Synthesis and Application of Novel Bifunctional Spin Labels

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Bioconjugate Chem. 1999, 10, 578−582

Synthesis and Application of Novel Bifunctional Spin Labels Ralf M. Lo¨sel,†,‡ Reinhard Philipp,† Tama´s Ka´lai,§ Ka´lma´n Hideg,§ and Wolfgang E. Trommer*,† Fachbereich Chemie, Universita¨t Kaiserslautern, P.O. Box 3049, D-67653 Kaiserslautern, Germany, and Institute of Organic and Medicinal Chemistry, University of Pe´cs, H-7643 Pe´cs, P.O. Box 99, Hungary. Received November 18, 1998; Revised Manuscript Received February 22, 1999

The synthesis of new bifunctional spin-labeled cross-linking reagents is described. Covalent attachment to papain was achieved via a thiol-specific thiosulfonate residue and, for the second anchor point, via a nonspecific photoreactive azido function. The thiosulfonate formed a reversible disulfide linkage, which could be cleaved again reductively by dithiothreitol. The spin label, a pyrroline-1-oxyl radical, was highly immobilized after attachment to papain by both functional groups and showed little if any relative motion with respect to the protein.

INTRODUCTION

Spin labeling has widely been used for studies of structure-function relationships in proteins. ESR investigations using spin labels may yield information about local polarities, segmental mobilities, and the conformational changes correlated with these parameters (1, 2). Moreover, precise intramolecular distances have been obtained in special cases (3-5). Interpretation of the data may, however, be hampered due to relative mobility of the spin label with respect to the macromolecule to which it is attached. In case of distance measurements, the error increases from several tenths of an angstrom by more than an order of magnitude (6, 7). This problem pertains even more to motional studies of the protein. Relative motion of the spin label with respect the macromolecule can be minimized by the use of bifunctional probes which allow for cross-links within, e.g., the protein. Such compounds have first been described by Wenzel et al. (8), but they contain a rather long spacer between the nitroxide and the reactive groups which are also of low specificity with respect to protein side chains. Intramolecular cross-linking of proteins with chemical reagents has become a well-established technique to give information on the proximity of amino acid residues. The reagents used for such purposes contain, separated by a spacer of defined length, two functional groups that may exhibit either equal (homobifunctional) or different (heterobifunctional) selectivity toward amino acid residues. Photoreactive groups are often favored as they remain silent, i.e., unreactive unless irradiated. Among the amino acids containing reactive side chains, amino and thiol (sulfhydryl) groups are most suitable for chemical derivatization as reactions usually take place under mild conditions. Whereas amino groups of lysines are quite abundant in most proteins, thiol-containing cysteines are usually present in small numbers, only. For this reason, the selective chemical modification of cysteines usually allows for rather precise determination of the site of attachment. There are, however, few reagents that are truly specific for thiols, as the commonly applied halo* To whom correspondence should be addressed. † Universita ¨ t Kaiserslautern. ‡ Present address: Labor Diagnostika Nord, PO Box 2180, D-48529 Nordhorn, Germany. § University of Pe ´ cs.

acetamides or maleimides may also react with other functional groups, particularly with amino groups of lysines. Among the specific sulfhydryl reagents are those forming disulfide bonds with the protein. This may be accomplished by disulfide exchange or by a substitution reaction with thiosulfonates (9). Cross-linking reagents with sulfhydryl-specific functionalities and additional photoreactive groups have been described (10-13). A thiol-specific, reversible methanethiosulfonate spin label has been described earlier (14-16). On the basis of this compound (1), we have synthesized several derivatives containing a photoreactive moiety and applied them to the test system papain. The latter is a thiol protease consisting of 212 amino acids with a molecular weight of 23 406 (17). Its essential thiol located at Cys 25 is very reactive and easily modified. In the following, we demonstrate the application of the new compounds for spin labeling of papain under stepwise intramolecular crosslinking as shown in Figure 1. EXPERIMENTAL PROCEDURES

Papain, twice crystallized (Sigma Lot no. 100H8075), was reductively activated at a concentration of 40 mg/ mL by incubation with 50 mM dithiothreitol (DTT) in 0.1 M acetate, pH 4.5, for 1 h. There was no difference in final enzyme activity compared to longer incubation periods (up to 12 h). Activation did not cause significant autolytic degradation of papain as judged from SDSPAGE (18). Activity measurements were done by photometric determination of liberated 4-nitroaniline from the substrate NR-benzoyl-L-arginine-p-nitroanilide [L-BAPA (19), commercially available from Merck]. Briefly, the assay mixture contained 0.1 M Tris, 0.2 M KCl, 1 mM EDTA, pH 7.0, supplemented with 1 mM L-BAPA. Nitroaniline formation was followed at 410 nm ( ) 8800 M-1 cm-1) for 2 min using a Beckman DU 640 UV spectrometer operating in the kinetics mode. Protein concentration was estimated by the Bradford procedure (20) using defatted bovine serum albumin as a standard rather than by A280 determination, as the aromatic moiety of incorporated spin-labels interfered at this wavelength. Photolysis was carried out by irradiating samples with 366 nm UV-light using a CAMAG source (254 or 366 nm, 4 W) or a Rayonet photoreactor (350 nm, 750 W).

10.1021/bc980138v CCC: $18.00 © 1999 American Chemical Society Published on Web 06/11/1999

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Figure 2. Chemical structures of reagents used in the modification experiments. (1) 1-Oxyl-4-methanethiosulfonylmethyl2,5-dihydro-2,2,5,5-tetramethyl-1H-pyrrole, (2) 1-oxyl-3-(4-azidoN-phthalimido)ethyl-4-methanethiosulfonylmethyl-2,5-dihydro2,2,5,5-tetramethyl-1H-pyrrole, (3) 1-oxyl-3-phthalimidoethyl4-methanethiosulfonylmethyl-2,5-dihydro-2,2,5,5-tetra-methyl-1H-pyrrole, (4) 1-oxyl-3-(4-azido-2-nitrobenzoyloxy)ethyl-4methanethiosulfonylmethyl-2,5-dihydro-2,2,5,5-tetramethyl-1H-pyrrole. Figure 1. Principle of the method. After selective reaction of the reagent with the thiol group of the protein under disulfide formation, irradiation effects covalent binding via decomposition of the azide group (at the other terminus). Finally, the disulfide may be cleaved reductively. At each step, ESR spectra may be recorded.

ESR spectra were recorded at 25 °C on a BRUKER ESP 300E ESR spectrometer operating in the X-band mode and employing a 100 kHz modulation of 1 G amplitude. To modify the enzyme, reductively activated aliquots (100 µL) were passed through centrifuge columns filled with Sephadex G25 (21) and equilibrated with the buffer used for activity measurements (0.1 M Tris, 0.2 M KCl, and 1 mM EDTA, pH 7.0). Some protein precipitated due to the different pH, but readily dissolved upon addition of an equal volume of buffer. To 100 µL of enzyme solution, 5 µL of the reagent stock solution (50 mM in dimethyl formamide) was added and thoroughly mixed. Reagents 2, 3, and 4 were not completely soluble under these conditions and formed turbid solutions. Samples were incubated for 1 h under exclusion of light and then separated from unbound reagent by column centrifugation over Sephadex G25 (21). Except for the thiosulfonate reagent 1, this procedure was insufficient to completely remove unspecifically bound spin-label, as indicated by an intense ESR signal of freely mobile label. Therefore, microdialysis against the same buffer (16 h at room temperature) was used in addition. Molecular modeling was performed using the QUANTA program running on a Silicon Graphics INDY. The structure data file used was 1pop found in the Protein Data Bank. Minimization was done in the CHARMm force field. Synthesis of 1-Oxyl-3-[(N-phthalimido)ethyl]-4methanethiosulfonylmethyl-2,5-dihydro-2,2,5,5-tetramethyl-1H-pyrrole (3). 1-Oxyl-3-[(N-phthalimido)ethyl]-4-bromomethyl-2,5-dihydro-2,2,5,5-tetramethyl1H-pyrrole (102 mg, 0.25 mmol), prepared analogously as described by Ka´lai et al. (22) for the 4-azido-Nphthalimido derivative, and NaSSO2CH3 (67 mg, 0.5 mmol) were dissolved in a mixture of dioxane (10 mL) and water (3 mL) and were refluxed for 30 min. Then the mixture was concentrated in vacuo, and the residue was dissolved in chloroform (20 mL), washed with brine (10 mL), dried over MgSO4, filtered, evaporated and, purified by preparative TLC on a silica gel plate (hex-

Figure 3. ESR spectrum obtained from papain modified with 2 as described in the Experimental Procedures. The protein concentration was 90 µM.

ane-ethyl acetate, 2:1). Yield 61 mg (56%); Rf 0.40 (CHCl3-Et2O 1:1); mp 128-129 °C. Anal. calcd for C20H25N2O5S2 (437.54): C, 54.90; H, 5.76; N, 6.40; S, 14.65. Found: C, 54.73; H, 5.77; N, 6.27; S, 14.32 (Heraeus Micro U/E, sulfur titrimetrically by Scho¨niger’s method). IR: 1700 cm-1 (CdO), 1610 cm-1 (CdC). MS m/z (rel int.%): 437 (M+, 68), 407(36), 310(66), 160(100), carried out on a VG Trio 2 instrument, EI mode 70 eV, direct inlet. Synthesis of 1-Oxyl-3-[(4-azido-2-nitrobenzoyloxy)ethyl]-4-methanethiosulfonylmethyl-2,5-dihydro2,2,5,5-tetramethyl-1H-pyrrole (4). To a stirred solution of 1-oxyl-3-hydroxyethyl-4-methanethiosulfonylmethyl2,5-dihydro-2,2,5,5-tetramethyl-1H-pyrrole (200 mg, 0.65 mmol) and pyridine (63 mg, 0.8 mmol) in dry CH2Cl2 (5 mL) was added 4-azido-2-nitrobenzoyl chloride [181 mg, 0.8 mmol, prepared according to Friebel et al. (11)], and the mixture was stirred for 3 h in the dark at room temperature. The mixture was washed with brine (5 mL), dried (MgSO4), evaporated, and purified by preparative TLC (Merck Silica Gel GF254, 20 × 20 × 0.2 cm), Rf 0.45 (CHCl3/Et2O 1:1), to give 1-oxyl-3-[(4-azido-2-nitrobenzoyloxy)ethyl]-4-methanethiosulfonylmethyl-2,5-dihydro2,2,5,5-tetramethyl-1H-pyrrole (4), yield 48 mg (15%), as a thick oil, which solidified upon cooling. The reaction is accompanied by formation of the corresponding biradical, Rf 0.6 (CHCl3/Et2O 1:1). Anal. calcd for C19H24N5O7S2 (498.55): C, 45.77; H, 4.85; N, 14.05; S, 12.86. Found:

580 Bioconjugate Chem., Vol. 10, No. 4, 1999

Figure 4. (A) Papain modified with reagent 4 in the dark, protein concentration 61 µM (17 scans, relative gain 1 × 106, resolution of x-axis 8192 points). (B) Rerecorded high and lowfield region of the sample from trace A after 10 min irradiation at 350 nm (17 scans, relative gain 2 × 106, resolution of x-axis 8192 points). (C) Sample from trace B after addition of DTT and NEM and microdialysis, protein concentration 24 µM (17 scans, relative gain 2 × 106, resolution of x-axis 2048 points).

C, 45.58; H, 5.00; N, 14.12; S, 12.50. MS: m/z (rel int.%) 498 (M+, 6), 468 (7), 442 (8), 196 (21), 165 (29), 134 (40), 65 (100). Compounds 1 and 2 were synthesized according to published procedures (14, 22). RESULTS

Papain was labeled at cysteine-25 with methanethiosulfonate spin label 1 under loss of activity and exhibited an ESR spectrum very similar to the one obtained by

Lo¨sel et al.

Berliner et al. (14) with the same reagent (data not shown). Likewise, the new spin-labeled reagents 2, 3, and 4 (structures shown in Figure 2) efficiently modified the active-site cysteine under complete inactivation of the enzyme. The spectrum of the phthalimide 2 (Figure 3), when bound to the protein but before irradiation, however, was strikingly different from that of the simple thiosulfonate 1. Whereas with 1, a 2Azz value of 52 G typical of weakly immobilized nitroxide radicals was observed, 2 exhibited a value of 67 G. Such large values are seen in highly immobilized radicals and are close to the rigid powder limit in a hydrophobic environment. Irradiation in a photoreactor at 350 nm for 10 min did not alter the spectrum, although 2 can be photolyzed readily in solution upon irradiation at 366 nm with a simple hand-held UV lamp (CAMAG) as demonstrated by concomitant changes in the UV spectrum (complete loss of absorbance at 261 and 344 nm within 15 min; isosbestic points at 231 and 375 nm). The almost identical, but not photoreactive label 3 after reaction with papain gave a spectrum that is indistinguishable from the one obtained with 2 (data not shown). ESR spectra obtained with the azidonitrophenyl compound 4, on the other hand, showed a significantly lower immobilization of the label (2Azz ) 64.8 ( 0.1 G, Figure 4a) as compared to phthalimides 2 and 3. After irradiation in a Rayonet photoreactor for 10 min, the maximum separation observed had increased to 67.1 ( 0.1 G (Figure 4b), thus, clearly indicating that an additional immobilization had occurred due to a second covalent linkage to the protein via the nitrene generated by photolysis. Upon addition of 3 mM DTT, the signal from freely tumbling radical increased, reaching equilibrium after about 30 min when N-ethyl maleimide in a final concentration of 20 mM was added to stop the disulfide

Figure 5. Molecular model of reagent 4 (liquorice model, colored by elements) bound in the substrate-binding cleft of papain (spacefilling model, yellowish) as obtained from the CHARMm calculations.

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reduction by DDT. At this point, the solution of modified protein was divided into two parts. One part was kept at room temperature without further treatment, while the other was dialyzed for 4 h against dialysis buffer as described in the Experimental Procedures. After this time, 2Azz was 65.5 ( 0.2 G in the dialyzed sample (Figure 4c), whereas the spectrum of the reference sample had remained unchanged. The residual bound signal in several experiments accounted for at least 50% crosslinking. For comparison, in a control sample modified with thiosulfonate 1, the reaction was complete in only 5 min, leaving no residual signal of immobilized radical. Molecular modeling using the protein structure and the azidophenyl compound 4 with subsequent energy minimization in a force field by the program CHARMm gave an excellent fit for location of the aromatic group of 4 in the S2 subsite of the protease (23) with the nitroxide moiety pointing outward, while the pyrroline ring to which the latter is attached is bound in the substratebinding cleft (Figure 5). DISCUSSION

The heterobifunctional spin label 4 was shown to react in a stepwise manner with papain under formation of a cross-link between the essential cysteine residue 25 and an amino acid located in the S2 subsite. Modification of the thiol group of Cys-25 was accompanied by the loss of enzymic activity. That cross-linking (at least 50%) had indeed occurred upon irradiation was seen clearly by the increase in the 2Azz value of the ESR spectrum of 4. Subsequent reduction with DTT of the disulfide linkage lead again to a smaller 2Azz value, indicating higher relative mobility. Upon double attachment, the label shows little if any relative motion with respect to papain (2Azz ) 67.1 G). According to Stoke’s eq 1 and a radius of papain of about 25 Å the rotational correlation time τr at 25 °C is 1.41 × 10-8 s (eq 1). On the basis of the 2A′zz value of 4 of 74 G in frozen solution at -25 °C (data not shown) a τr of 1.36 × 10-8 s can be calculated according to eq 2 for Brownian diffusion (24). Hence, 4 is well suited for motional studies of the protein by ESR and potentially, ST-ESR spectroscopy. The degree of cross-linking, however, is likely to vary from protein to protein.

τr ) 4πηr3/3kT

(1)

τr ) 5.4 × 10-10 (1 - A′zz/Azz)-1.36

(2)

Both reagents 2 and 3 exhibited highly immobilized ESR spectra after reaction with Cys-25 independent of subsequent irradiation. Apparently, the hydrophobic interaction of the aromatic moieties of these reagents with the S2 subsite sufficiently immobilizes these compounds even without a second covalent linkage. Although the azido function in 2 can be converted to a reactive nitrene by irradiation as shown by UV spectroscopy, it did not form a stable bond with any of the amino acids in its vicinity as shown by almost complete release of the label upon reduction of the disulfide linkage by DTT. However, 2 should prove as useful for a stepwise and double attachment to appropriate proteins as was shown for reagent 4 in this study. Due to the strong hydrophobic interaction of the aromatic moieties in 2 and 3 with papain, motional studies, e.g. by means of ST-ESR, can be caried out even without the second attachment. ACKNOWLEDGMENT

The authors express their gratitude to Anke Constantz and Elke Litmianski for excellent technical assistance. This work was supported by grants from the Hungarian

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