Development and Validation of a Fluorescence Method to. Follow the Build-up of Short Peptide Sequences on Solid. 2D Surfaces

Transcription

1 Supporting Information for Development and Validation of a Fluorescence Method to Follow the Build-up of Short Peptide Sequences on Solid 2D Surfaces Mischa Zelzer a* David J. Scurr, Morgan R. Alexander, Rein V. Ulijn* Corresponding author: mischa.zelzer@strath.ac.uk, Fax: +44 (0) , Tel: +44 (0) ; rein.ulijn@strath.ac.uk, Fax: +44 (0) , Tel: +44 (0)

2 1. Sample holder Supplementary Figure 1. (A) Model of the custom made sample holder for the measurement of solid state fluorescence. Dimensions are given in millimetres. (B) Photograph of the sample holder (left) and its position for the fluorescence measurements in the spectrophotometer (right). 1

3 2. Effect of Pbf on the Fmoc fluorescence The effect of the Pbf (2,2,4,6,7-pentamethyldihydro-benzofuran-5-sulfonyl) on the Fmoc fluorescence emission observed in solution for Fmoc-R(Pbf)-OH was investigated further. In particular, we were interested in whether the effect relies on the proximity of the Fmoc group to the Pbf group. This was investigated by preparing a dipeptide where the Fmoc is removed from arginine by an additional glycine, i.e. by comparing the fluorescence intensities of Fmoc-G-R(Pbf)-OMe and Fmoc-G-R-OMe Peptide synthesis Fmoc-G-R(Pbf)-OMe Arg(Pbf)-OMe (501 mg, 1.05 mmol) and Fmoc-Gly-OH (379 mg, 1.27 mmol, 1.2 equ.) were dissolved in DMF (10 ml). HBTU (482.4 mg, 1.27 mmol, 1.2 equ.) and DIPEA (456 μl, mg, 2.62 mmol, 2.5 equ.) were added. The solution turned orange within a few minutes. The reaction was stirred at room temperature over night. The reaction was washed with 1mmM NaHCO 3, brine 1M HCl and brine and dried over MgSO 4. The solvent was evaporated and the crude product was purified via column chromatography (CHCl 3 : MeOH = 97.5 : 2.5). The solvent was evaporated and after washing the product with diethyl ether it was obtained as a white powder (411 mg). 1 H NMR (400 MHz, MeOD): δ = 1.38 (s, 6 H, 2 x CH 3 -C, Pbf), 1.51 (m, 2 H, CH 2, Arg), 1.65 / 1.81 (m, 2 H, CH 2, Arg), 2.03 (s, 3 H, CH 3 -C ar, Pbf), 2.49 (s, 3 H, CH 3 -C ar, Pbf), 2.56 (s, 3 H, CH 3 -C ar, Pbf), 2.90 (s, 2 H, CH 2, Pbf), 3.14 (m, 2 H, CH 2, Arg), 3.64 (s, 3 H, O-CH 3, Arg), 3.81 (d, J = 4.44 Hz, 2 H, CH 2, Gly), 4.16 (t, J = 6.94 Hz, 1 H, CH, Fmoc), 4.32 (d, J = 7.04 Hz, CH 2, Fmoc), 4.41 (m, 1 H, CH, Arg), 7.25 (t, J = 7.44 Hz, 2 H, 2 x CH ar, Fmoc), 7.33 (t, J = 7.42 Hz, 2 H, 2 x CH ar, Fmoc), 7.61 (d, J = 7.40 Hz, 2 H, 2 x CH ar, Fmoc), 7.73 (d, J = 7.52 Hz, 2 H, 2 x CH ar, Fmoc). 13 C NMR (400 MHz, MeOD): δ = 12.5 (1 C, CH 3, Pbf), 18.4 (1 C, CH 3, Pbf), 19.6 (1, C, CH 3, Pbf), 28.7 (2 C, 2 x CH 3, Pbf), 29.8 (1 C, CH 2, Arg), 41.4 (1 C, CH 2, Arg), 43.9 (1 C, CH 2, Gly), 44.6 (1 C, CH 2, Pbf), 48.2 (1 C, CH, Fmoc), 52.8 (1 C, CH 3, Gly), 53.3 (1 C, CH, Arg), 68.1 (1 C, CH 2, Fmoc), 87.6 (1 C, C(CH 3 ) 2, Pbf), (1 C, C ar -CH 3, Pbf), (2 C, 2 x CH ar, Fmoc), (1 C, C ar -CH 3, Pbf), (2 C, 2 x CH ar, Fmoc), (2 C, 2 x CH ar, Fmoc), (2 C, 2 x CH ar, Fmoc), (1 C, C ar -CH 3, Pbf), (1 C, C ar -CH 2, Pbf), (1 C, C ar -S, Pbf), (2 C, 2 x C ar, Fmoc), (2 C, 2 x C ar, Fmoc), (1 C, HN=C(NH) 2, Arg), (1 C, O-C(=O)NH, Fmoc), (1 C, C ar -O, Pbf), (1 C, C(=O)OCH 3, Arg), (1 C, CH 2 -C(=O)NH, Gly). m/z: [M+H] + purity: 99 % 2

4 Fmoc-G-R(Pbf)-OMe Fmoc-Gly-Arg(Pbf)-OMe (300 mg, 0.63 mmol) was dissolved in a 9:1 (v/v) mixture of TFA and CH 2 Cl 2 (10 ml). The reaction was stirred over night at room temperature. After evaporation of the solvents the product was dissolved in methanol, precipitated by addition of diethyl ether, separated by filtration and recovered by washing the filter with methanol. The product was obtained as a white powder (89 mg). 1 H NMR (400 MHz, MeOD): δ = 1.63 / 1.77 / 1.88 (3 x m, 4 H, 2 x CH 2, Arg), 3.15 (m, 2 H, CH 2, Arg), 3.66, (s, 3 H, CH 3 -O, Arg), 3.87 (d, J = 7.40 Hz, 2 H, CH 2, Gly), 4.16 (t, J = 6.98 Hz, 1 H, CH-CH 2, Fmoc), 4.29 (d, J = 7.03 Hz, 2 H, CH 2 -CH, Fmoc), 4.44 (m, 1 H, CH, Arg), 7.26 (t, J = 7.10 Hz, 2 H, 2 x CH ar, Fmoc), 7.33 (t, J = 7.36 Hz, 2 H, 2 x CH ar, Fmoc), 7.62 (d, J = 7.28 Hz, 2 H, 2 x CH ar, Fmoc), 7.72 (d, J = 7.48 Hz, 2 H, 2 x CH ar, Fmoc). 13 C NMR (400 MHz, MeOD): δ = 26.1 (1 C, CH 2, Arg), 29.6 (1 C, CH 2, Arg), 41.8 (1 C, CH 2, Arg), 44.6 (1 C, CH 2, Gly), 48.2 (1 C, CH, Fmoc), 52.8 (1 C, CH 3, Arg), 53.3 (1 C, CH, Arg), 68.2 (1 C, CH 2, Fmoc), (2 C, 2 x CH ar, Fmoc), (2 C, 2 x CH ar, Fmoc), (2 C, 2 x CH ar, Fmoc), (2 C, 2 x CH ar, Fmoc), (2 C, 2 x C ar, Fmoc), (2 C, 2 x C ar, Fmoc), (1 C, HN-C(=NH)-NH 2, Arg), (1 C, O-C(=O)NH, Fmoc), (1 C, C(=O)OCH 3, Arg), (1 C, CH 2 -C(=O)NH, Gly). m/z: [M+H] + purity: 98% 2.2. Fluorescence spectra Supplementary Figure 2. Solution state fluorescence spectra (λ Ex = 290 nm) of Fmoc-GR-OMe with (dashed line) and without (solid line) Pbf protection of the arginine side group (c = 10 mm). 3

5 3. Chemicals 3.1. Amino acids Fmoc-Ala-OH Sigma-Aldrich Product Nr Fmoc-Arg(Pbf)-OH) Bachem Product Nr. B2375 Fmoc-Asp(OtBu)-OH Sigma-Aldrich Product Nr Fmoc-Cys(Trt)-OH Sigma-Aldrich Product Nr Fmoc-Glu(OtBu)-OH Fluka Product Nr Fmoc-Gly-OH Sigma-Aldrich Product Nr Fmoc-His(Trt)-OH Sigma-Aldrich Product Nr Fmoc-Leu-OH Sigma-Aldrich Product Nr Fmoc-Phe-OH Sigma-Aldrich Product Nr Fmoc-Thr(OtBu)-OH Sigma-Aldrich Product Nr Fmoc-Trp(Boc)-OH Sigma-Aldrich Product Nr Fmoc-Tyr(OtBu)-OH Sigma-Aldrich Product Nr H-Arg(Pbf)-OMe Bachem Product Nr Other chemicals HBTU, O-(benzotriazol-1-yl)- N,N,N,N -tetramethyluronium hexafluorophosphate DIPEA, N,N - diisopropylethylamine Novabiochem Product Nr Sigma-Aldrich Product Nr TFA, trifluoroacetic acid Sigma-Aldrich Product Nr. T6508 4