Spin-labeled sulfonyl fluorides as active site probes of protease structure. II. Spin label syntheses and enzyme inhibition.

Abstract The syntheses and enzyme inhibition results for the derivatives discussed in the preceding paper are given. Spin-labeled sulfonyl fluorides containing six-membered piperidinyl nitroxide rings were particularly susceptible to intramolecular hydrolysis. In some cases, however, this hydrolysis was retarded significantly by the enzyme environment.

The syntheses and enzyme inhibition results for the derivatives discussed in the preceding paper are given. Spin-labeled sulfonyl fluorides containing six-membered piperidinyl nitroxide rings were particularly susceptible to intramolecular hydrolysis. In some cases, however, this hydrolysis was retarded significantly by the enzyme environment.
The preceding paper has described the sensitivity to active site structure of the series of spin-labeled sulfonyl fluorides specifically SW & R R = spin label incorporated in cu-chymotrypsin and trypsin (1). The spectral results also show the power of applying such a method to structural comparison of active sites of new proteases whose threedimensional structures are not yet known. We present here the detailed syntheses and purifications of these spin labels and some of the specific problems encountered in preparing the inhibited sulfonylated enzyme derivatives. Indole and benzamidine hydrochloride were from Eastman Kodak Co., Rochester, N. Y. Protein chromatographic and ultra concentration methods were identical with those reported earlier (2).

Methods
Electron spin resonance (ESR) measurements were taken at X band on a Varian E-4 spectrometer at 26 f 2". Activity and protein concentration measurements were carried out on a Unicam SP 1800 spectrophotometer.
For protein concentrations the absorptivities used at 280 nm were 5.0 X lo4 Me1 cm-' and 3.7 X lo4 M-I cm-l for cr-chymotrypsin (3) and trypsin (4), respectively. The extinction coefficients were increased by less than 1% at 280 nm as a result of labeling. Activity measurements were made with N-acetyltyrosine ethyl ester (ATEE) and p-tosyl-L-arginine methyl ester (tosyl-AME) for oc-chymotrypsin (5) and trypsin (6), respectively, at 25". The stoichiometry of spin label incorporation was measured by hydrolyzing the spin label with strong base from a known concentration of each enzyme sample and comparing the resultant free nitroxide spectrum with that of a standard.

Preparation of Spin-labeled Enzymes
Lu-Chymotrypsin-The enzyme (-10 mg per ml) was reacted at room temperat,ure, pH 7 (0.1 M potassium phosphate buffer), with a 2-to 5-fold molar excess of an acetonitrile solution of spin label. After 30 min a similar concentration of inhibitor was added, as several of these spin labels were found to decompose in aqueous solution over this time course (probably via hydrolysis). The final organic solvent concentration in the reaction mixture was 9% (v/v).
After 30 min the solution was clarified where necessary by filtration through a Swinney filter, then dialyzed exhaustively at 4" against pH 3.5 di1ut.e aeet.ic acid (-0.006 M), and finally against pH 3.5 (acetic acid) 0.1 M KaCl.
Indole was added by dialyzing the spin label derivatives against saturated indole at pH 3.5, 0.1 N NaCl at 4".
Try&n--In a typical experiment approximately 28 mg of enzyme were dissolved in 2 ml of 0.1 M Tris-Cl buffer, pH 7.7, Containing 0.02 M CaC&.
Spin label was added in a 2-to 5-fold molar excess in two 0.5-ml dioxane portions over a 30.min period. This high concentration of organic solvent was necessary in order to insure sufficient solubility of the spin label.
It should be noted that dioxane concentrations up to 50% (v/v) do not affect enzyme activity, as shown by past workers (7). The room temperature inhibition reaction was stopped after 1 to 2 hours and subjected to dialysis at, 4" for 6 to 10 hours against dilute acetic acid (-0.006 M), pH 3.5, containing 0.02 M CaCh. The enzyme was then chromatographed on a Sephadex SP-50 column (1.5 x 11 cm, 30mlper hour flow rate) with0.1 ~Tris-Cl, pH 7.1, containing 0.02 M CaClt and none or 1 mM benzamidine as in earlier trypsin studies (2,8). The fractions were collected and mixed immediabely with 0.1 M citrate buffer (final pH was about 3.5) to reduce the probabilit.ies of both desulfonylation and autolysis. The latter half of the overlapping c~-and fl-trypsin peaks in the elution profile was consolidated and concentrated about lo-fold in a collodion bag apparatus (Schleicher and Schuell). A final protein concentration of 2 to 7 mg per ml resulted. In cases where benzamidine was included in the eluting buffer, the resulting solution remained at approximately 1 mM benzamidine after concentration.
All of the above purification steps were done at 4". ESR spectra were taken immediately after the final concentration step.
All melting points are uncorrected.
The structures of the starting fluorosulfonyl derivatives and nitroxides are shown in Figs. 1 and 2, respectively.
Refer to Fig. 2 of the previous paper for the structures of the inhibitors themselves.
Details of these syntheses are presented in the "Appendix" immediately following this paper.'

AND DISCUSSION
Synthetic Methods-The synthetic procedures used were relatively simple and straightforward procedures for ester and amide linkages as described in the "Appendix." On the surface, the syntheses appear to be quite easily accomplished; however, we encountered great difficulties in syntheses involving the sixmembered piperidinyl group in 1 or ,9. We found that the esters, amides, carbamide, and urethans containing this ring structure were extremely labile to hydrolysis.
The secondary alcoholic group 1 has also been reported by several groups to be quite sensitive to hydrolysis in ester and glycosidic linkages (lO).zp a Nevertheless, we were able to isolate the pure crystalline products in most instances by mutiple purifications on silica gel columns and careful drying of the pure products.
1 The synthetic details are presented as a miniprint supplement immediately following this paper. Material published in miniprint form can be easily read with the aid of a large field reading glass of a type readily available at most opticians.
For the convenience of those who prefer to obtain supnlementarv material in the form of a microf;che or full size phbtocopies, these same data are available as JBC Document No. 73M-1010.
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2 T. Frey and L. J. Berliner, unpublished results. t A similar difficulty was encountered with a pyrrolidinyl secondary alcohol in phosphatidyl esters (11). Reference: 9 9 9 9 9 10 Unfortunately, during an enzyme inhibition reaction in aqueous solution we were faced with this hydrolysis problem in that a significant fraction of inhibitor had decomposed to (diamagnetic) sulfonyl fluoride and nonreactive nitroxide. This created no problem in the electron spin resonance (ESR) spectral analysis, since the technique observes only those enzymes modified with the intact paramagnetic inhibitor.
Second, due to the quite limited solubility of all of the labels synthesized, and due to the minimal stability of several in aqueous solution, accurate kinetic measurements of the enzyme inhibition reaction were abandoned.
Nevertheless, we were able at pH 3.5 to isolate stable spin-labeled sulfonylated chymotrypsin and trypsin in every case but one.
Enzyme Inhibition-A typical a-chymotrypsin inhibition reaction required about 1,1/2 to 2 hours, compared to 30 min for phenyhnethane sulfonyl fluoride under similar reaction conditions (12).   The reactivity toward trypsin was much poorer, yet this was 'Oc expected from similar results obtained by earlier workers (12). The price paid for allowing longer reaction times for trypsin inhibition was the extensive autolysis of (already) labeled trypsin so species (2). Under any set of labeling conditions, there was y always a significant degree of autolysis of sulfonylated trypsin. & This appears to be consistent with results obtained by other workers, suggesting that a serine-substituted trypsin was conformationally more susceptible to autolysis than native trypsin (5). In any case, it was necessary to separate these autolyzed * forms from the intact forms by the specific procedures of Schroeder and Shaw (8). Since most of the labeled trypsin a0 samples still contained some free enzyme, particular care was necessary in the final ultraconcentration step, as the enzyme would still undergo limited autolysis at presumably stable pH TIME MS.) (3.5) values (2). In the cases where we included 1 mM benz- A and 0, free spin label and activity, respectively, for Fahrney (12,14,15) and Cardinaud and Baker (16) we can p-11 (p-SO*-GOH); A and e, free spin label and activity, respectively, for m-IV (m-CO-GNH). The activities and spin label confidentlyassume that the inhibition occurs specifically by modi-concentrations are normalized to reflect their concentrations as a fying the active Ser I95 of cy-chymotrypsin or trypsin4 Further-per cent of the total enzyme in the sample. Therefore, the free more, both diisopropylphosphoryl (DIP)-chymotrypsin and spin concentration curves will level off at a value corresponding to chymotrypsinogen A fail to react with a spin-labeled sulfonyl the original per cent spin label incorporated.

1680
fluoride under the identical conditions.5 Tables I and II show the results obtained for some typical label incorporation since, as we believe, a substantial percentage samples of labeled cY-chgmotrypsin and trypsin, respectively. of hydrolyzed (diamagnetic) sulfonyl fluoride inhibited the The data for chymotrypsin (Table I) (Table II) are quite similar, although the results display the significantly lower reactivity of this enzyme toward aromatic 4 Based on the chymotrypsinogen A numbering system (see fiuorosulfonyl compounds. Stroud el al. (17)).
A second problem that occurred to a much lesser degree was 6 S. S. Wong and L. J. Berliner, unpublished results.
the hydrolytic desuljonylaiion of the inhibitor from the enzyme.
This problem was studied in some detail by Gold for phenylmethane sulfonyl fluoride (12, 15). He found that the rate of desulfonylation was slow and independent of pH over the range 2 to 8.5, but, increased significantly at higher pH values. This phenomenon showed up at pH 3.5 as a finite but slow desulfonylation, which also resulted in the production of a sharp line spectrum for the liberated spin label sulfonate.
In order to eliminate this spectral impurity, it was necessary to measure the ESR spectrum of a labeled enzyme immediately after removal from the dialysis cell.
Both of these phenomena could, of course, occur simultaneously as well. In the only examples examined in any detail, we monitored the increase of free spin label with the increase in enzyme activity for two labeled chymotrypsin derivatives, m-IV (m-CO-6NH) and p-11 (p-S02-60H), respectively.
If the major process of liberation of spin label is desulfonylation, the free label release should approximately coincide or slightly lag behind the increase in enzyme activity (desulfonylation of diamagnetic sulfonates can also occur with no corresponding increase in spin label signal).
If intramolecular spin label hydrolysis is predominant, then the enzyme activity should remain rather constant.
The results are shown in Fig. 3. The derivative m-IV (m-CO-6NH) was remarkably stable to intramolecular hydrolysis on the enzyme (filled circles and triangles, Fig. 3). From the close correspondence of the ESR and activity curves, it was evident that this hydrolysis was comparable to, or only slightly faster than desulfonylation.
This was a particularly remarkable result when one notes that the stoichiometry data in Table I for this label showed that only 40 to 60% of the fully inhibited enzyme was paramagnetic.
Presumably, the environment of this highly vulnerable amide linkage was well protected from the solvent in its enzyme environment.
On the other hand the para-sulfonate derivative p-11 (p-SOS-60H) underwent intramolecular hydrolysis to a much greater extent than desulfonylation while on the enzyme (open circles and triangles, Fig. 3). Although we did not broaden this investigation to all of the derivatives in Tables I and II, we believe that, except for the case of o-11 (o-SO*-GOH), all of the hydrolytically susceptible labels were sufficiently well protected from intramolecular 1681 hydrolysis when on the enzyme. This is borne out by the fact that the free label spectrum appeared at a slow enough rate to obtain a "clean" ESR spectrum in the 5-to IO-min spectral scanning times. This approach to studying chemical decomposition on an enzyme may be valuable in the future as a convenient tool for comparing the chemical similarities of specific regions of enzyme active sites with the medium or other organic solvents.