Reversible Photocontrol of Peptide Helix Content: Adjusting Thermal

These cross-linkers are shown to permit photoregulation of helix content in a test peptide with half-lives for the cis conformation ranging from 11 s ...
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Bioconjugate Chem. 2004, 15, 1297−1303

1297

Reversible Photocontrol of Peptide Helix Content: Adjusting Thermal Stability of the Cis State Nina Pozhidaeva, Marie-Eve Cormier, Anita Chaudhari, and G. Andrew Woolley* Department of Chemistry, University of Toronto, 80 Saint George Street, Toronto M5S 3H6, Canada. Received June 21, 2004; Revised Manuscript Received September 7, 2004

Cross-linking reagents based on an azobenzene core can be used to reversibly photoregulate secondary structure when introduced as intramolecular bridges in peptides and proteins. Photoisomerization of the azobenzene core in the trans to cis direction is triggered by photon absorption but isomerization from cis to trans occurs thermally as well as photochemically. The rate of the thermal process effectively determines the half-life of the cis form as well as the extent to which the trans form can be recovered. We designed and characterized a series of methanethiosulfonate (MTS)-bearing thiol-reactive azobenzene-based cross-linkers. These cross-linkers are shown to permit photoregulation of helix content in a test peptide with half-lives for the cis conformation ranging from 11 s to 43 h at 25 °C. The cross-linkers described here thus broaden the range of reagents available for reversible photocontrol of peptide and protein conformation.

INTRODUCTION

Upon irradiation at appropriate wavelengths, azobenzene chromophores undergo reversible cis-trans isomerization (1). Kumita et al. introduced thiol-reactive azobenzene cross-linkers as tools for the control of peptide conformation (2-4). They designed a symmetrical reagent 4,4′-bis(iodoacetamide)azobenzene (Figure 1) in which two Cys-reactive iodoacetamide moieties are linked to the azobenzene chromophore. When the reagent was used to intramolecularly cross-link peptides with Cys residues spaced at either i, i+4, i, i+7 or i, i+11 positions, photoisomerization led to predictable changes in peptide secondary structure. Azobenzene isomerization in the cis-to-trans direction, in addition to being promoted by irradiation with light at >400 nm, occurs thermally. The half-life of the cis isomer of the bis(iodoacetamide)azobenzene cross-linker shown in Figure 1 is approximately 12 min at 25 °C (3). Half-lives for thermal isomerization are important for determining the practical usefulness of such photochemical switches. For instance, if structural studies are intended with the cis form, a much longer half-life than 12 min may be desired. Alternatively, if a pulsed conformational change is desired as part of a biochemical switch, then rapid return to the trans state would be preferable. Additionally, a rapid thermal relaxation to the trans form ensures virtually complete conversion to the trans isomer, important where a complete “off” state is desired for a biochemical switch. Because of the overlap in absorption spectra of cis and trans azobenzenes, irradiation never produces only one isomer. We note that natural photoswitches (e.g. photoactive yellow protein, phytochromes) also employ thermal relaxation to reset the switch (5). Several groups have shown that the rate of thermal isomerization of azobenzenes can be varied substantially by altering the nature of substituents on the aromatic rings (e.g. (6-9)). For instance, thermal stability can be * To whom correspondence should be addressed. E-mail: [email protected]; telephone/fax: (416) 978-0675.

Figure 1. Cis-trans isomerization of 4,4′-bis(iodoacetamide)azobenzene.

Figure 2. Two resonance forms of a substituted diaminoazobenzene.

adjusted by varying substituents that control the relative contribution of an N-N single bond resonance structure (Figure 2)(8, 10). Precisely how this operates to lower transition state energies is not clear, however, since the mechanism of isomerization (rotation or inversion) remains controversial (11). We wished to create a series of azobenzene crosslinkers analogous to the Kumita compound but showing a range of thermal stabilities. To enhance stability, we designed a derivative with sp3 carbon atoms in the para positions in the expectation that it would show stability similar to p-methyl azobenzene (τ1/2 2-3 days)(6). To increase the rate of thermal reversion we designed derivatives in which enhanced delocalization of the p-amino group lone pair electrons would be possible. High reactivity and selectivity toward Cys residues was achieved by introducing methanethiosulfonyl groups. These groups enable reversible attachment of the cross-linker to peptides and proteins via disulfide linkages (12). The effects of the cross-linkers on the conformation of a test peptide (FK-11)(3) were evaluated. EXPERIMENTAL PROCEDURES

The following includes general experimental procedures, specific details for representative reactions, and

10.1021/bc049855h CCC: $27.50 © 2004 American Chemical Society Published on Web 11/02/2004

1298 Bioconjugate Chem., Vol. 15, No. 6, 2004

isolation and spectroscopic information for the new compounds prepared. All 1H and 13C NMR spectra were recorded using a Varian Unity 400 or a Varian Gemini 300 spectrometer. All chemicals were purchased from Aldrich Chemical Co. except if specified otherwise. Highresolution mass spectra were obtained either by electron impact (EI) or electrospray (ESI) ionization. Peptide mass spectra were obtained either by electrospray (ESI) or MALDI ionization. Synthesis of 4,4′-Dimethylazobenzene (6). p-Toluidine (2.0 g, 18.7 mmol) was dissolved in dichloromethane (160 mL). Potassium permanganate (8.86 g, 56.1 mmol) and cupric sulfate pentahydrate (9.32 g, 37.3 mmol) were added, and the suspension was stirred at room temperature for 48 h. The suspension was filtered through a mixture of Celite and silica and eluted with dichloromethane. The solid obtained after evaporation of the filtrate was redissolved and purified on a silica column with dichloromethane as solvent to give 6 as an orange solid (780 mg, 40%). 1H NMR (300 MHz, CDCl3) δ 2.43 (s, 6H, CH3), 7.31 (d, 4H, J ) 8.1 Hz, Ar), 7.80 (d, 4H, J ) 8.1 Hz, Ar); 13C NMR (100 MHz, CDCl3) δ 21.5, 122.7, 129.7, 141.2, 150.8. HRMS-EI for C9H8O4S, calcd 210.1166, found 210.1157. Synthesis of 4,4′-Dibromomethylazobenzene (7). 4,4′-Dimethylazobenzene (6)(202 mg, 0.97 mmol), Nbromosuccinimide (343 mg, 1.9 mmol), and benzoyl peroxide (7.0 mg, 0.03 mmol) were suspended in carbon tetrachloride (4 mL) and refluxed for 2 h. Another aliquot of benzoyl peroxide (7.0 mg, 0.03 mmol) was added to the mixture and reflux continued for another 2 h. The mixture was then stirred at room-temperature overnight. The solution was filtered, and the orange solid obtained was redissolved in dichloromethane. The organic layer was then washed with water, dried over MgSO4, and concentrated under vacuum. The product was purified by flash chromatography using hexanes/EtO2 (1:1) as eluant to give 7 as an orange solid (175 mg, 49%). 1H NMR (300 MHz, CDCl3) δ 4.58 (s, 4H, CH2), 7.55 (d, 4H, J ) 8.4 Hz, Ar), 7.90 (d, 4H, J ) 8.4 Hz, Ar); 13C NMR (100 MHz, CDCl3) δ 32.7, 123.4, 129.9, 140.8, 152.3 HRMS-EI for C9H8O4S, calcd 365.9371, found 365.9367. Synthesis of 4,4′-Diiodomethylazobenzene (8). Sodium iodide (5.83 g, 39 mmol) was dissolved in dry acetone (50 mL) and stirred under nitrogen for 10 min. 4,4′-Dibromomethylazobenzene (7)(143 mg, 0.39 mmol) was added to the mixture in dry tetrahydrofuran (14 mL). The mixture was refluxed for 6 h and then stirred at room-temperature overnight. The solvent was evaporated and the solid resuspended in dichloromethane. The excess of sodium iodide and sodium bromide was filtered out, and the solution was evaporated under vacuum to give 8 as an orange solid (173 mg, 96%). 1H NMR (300 MHz, CDCl3) δ 4.53 (s, 4H, CH2), 7.52 (d, 4H, J ) 8.8 Hz, Ar), 7.84 (d, 4H, J ) 8.8 Hz, Ar); 13C NMR (100 MHz, CDCl3) not soluble enough in CDCl3 and DMSO for routine 13C; HRMS-EI for C9H8O4S, calcd 461.9090, found 461.9099. Synthesis of 4,4′-Methanethiosulfonatemethylazobenzene (1) (MTS-alkyl-azobenzene). 4,4′-Diiodomethylazobenzene (8) (28 mg, 0.062 mmol) and sodium methanethiosulfonate (Toronto Research Chemicals) (42 mg, 0.31 mmol) were dissolved in a mixture of ethanol (2 mL) and dichloromethane (2 mL) and refluxed overnight. Dichloromethane and water were added to the mixture. The organic layer was dried over MgSO4 and evaporated. The product was dissolved in dichloromethane and precipitated with a few drops of pentane to give 1 as an orange solid (12 mg, 45%). 1H NMR (300 MHz, CDCl3) δ trans isomer 2.99 (s, 6H, CH3), 4.45 (s, 4H, CH2),

Pozhidaeva et al.

7.57 (d, 4H, J ) 8.4 Hz, Ar), 7.93 (d, 4H, J ) 8.4 Hz, Ar); C NMR (100 MHz, CDCl3) δ 40.7, 51.6, 123.7, 130.2, 138.6, 152.2; HRMS-ESI for C9H8O4S, calcd 431.0222, found 431.0231. Synthesis of 4,4′-Dichloroacetamide Azobenzene (10). 4,4′-Diaminoazobenzene (9) (255 mg, 1.2 mmol) (Lancaster, Pelham NH) was dissolved in anhydrous tetrahydrofuran (20 mL) and stirred under nitrogen for 15 min protected from light. Three equivalents of triethylamine (500 µL, 3.61 mmol) was added, followed by the dropwise addition of 3 equiv of chloroacetyl chloride (287 µL, 3.61 mmol). The reaction mixture was left stirring under nitrogen for 30 min at room temperature. The reaction mixture was filtered via vacuum filtration to remove the precipitate that formed, and the tetrahydrofuran was removed by rotary evaporation. The crude product was redissolved in a solution of tetrahydrofuran and dichloromethane (1:1) and extracted with water acidified with a few drops of 1% HCl. The filtrate was dried over anhydrous MgSO4 and vacuum filtered, and solvents were removed by rotary evaporation producing a yellow-brown solid (10) (395 mg, 91%). 1H NMR (DMSO-d6, 300 MHz): δ 4.31 ppm (s, 4H), δ 7.79 ppm (d, J ) 9.0 Hz, 4H), δ 7.87 ppm, (d, J ) 9.0 Hz, 4H), δ 10.65 ppm (s, 2H). 13C NMR (DMSO-d6, 75 MHz): δ 43.6, 119.6, 123.5, 141.2, 148.0, 165.0; EI-HRMS: (C16H14Cl2N4O2), calcd 364.0494, found 364.0498. Synthesis of 4,4′-Diiodoacetamide Azobenzene (11). Anhydrous acetone (20 mL) and anhydrous tetrahydrofuran (15 mL) were combined and the solvent mixture degassed. Sodium iodide (1 g, 7.1 mmol) was dissolved in the degassed solvent and added to 150 mg (0.71 mmol) of dichloroacetamide azobenzene (10). The reaction mixture was stirred for 12 h under nitrogen and protected from light. After filtration to remove NaCl, the solvent was removed by rotary evaporation to produce a yellowbrown solid. The solid was redissolved in a solution of tetrahydrofuran and dichloromethane (1:1), and excess of NaI was removed by vacuum filtration. The filtrate was extracted with water, dried over anhydrous MgSO4, vacuum filtered, and solvents were removed by rotary evaporation to produce a yellow-brown solid (11) (330 mg, 85%). 1H NMR (DMSO-d6, 300 MHz): δ 3.87 ppm (s, 4H), δ 7.76 ppm (d, J ) 9.0 Hz, 4H), δ 7.86 ppm, (d, J ) 9.0 Hz, 4H), δ 10.67 ppm (s, 2H); 13C NMR (DMSO-d6, 100 MHz), δ 1.4, 119.3, 123.5, 141.5, 147.8, 167.0; EIHRMS: (C16H14 I2N4O2), calcd 547.9209, found 547.9199. Synthesis of 4,4′-Methanethiosulfonylacetamide Azobenzene (2). 4,4′-Diiodoacetamide azobenzene (11) (90 mg, 0.164 mmol) and sodium methanethiosulfonate (48.4 mg, 0.361 mmol) (Toronto Research Chemicals) were dissolved in a degassed mixture of anhydrous ethanol (15 mL) and anhydrous tetrahydrofuran (15 mL) and refluxed for 12 h. The solvents were removed by rotary evaporation to produce a yellow-brown solid (2) that was washed with water to remove excess sodium methanethiosulfonate and dried (77 mg, 85%). 1H NMR (DMSO-d6, 300 MHz): δ 3.61 ppm (s, 6H), δ 4.24 ppm (s, 4H), δ 7.78 ppm (d, J ) 9.0 Hz, 4H), δ 7.87 ppm, (d, J ) 9.0 Hz, 4H), δ 10.72 ppm (s, 2H); 13C NMR (75 MHz, DMSO-d6), δ 38.4, 50.6, 119.6, 123.5, 141.2, 148.0, 165.6; ESI-HRMS: (C18H21N4O6S4)(MH+), calcd 517.0343, found 517.0338. Synthesis of 4,4′-p-Nitrophenoxyacetamide Azobenzene (12). 4,4′-Diaminoazobenzene (9)(80 mg, 0.377 mmol) (Lancaster, Pelham NH) was dissolved in anhydrous dichloromethane (20 mL) and stirred under nitrogen for 15 min protected from light. Four equivalents of p-nitrophenyl chloroformate (304 mg, 1.509 mmol) (Flu13

Thiol-Reactive Azobenzene Cross-Linkers

ka) were dissolved in anhydrous dichloromethane (10 mL) and added to the diaminoazobenzene solution, followed by the dropwise addition of 4 equiv of pyridine (121.4 µL, 1.509 mmol). The reaction mixture was stirred under nitrogen at room temperature for 2 h. The volume of the solvent was reduced by half, and the solid precipitate was collected. The precipitate was purified by washing with water, 5% HCl, 5% NaOH, and water again to give 12 as a yellow-brown solid (165 mg, 81%). 1H NMR (DMSO-d6, 400 MHz): δ 7.58 ppm (d, J ) 9.0 Hz, 4H), δ 7.72 ppm, (d, J ) 9.0 Hz, 4H), δ 7.89 ppm (d, J ) 9.0 Hz, 4H), δ 8.32 ppm (d, J ) 9.0 Hz, 4H), δ 10.87 ppm (s, 2H); 13 C NMR (100 MHz, DMSO-d6), δ 118.9, 123.0, 123.6, 125.3, 141.0, 144.7, 147.8, 150.5, 155.3; Molecular ion was not observed with EI-MS. Synthesis of 4,4′-(Methanethiosulfonate-ethyloxy)acetamide Azobenzene (3). 4,4′-p-Nitrophenoxyacetamide azobenzene (12) (15.8 mg, 0.029 mmol) and 2-hydroxyethyl methanethiosulfonate (10 mg, 0.064 mmol) (Toronto Research Chemicals) were combined together and suspended in DMSO (3 mL). Diisopropylethylamine (2.2 equiv, 11.2 µL, 0.064 mmol) was added, and the reaction mixture was stirred at room temperature under nitrogen for 6 h. The solvent was removed under high vacuum. The crude product was washed with water, 5% sodium bicarbonate, and water and dried (10 mg, 60%). 1 H NMR (acetone-d6, 300 MHz): δ 3.470 ppm (s, 6H), δ 3.85 ppm (q, J ) 5.7 Hz, 4H), δ 4.31 ppm (t, J ) 5.7 Hz, 4H), δ 7.83 ppm (d, J ) 8.7 Hz, 4H), δ 7.96 ppm, (d, J ) 8.7 Hz, 4H), δ 9.80 ppm (s, 2H). 13C NMR (100 MHz, acetone-d6), δ 33.7, 38.9, 54.7, 120.7, 122.9, 140.4, 148.1, 154.0. ESI-HRMS: (C20H25N4O8S4)(MH+), calcd 577.0555, found 577.0549. Synthesis of 4,4′-(Methanethiosulfonate-ethylamino)acetamide Azobenzene (4). One equivalent of 4,4′-p-nitrophenoxyacetamide azobenzene (12) (63 mg, 0.116 mmol) and 2.5 equiv of 2-aminoethyl methanethiosulfonate hydrobromide (68.4 mg, 0.290 mmol) (Toronto Research Chemicals) were combined together and suspended in DMSO (4 mL). Diisopropylethylamine (2.5 equiv, 50 µL, 0.290 mmol) was added, the reaction mixture was stirred at room temperature under nitrogen for 6 h, and then the solvent was removed under high vacuum. The crude product was washed with water, 5% sodium bicarbonate, and water and then dried (53 mg, 80%). 1H NMR (DMSO-d6, 400 MHz): δ 3.35 ppm (t, J ) 6.4 Hz, 4H), δ 3.48 ppm (dt, J1 ) 6.4, J2)5.6 Hz, 4H), δ 3.57 ppm (s, 6H), δ 6.58 ppm (t, J ) 5.6 Hz, 2H), δ 7.58 ppm (d, J ) 7.8 Hz, 4H), δ 7.75 ppm, (d, J ) 7.8 Hz, 4H), δ 9.04 ppm (s, 2H); 13C NMR (100 MHz, DMSO-d6), δ 36.0, 38.7, 50.2, 117.7, 123.4, 142.9, 146.5, 154.8; ESIHRMS: (C20H27N6O6S4)(MH+), calcd 575.0874, found 575.0869. Cross-Linking of MTS-alkyl-azobenzene to FK-11. A 10 mM solution of the cross-linker 1 in DMSO and a 0.49 mM solution of FK-11 in water were prepared. The peptide solution (1.23 mL, 0.6 µmol), a first aliquot of the cross-linker solution (30 µL, 0.3 µmol), and 410 µL of DMSO were stirred at room temperature, in the dark, for 15 min. Another aliquot of cross-linker was added (30 µL, 0.3 µmol), and the solution was stirred for anther 15 min. The last aliquot of cross-linker (30 µL, 0.3 µmol) was added to the solution, and stirring was continued for another 40 min. The solvent was evaporated under high vacuum. The product was purified by HPLC on a preparative C18-column (Apex, ODS Prepsil, 25 cm 8 µm) with an ACN (0.1% TFA) and H2O (0.1% TFA) gradient (step 1: 15% to 90% ACN in 45 min; step 2: 90% ACN for 3.0 min; step 3: 90% to 15% ACN in 3.0 min), with

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detection at 360 nm. The retention time was 24 min (flow rate 10 mL/min). MS/ESI: calcd 2018, found 672.9 (3+), 1009.4 (2+). Cross-Linking of MTS-amide-azobenzene to FK-11. An 11.6 mM solution of the cross-linker 2 in DMSO and 0.549 mM solution of FK-11 in water were prepared. The peptide solution (1.09 mL, 0.6 µmol) and the first out of three aliquots of the cross-linker solution (55 µL, 0.6 µmol) were combined together followed by addition of extra 400 µL of pure DMSO. The reaction mixture was stirred at room temperature, in the dark, for 15 min. Another aliquot of cross-linker was added (55 µL, 0.6 µmol), and the solution was stirred for another 15 min. The last aliquot of cross-linker (55 µL, 0.6 µmol) was added, and stirring was continued for another 1.5 h. After completion the solvent was removed under high vacuum. The product was purified by HPLC on a semipreparative SB-C18 column (Zorbax, 9.4 mm ID × 25 cm) with an ACN (0.1% TFA) and H2O (0.1% TFA) gradient (step 1: from 15% to 90% ACN in 45 min; step 2: 90% ACN for 8 min; step 3: from 90% to 15% ACN in 8 min). Absorbance at 360 nm was monitored, and the pure product was eluted at 50% ACN with a retention time 14 min (flow rate 2.1 mL/min). MALDI-MS [MH+]: (C8113C2H128N31O26S4) calcd 2106 0.38, found 2106.96, 1053.96 (2+). Cross-Linking of MTS-carbamate-azobenzene to FK-11. A 4.5 mM solution of the cross-linker 3 in DMSO and a 0.55 mM solution of FK-11 in water were prepared. The peptide solution (1.09 mL, 0.6 µmol) and the first out of three aliquots of the cross-linker solution (132 µL, 0.6 µmol) were combined together followed by addition of extra 200 µL of pure DMSO. The reaction mixture was stirred at room temperature, in the dark, for 15 min. Another aliquot of cross-linker was added (132 µL, 0.6 µmol), and the solution was stirred for another 15 min. The last aliquot of cross-linker (132 µL, 0.6 µmol) was added, and stirring was continued for another 1.5 h. After completion the solvent was removed by high vacuum pumping. The product was purified by HPLC on a semipreparative SB-C18 column (Zorbax, 9.4 mm ID × 25 cm) with an ACN (0.1% TFA) and H2O (0.1% TFA) gradient (step 1: from 10% to 90% ACN in 45 min; step 2: 90% ACN for 5 min; step 3: from 90% to 10% ACN in 5 min), with monitoring at 360 nm. The pure product was eluted at 44% ACN with the retention time 25 min (flow rate 2.1 mL/min). MALDI-MS [MH+]: (C8313C2H134N31O28S4) calcd 2166.45, found 2165.97. Cross-Linking of MTS-urea-azobenzene to FK-11. A 5.92 mM solution of the cross-linker 4 in DMSO and a 0.549 mM solution of FK-11 in water were prepared. The peptide solution (1.09 mL, 0.6 µmol) and the first out of three aliquots of the cross-linker solution (100 µL, 0.6 µmol) were combined together followed by addition of extra 400 µL of pure DMSO. The reaction mixture was stirred at room temperature, in the dark, for 15 min. Another aliquot of cross-linker was added (100 µL, 0.6 µmol), and the solution was stirred for another 15 min. The last aliquot of cross-linker (100 µL, 0.6 µmol) was added, and stirring was continued for another 1.5 h. After completion, the solvent was removed under high vacuum. The product was purified by HPLC on a semipreparative SB-C18 column (Zorbax, 9.4 mm ID × 25 cm) with an ACN (0.1% TFA) and H2O (0.1% TFA) gradient (step 1: from 35% to 80% ACN in 45 min; step 2: 80% ACN for 5 min; step 3: from 80% to 35% ACN in 5 min) with monitoring at 360 nm. The pure product was eluted at 41% ACN with the retention time 6 min (flow rate 2.1

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Pozhidaeva et al. Scheme 1. Synthesis of MTS-alkyl-azobenzene

Figure 3. Structures of MTS-azobenzenes synthesized: MTSalkyl-azobenzene (1), MTS-amide-azobenzene (2), MTS-carbamate-azobenzene (3), MTS-urea-azobenzene (4).

mL/min). MALDI-MS [MH+]: (C8313C2H136N33O26S4) calcd 2164.48, found 2165.07. Synthesis of Glutathione Derivatives. One equivalent of each cross-linker was dissolved in 500 µL of DMSO. Ten equivalents of γ-glutathione were dissolved in 500 µL of TrisCl buffer (100 mM, pH 8) and added dropwise to each solution of cross-linker in DMSO. The reaction mixture was stirred at room temperature for 2 h in the dark. After completion, the solvent was removed under high vacuum. The products were purified by HPLC on a semipreparative SB-C18 column (Zorbax, 9.4 mm ID × 25 cm) as described above. Structures were confirmed by MALDI-MS. Photoisomerization. A 70 W Metal Halide Tri-Lite Lamp (World Precision Instruments Inc., FL) coupled with either a 340 ( 10 nm, 370 ( 10 nm, or 380 ( 10 nm band-pass filter (Harvard Apparatus Canada, PQ) was used to photoisomerize peptide solutions to the cis isomers. For photoisomerization to the trans isomer, a 455 nm cutoff filter (Harvard Apparatus Canada, PQ) was used coupled to the same lamp. Photoisomerization in both directions was completed within 10 min. UV/Vis and CD Spectroscopy and Relaxation Curves. Peptide samples were dissolved in a 5 mM phosphate buffer (pH 7). For the relaxation curves experiments, the spectra were taken using a BDS 100 Fiber Deuterium/Tungsten light source, a Quantum Northwest temperature control and an Oceanoptics UBS 2000 diode-array. Quartz cuvettes (0.1 cm path length) were used for the measurements. Relaxation curves were fit to single exponentials and time constants and activation energies evaluated in the standard way. All the circular dichroism (CD) experiments were performed on a Jasco Model J-710 spectropolarimeter using thermostated quartz cuvettes (0.1 or 1.0 cm path length). The samples were dissolved in a 5 mM phosphate buffer (pH ) 7). A 10 nm/min, 1.0 nm bandwidth and a 4 s response time were used. Corrected CD spectra for 100% cis isomers were obtained using the following equation: θ (100% cis) ) [θ (observed after irradiation) - (fraction trans × θ (dark-adapted))]/fraction cis. Calculations of Absorbance Spectra. Models of each chromophore were built in Spartan 04 for Windows (Wave function Inc.) with structures as shown in Figure 3 except that the CH3S(O)2SCH2 unit was replaced by a

methyl group. Geometry was optimized using the ab intio HF 6-31G* method in Spartan. Only trans geometries of the azo unit were studied. Wavelengths for absorbance maxima together with oscillator strengths were calculated using the DFT EDF1 6-31G method in Spartan. ZINDO/S calculations were performed on the HF 6-31G* minimized structures using Hyperchem v.6 (Hypercube Inc.). RESULTS AND DISCUSSION

Synthesis of MTS-azobenzene Cross-Linkers. A series of azobenzene-based thiol-reactive cross-linkers was synthesized with structures as shown in Figure 3. For convenience we will refer to these compounds according to the type of linkage between the azobenzene group and the thiol-reactive MTS moiety (e.g. MTScarbamate-azobenzene). It is this linkage that primarily affects the thermal stability of the compounds. The MTS-alkyl-azobenzene cross-linker (1) was obtained in a four-step synthesis (Scheme 1). First, ptoluidine 5 was oxidized using a mixture of potassium permanganate and cupric sulfate to give 6. Bromination of the benzylic methyl groups was achieved through a radical reaction using N-bromosuccinimide in carbon tetrachloride with benzoyl peroxide as an initiator. In our first trials, we attempted to install MTS on 7 by displacement of the bromide but the yield of this reaction was low. We therefore carried out a bromo/iodo exchange reaction on 7. Refluxing 8 in a mixture of dichloromethane and ethanol with the sodium methanethiosulfonate was sufficient to obtain the desired product 1. The MTS-amide-azobenzene cross-linker (2) was obtained by a simple displacement reaction between NaMTS and the Kumita cross-linker synthesized as described previously (Scheme 2). The MTS-carbamate-azobenzene cross-linker (3) and MTS-urea-azobenzene cross-linker (4) were obtained as outlined in Scheme 3. Reaction of diaminoazobenzene with p-nitrophenyl chloroformate produced the carbamate 12. This reacted rapidly with 2-aminoethyl methanethiosulfonate and more sluggishly with 2-hydroxyethyl methanethiosulfonate with DMSO as solvent to form cross-linkers 3 and 4, respectively. Peptide Cross-Linking with MTS-azobenzenes. On the basis of previous studies, we chose the peptide FK-11 (Acetyl-Glu-Ala-Cys-Ala-Arg-Glu-Ala-Ala-Ala-ArgGlu-Ala-Ala-Cys-Arg-Gln-amide) as a target for photo-

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Thiol-Reactive Azobenzene Cross-Linkers Scheme 2. Synthesis of MTS-amide-azobenzene

Figure 4. Spectra of dark-adapted (trans isomers) of crosslinked peptides: (‚‚‚) MTS-alkyl-azobenzene FK-11, (- ‚ -) MTS-amide-azobenzene FK-11, (- ‚‚ -) MTS-carbamate-azobenzene MTS, (s) MTS-urea-azobenzene FK-11. Scheme 3. Synthesis of MTS-carbamate-azobenzene and MTS-urea-azobenzene

Table 1. Experimental and Calculated Absorption Maxima of Chromophores

structure alkyl amide carbamate urea

control (3, 13). FK-11 has cysteine residues spaced 11 residues apart and is known to form stable R-helices in water (3, 14). Molecular models of cross-linked FK-11 indicated that when the peptide is in a helical conformation, the distance between the two Cys residues can accommodate the trans isomer of the cross-linkers with relatively little distortion of the helix. The end-to-end distance of the cis conformation of the cross-linker is less compatible with this Cys residue spacing. Trans-to-cis isomerization is thus is expected to lead to decreased peptide helical content in aqueous solution. Cross-linking of the peptides was achieved rapidly and efficiently by dissolving the peptide in water and slowly adding a solution of the cross-linker in DMSO. The modified peptides were purified by HPLC. To test for effects of peptide structure on the thermal stability of the cross-linker, the MTS-azobenzene cross-linkers were also reacted with glutathione to furnish a chemically similar adduct without secondary structure. Spectral Properties and Thermal Stability. Spectra were obtained for each of the MTS-azobenzene peptide conjugates after dark adaptation to produce pure trans isomers (Figure 4). Absorption maxima are collected in Table 1. The absorption maximum of the trans isomer of the MTS-alkyl-azobenzene is close to that observed for unmodified azobenzene (340 nm) and at a significantly shorter wavelength than that for the amide-, carbamate-, and urea-substituted compounds. The trend in absorption

λmax, nm, DFT EDF1 6-31G* λmax, nm λmax, nm (trans) (obsd) (trans) (ZINDO) (trans) 342 366 372 382

335 (1.018) 349 (1.190) 349 (1.198) 350 (1.212)

330 (0.817) 379 (1.519) 376 (1.555) 387 (1.567)

maxima is predicted by the ZINDO semiempirical method (9, 15, 16) although predicted λmax values are too small, as has been observed in related studies (16). The DFT EDF1 method gives a better match to experiment (Table 1). The trend in absorption maxima can be understood in terms of a greater degree of delocalization of electrons associated with the benzylic atom from alkyl through urea structures in the series. Spectra of pure cis forms for the alkyl and amide derivatives were similar to those reported previously for unmodified azobenzene (17) and the Kumita compound (3). Spectra for the pure cis isomers of the carbamate and urea compounds were not obtained in the present study due to complications arising from the rapid thermal reversion to the trans isomer (vide infra). In contrast to observations with some other some delocalized azobenzene derivatives ((16)), however, significant degrees of conversion to the cis isomer were obtained as can be seen from the spectra shown in Figure 5. Thermal stabilities of the cis isomers of cross-linked FK-11 peptides together with the corresponding glutathione adduct were measured. After irradiation, recovery of the trans form in the dark was monitored by following absorption at 370 nm for series of different temperatures (Figure 6). These data are collected in Table 2 together with calculated activation energies for the thermal isomerization process. As observed previously, half-lives depend on the nature of the attached peptide,i.e. whether it is unstructured glutathione or the highly structured FK-11 peptide (3). Nevertheless, a clear correlation is observed between the absorption wavelength maximum of the cross-linker and the thermal stability of the cis form. With more delocalization, absorption maxima increase and half-lives decrease. Effects of Photoisomerization on Peptide Conformation. Circular dichroism spectroscopy (CD) was employed to determine the effect of cross-linker isomerization on the FK-11 peptide structure. The CD spectrum of dark-adapted MTS-alkyl-azobenzene cross-linked

1302 Bioconjugate Chem., Vol. 15, No. 6, 2004

Pozhidaeva et al. Table 2. Half-lives and Activation Energies for Cross-Linked FK-11 Peptides

Figure 5. Spectra showing thermal relaxation at 4 °C of MTSurea-azobenzene FK-11 after irradiation at 370 nm. Spectra were recorded at 0.65, 1.3, 2.0, 3.3, 6.5, 12.3, and 61.3 min (solid line) after irradiation.

Figure 6. Time courses for thermal relaxation (25 °C) of FK-11 peptides cross-linked with linkers: (- ‚ -) MTS-amideazobenzene FK-11, (- ‚‚ -) MTS-carbamate-azobenzene MTS, (___) MTS-urea-azobenzene FK-11. The absorbance of the MTSalkyl-azobenzene cross-linked peptide shows no change over this time period.

structure

τ1/2@25 °C

τ1/2@37 °C

Ea (kJ/mol)

alkyl-FK11 amide-FK11 carbamate-FK11 urea-FK11 alkyl-glutathione amide-glutathione carbamate-glutathione urea-glutathione

43 h 8 min 96 s 26 s 43 h 12 min 80 s 11 s

12 h 3 min 33 s 10 s 12 h 4 min 30 s 5s

82 72 69 62 82 76 67 60

FK-11 peptide at 10 °C shows bands at 208 and 222 nm, characteristic of a helical peptide (Figure 7A). Upon irradiation at 340 nm, the intensity of the two bands decreases, the largest difference being for the band at 222 nm, consistent with a decrease in peptide helical content. The normalized spectrum for the pure cis form (calculated by subtracting the contribution of the remaining trans isomer) is shown in Figure 7A. Upon irradiation at >450 nm, the helical content returns to the level observed for the dark-adapted state. For each of the crosslinked peptides, photoisomerization of the cross-linker from trans to cis causes a decrease in peptide helicity as expected (Figure 7B-D). Thermal relaxation recovered the dark-adapted (trans) spectra. The size of the effect can be evaluated by measuring the percent change in ellipticity at 222 nm. However, as noted above, rapid thermal relaxation of the MTScarbamate and MTS-urea derivatives prevents isolation of the cis isomers for determination of their spectra. Nevertheless, the effect of irradiation on the absorption spectra of each of the derivatives is qualitatively similar (Figure 5) so that ∼75(5% conversion to the cis isomer is likely occurring in each case. Using this assumption, the change in percent helix content observed upon irradiation for each peptide was calculated (Table 3) The magnitude of the effect of trans-to-cis isomerization on peptide helix content follows the order MTS-alkylazobenzene > MTS-amide-azobenzene > MTS-carbamateazobenzene ≈ MTS-urea-azobenzene. This pattern can be qualitatively understood by examining distance ranges between atoms corresponding to Cys-γ-S atoms in models of the free linkers (Table 3). The ideal S-S distance range between Cys-γ-S atoms in positions i, i +11 of an R-helix is 14.1 to 19.2 Å. Whereas each linker is able to accommodate this distance range in the trans conformation,

Figure 7. CD spectra of MTS-alkyl-azobenzene cross-linked FK-11 (A), MTS-amide-azobenzene cross-linked FK-11 (B), MTScarbamate-azobenzene cross-linked FK-11 (C), MTS-urea-azobenzene cross-linked FK-11 (D). For A: Dark-adapted (s); 100% cis (- - -) (after irradiation at 340 nm and corrected for % conversion as described in the methods section); irradiated with a 450 long pass filter (- ‚ -); after treatment with TCEP (- ‚‚ -). For (B-D): Dark-adapted (s); after irradiation at 370 nm (- ‚ -), corrected for 100% cis (- - -). After thermal relaxation the dark-adapted spectra were recovered in each case.

Bioconjugate Chem., Vol. 15, No. 6, 2004 1303

Thiol-Reactive Azobenzene Cross-Linkers Table 3

structure

% helix change obsda

S-S range, Å (trans)b

S-S range, Å (cis)b

MTS-alkyl-azo-FK11 MTS-amide-azo-FK11 MTS-carbamate-azo-FK11 MTS-urea-azo-FK11

45 39 14 16

9.8-17.7 14.3-22.4 14.9-26.3 15.1-26.5

3.3-13.0 3.2-16.5 4.0-18.0 3.2-18.9

a Calculated as % helix (dark) - % helix (cis), where 100% helix is -30 000 deg‚cm2‚dmol-1. b S-S range is the distance between Cys-γ-S atoms. In an ideal R-helix this distance range is 14.119.2 Å for Cys residues spaced at i, i+11.

the alkyl linker is least compatible with this distance range when in the cis conformation. The MTS-ureaazobenzene and MTS-carbamate-azobenzene cross-linkers, in particular, are long and flexible enough to accommodate a significant part of the optimal distance range between Cys-γ-S atoms even when in the cis conformation. The length of these linkers may make them more suitable (in the trans conformation) for cross-linking between Cys residues spaced more than 11 residues apart in an R-helix. Alternatively, the cis conformations of the linkers may be used to link closely spaced Cys residues (e.g., i, i+4) in peptide sequence. In that case, photoisomerization in the trans-to-cis direction should stabilize an R-helical structure. Cleavage of the Cross-Linked Peptides. Under standard photoisomerization conditions the peptides appear chemically stable. Mass spectra of the samples after irradiation showed that the cross-linked peptides remained intact during at least 10-20 photocycles. Extensive irradiation at higher temperatures (50 °C) was found to cause some cleavage of the cross-linkers from the peptide. A possible advantage of the MTS-activated crosslinkers over the Kumita iodoacetamide linker is that they can be removed from the peptide upon addition of thiols or mild reducing agents. Addition of the water-soluble reducing agent triscarboxyethylphosphine (TCEP) to a solution of cross-linked peptide caused rapid and complete removal of the linker. Figure 7A shows the CD spectra obtained for FK-11 after treatment with TCEP. After cleavage, the only peak observed in the electrospray mass spectrum is the molecular ion for the un-crosslinked peptide. SUMMARY AND CONCLUSIONS

The new azobenzene-based cross-linkers described here can be covalently attached to peptides or proteins through cysteine residues and removed again by reduction using TCEP. When used to cross-link the test peptide FK-11 the linkers enabled photocontrol of peptide helical content in a manner similar to the iodoacetamide based crosslinker introduced by Kumita et al. (2, 3, 18) but with a large range of thermal stabilities observed for the cis forms from 11 s to 43 h at 25 °C. The cross-linkers described here thus broaden the range of reagents available for reversible photocontrol of peptide and protein conformation. ACKNOWLEDGMENT

The authors would like to acknowledge the financial support of the Natural Sciences and Engineering Re-

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