Supplementary information

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1 Supplementary information A capped dipeptide which simultaneously exhibits gelation and crystallisation behaviour Adam D. Martin, *a,b Jonathan P. Wojciechowski, a,b Mohan M. Bhadbhade, a,c and Pall Thordarson *a,b a School of Chemistry, The University of New South Wales, Sydney, 2052, NSW, Australia. b The Australian Centre for Nanomedicine and the ARC Centre of Excellence for Convergent Bio-Nano Science and Technology, The University of New South Wales, Sydney, 2052, NSW, Australia. c Mark Wainwright Analytical Centre, The University of New South Wales, Sydney, 2052, NSW, Australia s: adam.martin2@unsw.edu.au and p.thordarson@unsw.edu.au; Fax: ; Tel: Contents: Synthesis of benzimidazole acetic acid Synthesis of benzimidazole-diphenylalanine Fig. S1 1 H NMR spectrum of benzimidazole-diphenylalanine Fig. S2 13 C NMR spectrum of benzimidazole-diphenylalanine Fig. S3 1 H- 13 C NMR spectrum of benzimidazole-diphenylalanine Fig. S4 HPLC trace of benzimidazole-diphenylalanine after purification Analytical reverse-phase HPLC Preparation of hydrogels Circular dichroism measurements Attenuated total reflectance infrared spectroscopy measurements Rheology measurements Atomic force microscopy measurements Single crystal x-ray diffraction measurements Fig. S5 Single crystals of benzimidazole-diphenylalanine Fig. S6 ATR-IR comparison of benzimidazole-diphenylalanine crystals and hydrogel Fig. S7 Rheology frequency sweep of Fmoc-FF Fig. S8 Rheology strain sweep of Fmoc-FF Fig. S9 AFM of Fmoc-FF References S1 S1 S3 S4 S4 S5 S5 S5 S5 S5 S6 S6 S6 S7 S7 S8 S8 S9 S9

2 Synthesis of benzimidazole acetic acid 1 g (8.46 mmol) of benzimidazole and 2.35 g (17 mmol) of K 2 CO 3 was dissolved in 120 ml of dry acetonitrile. 1.5 ml (10.15 mmol) of tert-butyl bromoacetate was added and the mixture refluxed for 4 h. The suspension was filtered and the solvent removed under reduced pressure. The residue was dissolved in dichloromethane (150 ml), washed with water (2 x 100 ml) and dried over MgSO 4. The solvent was removed under reduced pressure and the resultant oil purified by column chromatography (3:2 hexane:dichloromethane) to give 1.9g (94%) of the tert-butyl benzimidazole acetate. Characterisation data for tert-butyl benzimidazole acetate: IR: 3059 (w), 2977 (w), 2940 (w), 1733 (s), 1617 (w), 1590 (w), 1497 (m), 1465 (m), 1421 (w), 1389 (w), 1367 (m), 1358 (m), 1337 (m), 1296 (w), 1272 (m), 1231 (s), 1204 (m), 1193 (m), 1157 (s), 1011 (m), 973 (m), 984 (w), 891 (w), 881 (m), 856 (m), 795 (w), 775 (m), 765 (w), 741 (s) ; 1 H NMR (CDCl 3, 400 MHz): 7.91 (s, 1H, ArH), 7.82 (dd, 1H, ArH), (m, 3H, ArH), 4.79 (s, 2H, CH 2 ), 1.44 (s, 9H, CH 3 ); 13 C{1H} NMR (DMSO-d 6, 100 MHz): , , , , , , , , 83.32, 46.95, 27.92; HR-MS (ESI): calcd for C 13 H 16 N 2 O 2 + : , found g of tert-butyl benzimidazole acetate was stirred with 40 ml of a 20% (v/v) solution of trifluoroacetic acid in dichloromethane overnight. The solvents were then removed under reduced pressure, the residue dissolved in a 1:1 mixture of water:acetonitrile and lyophilised. Diethyl ether was added to the resultant powder and the mixture filtered and washed with ethyl acetate, giving 0.72g (95%) benzimidazole acetic acid. Characterisation data for benzimidazole acetic acid: IR: 3164 (w), 2733 (w), 1919 (w), 1708 (s), 1638 (w), 1611 (m), 1557 (m), 1502 (w), 1485 (w), 1454 (m), 1425 (m), 1360 (w), 1336 (w), 1273 (w), 1254 (m), 1175 (s), 1128 (s), 1090 (m), 1012 (w), 986 (w), 947 (w), 905 (m), 886 (m), 831 (m), 795 (m), 786 (m), 748 (s), 720 (s), 672 (m); 1 H NMR (DMSO-d 6, 400 MHz): (br. s, 1 H, COOH), 9.26 (s, 1H, ArH), (m, 2H, ArH), (m, 2H, ArH), 5.39 (s, 2H, CH 2 ); 13 C{1H} NMR (DMSO-d 6, 100 MHz): , , , , , , , , , , , 46.86; HR-MS (ESI): calcd for C 9 H 8 N 2 O 2 + : , found Synthesis of benzimidazole-diphenylalanine Initial amino acid loading 2-chlorotrityl chloride resin ( mesh; 1% DVB; 1.41 mmol/g) (400 mg, 0.56 mmol) was weighed into a 10 ml polypropylene syringe equipped with a porous polypropylene frit (Torviq SF-1000), which was used as the reaction vessel. The resin was washed with dichloromethane (3 5 ml) before being allowed to swell in dichloromethane (5 ml) for at least 0.5 h prior to the loading of the first amino acid.

3 A solution of Fmoc-AA-OH (3 equiv.) was dissolved in dry dichloromethane (4 ml) and N,N-diisopropylethylamine (DIPEA) (0.8 ml, 8 equiv.) and taken up into the syringe with resin and stirred overnight using an orbital shaker. The resin was then washed with dichloromethane (3 x 4mL) and N,N-dimethylformamide (DMF) (3 4 ml). N-terminal Fmoc deprotection A solution of 20% (v/v) piperidine in DMF (2 4 ml) was added to the resin once for 5 mins, then a fresh aliquot was taken up again and left for 10 mins. The solution was subsequently expelled and the resin washed with DMF (5 4 ml). The resulting resin-bound amine was used immediately in the next peptide coupling step. Amino acid coupling Fmoc-AA-OH (655 mg, 3 equiv.) was dissolved in a 0.45 M solution of 1- hydroxybenzotriazole hydrate (HOBt H 2 O)/N,N,N,N -tetramethyl-o-(1h-benzotriazol-1- yl)uronium hexafluorophosphate (HBTU) (3.4 ml, 3 equiv.) and DIPEA (0.6 ml, 6 equiv.) and this coupling solution added to the resin and stirred for 45 mins using an orbital shaker. The solution was expelled and the resin washed with DMF (5 x 4 ml). After another N-terminal Fmoc deprotection, iterative couplings were performed in order to build up the required peptide sequence. Benzimidazole coupling The benzimidazole capping group was introduced to the peptide in the same manner as for the amino acids, however a double coupling was required. Benzimidazole acetic acid (300 mg, 3 equiv.) was dissolved in a 0.5M solution of 1-hydroxybenzotriazole hydrate (HOBt H 2 O)/ N,N,N,N -tetramethyl-o-(1h-benzotriazol-1-yl)uronium hexafluorophosphate (HBTU) (3.4 ml, 3 equiv.) and DIPEA (0.6 ml, 6 equiv.) and this coupling solution added to the resin and stirred for 45 mins using an orbital shaker. Coupling was confirmed through the Kaiser test. 2 Cleavage of the peptide After the final coupling step, the resin was washed with DMF (3 x 4 ml) and dichloromethane (3 x 4 ml). A solution of 7:3 dichloromethane: trifluoroacetic acid was then added to the resin and the resin stirred for 15 minutes using an orbital shaker. The cleavage solution was then expelled, the resin washed with dichloromethane (2 x 4 ml) and the solvents evaporated under a stream of nitrogen. The resulting orange residue was lyophilised and purified by semi-preparative HPLC using an acetonitrile/water gradient, giving a white fluffy solid. Characterisation data for benzimidazole diphenylalanine (BzIm-FF): IR: 3292 (m), 3065 (w), 3031 (w), 2927 (w), 1715 (w), 1655 (s), 1606 (w), 1546 (s), 1498 (m), 1461 (w), 1454 (w), 1433 (w), 1380 (m), 1333 (w), 1288 (w), 1263 (m), 1231 (w), 1190 (s), 1114 (m), 1083 (w), 1032 (m), 971 (m), 916 (w), 876 (w), 798 (m), 773 (w), 736 (s), 697 (s); 1 H NMR (DMSO-d 6, 400 MHz) δ (s, 1H, COOH), 8.63 (d, J = 8.7 Hz, 1H, NH), 8.48 (d, J = 7.8

4 Hz, 1H, NH), 8.02 (s, 1H, ArH), 7.60 (dd, J = 6.7, 2.1 Hz, 1H, ArH), (m, 12H, ArH), 7.02 (dd, J = 6.7, 2.0 Hz, 1H, ArH), 4.84 (dd, J = 42.8, 16.4 Hz, 2H, CH 2 ), 4.62 (td, J = 9.9, 4.0 Hz, 1H, CH), 4.46 (td, J = 8.4, 5.4 Hz, 1H, CH), 3.00 (ddd, J = 22.8, 13.9, 7.1 Hz, 2H, CH 2 ), 2.88 (ddd, J = 23.9, 14.2, 7.6, 2H, CH 2 ). 13 C{1H} NMR (DMSO-d 6, 101 MHz) δ , , , , , , , , , , , , , , , , , , 53.63, 53.60, 46.59, 37.92, 36.62; HR-MS (ESI): calcd for C 27 H 26 N 4 O 4 + : , found Figure S1 1 H NMR spectrum for benzimidazole-diphenylalanine.

5 Figure S2 13 C NMR spectrum for benzimidazole-diphenylalanine. Figure S3 1 H- 13 C HSQC spectrum for benzimidazole-diphenylalanine.

6 Figure S4 HPLC trace of benzimidazole-diphenylalanine. Crystal data for benzimidazole diphenylalanine: C 27 H 26 N 4 O 4, M = , monoclinic P2 1 (No. 4), a = 5.010(10), b = (4), c = (3), α = γ = 90, β = 91.32(3), V = (4) Å 3, Z = 2, T = 100(2) K, D calcd = gcm -3, µ = mm -1, N(unique) = 6237 (merged from 6664), R int = , R 1 = , wr 2 (all data) = , GOF = 1.099, max F peak = 0.38 e Å -3 Analytical reverse-phase HPLC High Performance Liquid Chromatography (HPLC) was recorded on a Shimadzu Prominence LC-20A HPLC system using a Waters XBridge C18 10 µm 150 x 4.60 mm analytical column at a flow rate of 1 ml/min. The mobile phase consisted of eluents: A (water with 0.1% formic acid) and B (acetonitrile with 0.1% formic acid). The HPLC trace was obtained using a gradient from 5% B to 95% B over 20 minutes. Preparation of hydrogels 32 µl 0.1 M NaOH (1.5 equiv.) was added to 1 mg of benzimidazole diphenylalanine and 68 µl milliq water added to make the suspension up to 1% (w/v). This suspension was sonicated until homogenous, upon which time 3 molar equivalents of glucono-δ-lactone (GdL, 1.1 mg) was added to lower the ph, resulting in a final ph of approximately 4 and gelation after 60 minutes. Hydrogels of Fmoc-FF were prepared in the same manner. Circular dichroism measurements CD measurements were performed using a ChirascanPlus CD spectrometer, with data collected between wavelengths of nm with a bandwidth of 1 nm, sample ratio of 0.1 s/point and step of 1 nm. In a typical experiment, 1% (w/v) hydrogels were prepared and diluted 1:8 (v/v) in water. Temperature was kept constant at 20 C and all experiments were repeated three times and averaged into a single plot. Attenuated Total Reflectance-Infrared Spectroscopy measurements Fourier transform infrared spectroscopy (FTIR) measurements were made on a Perkin Elmer Spotlight 400 FT-IR spectrophotometer equipped with a diamond crystal attenuated total

7 reflectance (ATR) accessory. Hydrogels were prepared at 1% (w/v) and pressed between the diamond crystal and substrate. All spectra were scanned 16 times over the range of cm -1. Rheology measurements Rheological measurements were performed on an Anton Paar MCR 302 rheometer using a 25 mm stainless steel parallel plate geometry configuration and analysed using RheoPlus v3.61 software. Typical rheology measurements involved casting 550 µl of a 1% (w/v) sol onto one of the stainless steel plates, lowering the other plate to the measurement position, and allowing two hours for the gel to form via the ph switch method described above. A Peltier temperature control hood and solvent trap was used to reduce evaporation and maintain a temperature of 25 C for frequency and amplitude sweeps. Frequency sweeps were performed with a log ramp frequency (f) = Hz and constant strain (γ) = 0.5%. Amplitude Sweeps were performed with constant frequency (f) = 1 Hz and log ramp strain (γ) = %. The rheology plots displayed are an average of at least three repeats for each point and error bars denote two standard deviations from the log-averaged mean. AFM measurements 0.1% (w/v) hydrogels of 1 and 2 were prepared using the ph switch method described above. Upon addition of GdL and before gelation, one drop of the hydrogel solutions was cast onto a freshly cleaved mica substrate, followed by spreading of the drop over the mica using a glass slide, with the excess liquid wicked away using capillary action. These samples were left to dry in air overnight, or for three days, as the figure caption indicates. Imaging was undertaken on a BrukerMulitmode 8 atomic force microscope in Scanasyst mode in air, whereby the imaging parameters are constantly optimised through the force curves that are collected, preventing damage of soft samples. Bruker Scanasyst-Air probes were used, with a spring constant of N/m and a tip radius of 2 nm. Single crystal x-ray diffraction X-ray diffraction measurements were carried out at MX2 beamline at the Australian Synchrotron Facility, Melbourne. The crystal was mounted on the goniometer using a cryo loop for diffraction measurements, was coated with paraffin oil and then quickly transferred to the cold stream using Cryo stream attachment. Data were collected using Si<111> monochromated synchrotron X-ray radiation (λ = Å) at 100(2) K and were corrected for Lorentz and polarization effects using the XDS software. 1 The structure was solved by Direct methods and the full-matrix least-squares refinements were carried out using SHELXL 2 with the program Olex-2. 3 Hirshfeld surface analysis was undertaken using CrystalExplorer

8 Figure S5 Single crystals of benzimidazole-diphenylalanine, displaying needle-like morphology. Figure S6 ATR-IR comparison of benzimidazole-diphenylalanine crystals (black trace) and hydrogel (red trace). Inset, zoomed in region of the spectrum corresponding to the Amide I and II regions.

9 Figure S7 Frequency sweep for a 1% (w/v) hydrogel of Fmoc-FF, performed at 25 C. Figure S8 Strain sweep for a 1% (w/v) hydrogel of Fmoc-FF, performed at 25 C.

10 Figure S9 Representative AFM images for Fmoc-FF, prepared at 0.05% (w/v) using a ph switch method and dried in air overnight. References 1. Kabschi, W. Automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constants. J. Appl. Cryst. 1993, 26, Sheldrick, G. M. A short history of SHELX. Acta Cryst., 2008, A64, Dolomanov, O. V.; Bourhis, L. J.; Gildea, R. J.; Howard, J. A. K.; Puschmann, H., OLEX2: A complete structure solution, refinement and analysis program. J. Appl. Cryst., 2009, 42, Wolff, S. K.; Grimwood, D. J.; McKinnon, J. J.; Turner. M. J.; Jayatilaka, D.; Spackman, M. A. CrystalExplorer 3.1, University of Western Australia, 2012.