Protocol


Nature Protocols 2, 161 - 167 (2007)
Published online: 8 February 2007 | doi:10.1038/nprot.2006.488

Subject Categories: Isolation, purification and separation | Chemical modification | Synthetic chemistry | Spectroscopy and structural analysis | Biochemistry and protein analysis

Synthesis and application of an azobenzene amino acid as a light-switchable turn element in polypeptides

Andreas Aemissegger1 & Donald Hilvert1

The synthesis of an azobenzene amino acid (aa) for use as a photo-inducible conformational switch in polypeptides is described. The compound can be easily incorporated into an aa sequence by solid-phase peptide synthesis using standard 9-fluorenylmethoxycarbonyl methods. A reversible conformational change of the peptide backbone is induced by switching between the cis and trans configurations of the azobenzene moiety by irradiation with light of suitable wavelength. Thermal cistrans isomerization of this azobenzene aa is slow, enabling detailed structural investigations of the modified peptides, e.g., using NMR techniques. The total time for the synthesis of the photoswitch is typically 4 d, with an overall yield of 40–50%.

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Introduction

Strategic incorporation of molecular switches into (bio)polymers potentially represents a powerful strategy for modulating their structural and functional properties in a controlled way. The ability to transform peptides and proteins from one unique structure into another unique structure on demand, for instance, could be exploited to trigger reversible (un)folding or to regulate receptor function or enzyme activity. The storage of information at the molecular level, in the form of 'on' and 'off' conformational states, might also be utilized for diagnostics applications and for the design of nanoscale devices1, 2.

Photochromic compounds that undergo large conformational changes when exposed to light of appropriate wavelength are particularly attractive as molecular switch elements3. Among the many chromophores that fulfill this criterion, azobenzenes have become a popular choice4. They are readily synthesized, they have been extensively characterized and their isomerization occurs with high quantum efficiencies and gives rise to large geometrical changes. The thermodynamically favored trans isomer is rapidly converted to the cis isomer by irradiation at the wavelength of the ππ* transition (λ2 for aa 1, see Fig. 1), whereas the reverse process is achieved either (slowly) by thermal relaxation in the dark or (quickly) by irradiation at the wavelength of the nπ* transition (λ1 for aa 1, see Fig. 1).


For the purpose of photoregulation, azobenzene derivatives have been incorporated into numerous peptides and proteins, either as components of specific side chains4, 5, 6, 7, 8, 9, 10, 11, 12 or as part of the backbone itself13, 14, 15, 16. Backbone modification is particularly useful for inducing large structural rearrangements in a polypeptide chain, which can be used to probe folding mechanisms and conformational dynamics17. The azobenzene aa 1 (Fig. 1), for example, was developed as a reversible β-turn mimetic18. In its cis configuration (1a), this aa facilitates formation of stable β-hairpin structures by bringing together peptide segments attached to its amino and carboxylate groups via amide bonds19, 20. The interactions between the peptides are rapidly lost upon isomerization to the trans form of the linker 1b (i.e., unfolding). This linker is not restricted to β-sheet-like structures; it has also been successfully used to switch photochemically the relative alignment of helical substructures in a derivative of avian pancreatic polypeptide21. The synthesis of this useful azobenzene derivative, and its straightforward incorporation into peptide sequences, are described in this protocol.

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Experimental design

Figure 2 shows the synthetic route to 9-fluorenylmethoxycarbonyl (Fmoc)–protected 1 (i.e., compound 9). This aa can be used for solid-phase peptide synthesis with standard Fmoc conditions. Amounts produced following this protocol are adequate for preparative runs on an Applied Biosystems 433A automated peptide synthesizer.

Figure 2: Synthesis of protected azobenzene aa 9.
Figure 2 : Synthesis of protected azobenzene aa 9.

(a) Di-tert-butyl dicarbonate (Boc2O), cat. 4-(dimethylamino)pyridine (DMAP), tert-butanol; (b) NH4Cl, Zn, then FeCl3; (c) Fmoc chloride (Fmoc-Cl)N,N-diisopropylethylamine (DIPEA); (d) H2, cat. PtO2; (e) AcOH; (f) trifluoroacetic acid (TFA).

Full size image (37 KB)

Note that all solvents used in the procedure detailed below are HPLC grade or puriss p.a. unless stated otherwise.


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Materials

Reagents

  • Ammonium chloride (Fluka, cat. no. 09700)
  • tert-Butanol (Acros, cat. no. 107710010)
  • Celite 521 (Sigma-Aldrich, cat. no. 221791)
  • Di-tert-butyl dicarbonate (Boc2O; Fluka, cat. no. 34660)
  • N,N-diisopropylethylamine (DIPEA; Fluka, cat. no. 03440)
    Caution Highly flammable and corrosive
  • 4-(Dimethylamino)pyridine (DMAP; Fluka, cat. no. 29224)
    Caution Highly toxic
  • Fmoc chloride (Fmoc-Cl; Acros, cat. no. 170940250)
  • Hydrogen (5.0 grade)
    Caution Extremely flammable; can form explosive mixture with air; may react violently with oxidants
  • Iron (III) chloride hexahydrate (Fluka, cat. no. 44944)
  • 2-Methoxyethanol (Acros, cat. no. 180790010)
  • 3-Nitrobenzylamine hydrochloride (Sigma-Aldrich, cat. no. 191663)
  • 3-Nitrophenylacetic acid (Aldrich, cat. no. 103608)
  • Platinum (IV) oxide (Aldrich, cat. no. 206032)
  • Trifluoroacetic acid (TFA; Acros, cat. no. 139721000)
    Caution Toxic and corrosive
  • Zinc dust (Aldrich, cat. no. 209988)
  • Hydrochloric acid (HCl)
    Caution Toxic and corrosive
  • Magnesium sulfate, anhydrous
  • Sand
  • Silica gel 60 (Fluka, cat. no. 60752)
  • Sodium hydrogencarbonate
  • Thin layer silica gel plates (Merck, cat. no. 105715)
  • Acetic acid
  • Dichloromethane
  • 1,4-Dioxane
    Caution Toxic and highly flammable
  • Ethanol
  • Ethyl acetate
  • Hexane
  • Triisopropylsilane (TIPS; Fluka, cat. no. 92095)
  • N,N-dimethylformamide (DMF, peptide synthesis grade)
    Caution Toxic
  • Piperidine (peptide synthesis grade)
    Caution Toxic and corrosive
  • 1-Hydroxy-benzotriazole (HOBt, peptide synthesis grade)
  • Benzotriazol-1-yl-N-tetramethyluronium hexafluorophosphate (HBTU, peptide synthesis grade)
  • N-methylpyrrolidinone (NMP, peptide synthesis grade)

Equipment

  • Addition funnels
  • Balloons
  • Dewar dish
  • Disposable hypodermic syringe needles
  • Filter paper
  • Fritted chromatographic column (5.6 cm i.d., length 50 cm)
  • Fritted funnels with standard glass joints and medium porosity
  • Heat gun
  • Keck clips
  • Plastic syringes (polypropylene)
  • Quartz or fused silica glassware, e.g., fluorescence cuvettes
  • Rotary evaporator
  • Round-bottom flasks, one- and two-necked
  • Separatory funnels
  • Sonicator
  • Teflon-coated magnetic stir bars
  • Gas inlet tube (glass)
  • Light sources (see EQUIPMENT SETUP)

Equipment setup

  • For trans → cis isomerization Irradiation can be performed in a standard Rayonet photoreactor (Southern New England Ultra Violet Company, Branford, CT) equipped with RPR 3 500 lamps (εmax 350 nm) or Philips PL-S 9W/12 compact fluorescent tubes (εmax 320 nm) with matching fluorescent ballast (e.g., Conrad Electronics, Hirschau, Germany, cat. no. 618896–62).
  • For cis→trans isomerization A blue light source, e.g., Osram Dulux-S/67 9W or Coralife True Actinic 03 9W compact fluorescent tubes (εmax 430 nm) with matching fluorescent ballast, is used.
  • For automated peptide synthesis ABI 433A solid-phase peptide synthesizer.
    Caution All chemical operations should be performed in a fume hood and standard laboratory apparel must be worn (safety glasses/goggles, lab coat, disposable nitrile gloves, long pants and closed-toed shoes).
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Procedure

  1. Synthesis of 3Weigh 2.00 g 3-nitrophenylacetic acid 2 into a 100 ml round-bottom flask equipped with a Teflon-coated magnetic stir bar.
  2. Add 50 ml tert-butanol. Turn the magnetic stirrer on and wait until a clear solution is obtained.
  3. Weigh 4.82 g (2 eq.) Boc2O and add to the round-bottom flask. Wait until completely dissolved.
  4. Add 0.40 g (30 mol%) DMAP and stir for 1 h at room temperature (18–25 °C).
    Caution CO2 gas evolves from the solution.
  5. Evaporate the solvent on a rotary evaporator at 30 °C to obtain a dark brown oil.
  6. Pack a fritted glass funnel (diameter approximately 8 cm) on a 1 l round-bottom flask with silica gel to a height of approximately 6 cm using a 1:9 (vol/vol) mixture of ethyl acetate and hexane. Place a filter paper (diameter approximately 7 cm) on top of the silica gel layer.
  7. Dissolve the crude product obtained in Step 5 in 20 ml dichloromethane and apply it to the top of the silica bed. Let the sample soak into the silica bed.
    Critical step Make sure that the entire surface of the silica layer is covered.
  8. Filter the mixture through the silica by continuously adding solvent (a 1:9 (vol/vol) mixture of ethyl acetate and hexane) to the top of the silica bed while applying light vacuum to the receiver flask.
    Critical step Do not elute the dark-colored byproducts.
  9. Evaporate the solvents using a rotary evaporator at 30 °C to obtain 3 as a pale yellow oil. Dry the product on a vacuum pump; it may solidify during this process. The yield is quantitative.
  10. Synthesis of 6Weigh 2.50 g 3-Nitrobenzylamine hydrochloride 5 into a 100 ml round-bottom flask equipped with a Teflon-coated magnetic stir bar.
  11. Add 50 ml dichloromethane. Turn the magnetic stirrer on. A suspension will form.
  12. Weigh 3.43 g (1 eq.) Fmoc-Cl and add it to the flask.
  13. Add 7 ml (3 eq.) DIPEA using a graduated glass pipette to obtain a clear solution.
    Caution Exothermic reaction. Add the DIPEA slowly to avoid excessive heating.
  14. Stopper the flask with an inlet adapter fitted with a nitrogen-filled balloon and stir at room temperature until the starting material has been consumed (check by thin layer chromatography (TLC) with a 1:2 (vol/vol) mixture of ethyl acetate and hexanes as solvent system) or overnight.Pause Point The reaction mixture can be left stirring at room temperature overnight.
  15. Transfer the solution into a 250 ml separatory funnel. Rinse reaction flask twice with 10 ml dichloromethane and add it to the separatory funnel.
  16. Extract the dichloromethane solution with 10% HCl (3 × 50 ml), then with saturated NaHCO3 solution (1 × 50 ml), then with brine (1 × 50 ml) and dry over anhydrous magnesium sulfate (approximately 5 g, stir vigorously for 2 min).Troubleshooting
  17. Filter the mixture through a fritted funnel into a 250 ml round-bottom flask to remove the magnesium sulfate. Wash the filter cake with 2 × 10 ml dichloromethane. Evaporate the solvent on a rotary evaporator at 30 °C and dry the residual white solid on a vacuum pump. The yield is quantitative.
  18. Synthesis of 7Weigh 4 g compound 6 (Step 17) into a 500 ml two-necked round-bottom flask equipped with a Teflon-coated magnetic stir bar.
  19. Add 200 ml of a 2:1 (vol/vol) mixture of ethanol and 1,4-dioxane. Turn the magnetic stirrer on.
  20. Inflate a thick-walled balloon with hydrogen gas and attach it to a flushing adapter. Grease the joint of the adapter lightly and secure it to the straight neck of the reaction flask using a Keck clip.
    Caution Hydrogen gas is highly flammable. Avoid open flames and hot surfaces. Use only in well-ventilated explosion-safe fume hoods.
  21. Attach a three-way flushing adapter to the angled neck of the reaction flask. Grease the joint lightly and secure with a Keck clip. Connect vacuum and inert gas lines to the adapter.
  22. Purge the reaction flask with inert gas.
  23. With gentle inert gas flow, briefly remove the hydrogen flushing adapter and add 100 mg platinum (IV) oxide. Reattach the flushing adapter and secure it with the Keck clip. Purge thoroughly three times with inert gas.
    Critical step The use of Pd/C as hydrogenation catalyst instead of PtO2 leads to Fmoc cleavage.
  24. Purge the flask with hydrogen gas. Stir the mixture vigorously for 2 h.
    Caution The presence of oxygen in the reaction flask must be avoided. Hydrogen and oxygen can react violently, especially in the presence of hydrogenation catalysts.Troubleshooting
  25. Purge the reaction flask with inert gas.
  26. Remove the catalyst by filtration through a pad (height approximately 1 cm) of Celite in a fritted glass funnel into a 500 ml round-bottom flask. Wash the pad twice with 20 ml ethanol.
  27. Evaporate the solvent on a rotary evaporator at 30 °C and dry the residual white solid on a vacuum pump. The yield is quantitative.Pause Point Product 7 can be stored overnight at room temperature.
  28. Synthesis of 4Weigh 2.5 g 3 (Step 9) into a 250 ml two-necked round-bottom flask equipped with a Teflon-coated magnetic stir bar.
  29. Fit the angled neck of the reaction flask with a gas inlet tube (glass) and connect it to an argon line.
  30. Add 60 ml 2-methoxyethanol. Turn the magnetic stirrer on.
  31. Add a solution of 0.85 g NH4Cl in 20 ml H2O.
  32. Lower the gas inlet tube so that its outlet is immersed in the reaction solution.
  33. With a gentle flow of inert gas and vigorous stirring, degas the reaction solution for 1 h.
  34. Add a total of 2 g zinc dust in aliquots of approximately 200 mg over the course of 1 h and stir for another hour. If TLC (1:3 (vol/vol) mixture of ethyl acetate and hexane, R f = 0.23) still shows starting material, stir for another hour or until starting material disappears. Note that sonication will considerably accelerate this reaction step.
  35. In a 500 ml round-bottom flask equipped with a Teflon-coated magnetic stir bar prepare a solution of 5.70 g iron (III) chloride hexahydrate in 80 ml 2:1 (vol/vol) mixture of H2O and EtOH. Cool to −10 °C with the aid of a sodium chloride/ice bath (Dewar dish).
  36. Filter the reaction mixture of Step 34 through a Celite pad (height approximately 1 cm) in a fritted glass funnel directly into a 250 ml addition funnel fitted onto the flask prepared in Step 35. Wash the filter cake twice with 10 ml methoxyethanol.
  37. Add the solution dropwise (3–4 drops per second). Stir for 1 h at −10 °C.
  38. Pour the reaction mixture into 500 ml ice-cold water with vigorous stirring.
    Caution Nitroso compounds such as the desired product are carcinogenic and very toxic.
  39. Transfer the mixture into a 1,000 ml separatory funnel and isolate the green colored nitroso compound by extracting with three 100 ml portions of ethyl acetate.
  40. Dry the pooled ethyl acetate solutions with anhydrous magnesium sulfate (10–20 g, stir for 2 min).
  41. Filter off the magnesium sulfate through a fritted glass funnel into a 500 ml round-bottom flask. Wash the filter cake twice with 10 ml ethyl acetate. Evaporate the solvent on a rotary evaporator at room temperature and dry the residual oil for 1–2 h on a vacuum pump.Pause Point The compound can be stored under argon at −20 °C overnight.
  42. Synthesis of 8Weigh 1 eq. (relative to crude 4) of compound 7 into a 250 ml round-bottom flask equipped with a Teflon-coated magnetic stir bar.
  43. Add 50 ml glacial acetic acid. Turn the stirrer on.
  44. Mount a 100 ml addition funnel onto the round-bottom flask and secure the adapter with a Keck clip.
  45. Dissolve the crude 4 (Step 41) in 50 ml glacial acetic acid and add the solution into a 100 ml addition funnel. Stopper the funnel with a flushing adapter fitted with a nitrogen filled balloon.
  46. Add the solution of 7 at approximately 3–4 drops per second. Let the solution stir for 12 h at room temperature.Pause Point The reaction mixture can be left stirring overnight at room temperature.
  47. Remove the solvent on a rotary evaporator at 30 °C.
  48. Pack a chromatography column (5.6 cm i.d. × 50 cm length) with approximately 600 ml silica gel using a 1:3 (vol/vol) mixture of ethyl acetate and hexane. Cover the top of the silica layer with a flat filter paper of 5.5 cm diameter.
  49. Dissolve the residue of Step 47 in a minimal amount of dichloromethane (10–20 ml). Some precipitate may remain. Apply the mixture to the top of the silica bed using a glass pipette. Let it soak into the silica and cover the top of the column with approximately 1 cm sand.
  50. Elute the column under light pressure (according to Still22) using a 1:3 (vol/vol) mixture of ethyl acetate and hexane. The product will be visible as a bright orange band. Collect 20 ml fractions.
  51. Evaporate the solvents from the pooled fractions containing 8 and dry the product further using a vacuum pump. The yield is typically 55–60%.Pause Point Product 8 can be stored overnight at room temperature.
  52. Synthesis of 9Add 8 (Step 52) to a 100 ml round-bottom flask equipped with a Teflon-coated magnetic stir bar.
  53. Add 50 ml dichloromethane. Turn the magnetic stirrer on.
  54. Add 10 ml TFA from a graduated glass pipette.
    Caution TFA is toxic and corrosive.
  55. Stopper the flask with a flushing adapter fitted with a nitrogen-filled balloon and secure the adapter with a Keck clip. Stir at room temperature until the starting material disappears (check by TLC with a 1:2 (vol/vol) mixture of ethyl acetate and hexanes as solvent) or overnight.Pause Point The reaction solution can be left stirring overnight at room temperature.
  56. Transfer the solution into a 250 ml separatory funnel containing 50 ml H2O. Wash the organic layer with 50 ml portions of H2O until neutral, and then once with 50 ml brine. Dry by adding anhydrous magnesium sulfate (5 g, stir vigorously for 2 min).
  57. Filter the mixture through a fritted funnel into a 250 ml round-bottom flask to remove the magnesium sulfate. Wash the filter cake with 2 × 10 ml dichloromethane. Evaporate the solvent on a rotary evaporator at 30 °C and dry the brown–orange solid on a vacuum pump. The product can be used without further purification for solid-phase peptide synthesis.
  58. Peptide synthesisThe protected azobenzene aa 9 is compatible with standard FastMoc protocols for an ABI 433A solid-phase peptide synthesizer. These methods generally involve the use of 20% piperidine in DMF for Fmoc deprotection, HOBt/HBTU for activation, DIPEA as a base and NMP as solvent. Compound 9 is also likely to be compatible with other activating agents and solvents. The reader is referred to the literature23, 24 for detailed protocols for (manual) peptide synthesis.
  59. Deprotection and cleavage from the resin is best carried out using a 95:5 (vol/vol) mixture of TFA and water only as azobenzenes can be reduced by the silanes normally used as scavengers15. If the presence of functionalized aa side chains in the peptide sequence requires the addition of scavengers to avoid alkylation by cleavage products, a cleavage cocktail of TFA:water:TIPS 95:2.5:2.5 (vol/vol) has been found to produce only minor amounts of byproducts. The peptide is typically cleaved within 1 h from the Rink amide MBHA (4-methylbenzhydrylamine) resin. Arg should be incorporated with a 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf)–protected side chain as it is cleaved at a much faster rate than other protecting groups. This allows exposure of the azobenzene linker to the reducing silane to be minimized.
    Caution The linker has not been tested with cleavage cocktails commonly used for deprotection of peptide sequences containing Cys or Met23, 24.
  60. IrradiationIsomerization of the azobenzene chromophore is typically carried out by irradiating a peptide sample (1 mg ml−1) with near-UV (320 nm, trans → cis) or blue light (420 nm, cis → trans) for 2–3 min. Quartz glassware should be used for all irradiation experiments involving UV light. The use of compact fluorescent tubes as light sources (see MATERIALS for suitable models) has been found to produce the best results. The aqueous sample is placed in a suitable reaction vessel (stirring may be necessary for large sample volumes) at a distance of approximately 5 cm from the fluorescent tube; irradiation is conveniently performed in a standard Rayonet photochemical reactor (Model RPR-100 or RPR-200) but other setups can also be used. Isomerization is typically complete within 2–3 min. Small sample volumes can also be irradiated in instruments equipped with a suitable lamp and a monochromator, e.g., a fluorescence spectrophotometer.
    Critical step Irradiated samples should be handled in dimmed light to avoid relaxation to the more stable isomer. The photostationary state typically contains 80–85% of the cis isomer. If a higher cis content is required, the isomers can be separated by preparative HPLC, although success depends on the specific peptide sequence.Pause Point Lyophilized samples can be stored at −80 °C in the dark for at least 1 year without noticeable isomerization.
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Timing

Steps 1–13: 2 h
Step 14: 4–12 h
Steps 15–27: 5 h
Steps 28–46: 9 h
Steps 47–54: 5 h
Step 55: 6–12 h
Steps 56 and 57: 1 h

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Troubleshooting

Troubleshooting advice can be found in Table 1.


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Anticipated results

Typical yields

Fmoc and tert-butyl protection, reduction of compound 6 and tert-butyl ester deprotection are quantitative. Generation of the nitroso compound 4 is quantitative as judged by TLC. Coupling to the diazo compound typically proceeds in 55–60% yield. An overall yield of 40–50% can be expected for 9.

Analytical data

tert-Butyl (3-nitrophenyl)acetate (3).

1H NMR (CDCl3, 400 MHz): 8.17–8.12 (m, 2 H), 7.63–7.60 (m, 1 H), 7.50 (t, J = 7.9 Hz, 1 H), 3.65 (s, 2 H), 1.46 (s, 9 H). 13C NMR (CDCl3, 100 MHz): 169.6, 148.3, 136.6, 135.6, 129.3, 124.4, 122.1, 81.8, 42.0, 28.0. MS (EI) m/z [M]+ 237.2.

9H-Fluoren-9-ylmethyl (3-nitrobenzyl)carbamate (6).

Mp 152–153 °C. 1H NMR (DMSO-d6, 400 MHz): 8.13 (s, 1 H) 8.12 (d, J = 7.2 Hz, 1 H), 8.01 (t, J = 6.1 Hz, 1 H), 7.89 (d, J = 7.5 Hz, 2 H), 7.70–7.61 (m, 4 H), 7.42 (t, J = 7.4 Hz, 2 H), 7.32 (t, J = 7.1 Hz, 2 H), 4.38 (d, J = 6.8 Hz, 2 H), 4.32 (d, J = 6.1 Hz, 2 H), 4.24 (t, J = 6.7 Hz, 1 H). 13C NMR (DMSO-d6, 100 MHz): 156.3, 147.7, 143.7, 142.1, 140.7, 133.7, 129.8, 127.5, 126.9, 125.0, 121.7, 121.5, 120.0, 65.4, 46.7, 43.0. HRMS (MALDI, DHB) m/z [M+Na]+ calc. 397.1159, found 397.1160.

9H-Fluoren-9-ylmethyl (3-aminobenzyl)carbamate (7).

Mp 142–143 °C. 1H NMR (DMSO-d6, 400 MHz): 7.89 (d, J = 7.5 Hz, 2 H), 7.75 (t, J = 6.1 Hz, 1 H), 7.71 (d, J = 7.4 Hz, 2 H), 7.42 (t, J =7.4 Hz, 2 H), 7.33 (td, J = 7.4, 0.9 Hz, 2 H), 6.94 (t, J = 7.7 Hz, 1 H), 6.46 (d, J = 1.6 Hz, 1 H), 6.43 (d, J = 7.9 Hz, 1 H), 6.38 (d, J = 7.5 Hz, 1 H), 5.00 (s, 2 H), 4.31 (d, J = 6.9 Hz, 2 H), 4.22 (t, J = 6.9 Hz, 1 H), 4.05 (d, J = 6.1 Hz, 2 H). 13C NMR (DMSO-d6, 100 MHz): 156.2, 148.5, 143.8, 140.6, 140.2, 128.6, 127.5, 127.0, 125.1, 120.0, 114.4, 112.4 (2 isochronous signals), 65.3, 46.7, 44.0. HRMS (MALDI, DHB) m/z [M+Na]+ calc. 367.1417, found 367.1417.

tert-Butyl (3-{[3-({[(9H-fluoren-9-ylmethoxy)carbonyl]amino}methyl)-phenyl]diazenyl}phenyl)acetate (8).

Mp 64–66 °C. 1H NMR (CDCl3, 500 MHz): 7.84–7.80 (m, 4 H), 7.75 (d, J = 7.4 Hz, 2 H), 7.60 (d, J = 7.4 Hz, 2 H), 7.49–7.45 (m, 2 H), 7.41–7.37 (m, 4 H), 7.30 (t, J = 7.3 Hz, 2 H), 5.20 (bs, 1 H,), 4.49–4.47 (m, 4 H), 4.24 (t, J = 6.9 Hz, 1 H), 3.63 (s, 2 H), 1.45 (s, 9 H). 13C NMR (CDCl3, 125 MHz): 170.5, 156.5, 152.9, 152.7, 143.9, 141.3, 139.6, 135.9, 132.0, 130.0, 129.5, 129.2, 127.7, 127.1, 125.0, 123.5, 122.4, 121.8, 121.4, 120.0, 81.1, 66.8, 47.3, 44.9, 42.4, 28.1. HRMS (MALDI, DHB) m/z [M+Na]+ calc. 570.2363, found 570.2371.

(3-{[3-({[(9H-Fluoren-9-ylmethoxy)carbonyl]amino}methyl)phenyl]-diazenyl}phenyl)acetic acid (9).

Mp 168–169 °C. 1H NMR (DMSO-d6, 400 MHz): 12.43 (bs, 1 H), 8.00 (t, J = 6.1 Hz, 1 H), 7.89 (d, J = 7.5 Hz, 2 H), 7.80–7.77 (m, 4 H), 7.72 (d, J = 7.5 Hz, 2 H), 7.58–7.53 (m, 2 H), 7.49–7.39 (m, 4 H), 7.32 (t, J = 7.4 Hz, 2 H), 4.38 (d, J = 6.8 Hz, 2 H), 4.33 (d, J = 6.1 Hz, 2 H), 4.25 (t, J = 6.8 Hz, 1 H), 3.74 (s, 1 H). 13C NMR (DMSO-d6, 100 MHz): 172.4, 156.3, 151.9, 151.8, 143.8, 141.3, 140.7, 136.5, 132.6, 130.1, 129.3, 129.2, 127.5, 126.9, 125.0, 123.0, 121.5, 121.3, 120.4, 120.0, 65.3, 46.7, 43.4, 40.2. HRMS (MALDI, DHB) m/z [M+Na]+ calc. 514.1737, found 514.1731.

Representative example

The 11-residue peptide 10 derived from protein GB1 has been shown to adopt a monomeric hairpin structure in aqueous solution with 1 in its cis configuration, while the corresponding trans isomer forms higher-order aggregates and precipitates upon repeated isomerization19.

H — Arg — Trp — Gln — Tyr — Val — 1 — Lys — Phe — Thr — Val — Gln — NH2 (10)

After solid-phase peptide synthesis and purification, 10 displayed a characteristic maximum in the UV spectrum at 314 nm (Fig. 3a, solid line). The corresponding HPLC chromatogram (Fig. 3b) shows the dominant trans species (t R = 34.3 min) and the presence of approximately 5% cis isomer (t R = 32.5 min).

Figure 3: Cis and trans isomers of peptide 10.
Figure 3 : 
					Cis and trans isomers of peptide 10.

(a) UV spectra in the thermodynamic (solid line) and photostationary (dotted line) state in 50 mM sodium acetate buffer, pH 3.8. (b) Corresponding HPLC traces (Macherey Nagel Nucleosil 100-5 C18 250 × 4.6 mm2, 5–60% acetonitrile containing 0.05% TFA over 45 min in water containing 0.1% TFA at a flow rate of 1 ml min−1).

Full size image (24 KB)

A 0.1 mM solution of 10 in H2O was then placed in a quartz reaction vessel and irradiated for 10 min with a Philips PL-S 9W/12 compact fluorescent tube (εmax = 320 nm). The conformational change of the azobenzene moiety is reflected in the UV spectrum (Fig. 3a, dotted line). HPLC analysis shows an increased cis content of approximately 85% (Fig. 3b, dotted line).



Competing interests statement: 

The authors declare no competing financial interests.

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References

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  1. Laboratory of Organic Chemistry, ETH Zurich, CH-8093 Zurich, Switzerland.

Correspondence to: Donald Hilvert1 e-mail: hilvert@org.chem.ethz.ch