Electronic Supplementary Material (ESI) for rganic & Biomolecular Chemistry. This journal is The Royal Society of Chemistry 2015 Supplementary Information Design and Solid Phase Synthesis of New DTA Conjugated (+)-Biotin Dimers Planned to Develop Molecular Weight-Tuned Avidin ligomers Alessandro Pratesi, Mauro Ginanneschi, Fabrizio Melani, Marco Chinol, Angela Carollo, Giovanni Paganelli, Marco Lumini, Mattia Bartoli, Marco Frediani, Luca Rosi, Giorgio Petrucci, Luigi Messori, and Anna Maria Papini Contents: Potential energy data for molecular modelling studies Sinthesis and characterization of Fmoc-Lys-tBu Bis-biotins characterization via HPLC-ESI-MS MALLS correlation functions S1
Figure 1S. Structures of bis-biotins designed for the molecular modeling study. In brackets is reported the linker arm length (Å) between the two amide C= of the biotin residues, calculated on the fully extended planar conformation. S2
Table 1S. Potential energy data for type 1 complex. 150 K 300 K 450 K Entry Ep Ep Ep sd sd (Kcal/mol) (Kcal/mol) (Kcal/mol) sd bis-biotins with non-peg spacers 1-6734 24-5180 43-3481 65 2-6792 23-5231 43-3529 69 3-6766 24-5209 45-3504 68 4-6794 25-5208 46-3502 67 5-6766 25-5190 43-3509 67 6-6781 24-5177 43-3427 68 7-6731 23-5145 46-3393 71 8-6723 22-5111 45-3354 72 bis-biotins with PEG spacers 1-6730 24-5180 41-3506 64 2-6775 29-5215 41-3549 72 9-6795 23-5229 45-3538 69 10-6773 24-5224 41-3528 67 11-6810 22-5244 45-3521 77 12-6795 26-5211 41-3519 68 13-6772 24-5185 47-3468 63 14-6744 23-5160 44-3439 67 15-6744 25-5141 44-3422 73 Type 1 Complex: potential energy average from 50 conformations recorded at a constant temperature (Ep) and standard deviation (sd) recorded during dynamic simulations. The structures 1 and 2 are the same in both series. S3
Table 2S. Potential energy data for type 2 complex. 150 K 300 K 450 K Entry Ep Ep Ep sd sd (Kcal/mol) (Kcal/mol) (Kcal/mol) sd bis-biotins with non-peg spacers 1-6719 21-5162 43-3455 76 2-6751 26-5189 45-3502 67 3-6754 24-5178 44-3473 68 4-6743 24-5173 46-3494 68 5-6728 25-5140 44-3406 69 6-6692 24-5105 49-3373 67 7-6711 23-5103 43-3363 77 8-6663 28-5070 44-3322 72 bis-biotins with PEG spacers 1-6719 27-5175 45-3497 64 2-6762 22-5207 43-3508 67 9-6786 26-5234 41-3535 64 10-6767 24-5194 44-3494 70 11-6755 22-5180 41-3509 65 12-6730 23-5165 42-3439 66 13-6734 22-5158 44-3451 72 14-6673 25-5073 45-3344 65 15-6637 24-5026 46-3292 69 Type 2 complex: potential energy average from 50 conformations recorded at a constant temperature (Ep) and standard deviation (sd) recorded during dynamic simulations. The structures 1 and 2 are the same in both series S4
Scheme 1S. Synthetic route for compound 3. H N CH (Boc) 2 DMAP tbuh H N CtBu AcEt TFA H N CtBu 1 HN HN 2 NH 2 3 Synthesis of tert-butyl 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-aminohexanoate (Fmoc-Lys-tBu, 3). 2.0 g of Fmoc-L-Lys(Boc)-H (1) were added with 2.33 g of di-terbuthyl dicarbonate, Boc 2, and 156.5 mg of 4-dimethylaminopiridine, DMAP, in 30 ml of anhydrous tert-buthanol. The reaction was stirred for 30 min at 35 C and for 15h at room temperature. At the end of the reaction the product, 2, was checked by TLC (AcEt/exane 2:1), R f = 0.75. The solvent was removed under vacuum and the solid residue was dissolved 30 ml of ethyl acetate and 5 ml of water; the organic layer was washed with a saturated solution of NaHC 3 (20 ml for 3 times), and with water (20 ml for 3 times). The organic phase was dried with anhydrous Na 2 S 4, the solvent was removed under vacuum and the crude product was purified by direct-phase LC on silica gel using AcEt/exane 30:70. 1.70 g of the purified product 2 were dissolved in 56 ml of a solution containing 28 of TFA in AcEt. The solution was stirred at 45 C for 1h. After, another 10 ml of TFA were added. The reaction was stirred for 2h and at the end TFA was removed under nitrogen flow. The crude compound 3, was solubilized in 10 ml of CH 3 CN/H 2 2:3 and purified by solid phase extraction on reverse-phase C18 silica gel, obtaining the pure compound Fmoc-Lys-tBu (3) (311 mg, 57 yield). 1 H NMR (298 K), δ (DMS, 200 MHz): 7.78-7.75 (m, 2H), 7.54-7.50 (m, 2H) 7.41-7.26 (m, 4H), 4.01-3.90 (m, 2H), 1.65 (m, 2H), 1.56-1.52 (m, 15H). ESI-MS: calculated m/z 425.5 [M+H] + ; found 425.5. S5
Figure 2S. 1 H-NMR spectrum of compound 3 (200 MHz, DMS). S6
AP_BB-1_2_10 3.12 2: Diode Array Range: 1.188 7.5e-1 AU 5.0e-1 2.5e-1 0.0 0.97 AP_BB-1_2_10 85 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 3.12 TIC 7.93e6 0.88 7.74-15 Time 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 AP_BB-1_2_10 296 (3.082) Cm (289:304) 100 807.44 1.59e6 538.53 807.36 538.46 0 808.03 538.60 236.05 404.67 539.20 726.18 808.18 1613.34 200 400 600 800 1000 1200 1400 1600 1800 1936.85 m/z Figure 3S. HPLC-MS analysis of 6. Method: 15 to 50 of eluent B in 5 min; flow 0.6 ml/min. Rt= 3.12 min HPLC purity >98.8 ESI-MS analysis - m/z: [M+H] + 1613.34; [M+2H] 2+ 807.44; [M+3H] 3+ 583.53 S7
AP_BisBio-6a 3.46 2: Diode Array Range: 9.816e-1 AU 5.0e-1 0.0 AP_BisBio-6a 85 0.99 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 3.40 3.45 3.49 3.52 TIC 6.00e6 7.72-15 Time 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 AP_BisBio-6a 324 (3.364) Cm (320:332) 537.89 100 8.62e5 806.33 806.40 0 537.82 537.96 806.26 312.19 538.45 650.57 806.54 805.28 433.92 807.59 1611.07 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 m/z Figure 4S. HPLC-MS analysis of 9. Method: 15 to 50 of eluent B in 5 min; flow 0.6 ml/min. Rt= 3.46 min HPLC purity >98.5 ESI-MS analysis - m/z: [M+H] + 1611.07; [M+2H] 2+ 806.33; [M+3H] 3+ 537.89 S8
AP_BisBio-3 1.0 3.54 2: Diode Array Range: 1.463 AU 0.0 AP_BisBio-3 85 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 3.49 3.54 TIC 1.49e7 3.61 7.84-15 Time 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 AP_BisBio-3 332 (3.448) Cm (327:348) 666.74 100 3.17e6 0 999.82 666.67 999.68 500.39 312.19 459.04 562.87 667.30 1000.73 844.00 999.54 1133.65 1998.24 m/z 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 Figure 5S. HPLC-MS analysis of 11. method: 20 to 60 of eluent B in 5 min; flow 0.6 ml/min. Rt = 3.54 min HPLC purity >99 ESI-MS analysis - m/z: [M+H] + 1998.24; [M+2H] 2+ 999.82; [M+3H] 3+ 666.74 S9
AP_BisBio-4 5.0 3.61 2: Diode Array Range: 5.598 AU 2.5 0.0 AP_BisBio-4 85 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 3.55 TIC 3.51 3.59 1.09e7 3.70 7.75-15 Time 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 AP_BisBio-4 338 (3.505) Cm (332:361) 100 716.09 1.10e6 716.16 0 1073.88 1073.81 1074.02 537.26 312.26 459.25 537.19 716.02 716.79 804.58 1073.74 1074.65 924.85 1073.18 1176.26 1714.61 1879.63 1918.48 m/z 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 Figure 6S. HPLC-MS analysis of 14. method: 20 to 60 of eluent B in 5 min; flow 0.6 ml/min. Rt = 3.61 min HPLC purity >99 ESI-MS analysis - m/z: [M+2H] 2+ 1073.88; [M+3H] 3+ 716.09 S10
AP_BB7_170214 3.36 2: Diode Array Range: 1.461 1.0 AU 5.0e-1 7.82 0.0 AP_BB7_170214 85 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 3.47 TIC 1.62e8 3.60-15 Time 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 AP_BB7_170214 331 (3.435) Cm (324:340) 721.00 100 3.66e7 721.07 0 283.98 565.31 721.88 720.03 1441.25 480.83 1015.83 1441.18 1442.80 200 400 600 800 1000 1200 1400 1600 1800 m/z Figure 7S. HPLC-MS analysis of 16. method: 20 to 50 of eluent B in 5 min; flow 0.6 ml/min. Rt = 3.36 min HPLC purity 99 ESI-MS analysis - m/z: [M+H] + 1441.25; [M+2H] + 721.00 S11
AP_BB8_170214 3.42 2: Diode Array Range: 1.452 1.0 AU 5.0e-1 0.0 1.31 7.82 AP_BB8_170214 85 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 3.54 TIC 1.46e8-15 Time 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 AP_BB8_170214 337 (3.498) Cm (330:347) 664.01 100 3.48e7 663.94 0 283.98 1327.28 508.13 748.38 958.63 1326.17 1328.84 1991.78 m/z 200 400 600 800 1000 1200 1400 1600 1800 Figure 8S. HPLC-MS analysis of 18. method: 20 to 50 of eluent B in 5 min; flow 0.6 ml/min. Rt = 3.42 min HPLC purity 98 ESI-MS analysis - m/z: [M+H] + 1327.28; [M+2H] + 664.01 S12
Figure 9S. Correlation function of native avidin, experimental data are showed as black dots, while the result of interpolation is showed as red line. S13
Figure 10S. Correlation function for Av/16 complexes, experimental data are showed as black dots, while the result of interpolation is showed as red line. A) t R 10.76 min; B) t R 9.44 min; C) t R 8.54 min; D) t R 8.11 min. S14
Figure 11S. Correlation functions of Av/18 polymer at different incubation time: A) 0 min.; B) 45 min; C) 150 min.; D) 360 min. S15