NMR Studies of Fluorinated Serine Protease Inhibitors*. (Received for publication, August 27, 1984). J. T. GerigS and J. D. Reinheimers. From the Department of ...
Vol. 260, No. 8, Issue of April 25. pp. 4713-4717, 1985 Printed in U.S.A.
THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1985 by The American Society of Biological Chemists, Inc.
NMR Studies of Fluorinated Serine ProteaseInhibitors* (Received for publication, August 27, 1984)
J. T. GerigS and J. D. Reinheimers From the Department of Chemistry, Universityof California, Santa Barbara, Santa Barbara, California 93106
Powers and co-workers have provided evidence that thiobenzyl N-heptafluorobutyrylanthranilate (I) is an extremely potent inhibitor of serine proteases, especially a-chymotrypsin (Teshima,T., Griffin, J. C., and Powers, J. C. (1982) J. Biol. Chern. 257,5085-5091). We have prepared additional derivatives of this structure inwhich fluorine substitutions have been made on the aromatic rings and have attempted to carry out fluorine NMR studies of the interaction of Powers’ compound and thesenew derivatives with chymotrypsin. The solubility of all inhibitors examined in solvent systems compatiblewith the retention of native enzyme structure is extremely low. While some nmr evidence for complex formation could be obtained, preparations of the complexes examined were metastable and precipitation of the inhibitor eventually limits the amount of complex that can be present in solution to such low levels that nmr experimentsare impractical. An unusual effectof solvent composition on fluorine chemical shifts suggests that theconformation of the inhibitors in aqueous solution and when bound to the enzymeis different from that in organic solvents.
In searching for new and more selective inhibitors of human leukocyte elastase, Powers and his collaborators found that thiobenzyl N-heptafluorobutyrylanthranilate, I, was especially effective in inhibiting a-chymotrypsin, showing an apparent dissociating constant, KI, for the enzyme-inhibitor complex of 81 nM (Teshima, et al., 1982). Fluorine nuclei in biological systems provide useful probes for examination of structure by fluorine nuclear magnetic resonance (Gerig, 1982) and the reported high stability of the complex formed between I and chymotrypsin was of particular interest as it appeared that it would provide a system for exploring the
ON C,S-CH
0 \ /
F STRUCTURE I1
F
STRUCTURE 111
active site of this enzyme by means of proton-fluorine nuclear Overhauser effects. We therefore prepared other fluorinated analogs of Powers’ compound (I1 and 111) in which fluorine probes were placed on the aromatic rings. Replacement of one or more aryl hydrogens in these structures by fluorine would not be expected to alter thebinding constant drastically and, given the enhanced hydrophobicity of aryl fluorine over aryl hydrogen (Hansch andLeo, 19791, could evenlead to enhancement of binding. A reasonable anticipation was that one of the aromatic rings of I would reside in thetosyl pocket of the enzyme and should this be the case, an appreciable, downfield, enzyme-induced shift of the resonance of the fluorine attached tothe ring so situated should beobserved (Gerig, 1978 Amshey and Bender, 1983; Gorenstein and Shah, 1982). We describe here fluorine nmr studiesof the interaction of Structures 1-111 with a-chymotrypsin; the results are dominated by the very limited solubility of all structures under the experimental conditions and suggest that the free energy change upon binding to the enzyme is smaller than the free energy difference between crystalline and solution states of the inhibitor.
STRUCTURE I
MATERIALS AND METHODS
* This investigation was supported by Grant GM-25975 from the National Institutes of Health. The costsof publication of this article were defrayed in part by the payment of page charges. This article must thereforebe hereby marked“aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ To whom correspondence should beaddressed. On research leave from the Department of Chemistry, The College of Wooster, Wooster, OH 44677.
Anthranilic acid, heptafluorobutyricanhydride, 4-fluorobenzoic acid,4-fluorobenzylbromide, and Woodward’s reagent K were obtained from Aldrich. a-Chymotrypsin ( 3 recrystallized, ~ salt-free) was obtained from Sigma. Compound I was prepared as described by Teshima et al. (1982). Tosylchymotrypsin was prepared following the procedure of Sigler et al. (1966). Lysozyme was purchased from Sigma. Thiobenzyl N-heptafluorobutyryl-5-fluoroanthranilate(11) was prepared from2-amino-5-fluorobenzoic acid bythe general procedure outlined below. The acid was obtained by catalytic reduction of 2-
4713
NMR Studies of Fluorinated Serine Protease Inhibitors
4714
nitro-5-fluorobenzoic acid, which was prepared from 3-fluorobenzoic and approximately 450 nM in 10% Me2S0,' 90% water. For the last determination an NMR method of analysis employing an internal acid by the procedure of Slothouwer (1914). 4-Fluorothiobenzyl N-heptafluorobutyryl-5-fluoroanthranilate reference was used since the absorbance of dimethyl sulfoxide at 285 (111) was synthesized using 2-amino-5-fluorobenzoic acid and 4-fluo- nm precluded use of the optical technique. Enzymatic Activity-Enzymatic activity was assayed using the robenzyl mercaptan, prepared from 4-fluorobenzylbromide and thihydrolysis of N-glutarylphenylalanine p-nitroanilide (Sigma) essenourea by the method of Paquette et al. (1968). General Procedure for the Synthesis of N-Heptafluorobutyryln- tially as described previously (Gerig and Hammond, 1984) except thranilic Acids-Under anhydrous conditions, an excess of hepafluo- that 10% methanol was the co-solvent. NMR Spectroscopy-Proton and fluorine NMR experiments were robutyric anhydride was added to 2-24 mmol of anthranilic acid suspended in 25mlof methylene chloride by means of magnetic carried out on a Nicolet NT-300 spectrometer (fluorine frequency stirring at room temperature. The anthranilic acid went into solution 282.3 MHz) using 10-mm sample tubes. Sample temperatures were after a few minutes and then a new precipitate began to form. After regulated at 25"C using the controller of the instrument. A thin 1.5 h, the reaction mixture was filtered and thefiltrate evaporated to capillary containingp-fluorobenzaldehydein acetone provided a condryness. The precipitate and the residue were combined and washed venient external reference signal. No corrections were made for several times with water. One mmol of N-heptafluorobutyryl acid and differences in bulk susceptibility as these were expected to be small 1.1 mmolof benzyl mercaptan were dissolved in 2 mlof dry ethyl relative to the effects observed (Muller, 1977). The spectral acquisiacetate. The magnetically stirred reaction mixture wascooled to tion parameterswere such as togive a digital resolution of 0.003 ppm dicyclohexylcarbodiimide (Aldrich) was in the fluorine spectra; chemical shifts were relative to the external -5 "C, then 1.2mmolof added. A precipitate appeared as rapidly as the dicyclohexylcarbodi- reference were reproducible to better than 0.05 ppm. Spin-lattice imide dissolved. The reaction mixture was stirred for 1 h whereupon relaxation time (Tl) and "F('H] NOE determinations were carried the reaction mixture was filtered and the precipitate washed with out as described previously (Gerig and Hammond, 1984). It was ethyl acetate. The ethyl acetatewas allowed to evaporate (hood). The determined that solutions approximately 10p M in N-heptafluorobutysolid residue was dissolved in dry chloroform and chromatographed rylanthranilic acid wouldgive signals with signal to noise ratio of through a small silica gel column (Woelm, activity III/30 mm, 10 X about 10 after accumulation of 3000 transients. Samples for fluorine NMR experiments with enzyme wereprepared 0.5 cm). The product appeared in the early fractions. The product dissolving the appropriate amount of enzyme to give a 1 or 2 mM was recrystallized from petroleum ether/ethyl acetate or methanol/ water. Materials were characterized by their proton and fluorine nmr solution in 0.11 M sodium acetate buffer, made up in deuterium oxide spectra and by their mass spectra under conditions of chemical (Stohler) at an apparent pH of 4.6 to the glass electrode. T o 3 ml of enzyme solution was added over 1min, 0.3 mlof a solution of inhibitor ionization using methane as the reactant gas; no parent ion was in Me2S0, acetonitrile, or acetone at a concentration sufficient to detected using electron impact ionization. The physical properties of give a concentration that upon dilution was stoichiometric with the 1-111 and the spectral data are summarized in Table I. enzyme, while the mixture was vigorouslystirred with a vortex mixer. Modification of Tosylchymotrypsin with N-Heptafluorobutyrylun- A soapy appearing suspension was obtained. This was centrifuged on thranilic Acid-The acid (0.1 mmol) and 13 p l of triethylamine were a clinical centrifuge to remove a small amount of precipitate and the dissolved in 2 ml of acetonitrile and activated with 27 mg of Wood- supernatant, which still scattered light, was transferred to an NMR ward's reagent K at 0 "C. After 2 h, a solution of 82 mg of tosylchy- tube. The nominal concentration of the inhibitor in this sample was motrypsin in 8 ml of 0.1 M carbonate buffer at pH 9.2 was added and approximately equal to that of the enzyme. the mixture stirred for 50min. The solution was then dialyzed against water repeatedly and lyophilized. An approximately 0.3 mM solution RESULTS of the product in D20was prepared for fluorine nmr experiments. Modification of Chymotrypsin with 4-Fluorobenzenesulfonyl ChbSamples of the inhibitors 1-111 in the presence of the ride-A solution of 4-fluorobenzenesulfonyl chloride (Aldrich) in enzyme were prepared by adding solutions of these materials acetonitrile was prepared (57 mg/ml). An aliquot of this solution (0.3 ml) was added to 3 ml of a solution of chymotrypsin (29 mg/ml) in in an organic solvent to a concentrated solution of chymo0.1 M carbonate buffer at pH 9.8. After stirring for 1 h a t room trypsin. Given the presumed very strong affinity of these temperature, the mixture was brought to pH 5.3 with HCI, dialyzed materials for this protein, it was anticipated that a stoichiorepeatedly against water and lyophilized. A solution approximately metric interaction between inhibitor and protein would take 0.3 mM in D20 of the product was prepared for NMR observation. place such that theinsolubility of the inhibitor in water would Solubility Determinations-An excess of solid inhibitor was added be overcome and a high concentration of soluble enzymeto 500 ml of the solvent and magnetically stirred overnight at 25 "C. The mixture was filtered through a sinteredglass filter and thefiltrate inhibitor complex would be formed. Nevertheless some preextracted with methylene chloride (3 X 25 ml). The extract was dried cipitation of inhibitor was noted as theenzyme and inhibitor centrifugation clarified over sodium sulfate and evaporated to dryness. The residue was solutions weremixed.Low-speed these mixtures but asignificant amount of presumably colloitransferred from the flask by washing 4-5 times with 0.5-1.0 ml of methylene chloride. The combined washings were brought to 5.0 ml dal inhibitor remained in the samples that were examined by in a volumetric flask and the optical density at 285 nm was deter- fluorine NMR spectroscopy. mined. The concentration of inhibitor was estimated using the exFluorine spectra of samples of 1-111 with chymotrypsin tinction coefficient 12,400cm" "'. A control determination, without obtained shortly aftersample preparation exhibited signals at the inhibitor, was also made. The solubility of I in water was thus determined to be 72 nM in water, 140 nM in 10%acetone 90% water the expected chemical shifts for both the heptafluorobutyryl
TABLE I Properties of inhibitors Compound
Fluorine nmr shifts
m.p."
Mass spectrum mlz
CF2 CF,
CFP 4-F 5-F ppm
21.4 -18.0 -24.6 61-62' 21.3-18.5-24.9 68-69440 (M + 1) 316 (M-SCH2CsHs) 22.3 -17.3 -24.2 I11 79-80458 (M + 1) 334 (M-SCHzCeH9) a Recrystallized from methanol. Literature: 50-51 "C (Teshima et ai., 1982). e In acetone-d6. In dimethyl sulfoxide-d6.
I I1
-' -12.9
-' -'
-7.8 -10.Ijd
group and the aromatic fluorines, the ease of signal detection being consistent with a sample containing the inhibitor at a concentration of 0.5-1 mM. These signals were broadened somewhat (2-5 Hz). Determination of fluorine spin-lattice relaxation times (Tl)and "F{'H] NOES showed that fluorine relaxation was appreciably accelerated in the presence of the enzyme (Table 11). Over the course of 24 h, signals disappeared from these spectra at a rate dependent on the structure of the inhibitor and the organic co-solvent present in the solutions. The disappearance of signal correlated with the appearance of a fine white precipitate in the NMR samples; this precipitate 'The abbreviations used are: Me2S0, dimethyl sulfoxide; NOE, nuclear Overhauser effect.
NMR Studies of Fluoridated Serine Protease Inhibitors
4715
TABLEI1 Fluorine nmr effects in chymotrypsin inhibitor systems CFz
CFa
Inhibitor
5-F
4-F
~~
Inhibitor alone" I Shifts
TI, s
I1 Shifts
111 Shifts
Inhibitor + enzyme* I Shifts
I1 Shifts
0.5
0.5
0.6
-0.16
-0.19
-0.18
-0.53-0.15
-0.09
-0.10
0.6
0.6
-11.6
-24.0
-17.8
21.8
-11.9
-24.4
-18.0
21.5
-7.8 -7.8
-23.9
-17.6
21.9
1.o
-22.3 -10.6-22.3
-15.3 -15.3
24.3 24.3
NOE 111 Shifts TI. s -0.16 NOE
0.6
1.2
TI, s NOE
-22.5
-16.1
24.1
0.5
0.6
-11.2 0.5
-0.13
Solutions were about 1mM in 83% dimethyl sulfoxide/deuterium oxide. No "F(H] nuclear Overhauser effect could be detected in these samples. Chemical shifts are given in parts/million. Solutions were approximately 0.8 mM inhibitor (initially) and 2 mM chymotrypsin in a solvent consisting of 90% 0.1 M acetate buffer at pH 4.2 and 10% dimethyl sulfoxide.
was identified spectrally asrecovered inhibitor. Therewas no motrypsin shouldproduce detectable changesin the chemical shifts of fluorine nuclei contained in inhibitors 1-111. In evalevidencefor precipitation of protein. Given thedetection limits of our instrumentation, the concentrationof inhibitor uating the magnitude of these shifts one needs the chemical solution without enzyme present under remaining in solution after precipitation had taken place was shift of the inhibitor in not greater that 10 pM. The precipitation process was slow conditions comparable to thoseused to obtain the spectraof enough that there was no significant change of signal intensity the inhibitor-enzyme complex. Unfortunately, the very low over the first few hours after sample preparation andenough solubility of the present inhibitorsprecludes that determinatime was available for completion of the T I and NOE experi- tion and we decided instead to examine thesefluorine chemical shifts in mixed organic solvent/water solutions, hoping ments. Enzymaticactivityasmeasured by therate assay was that a sensible extrapolation to100%water would provide the identical within experimental error for native enzyme alone, needed information. Fig. 1 shows that the fluorine shifts of for enzyme plus the inhibitorimmediately after mixing, or for the inhibitors aresubject to large, nonlinear variations as the enzyme and inhibitor after20 h, when the precipitation proc- amount of water in the solventincreases. An extrapolation to pure water under these conditions isimpractical; the data in ess appeared to becomplete. Experiments were carried out in which the amount of co- Fig. 1 suggest that the enzyme-induced chemical shift effects solvent, the natureof the co-solvent, and the nature and pH are probably small at all positions in the inhibitor but these of the buffer were changed. These variables did not alter the appearance of the spectrum as long as theco-solvent was 10% or less, andthetime of precipitation was notessentially changed. Attempts to use a higher percentage of organic cosolvent (Clement and Bender, 1962) were also unsuccessful inpreventingprecipitation.Inthese cases, the precipitate that occurred after the sample had aged overnight was of a different character and, because of its insolubility in water and organic solvents,was assumed t o be denatured enzyme. Several experiments indicate that native a-chymotrypsin is .c necessaryfor the solubilization of the inhibitor. When no 0.0 protein is present the concentration of inhibitor remaining after sample preparation and centrifugation is below the limits " wlth ; studies of detection by fluorine NMR, about 10 p ~solubility enzyme put the concentration of inhibitor in this case in the 100 nM -2.0 range. When native enzyme is replaced by tosylchymotrypsin, I I I , I 1 0 (0 20 30 40 50 60 70 00 90 in which the active site is presumably blocked by the tosyl Volume % Ma2S0 group (Birktoftand Blow, 1972; Segal, et al., 1971) or by lysozyme, the NMR samples did not exhibit the characteristicFIG. 1. Fluorine chemical shift effects in I11 in dimethyl soapy appearance and, again, there were no detectablefluorine sulfoxide/water mixtures; using the shift observed in pure dimethyl sulfoxide as the reference point, the shift changes NMR signals, indicating that the concentration of inhibitor for solvent mixtures are given. A positive number corresponds to in the presence of these proteins isless than 10 p ~ . an upfield shift relative to the shift in pure organic solvent. The top Teshima et al. (1982) anticipated that StructureI would be curve (e) indicates the shifts for the 5-fluOrO group, the next (B) a substrate for elastase but reportedonly competitive inhibi- represents the data for the 4-fluor0 group and the lower curves show tion of this enzyme as well as chymotrypsin. We have made the effects on the CFa (O),central CFz ( O ) ,and a-CFz (0)groups. n o NMR observations that would indicate enzyme-catalyzed The dashed line indicates the chemical shift of 6,6,6-trifluorohexanoI the same conditions (Muller, 1977). Also shown on the far left hydrolysis of the inhibitors1-111 a t either theamide or thioes- under are the chemical shifts foreach fluorine of I11 observed inthe presence ter linkages. of chymotrypsin in 10% dimethyl sulfoxide/water mixtures. Shift Based on previous work one expects that binding to chy- data for I1 were very similar to those represented here. v)
I
I
I
1
4716
NMR Studies of Fluorinated Serine Protease Inhibitors
cannot be expressed quantitatively at this point. In order toprovide some indicationof the relaxationeffects
tively, a colloid involving inhibitor particles and the protein may form. The latter possibility seems unlikely, given the inability of tosylchymotrypsin or lysozyme to participate in such a system. In any event, the system is metastable and over a period of 1 day reaches equilibrium in which there is no longer anyNMR-detectableinhibitor insolutioneven though the protein concentration in solution is unaltered. Under thisequilibrium chymotrypsin remains insolution and retains essentially 100%activity. There are qualitative indications fromour NMR observaSTRUCTURE IV tions that in the initially formedmixturestheinhibitors of enzyme binding on the fluorine nuclei of the inhibitors, interact with the native enzyme. Although no reliable inforacid IV was covalently attached to tosylchymotrypsin. The mation on the natureof enzyme-induced chemical shifts was fluorine nmr spectraof this modified enzyme(Fig. 2) indicated obtained, it is clear from relaxation effects (line widths, T,, that under the conditions used at least four distinguishable NOE data) that the inhibitors interact strongly enough with sites on the protein hadreacted. The line widthsobserved for the protein that the motion(s) of the nuclei attached to them protein-bound IV a t every site of reaction are larger than aresignificantly affected by a slowly tumblingstructure. those observed with I in the presenceof the enzyme. Comparison to the model systems involving covalent attachChymotrypsin was treated with4-fluorobenzenesulfonyl ment of IV and the 4-fluorobenzenesulfonylgroup to the chloride to attach 4-fluorophenyl groups to the protein; the enzyme suggest that this interaction isfairly weak, however. spectra of this modified enzyme should suggest the spectral Fluorine-proton nuclear Overhauser data can be used to effects of binding the fluoroaromatic ringsof I1 and I11 to the indicate the importance of proton-fluorine dipolar interacenzyme. Five signalswith chemical shifts over a 9-ppm range tionsin relaxing a fluorinenucleus. If bound entirelyto and with line widths 100-300 Hz at 282 MHz were observed. chymotrypsin we expect this NOE to be very nearly -1 for Again, theprotein-inducedlinebroadenings were much aromatic fluorine nuclei (Gerig, 1977). The NOE for CF2 and greater than the line width effects observed for the inhibitors CF3 groupsis more difficult to estimatesince proton-fluorine in the presenceof the native enzyme. interactions will play a smaller role in relaxationof the nuclei of these groups but surely these NOES will be smaller in DISCUSSION magnitude than the NOE on aromatic fluorine. Should chemThe goals of this study havebeen largely frustrated by the ical exchange between freeand enzyme-bound forms of inhibextreme insolubility of the inhibitors examined. The nature itors be present, the magnitude of the NOE will be reduced, of the solutions formed with them in the presence of the the extent of the reduction being dependent on details such enzyme by our proceduresis not well-defined. The cloudiness as the fractionof inhibitor complexed to theenzyme, the rate of solutions we observe couldresult from particlesof inhibitor of exchange between the free and bound forms and the nature that have not been removed by the centrifugationwhile much of inhibitor motions in thefree and bound states. Inrecently of the inhibitor is bound to the (soluble) enzyme. Alterna- reported experiments, an HF-NOEof -0.34 was observed for
FIG. 2. Fluorine nmr spectrum of I in the presence of chymotrypsin ( A ) and of chymotrypsin to which acid IV had been covalently attached ( B ) .From left to right the panels correspond to theCF3,internal CF2, and a-CF2 signals.
NMR Studies of Fluoridated Serine Protease Inhibitors 4-fluorocinnamic acid underconditions where 42% of the inhibitor was bound to chymotrypsin (Cairi and Gerig, 19851, a result not out of line with those reported herein. After preparation of samples, at least two competing processes must be present in these systems, as represented by Equations 1 and 2. Native enzyme ( E ) and an inhibitor (0 may interact to form a complex (ET) whose dissociation is governed by the constant Kr. Concurrently, the solubility equilibrium represented by Equation 2 must be achieved; in this case I, corresponds to theinsoluble form of the inhibitor. EISE+I
I.
(1)
*I
(2)
After equilibrium is reached it can be shown that thefraction of enzyme complexed to inhibitor, x,, is given bythe following. =
tEII/[Emta~l= [4/([4 + K )
(3)
[a
The concentration is defined by Equation 2. Given our detection limits and the concentration of enzyme present in the experiments, the fraction bound at equilibrium must be less than 0.01. Assuming a solubility of 100 nM, our observaor more than tions suggest that KTmust be greater than 2 orders of magnitude larger than the value for I given by Teshima et al. (1982). Theirestimate was obtained using inhibition kinetics under conditions where the enzyme concentration was much lower than in our NMR experiments and thediscrepancy may be due in some way to theeffects of protein association. Changes in fluorine chemical shifts asa function of solvent composition in dimethyl sulfoxide/water mixtures has been examined by Muller (1977) and is represented in Fig. 1by the dotted line. To the extent that Muller’s results define the “normal” solventeffects in thissystem, the solvent effects on the fluorine shifts of1-111 are unusually large. These effects suggest to us the possibility of a change of conformation of these molecules as they enter a water-rich environment. A study of models of the inhibitors indicates the possibility of an internal hydrogen bond between the amide hydrogen and the oxygen of the carboxylic acid as indicated in the drawing below. If this hydrogen bond forms, the outlined part of the molecule will tend to be planar, and free rotation about the remaining C-C and C-Sbonds will be important in determining the conformation.
Q,p--s. I
.
40..H C
-1 I
I
The perfluorinated side chain cannot reach either of the benzene rings, and all fluorines are likely to be exposed similarly to their solution environment. Rotation about the remaining bonds would lead to folded (coiled) or extended structures. In the coiled structure, the two benzene rings are approximately face to face and fluorine nuclei on either ring would be expected to experience appreciable upfield shifts due
4717
to ring current effects (Perkins, 1982). Alterations of the shifts for the perfluorinated side chain of the inhibitor might also be expected on the same grounds although the shifts observed for these are not as dramatic as the changes in the shifts of the aromatic fluorines. An initial interaction between 1-111 and native chymotrypsin is indicated by NMR observations of fluorine nuclei attached to these structures. In particular, the relaxation time and Overhauser data areconsistent with these molecules becoming part of a larger structure. It appears on the basis of the lack of enzyme-induced chemical shift changes and the lack of kinetic inhibition that these molecules interact with the enzyme in a mannerthat does not insinuate a fluorinated aromatic ring into thetosyl pocket. Consideration of Kendrew models of the enzyme and inhibitor suggest that the folded conformation of the inhibitor discussed above will not be able to enter thepocket at theactive site of the enzyme, although the extended conformation would be able to do so. The lack of reactivity of these molecules with the enzyme may thus be due to binding of a conformation that has potential leaving groups improperly oriented for attack by the catalytic groups of the protein. There seems to be a disagreement between our observation and those reported by Teshima et al. (1982) as regards inhibition of the enzyme although the two sets of experiments are not directly comparable. Should Structures 1-111 be competitive inhibitors, they may inactivate by simply sticking tightly to the active site region of the enzyme in a rather nonspecific way; the highly hydrophobic nature of the heptafluorobutyryl side chain may aid in this process. Regardless of the natureof the initially formed agglomerate of chymotrypsin and theinhibitors, the systems are metastable and eventually establishment of equilibrium (Equation 2) in competition with the binding process(es) represented by Equation 1render the system unsuitable for nmr studies. Acknowledgments-We thank Professor J. C . Powers for eliciting our interest in these systems and for useful conversations. REFERENCES Amshey, J. W., and Bender, M. L. (1983) Arch. Biochem. Biophys. 224,378-381 Birktoft, J. J., and Blow, D. M. (1972) J. Mol. Biol. 68, 187-240 Cairi, M., and Gerig, J. T. (1985) J . Magn. Reson., in press Clement, G. E., and Bender, M. L. (1962) Biochemistry 2,836-843 Gerig, J . T. (1977) J. A m . Chem. SOC.99, 1721-1725 Gerig, J. T. (1978) Biol. Magn. Reson. 1, 139-203 (Filler, Gerig, J. T. (1982) in BiomedicalAspects ofFluorine Chemistry R., and Kobayashl, Y., eds) pp. 163-189, Elsevier, New York Gerig, J. T., and Hammond, S. J. (1984) J. Am. Chem. SOC. 106, 8244-8251 Gorenstein, D. G., and Shah, D. 0. (1982) Biochemistry 2 1 , 46794686 Hansch, C., and Leo, A. (1979) Substituent Constantsfor Correlation Analysis i n Chemistry and Biology, p. 13, John Wiley and Sons, New York Muller, N. (1977) J . Magn. Reson. 28, 203-216 Paquette, L. A., Wittenbrook, L. S., and Schreiber, K. (1968) J. Org. C k m . 33,1080-1083 Perkins, S. J. (1982) Biol. Magn. Reson. 4, 193-336 Segal, D. M.,Powers, J. C., Cohen, G. H., Davies, D. R., and Wilcox, P. E. (1971) Biochemistry 10,3728-3738 Slothouwer, J. H. (1914) Rec. Trau. Chim. Pays-Bas Belg. 33, 32442; 9, 1314 Sigler, P. B., Jeffery, B. A., Mathews, B. W., and Blow, D. M. (1966) J. Mol. Biol. 15,175-192 Teshima, T., Griffin, J. C., and Powers, J. C. (1982) J . Biol. Chem. 267,5085-5091
