for alkylation as the bromomethylketone.

The ratio kz/K,, which may be termed the inactivation potency, is often used to characterize the effectiveness of compounds as enzyme inactivators. The lactones we have studied present a very wide range of kz/Ki values, ranging from 6.23 M" s-l for lactone 1 to 292,000 M" s" for lactone 9. The major structural parameter that affects this ratio is the lactone ring size; aryl substituent and halogen have, at most, a 3- and 2-fold effect, respectively. The inactivation potency of the lactone inactivators compare very favorably with the kz/Ki values of other better known inactivators of chymotrypsin (cf. Table 11).

Enzyme-catalyzed Hydrolysis Constant (k,)--The depend- ence of this constant on structural changes parallels that of the inactivation rate constant kz in that the 6-membered ring lactones are 30-160-fold more active than the 5-membered ones. In contrast, however, the nature of the aryl substituent and the halogen have a greater effect on kc and on kz. Since enzyme-catalyzed hydrolysis of ester-type substrates (e.g. lac- tones) is generally limited by the rate of dissociation of the acid component (which is thought to be governed by the rate of deacylation), one might imagine that the more lipophilic a-naphthyl and iodo bearing lactones could be slower to dissociate and thus would be less actively hydrolyzed.

The ratio ( k c + kz ) /kz , termed turnovers per inactivation, is often used to characterize the efficiency or selectivity of a suicide substrate. With the compounds we have studied, this ratio varies from 91 to 1.7. Both lactone ring size and the nature of the aryl substituent play a major role in determining the value of this ratio. An ideal suicide substrate would inactivate an enzyme stoichiometrically, that is, the ratio of ( k , + k2)/k2 would be 1. Three of the lactones we have studied

TABLE Ir Potencies of irreversible inhibitors of chymotrypsin

Inhibitor k d K

"S"

Phenylalanine chloromethylketone" 0.041 N-Formylphenylalanine chloromethylke- 1.35

N-Tosylphenylalanine chloromethylketone" 7.7 N-Formylphenylalanine bromomethylke- 26

Diisopropylphosphofluoridateb 45 Benzyloxycarbonylphenylalanine chloro- 69

Benzyloxycarbonyl-Ala-Gly-Phe-chloro- 100

3,4-Dihydro-6-chloromethylcoumarin [151d 170 Phenylmethanesulfonyl fluorideb 250 N,N-Diphenylcarbamoyl chloride' 480 Benzyloxycarbonylphenylalanine bromo- 790

3,4-Dihydro-3,4-dibromo-6-bromomethyl- 3,000

3,4-Dihydro-3-benzyl-6-chloromethylcou- 720,000

tone"

tone"

methylketone"

methylketone'

methylketone"

coumarin [5Jd

marin [21d

' Reference 19. Reference 20. Reference 21. Reference 10. Numbers in brackets are values for turnovers per

N P ~ I (9) 292,000

inactivation ( (k2 + k,) /k , ) estimated from the data in Reference 10. e Reference 22.

(5, 8, and 9) approach this ideal value within an order of magnitude.

Spontaneous Hydrolysis Rate Constant (kh)"This rate proved to be much less sensitive to structural alterations than any of the other, enzyme-related, constants. The valeroIac- tones hydrolyzed, on the average 3-6-fold slower than the butyrolactones, but the aryl substituent and halogen had no greater than a 3-fold effect.

In all cases, the rates of spontaneous hydrolysis are low; half-times vary from 73 min (for 5 ) to 710 min (for 8). The fact that the spontaneous hydrolysis rates have a relatively insignificant effect on the inactivation efficiency can be seen from the ultimate activity assay data (cf Fig. 5). In this assay, the effect of spontaneous hydrolysis of the suicide substrate is manifest by a decreased slope and an outward curvature of the line relating E/Eo to Io/&. This curvature is apparent only at very low levels of E/E,. Furthermore, the n intercept projected from the linear portions of these curves differs from the values of (kc + k z ) / k ~ calculated from the data in Table I by only small factors.

The reduced hydrolytic lability of the 6-membered ring lactones relative to the corresponding 5-membered ring ones is in contrast to the reports that simple valerolactones are considerably more easily hydrolyzed than butyrolactones (23, 24). It is possible that the a-aryl substituent or the enol nature of our lactones is responsible for the reversal in the reactivity trend.

One factor that we have not considered explicitly in any of our studies is that all the lactones we have utilized are racemic mixtures. In general, chymotrypsin acts on only one enan- tiomer of a racemic pair (25), while the other will often bind with considerable affinity, and act as a competitive inhibitor. The effect of the racemic nature of our suicide substrates on our estimates of the kinetic parameters is somewhat difficult to predict. It is most likely that the values for kz and kc of the active enantiomer will be greater, and for Ki, smaller than that of the racemate; k h should be unaffected. Also, the

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15052 Chymotrypsin Enzyme-activated Irreversible Inactivators

ultimate activity assay (cf. Fig. 5) could be affected in a way that would make the abscissa scale differ by a factor of 2. All these changes would have the effect of making the active enantiomer a more potent and selective inactivator as com- pared to the racemate. We are currently preparing the pure enantiomers of the more effective haloenol lactones for de- tailed kinetic analysis.

CONCLUSIONS AND IMPLICATIONS FOR FURTHER DESIGN

It is evident from our studies of the inactivating behavior of haloenol lactones toward chymotrypsin that the parameters of inactivation are very dependent upon certain structural features, primarily lactone ring size, and secondarily, the nature of the aryl and halogen substituents. Within the lim- ited series of structural variants that we examined, we have compounds that approach, within an order of magnitude, the behavior of an ideal suicide inactivator.

Our results have implications in the further design of in- activators. Since the nature of the halogen has such a minor effect, it may prove useful to examine less reactive leaving groups, such as chloride and perhaps even fluoride. The greater effectiveness of the 6-membered ring lactones uersus the 5-membered ones in terms of Ki, k2, and kh indicates the importance of an appropriate fit to the active site and acces- sibility of the alkylating function released and the reactive nucleophilic residue. This suggests that even larger ring size lactones or lactones where the dimensions between the acyl group (to become affixed to the active site serine), the alkyl- ating group, and the aromatic substituent should be explored systematically. Finally, the effect of the aryl substituent is considerable; affinity is enhanced and enzymatic hydrolysis retarded by the larger naphthyl substituent. This suggests that lactones bearing a larger variety of aryl groups should be explored in the search for inactivators of yet better potency and efficiency.

REFERENCES

1. Sandler, M., ed (1980) Enzyme Inhibitors as Drugs, University Park Press, Baltimore, MD

2. Kalman, T. I., ed (1979) Drug Action and Design: Mechunism- Bused Enzyme Inhibitors, Elsevier/North-Holland Biomedical

Supplemental Plaferlals to

HAMENOL LIICTONES: POTENT ENZYME-ACTIVATED IRREVERSIBLE

INHIBITORS OF o-CHYMOTRYPSIN

Scott 8 . Danlels, Edward Cooney. Michael J. Sofla,

Prasun X . Chdkravarey, and John A. Xatrenellenbogen

EXPERIMENTAL SECTION

m e l t L m apparatus and are uncorrected. Proton llH-NHRl spectra Were Chemical - Beltmg polnts were determlned with a Thomas Hoover capillary

obtalned on Varian Associates models HR-220 1200 H H Z J and EM-390 190 MHzl , O r

mi1l.o" downfield from tetramethylnilane as an Internal Standard 1 6 scale). Nicolet nodel NT 360 1360 M I Z J spectrometers, and are expressed as parts per

The f~-NMR data are presented in the form: 6 value of s l g n a l (peak multipli- city, coupling Constant (If applicable1,lnteqrated number of plOton9J. Mass spectra were obtalned on a Varlan AsSoClates MAT CH-5 spectrometer at 10 eV or lo e". nata are reported ~n the form: m / r (intensity relative to base peak= 1 O O J . Elemental analyses were performed by the Mlcroanalytical Service Labor- atory of the Un~verslty of Illinols School of Chernlcai Sciences. Inactivation assays were performed using a Varian 635 UV-VIS or Beckman DU-8 spectrometec.

and was tltrdted for oeganlc base content by the method of Kofron 127). 4.4"Dlmethoxybenzophenone and praparqyl chloride were purchased from Aldrlch. 3-Butvn-1-01 was ourchased from Farchan Labs. ALT and molsture sensitive

"-Butyl llthlurn as a hexane solution was purchased from Alfa IVentronI

Press, Amsterdam 3. Seiler, N., Jung, M. J., and Koch-Weser, J., eds (1978) Enzyme-

activated Irreversible Inhibitors, Elsevier/North Holland Bio- medical Press, Amsterdam

4. Walsh, C. (1982) Tetrahedron 38 , 871-909 5. White, E. H., Jelinski, L. W., Perks, H. M., Burrows, E. P., and

6. White, E. H., Roswell, D. F., Politzer, I. R., and Branchini, B. R.

7. Westkaemper, R. B., and Abeles, R. H. (1983) Biochemistry 22,

8. Wakselman, M., Hamon, J. F., and Vilkas, M. (1974) Tetrahedron

9. Nicolle, J. P., Hamon, J. F., and Wakselman, M. (1977) Bull. SOC. Chim. Fr. 1977,83-88

10. BBchet, J.-J., Dupaix, A., and Blagoeva, I. (1977) Biochimie (Paris) 59,231-239

11. BCchet, J.-J., Dupaix, A., Roucous, C., and Bonamy, A.-M. (1977) Biochimie (Paris) 59,241-246

12. Vilkas, M. (1978) in Enzyme-activated Irreversible Inhibitors (Seiler, N., Jung, M. J., and Koch-Weser, J., eds) pp. 323-335, Elsevier/North Holland, Amsterdam

13. Krafft, G. A., and Katzenellenbogen, J. A. (1981) J. Am. Chetn.

14. Chakravarty, P. K., Krafft, G. A., and Katzenellenbogen, J. A.

15. Kitz, R. J., and Wilson, I. B. (1962) J. Bwl. Chem. 2 3 7 , 3245-

16. Main, A. R. (1973) in Essays in Toxicology (Hays, W. J., ed) Vol.

17. Rando, R. R. (1974) Science (Wash. D. C.) 185 , 320-324 18. DelMar, E. G., Largman, C., Brodrick, J. W., and Geokas, M. C.

19. Shaw, E., and Ruscica, J. (1971) Arch. Biochem. Biophys. 145,

20. Fahrney, D. E., and Gold, A. M. (1963) J. Am. Chem. SOC. 8 5 ,

21. Morihara, K., and Oka, T. (1970) Arch. Biochem. Biophys. 138,

22. Erlanger, B. F., Vratsanos, S. M., Wassermann, N. H., and

23. Hall, H. K., Jr., Brandt, M. K., and Mason, R. M. (1958) J. Am.

24. Brown, H. C., Brewster, J. H., and Schechter, H. (1954) J. Am.

25. Zeffren, E., and Hall, P. L. (1973) The Study of Enzyme Meehu-

Additional references are found on p. 15053.

Roswell, D. F. (1977) J. Am. Chem. SOC. 99 , 3171-3173

(1975) J. Am. Chem. SOC. 9 7 , 2290-2291

3256-3264

30,4069-4079

SOC. 103,5459-5466

(1982) J. Biol. Chem. 257 , 610-612

3249

4, pp. 59-105, Academic Press, New York

(1979) A d . Biochem. 99,316-320

484-489

997-1000

526-531

Cooper, A. G. (1970) Biochem. J. 118,421-425

Chem. SOC. 8 0 , 6420-6427

Chem. SOC. 76,467-474

nism, pp. 179-186, John Wiley & Sons, New York

buffer used I" all chymotrypsin inactivation studles was 0.1 M KH~PO~-I(~HPO~, pH 1.2. The preparatlon of lactones &, 2 , and 2 and acld &z Eias been reported I n previous publlcatlons 113.141.

Chernxeal Synthenls

I-Bromo-3-butyne - A s~lutlon of phosphorus trlbromide 16.1 mL, 0.071 m o l l in dry ether 125 m L 1 was added dr-opwlse at O'C over a period of 30 m m to a solutlon of 3-butyn-1-01 110 g , 0.14 moll in dry ether 114 mL1. The reaction rnlxture was then browht to reflux for 2h. After belncl cooled. the reactlon mlxrure was poured lnto a beaker contalning crushed rce and Stirred v ~ ~ o ~ o u I I ~ . The ether layer vas collected, and the aqueous layer was extracted wlth ether

saturated NaHCO3, water and saturated NaCl, dried lPlgSO4). and evaporated I50 mLJ. The combxned ether extracts were washed successlveiy with water,

leavxnq an oil that was purlfled by dlstlllatlon: Yleld 13.5 q 171%); bp 110- 112'C 1 I l t . 1291 10?°C/140 m l , IH-NMR (Neat): 6 2 . 2 5 it, 3 ; l . O Hz, 1"). 2.18 ldt. 5=7.5. 3.0 HZ, 2H1, 3.49 It, 517.5 Hz, 2Hl.

mlxrure was poured lnto a beaker contalning crushed rce and Stirred v ~ ~ o ~ o u I I ~ . The ether layer vas collected, and the aqueous layer was extracted wlth ether

saturated NaHCO3, water and saturated NaCl, dried lPlgSO4). and evaporated I50 mLJ. The combxned ether extracts were washed successlveiy with water,

leavxnq an oil that was purlfled by dlstlllatlon: Yleld 13.5 q 171%); bp 110- 112'C 1 I l t . 1291 10?°C/140 m l , IH-NMR (Neat): 6 2 . 2 5 it, 3 ; l . O Hz, 1"). 2.18 ldt. 5=7.5. 3.0 HZ, 2H1, 3.49 It, 517.5 Hz, 2Hl.

dropwlse to a solution of diisopropylamlne (3.1 mL. 22 ml1 In 20 mL of THF at 0'C under nitrogen. ThLs solufron Of lxthlum dllsopropyldmrde was added dropwse over Ih to 1-naphthylacetic acid 11.9 9 , 10 mol) in 50 mL of THF at -18OC under nitrogen and the reaction mlxture strrred for 2h. I-Chlaro-2- propyne 10.15 4 , 10 moll ~n 10 m L of THF was added dropwlse and khe reaction was allowed to s lowly warm to room temperature. After 14h the reactlon Was quenched with 25 mL of 1 N hydrachlorlc acld: 50 mL of ether was added and the organlc layer was collected and washed wlth another 25 mL-portlo" of acld.

The comblned ether extracts were washed wrth saturated sodium chlorlde and The comblned acld phases were extracted with three SO mL-portions of ether.

drled over anhydrous magneslurn Sulfate. Thls IOlUtlOn was filtered through a 3 cm pad of sllica g e l , and the solvent was removed to g i v e the crude product as a yellow solzd. Repeated recrysralllzatlon Of the crude product from 1:s- ethe1:hexane qave 1.6 g (6981 of whlte crysta ls wrth a rnelr lng polnt Of 135-

ppm, J=17 Hz, wlth each line appearing as d doublet Of doublets. 5-1.5, 3.0 H I , 131'C. IH-NMR lCDc13J: 6 1.90 I t , 5=3.0 H z , 1 1 1 ) . 2.88 118 quarte t , Cv~0=0.30

2H1, 4 . 6 4 ldd, J = 8 . 0 , 1.0 H z , IS), 7.21-1.60 im. 4 H I . 7.66-7.92 l m , 2Hl. 7.95-

M + l , 186 11E.BJ. 185 146.0). 180 111.61. 179 IlDOI. M a l . Calcd. for C15H1202: 8.26 lm, IN), 11.27 1s. 1 H ) . Mas8 spec. i i 0 eVJ: m/n. 225 110.81. 224 16S.El

c, 80.34: H, 5.39. Found: C, 80.08: H , 5.44.

2-Phen 1-5-hex ~ O I C acld I J - D~lSopropylamine 11.5 mL, 1 1 moll was added to a ~olutionYaf n-BuLi 1@9 mL, 10 mo1J ~n dry THF I S mL1 at -20*C.

2-l l -NaphthulJ-4-pentrnoic Acid 14.21 - n-Butylllthlum 120 m o l ) was added

After the additlon was %mplete. the reactlon mixture-was warmed up to 0-C and stlrred for 30 mln. TO this, a solutlon Of phenyl acetlc acld 10.68 9 . 5.0 mll in dry THF 15 mLJ was added dropwise at ODC. and the rnlxeure wa5 Stlrred

wrrh the addltlon of HMPA 10.5 m L 1 . followed by the addltlon of l-bramo-3- at that temperature for 1.5h. The yellow precipitate that formed was dissolved

butyne 10.69 g , 5.2 -1). The resulting lrght yellow solutlon was Stirred a t O°C for 2h and at 2SrC for 12h. The IeaCtlOn mlXtUle Was acldilred wlth 3 N HC1, extracted wlrh ether. and the ether layer vas washed n t h water, drred-

Yleld 0.1 q 114hJ; mp 91-93°C IIecrystalllred from ether-hexanel: IH-NMR lPlqS04), and punfred by flash column chromatography u s l n q ether-hexane 11:21:

ICDC13J: 6 1.96 Id. J = 3 H z . 1H. acetylenlcl, 2.25 l m , 4HJ. 3.89 It. J=8 Hz,

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Chymotrypsin Enzyme-activated Irreversible Inactivators 15053 1 ~ ) 7.35 15 5 ~ 1 ; M ~ S S spec. I10 e v ) : m / n 189 110, Mi), 148 191, 143 IlOOl I 136'1381, 12; (191, 91 154): Anal. Calcd. for C12H1202: C, 76.57: H, 6.43. Found: C, 76.39: H. 6.44.

removed and ;he Crude 9olLd prod& iecrystalllzed from 1:10-ether:hexane to

YH-NMR lCDCl3i : 6 3.04 lddd, J=17.8, 7.07, 2.51 HZ. IHI, 3.59 lddd, J=17.8, 1ve 0.53 g of 2 188%) as whlte crystals vlth a meltlng polnt of 74.5-76.0'C.

302 1100. M ' l , 234 t49.11, 232 140.0l. 223 120.21. 154 147.51. Anal. Calcd.

Br, 2&.9&. for C gH 1Br02: C, 59.43; H, 3 . 6 6 : Br, 26.36. Found: C, 59.23: H, 3.54:

bicarbonate 7 0 . 2 0 9, 2.0 mmoli, N-lodosuccln~rnide 10.45 q, 2.0 moll, 2-11- naphthyl)-4-pentynolc acid 42 10.45 g , 2.0 m i ) , and 0.5 mL of a 0.4 n aqueous B o l u t l o n of tetrabutylamnlum hydroxide were Stlrred In 25 mL of methylene chlorlde at roam temperature for lh. Work up and purlf~catlon as descrlbed for

xng poxnt of 90.5-91.5°C. IbNMR (CDc13): 6 3.10 lddd, J=17.7, 7.07, 2.40 HZ, lactone 4 ylelded 0 . 5 7 g of 182%) as a whlte crystalllne product vlth a melt-

5.98 It, J=2.24 Hz, 1HI. 7.26-1.51 l m , 4 H I . 7.62-7.81 Im, 3 H l . M S S spec. I H I , 3.49 lddd, 5=17.7. 10.8. 2.08 H z , 1HI. 4.78 ldd, J=IO.B, 7.18 Hz, I H ) ,

110 e V ) : m/z 3 5 1 117.11, 350 1100, M + I , 213 (21.91, 195 116.0. 154 149.1). Anal. Calcd. for C15HllIO2: c. 5 1 . 4 5 ; H. 3 . 1 7 ; I, 36.24. Found: C. 51.50; H, 2.96; I, 36.24.

3-ll-Naphthyll-5IEi-llodomethylenel-fetrahydrofuran"one 121 - Potasslum

J - P h e n y l - 6 l E ~ - ~ b r 0 m m ~ t h y l ~ ~ ~ l - t ~ t ~ ~ h dro ran-2-one I 1 Potassium blcdrbonate P7 mg, 0.27 mmoli, N-bIOmOSUC$lnl%de 147 mg, 8.2; nrmall, 2- phenyl-5-hexynoic aczd ( 5 0 mq. 0.27 moll, and 0.1 mL of a 0.4 M aqueous solution of tetrabutylammonium hydroxlde were Stirred ~n 4 mL of Gthylene chloride at room temperature for 12h. work up as deecrlbed for lactone and puxxflcatlon by flash chromatoqraphy using 258 ether-hexane yaYe 36 mg O i 0 150%) as a Semisolid. IH-NIUI lCDC13): 6 2.20 In. 2H1, 2.15 l m , 2Hi, 3 . 8 0 l m . I H I , 6.05 I t , 5=2.0 Hz, 1H1, 7.32 lm , 5Hl : Mass spec. I10 eVI : m / r 268 16. M + ) . 266 17. M + ) , 239 (41, 231 141. 187 187). 159 1100). 143 (711, 128 1151: Anal. Calcd. for C12HllBr02: C, 51.99; H, 3.97; B r , 29.91. Found: C, 51.88: H. 4 . 0 3 : Br. 30.09.

3 - P h e n y l - 6 l ~ ) - l l o d o m t h y l e n e l - r e t r a h y d r o ~ y ~ ~ ~ - 2 - ~ ~ ~ 111 - Iodine 10.14 g , 0.55 moli was aaaed to a mlxture of 2-phenyl-5-hexynolc acid 10.10 9. 0 . 5 5 mmoll and KHCO) (0.055 9. 0.55 mmll In dry CHlCN 112 mLJ at room tempera-

crlbed for lactone $ furnished a crude product that was purlfled by flash ture, and the mrxture was stlrred I" dark for 16h. Product 1solatlOn ais des-

chronatoqraphy urinq 15% ether-hexane yleldlng 0.070 4 Of 2 141%): mp 83-84Y ICryStalliEatlOn from ether-hexane): Rf 0.29 ~n 15% ether-hexane. lH-NHR lCDcl31: 6 2.20 l t , J=6 Hz, 2Hl. 2.15 lq, J=6 Hz, and each peak further split i n t o a d, J=2.0 HI. 2H1, 3.78 ldd, 5=6. 9 HZ. IHI. 5.98 It. 3=2.0 Hz, 1HI. 7.28

128 1301, 117 1361. Anal. Calcd. for C12H11102: c. 4 5 . 8 8 ; H . 3 . 5 3 ; I. 40.40. Found: c, 45.95: H, 1.62; I, 40.25.

I", SH). mass spec. 110 N: m/r 314 123, M + ) , la7 151). 159 1 5 7 1 . 143 1100).

blcarbanate 10.10 q , 1.0 -11, N-bromasuceinirnide (0.18 4 , 1.0 mol), 2-ll- naphthyl)-5-hexynox acld 10.24 4 , 1.0 m o l ) , and 0.25 mL of a 0.4 M aqueous SOlutlOn of tetrabutvlamnlum hvdroxlde were stirred I" 15 mL of mthvlene

3- 11-Naphthyl-6 lE)-lbromomethylene)-t~~~~hydro~~ran-2-o~~ 1@ - PotassLYm

chlorlde a f room ternberature for- Jh. WoEk up 1s descr;bed fox lactone and purlfrcatlon by flash chromatography using 1 0 8 ether-hexane produced 0.81 g Of

f.48 ldd, 5=6, 9 H z , 1HI. 6.05 lt 3=2 Hz 1Hl 1 . 4 5 (n, 4H) and 1.90 l m , 3 H ) . 166%) as a semlsolld. NMR lCoC1~1: 6 2.32 lq, J=6.0 Hz, 2H). 2.84 Im, 2HI.

M E S spec. 110 e V i : W z 318 174,'M'). 31; 186: Mil. 290 122). 288 1211, 237 1 8 0 ) . 209 11001. 193 1911, 153 1751. linal. Calcd. for C 6H13Br02: C, 60.59: H. 4.13; ET. 25.19. Found: C , 60.52; H, 4.25; B r , 25.0i.

~ ~~ 1~

chlorlde a f room ternberature for- Jh. WoEk up 1s descr;bed fox lactone and purlfrcatlon by flash chromatography using 1 0 8 ether-hexane produced 0.81 g Of

f.48 ldd, 5=6, 9 H z , 1HI. 6.05 lt 3=2 Hz 1Hl 1 . 4 5 (n, 4H) and 1.90 l m , 3 H ) . 166%) as a semlsolld. NMR lCoC1~1: 6 2.32 lq, J=6.0 Hz, 2H). 2.84 Im, 2HI.

M E S spec. 110 e V i : W z 318 174,'M'). 31; 186: Mil. 290 122). 288 1211, 237 1 8 0 ) . 209 11001. 193 1911, 153 1751. linal. Calcd. for C 6H13Br02: C, 60.59: H. 4.13; ET. 25.19. Found: C , 60.52; H, 4.25; B r , 25.0i.

~ ~~ 1~

Of acid IO 0.50 mmo ? ~n dry CHJCN (15 mL) was eflrred at room temperature in dark for

.I2 9, 0.49 moll. XCHOJ 10.050 y , 0.50 mol) and 12 10.127 q ,

24h. The product was Isolated a6 whlte solld after work up, as descrrbed for

hexane: Y l e d 0 11 g 162%); mp 109-llODC lrecrystalllzed from ether-hexanel the lactone 4 an! was purlfled by flash column chromataqraphy usln9 20% ether-

Rf 0 . 1 1 ~n 108 ether-hexane. NHR lCDc13I: 6 2.30 l q , J=6.0 Hz, 2Hi. 2.79

J.7.5 HZ, 1H). 6.05 It, J=2.0 H a , 1Hi. 7.40 lm, 4H1, 7.60 lm, ? H I . Mass spec. lqulntet, J=6.0 Hz, and each peak further spllt LntO d, 5=2 H Z . 2H). 4.50 I t .

Calcd. for C16H13102: C, 52.77: H, 1 . 6 0 : I, 34.85. Found: C, 52.70; H, 110 eV1: m/r 364 122, Mil, 237 160) . 209 1 4 4 1 , 181 1141. 161 11001. Anal.

3.81; I , 15.03.

3 - l l - N a p h t h y l l - 6 l E ) - l ~ ~ d ~ ~ t h y l e n e ) - t e t r ~ h y d ~ ~ p y ~ ~ " - 2 - ~ ~ ~ 1,9j - A mixture

hydrorysis of the inhibitor is a firse-order process deacr Deternunation of the Hydrolysis Rate Constant, k h q i PPontaneOUs

= -khiIl.

Integration and substitution of A/E~, where A ia the absorbance, c the malar absorprlvlty, and 1 the cell path lenqth, gives

A = A0E-*ht

where Is the absorbance at Inltial time. The difference Spectrum of each

length where there was an appreciable difference between the two. The dls- inhrbitor wa8 taken in pH 7.2 phosphste buffer ~n order to determine a wave-

"ai8 m the region of 230 nm and 215 nm for the phenyl- and napthyl-substituted appearance of each lactone was followed at the appropriate wavelenath, which

nentlal function qave the hydrolyeis rate constant, kh. inhibitors, respectively. Fittinq the resulting PTDOI~SO curves to the expo-

gUOt of a Ca . 2 % solution of lactone I n acetonltrlle was added lnfo a 10 BL- Deterrmnatlon of the Hydrolyrls Rate Constant kh by HPLC - An 80 y ~ - a l l -

reactlo" vessel, equllzbrafed I n a thermostated water bath a t 25-, contalnlnq 4 mL of 0.1 M DhoSDhate buffer oH 7.4 and 80 uL of a 1.0 mM solur~on of 4.4'-

HLfEWNCES

26. Stlll, W. C., Kahn, M . , and Mlrra. A. 119781, J. Org. Chem. 9, 2923-2325. 27. Kofron. W. G. and Baclovks1, L . M . 119761, J. Org. Chem. ", 1819-1890. 28. Schonbaum, G . R., Zerner. 8 . . and Bender, M. L. 119611. J. 9101. Chem.

29. Eglintan, G. and irhltlnq, M . C. 119501. J. Chem. SDC. 9, 3650-3656. 236, 2930-2935, -

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Page 9: Haloenol Lactones

S B Daniels, E Cooney, M J Sofia, P K Chakravarty and J A Katzenellenbogenalpha-chymotrypsin.

Haloenol lactones. Potent enzyme-activated irreversible inhibitors for

1983, 258:15046-15053.J. Biol. Chem. 

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