Electronic Supplementary Material (ESI) for Organic & Biomolecular Chemistry. This journal is The Royal Society of Chemistry 2018 Facile Cu(II) mediated conjugation of thioesters and thioacids to peptides and proteins under mild conditions Yao Sun a, Zhenbin Lyu b, Zhiqiang Wang b, c, Xiaodong Zeng b, Hui Zhou b, Fuchun Xu d, Ziyang Chen b, Yuling Xu a, Ping Xu c*, Xuechuan Hong b,d* a. Key Laboratory of Pesticides and Chemical Biology, Ministry of Education, Hubei International Scientific and Technological Cooperation Base of Pesticide and Green Synthesis, Chemical Biology Center, College of Chemistry, Central China Normal University, Wuhan430079 b. State Key Laboratory of Virology, Zhongnan Hospital of Wuhan University, Wuhan University School of Pharmaceutical Sciences, Wuhan 430071, China. c. State Key Laboratory of Proteomics, National Center for Protein Sciences Beijing, Beijing Institute of Radiation Medicine, Beijing102206, China d. Medical College, Tibet University, Lasa, 850000, China Corresponding Authors: Xuechuan Hong, PhD; PingXu, PhD Email: xhy78@whu.edu.cn; xuping@mail.ncpsb.org These authors contributed equally to this work.
Table of Contents Figure S1, the cell viability of Cu(OAc) 2 and HOBt Figure S2-S6, MS spectrum of peptide-based fluorescent probes General procedure for the modification of Ub protein with thioester and the sample preparation for MS analysis. Figure S7-S14, MS analysis of Ub protein modification with thioester General procedure for the modification of Ub protein with thioacid and the sample preparation for MS analysis. Figure S15-S22, MS analysis of Ub protein modification with thioacid General methods S3 S3-S8 S9-S15 S16-S23 S24 Chemical synthesis and characterization S22-34 1 H and 13 C NMR for obtained compounds S35-45
Figure S1. The MTT experiment on L929 cell lines for Cu(OAc) 2 and HOBt.
Figure S2. ESI-MS of Cy5.5COSH
Figure S3. ESI-MS of Cy5.5-RGDfK
Figure S4. ESI-MS of Cy5.5-JMV594
Figure S5. MALDI-TOF-MS of Cy5.5-AE105
Figure S6. ESI-MS of Cy5.5-E[c(RGDyK)2]
General procedure for the modification of Ub protein with thioester and the sample preparation for MS analysis. The thioester (100 to 200 eq) and Ub (86.0 µg, 0.01 µmmol, 1 eq) were dissolved in the mixture of PBS/DMF (5:5 to 7:3, total 150 µl). Then Cu(OAc)2 (2.00 µg to 4.00 µg, 1 eq to 2 eq) and HOBt (13.5 µg, 10 eq) were added and the reaction mixture was stirred at room temperature for 6 h to 16 h. The crude product was in-gel digested with trypsin overnight. The peptides mixtures were extracted with extraction buffer (5 % FA and 50 % ACN in water) and analyzed through LC-MS/MS. The crude protein was purified by SDS-PAGE. The gel was stained with Coomassie Blue G-250 and cut into about 1 mm 3 cubes, followed by destaining and in-gel digestion with 10 ng/µl of trypsin at 37 C overnight. The peptides were extracted with extraction buffer (5 % FA and 50 % ACN in water) and then ACN and finally dried. Peptide mixtures were loaded onto an in-house packed capillary column (75 μm I.D. and 15 cm) with 3 µm C18 reverse-phase fused-silica (Michrom Bioresources, Inc., Auburn, CA) with a flow rate of 0.3 μl/min, and eluted with a 100 minutes gradient developed as follows: 0-5 % B for 10 minutes, 5-10 % B for 10 minutes, 10-20 % B for 30 minutes, 20-45 % B for 40 minutes, and 45-80 % B for 10 minutes (Buffer A: 0.1 % FA; Buffer B: 0.1 % FA and 100 % ACN). Figure S7. The result of MALDI-TOF-MS spectrum of Ub modification with 100 eq of Cbz-L-Phe thioester under 1eq Cu(OAc) 2 and 10eq HOBt
300 8564.7046 250 1+ (A) 8846.8628 200 150 100 50 8400 8500 8600 8700 8800 8900 9000 9100 Figure S8. The result of MALDI-TOF-MS spectrum of Ub modification with Cbz-L-Phe thioester under 2eq Cu(OAc) 2, 200 eq thioester and 16 h conditions. Figure S9. LC-MS spectrum of the resulting peptide fragment of trypsinized Ub conjugated with Cbz-L-Phe thioester.
Relative Intensity (%) 0 10 20 30 40 50 60 70 80 90 100 Peptide sequence Mass() Mass() QDK 33EGIPPDQQR 1805.761 903.380 Ub-Phe #3583 RT: 19.95 AV: 1 NL: 2.00E5 T: FTMS + c NSI Full ms [300.00-1600.00] 100 903.450 Relative Abundance 90 80 70 60 50 40 30 20 10 0 321.131 383.102 445.121 536.166 619.274 K 33 modification 849.887 922.423 762.396 z=3 1025.116 1143.090 1528.658 z=3 1268.177 1380.717 400 600 800 1000 1200 1400 1600 Relative Intensity (%) 0 10 20 30 40 50 60 70 80 90 100 y3+ 431.26 y5+ 643.37 y10++ 724.85 y6+ 740.40 b4+ 766.48 y7+ y12++ 853.53 846.53 y8+ 910.59 b6+ 952.49 500 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 y9+ 1039.59 b7+ 1065.49 2+ I y12 Q y11 D y10 K b4 y9 E y8 G b6 y7 I b7 y6 P y5 P D y3 Q y11+ 1563.81 Q R Figure S10. MS and MS 2 spectrums of the peptide fragment of Ub K 33 modified with Cbz-L-Phe thioester.
Peptide sequence Mass() Mass() LIFAGK 48QLEDGR 1628.645 814.826 Ub-Phe #4415 RT: 24.47 AV: 1 NL: 1.08E6 T: FTMS + c NSI Full ms [300.00-1600.00] Relative Intensity (%) 0 10 20 30 40 50 60 70 80 90 100 100 814.429 90 Relative Abundance 80 70 60 50 40 30 20 10 0 394.223 536.166 610.185 789.919 833.903 956.942 1085.904 z=3 K48 modification 1206.637 1285.671 1541.954 400 600 800 1000 1200 1400 1600 Relative Intensity (%) 0 10 20 30 40 50 60 70 80 90 100 y2+ 232.04 y3+ 347.36 b3+ 374.38 y4+ 476.16 y5+ y8++ 589.24 592.70 y10++ 701.59 y6+ 717.51 b11++ 727.51 y11++ 758.35 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 b6+ 911.72 2+ L b7+ 1039.51 y11 I y10 F b3 y9 A b8+ 1152.62 y8 G y8+ 1183.58 K b6 y6 Q b7 y9+ 1254.53 b9+ 1281.80 y5 L b8 y4 E b9 y3 D b10 b10+ 1396.74 y10+ 1401.59 y2 G R b11 Figure S11. MS and MS 2 spectrums of the peptide fragment of Ub K 48 modified with Cbz-L-Phe thioester.
Peptide sequence Mass() Mass() MQIFVK 6TLTGK 1492.658 746.829 Ub-biotin #3197 RT: 21.06 AV: 1 NL: 1.12E6 T: FTMS + c NSI Full ms [300.00-1600.00] 746.408 100 90 80 Relative Abundance 70 60 50 40 30 20 10 0 355.173 536.165 Relative Intensity (%) 0 10 20 30 40 50 60 70 80 90 100 672.409 791.748 z=3 883.939 969.133 z=3 K 6 modification 1187.119 1349.214 1453.197 400 600 800 1000 1200 1400 1600 Relative Intensity (%) 0 10 20 30 40 50 60 70 80 90 100 b2+ 260.13 y5++ 260.13 y3+ 305.22 b5++ 310.29 b3+ 373.23 2+ M y4+ 418.31 y10 Q y7++ 486.72 b2 y9 I b4+ 520.27 y5+ 519.28 b3 y8 F y8++ 560.29 b4 y7 V b5 y9++ 617.02 b5+ 619.33 y6 K b6 y5 T y10++ 681.07 b7 y4 L b8 y3 T b9 G 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 K b10 y6+ 873.50 y7+ 972.57 b6+ 973.55 b7+ 1074.47 y8+ 1119.60 b8+ 1187.56 y9+ 1232.63 b9+ 1288.59 b10+ 1345.70 Figure S12. MS and MS 2 spectrums of the peptide fragment of Ub K 6 modified with biotin thioester.
Peptide sequence Mass() Mass() IQDK 33EGIPPDQQR 1750.733 875.87 Ub-biotin #1961 RT: 15.19 AV: 1 NL: 3.04E6 T: FTMS + c NSI Full ms [300.00-1600.00] 100 Relative Intensity (%) 0 10 20 30 40 50 60 70 80 90 100 875.433 90 Relative Abundance 80 70 60 50 40 30 20 10 0 435.773 324.707 511.256 583.957 z=3 648.407 822.386 894.405 K 33 modification 1021.505 1167.576 z=3 1313.146 z=4 1400.689 1564.053 400 600 800 1000 1200 1400 1600 Relative Intensity (%) 0 10 20 30 40 50 60 70 80 90 100 b2+ 242.15 y2+ 303.18 b3+ 357.18 y3+ 431.24 y4+ 546.26 y5+ 643.32 b4+ 711.35 b11++ 724.34 y6+ 740.37 b5+ 840.39 y7+ 853.46 b6+ 897.41 y8+ 910.47 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 2+ I b7+ 1010.50 y9+ 1039.52 Q b2 y11 D b3 y10 K b4 y9 E b5 y8 G b6 y7 I b7 y6 P y10+ 1393.69 y5 P y4 D y3 Q y11+ 1508.72 b11 y2 Q R Figure S13. MS and MS 2 spectrums of the peptide fragment of Ub K 33 modified with biotin thioester.
Peptide sequence Mass() Mass() LIFAGK 48QLEDGR 1573.618 787.310 Ub-biotin #2914 RT: 19.75 AV: 1 NL: 4.81E6 T: FTMS + c NSI Full ms [300.00-1600.00] 100 786.912 90 Relative Intensity (%) 0 10 20 30 40 50 60 70 80 90 100 Relative Abundance 80 70 60 50 40 30 20 10 0 343.172 537.592 z=3 647.364 734.406 805.884 936.485 1049.214 z=3 K 48 modification 1166.116 1379.201 1572.817 400 600 800 1000 1200 1400 1600 Relative Intensity (%) 0 10 20 30 40 50 60 70 80 90 100 y2+ 232.21 y3+ 347.34 b3+ 374.39 b4+ 445.40 y4+ 476.34 y7++ 536.57 y8++ 565.03 y5+ 589.38 y9++ 600.55 y10++ 674.15 b11++ 699.46 y6+ 717.47 y11++ 730.68 b6+ 856.57 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 2+ L b7+ 984.62 y11 I y10 F b3 y9 A y7+ 1071.59 b8+ 1097.66 y8+ 1128.58 b4 y8 G y7 K b6 y9+ 1199.55 b9+ 1226.62 y6 Q b7 y5 L b8 y4 E b9 y3 D b10+ y10+ 1341.68 1346.65 b10 y2 G R b11 Figure S14. MS and MS 2 spectrums of the peptide fragment of Ub K 48 modified with biotin thioester.
General procedure for the modification of Ub protein with thioacid and the sample preparation for MS analysis. The thioacid (100 to 200 eq) and Ub (86.0 µg, 0.01 µmmol, 1 eq) were dissolved in the mixture of PBS/DMF (5:5, total 150 µl) or PBS. Then Cu(OAc)2 (2.00 µg to 4.00 µg, 1 eq to 2 eq) and HOBt (2 eq to 4 eq) were added and the reaction mixture was stirred at room temperature for 6 h. The crude product was in-gel digested with trypsin overnight. The peptides mixtures were extracted with extraction buffer (5 % FA and 50 % ACN in water) and analyzed through LC-MS/MS. The crude protein was purified by SDS-PAGE. The gel was stained with Coomassie Blue G-250 and cut into about 1 mm 3 cubes, followed by destaining and in-gel digestion with 10 ng/µl of trypsin at 37 C overnight. The peptides were extracted with extraction buffer (5 % FA and 50 % ACN in water) and then ACN and finally dried. Peptide mixtures were loaded onto an in-house packed capillary column (75 μm I.D. and 15 cm) with 3 µm C18 reverse-phase fused-silica (Michrom Bioresources, Inc., Auburn, CA) with a flow rate of 0.3 μl/min, and eluted with a 100 minutes gradient developed as follows: 0-5 % B for 10 minutes, 5-10 % B for 10 minutes, 10-20 % B for 30 minutes, 20-45 % B for 40 minutes, and 45-80 % B for 10 minutes (Buffer A: 0.1 % FA; Buffer B: 0.1 % FA and 100 % ACN). Figure S15. MALDI-TOF-MS spectrum result of Ub modification with thioacid under 1eq Cu(OAc) 2, 2 eq HOBt, 100 eq thioester and 6 h conditions.
Figure S16. MALDI-TOF-MS spectrum result of Ub modification with thioacid in PBS solution.
Peptide sequence Mass() Mass() *MQIFVK 6 849.453 425.230 Ub-CH3COOSH #15196 RT: 54.42 AV: 1 NL: 2.24E5 T: FTMS + c NSI Full ms [300.00-1600.00] 100 849.458 Relative Abundance 90 80 70 60 50 40 30 20 10 0 371.103 407.206 445.122 519.141 536.168 683.379 725.064 z=3 871.440 747.387 z=3 1087.092 906.807 z=3 1278.675 1418.790 1586.644 400 600 800 1000 1200 1400 1600 Relative Intensity (%) 0 10 20 30 40 50 60 70 80 90 100 y2++ 145.01 b1+ 174.10 y1+ 189.13 Relative Intensity (%) y4++ 274.24 b4++ 281.24 y2+ 288.36 0 10 20 30 40 50 60 70 80 90 100 M and K 6 modification b2+ 302.12 y5++ 338.63 b5++ 331.09 100 200 300 400 500 600 700 [M]++ 425.26 y3+ 435.22 y4+ 548.31 b4+ 562.26 2+ M b1 y5 Q b2 y4 I b5+ 661.39 y3 F y5+ 676.38 b4 y2 V b5 y1 K Figure S17. MS and MS 2 spectrums of the peptide fragemnts of Ub N-terminal and K 6 modified with thioacid.
Peptide sequence Mass() Mass() TLTGK 11TITLEVEPSDTIENVK 2330.233 1165.620 Ub-CH3COOSH #12429 RT: 44.77 AV: 1 NL: 1.55E7 T: FTMS + c NSI Full ms [300.00-1600.00] 100 1166.124 Relative Abundance 90 80 70 60 50 40 30 20 10 0 321.132 470.276 536.167 654.375 759.418 z=3 777.752 z=3 795.389 z=3 977.916 1138.624 1192.579 1307.742 1554.498 1473.684 z=3 400 600 800 1000 1200 1400 1600 Relative Intensity (%) 0 10 20 30 40 50 60 70 80 90 100 Relative Intensity (%) b8++ 429.37 0 10 20 30 40 50 60 70 80 90 100 K 11 modification y4+ 489.19 b5+ 543.33 y5+ 602.43 b6+ 644.17 y6+ 703.48 b7+ 757.62 y14++ 787.60 y7+ 818.39 b8+ 858.53 y8+ 905.63 b9+ b18++ 971.53 985.64 y9+ 1002.59 y18++ 1008.26 2+ T y10+ 1131.53 b20++ b10+ 1100.63 1092.81 y20++ 1115.53 b11+ 1199.66 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 y20 y18 y11+ 1230.62 b5 y16 b12+ 1328.77 y12+ 1359.64 L T G K T I b6 y15 b7 y14 T b8 y13 L b9 y12 E b10 y13+ 1472.79 b14+ 1512.60 y11 V b11 y14+ 1573.78 y10 E b12 y9 P b15+ 1627.85 y8 S y15+ 1687.00 b14 y7 D b16+ 1728.65 b15 y6 T b16 y16+ 1787.99 y5 I b17 b17+ 1842.07 y4 E N b18 V b20 b18+ 1970.89 K Figure S18. MS and MS 2 spectrums of the peptide fragments of Ub K 11 modified with thioacid.
Peptide sequence Mass() Mass() TITLEVEPSDTIENVKAK 29 2029.069 1015.038 Ub-CH3COOSH #11600 RT: 42.39 AV: 1 NL: 4.50E7 T: FTMS + c NSI Full ms [300.00-1600.00] 100 1015.540 Relative Intensity (%) 0 10 20 30 40 50 60 70 80 90 100 90 Relative Abundance 80 70 60 50 40 30 20 10 0 321.131 415.212 987.506 K 29 modification 619.273 690.012 1041.995 1353.719 803.949 908.466 1165.116 z=3 1522.561 z=4 400 600 800 1000 1200 1400 1600 Relative Intensity (%) 0 10 20 30 40 50 60 70 80 90 100 y3+ 388.35 b7++ 393.41 2+ T I y4+ 487.21 y16 T y9++ 530.40 b5+ 558.29 y15 L y14 E b5 b11++ y5+ 601.53 593.32 y11++ 622.73 y13 V b6+ 657.44 b6 y12 E b7 y11 P y6+ 730.51 b8 y10 S b9 b7+ 786.51 y9 D y8 T I b11 b12 y15++ 857.63 b8+ 883.46 y16++ 908.07 y6 E y5 N y8+ 944.54 b9+ 970.58 y4 V [M]++ 1015.08 b15 y3 K y9+ 1059.75 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 A K y10+ 1146.70 b11+ 1186.79 y11+ 1243.58 b12+ 1299.70 y12+ 1372.62 y13+ 1471.69 y14+ 1600.83 b15+ 1641.76 Figure S19. MS and MS 2 spectrums of the peptide fragments of Ub K 29 modified with thioacid.
Peptide sequence Mass() Mass() Relative Intensity (%) 0 10 20 30 40 50 60 70 80 90 100 IQDK 33EGIPPDQQR 1565.791 783.399 Ub-CH3COOSH #7812 RT: 31.64 AV: 1 NL: 3.42E7 T: FTMS + c NSI Full ms [300.00-1600.00] 783.401 100 Relative Abundance 90 80 70 60 50 40 30 20 10 0 379.176 561.296 758.355 z=3 809.855 903.971 1044.533 z=3 K 33 modification 1121.584 1303.203 1479.330 1565.795 400 600 800 1000 1200 1400 1600 Relative Intensity (%) 0 10 20 30 40 50 60 70 80 90 100 y2+ 303.33 y5++ 322.18 b3+ 357.21 b6++ 357.21 b7++ 414.03 y9++ 519.87 y5+ 643.24 b5+ y11++ 656.28 662.37 b6+ 713.27 y12++ 727.11 y6+ 740.36 [M]++ 783.48 b7+ 826.36 y7+ 853.40 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 2+ I y12 Q y8+ 910.52 b8+ 923.11 y11 D b3 K y9+ 1039.87 y9 E b5 y8 G b6 b10+ 1135.57 y7 I b7 y6 P b8 y5 P D b10 Q y11+ 1324.62 y2 Q R Figure S20. MS and MS 2 spectrums of the peptide fragments of Ub K 33 modified with thioacid.
Peptide sequence Mass() Mass() LIFAGK 48QLEDGR 1388.753 694.880 Relative Intensity (%) 0 10 20 30 40 50 60 70 80 90 100 Ub-CH3COOSH #10805 RT: 40.09 AV: 1 NL: 2.29E8 T: FTMS + c NSI Full ms [300.00-1600.00] 694.879 100 90 Relative Abundance 80 70 60 50 40 30 20 10 0 321.130 463.588 z=3 633.821 710.872 854.387 926.503 z=3 999.980 K 48 modification 1203.680 1388.750 1554.002 400 600 800 1000 1200 1400 1600 Relative Intensity (%) 0 10 20 30 40 50 60 70 80 90 100 b2+ y2+ 227.15 232.13 y3+ 347.23 b3+ 374.29 b4+ 445.16 y8++ y4+ 472.84 476.18 b5+ y9++ 502.26 508.27 y10++ 581.88 y5+ 589.25 y11++ 638.37 [M]++ 694.42 y6+ 717.37 200 300 400 500 600 700 800 900 1000 1100 1200 1300 2+ L b7+ 800.40 y11 I b2 y10 F b3 y9 A y7+ 887.48 b8+ 913.56 b4 y8+ 944.47 y8 G b5 y7 K y9+ 1015.46 b9+ 1042.58 y6 Q b7 y5 L b8 y4 E b10+ 1157.49 y10+ 1162.49 b9 y3 D b11+ 1214.65 b10 y2 G R b11 Figure S21. MS and MS 2 spectrums of the peptide fragments of Ub K48 modified with thioacid.
Peptide sequence Mass() Mass() TLSDYNIQK 1123.562 562.285 Ub-CH3COOSH #9257 RT: 35.61 AV: 1 NL: 3.45E6 T: FTMS + c NSI Full ms [300.00-1600.00] 100 90 829.403 1123.567 Relative Abundance 80 70 60 50 40 30 20 10 0 562.287 357.142 695.846 846.434 1098.520 679.363 530.787 z=3 413.248 z=3 719.342 935.452 1018.039 z=3 1145.549 1241.094 1391.685 1486.897 400 600 800 1000 1200 1400 1600 Relative Intensity (%) Relative Intensity (%) 0 10 20 30 40 50 60 70 80 90 100 K 63 modification 0 10 20 30 40 50 60 70 80 90 100 y1+ 189.08 b2+ 215.09 b3+ 302.26 y2+ 317.20 b7++ 404.46 b4+ 417.28 y3+ 430.27 [M]++ 562.37 b5+ 580.18 200 300 400 500 600 700 800 900 1000 b6+ 694.25 y5+ 707.44 2+ T L b7+ 807.36 b2 y6+ 822.40 y7 S b3 y6 D b4 y7+ 909.40 y5 Y b8+ 935.41 b5 N b6 y3 I b7 y2 Q b8 y1 K Figure S22. MS and MS 2 spectrums of the peptide fragments of Ub K63 modified with thioacid.
General methods All chemicals were purchased from commercial sources (such as Aldrich, conju-probe, Lumiprobe and peptide international). The 1 H and 13 C NMR spectra were acquired on a Bruker 400 MHz magnetic resonance spectrometer. Data for 1 H NMR spectra are reported as follows: chemical shifts are reported as δ in units of parts per million (ppm) relative to chloroform-d (δ 7.26, s); multiplicities are reported as follows: s (singlet), d (doublet), t (triplet), q (quartet), dd (doublet of doublets), m (multiplet), or br (broadened); coupling constants are reported as a J value in Hertz (Hz); the number of protons (n) for a given resonance is indicated nh, and based on the spectral integration values. MALDI-MS spectrometric analyses were performed on an Applied Biosystems 4700 MALDI TOF mass spectrometer. HPLC was performed on a Dionex HPLC System (Dionex Corporation) and a reversed-phase C18 column was used for analysis (Phenomenax, 5 μm, 4.6 mm 250 mm) and semi-preparation (Agilent, 5 μm, 10 mm 250 mm).
Chemical synthesis and characterization Synthesis of 1a: The mixture of Boc-Phe-L-amino acid (265.0 mg, 998.9 µmol), 4-Methylbenzenethiol (136.4 mg, 1.098 mmol), diisopropylcarbodiimide (189.3 mg, 1500 µmol) and 1-hydroxybenzotriazole (202.6 mg, 1500 µmol) were dissolved in ethyl acetate (2000 µl). The reaction mixture was stirred for overnight and purified by flash chromatography affording the desired product as a white solid (315.4 mg, 85 %). 1 H NMR (400 MHz, CDCl3) δ = 7.37-7.21 (m, 9H), 5.00 (d, J=12.0, 1H), 4.79-4.77 (m, 1H), 3.20-3.14 (m, 2H), 2.40 (s, 3H), 1.46 (s, 9H); 13 C NMR (101 MHz, CDCl3) δ = 199.7,154.9, 139.8, 135.6, 134.5, 130.1, 129.4, 128.6, 127.1, 123.7, 119.0, 80.4, 60.8, 38.4, 28.3, 21.3; HRMS (ESI) Calcd for: C21H26NO3S: 372.1628. Found: 372.1622 ([M+H] + ). Synthesis of 1b: The mixture of biotin (244.0 mg, 998.9 µmol), 4-Methylbenzenethiol (136.4 mg, 1.098 mmol) diisopropylcarbodiimide (189.3 mg, 1500 µmol) and 1-hydroxybenzotriazole (202.6 mg, 1500 µmol) were dissolved in DMF (2000 µl). The reaction mixture was stirred for overnight and purified by flash chromatography affording the desired product as a white solid (273.1 mg, 78 %). 1 H NMR (400 MHz, CDCl3) δ = 8.27 (br, 1H), 7.62 (d, J=8.0, 1H), 7.40 (d, J=8.0, 1H), 7.27-7.14 (m, 6H), 7.06 (br, 1H), 5.16 (d, J=8.0, 1H), 4.86-4.81 (m, 1H), 3.45-3.29 (m, 2H), 2.39 (s, 3H), 1.48 (s, 9H); 13 C NMR (101 MHz, CDCl3) δ = 200.6, 155.2, 139.7, 136.1, 134.6,
130.0, 127.6, 123.8, 123.2, 119.8, 119.0, 111.2, 109.7, 80.4, 60.5, 28.3, 28.1, 21.3; HRMS (ESI) Calcd for: C17H23N2O2S2: 351.1195. Found: 351.1193 ([M+H] + ). Synthesis of 1c: The mixture of Cbz-Phe-L-amino acid (299.0 mg, 999.0 µmol), 4-Methylbenzenethiol (136.4 mg, 1.098 mmol) diisopropylcarbodiimide (189.3 mg, 1500 µmol) and 1-hydroxybenzotriazole (202.6 mg, 1500 µmol) were dissolved in ethyl acetate (2000 µl). The reaction mixture was stirred for overnight and purified by flash chromatography affording the desired product as a white solid (344.3 mg, 85%). 1 H NMR (400 MHz, CDCl3) δ = 7.29-7.08 (m, 14H), 5.17 (d, J=8.0, 1H), 5.04 (s, 2H), 4.78-4.73 (m, 1H), 3.12-3.02 (m, 2H), 2.30 (s, 3H); 13 C NMR (101 MHz, CDCl3) δ = 199.1, 155.6, 140.0, 136.0, 135.3, 134.5, 130.1, 129.4, 128.7, 128.5, 128.3, 128.1, 127.3, 123.4, 67.3, 61.3, 38.4, 21.4,; HRMS (ESI) Calcd for: C24H24NO3S: 406.1471. Found: 406.1471 ([M+H] + ). Synthesis of 1d: The mixture of Boc-L-Phe-Gly-OH (322.0 mg, 999.1 µmol), 4-Methylbenzenethiol (136.4 mg, 1.098 mmol) diisopropylcarbodiimide (189.3 mg, 1500 µmol) and 1-hydroxybenzotriazole (202.6 mg, 1500 µmol) were dissolved in ethyl acetate (2000 µl). The reaction mixture was stirred for overnight and purified by flash chromatography affording the desired product as a white solid (325.4 mg, 76 %). 1 H NMR (400 MHz, CDCl3) δ = 8.21(s, 1H), 7.61 (d, J=8, 1H), 7.41 (d, J=8, 1H), 7.26-7.08 (m, 7H), 5.14 (d, J=12, 1H), 4.85-4.80 (m, 1H), 3.45-3.28 (m, 2H), 2.39 (s, 3H), 1.47 (s, 9H); 13 C NMR (101 MHz, CDCl3) δ = 199.8,139.8, 135.6,
134.5, 130.1, 129.4, 128.7, 127.1, 123.7; HRMS (ESI) Calcd for: C23H28N2O4S: 429.1843. Found: 429.1843 ([M+H] + ). Synthesis of Cy5.5COSH: The Cy5.5-NHS (2.100 mg, 2.932 µmol) was dissolved in distilled DMF (300.0 µl). NaSH (300.0 µg, 5.355 µmol) was added to this stirring solution under N2 atmosphere. The reaction mixture was allowed to stir for 4h. Then the reaction mixture was purified by HPLC to get the desired Cy5.5 thioacid as a blue solid. ESI-MS Calcd for:c40h43n2os: 599.3,Found: 599.5 ([M+H] + ). Synthesis of 3a: Boc-Phe-p-toluene thiol ester (371.0 mg, 998.7 µmol) was added to DMF (2000 µl) or DMF/PBS (2000 µl, 1:1) in round-bottom flask, then added catalyst (1 eq) or catalyst and HOBt (1eq or 2eq) and Try-OMe (327.0 mg, 1.498 mmol) in it. The mixture was stirred at 37 o C, then cooled to room temperature. The solvent was evaporated under reduced pressure, and the residue dissolved in ethyl acetate (15.00 ml) then washed successively with 1M aqueous HCl, 2M aqueous NaOH and brine. The organic layer was dried with Na2SO4, filtered, and concentrated to dryness. The crude product was purified by flash chromatography (first use 25% ethyl acetate in petroleum ether, then use 10% MeOH in DCM wash the chromatography to get the product. The title compound was prepared as a white solid.
1 H NMR (400 MHz, CDCl3) δ = 8.20 (br, 1H),7.36-7.09 (m, 10H), 6.41 (br, 1H), 4.84-4.76 (m, 2H), 4.36 (br, 1H), 3.64 (s, 3H), 3.28-3.27 (m, 2H), 3.04-3.03 (m, 2H), 1.38 (s, 9H); 13 C NMR (101 MHz, CDCl3) δ = 171.7, 170.8, 136.5, 136.0, 129.4, 128.6, 126.9, 122.9, 122.2, 119.6, 118.4, 111.3, 109.7, 52.9, 52.3, 38.3, 28.2, 27.6; HRMS (ESI) Calcd for: C26H32N3O5: 466.2336. Found: 466.2347 ([M+H] + ). Synthesis of 3b: Boc-Phe-p-toluene thiol ester (371.0 mg, 998.7 µmol) was added to DMF/PBS (2000 µl, 1:1) in round-bottom flask, then added Cu(OAc)2/HOBt (1 eq) and Gly (116.0 mg, 1.125 mmol) in it. The mixture was stirred at 37 o C for 1h, then cooled to room temperature. The solvent was evaporated under reduced pressure, and the residue dissolved in ethyl acetate then washed successively with 1M aqueous HCl, 2M aqueous NaOH and brine. The organic layer was dried with Na2SO4, filtered, and concentrated to dryness. The crude product was purified by flash chromatography (first use 25% ethyl acetate in petroleum ether, then use 10% MeOH in DCM wash the chromatography to get the product). The title compound was prepared as a white solid (308.9 mg, 88 %). 1 H NMR (400 MHz, CDCl3) δ = 7.22-6.79 (m, 5H), 6.79 (br, 1H), 5.25-5.22 (m, 1H), 4.40 (br, 1H), 4.13 (dd, J1=8.0, J2=16.0, 2H), 4.38 (br, 1H), 3.92-3.86 (m, 2H), 3.09-2.91 (m, 2H), 1.30 (s, 9H), 1.20 (t, J=4.0, 3H), 13 C NMR (101 MHz, CDCl3) δ = 171.5, 170.4, 169.4, 136.6, 129.2, 128.5, 126.9, 61.5, 54.2, 41.3, 38.2, 23.0, 14.1; HRMS (ESI) Calcd for: C16H23N2O5: 351.1914. Found: 351.1914 ([M+H] + ).
Synthesis of 3c: Biotin-p-toluene thiol ester (350.0 mg, 998.6 µmol) was added to DMF/PBS (2000 µl, 1:1) in round-bottom flask, then added Cu(OAc)2/HOBt (1 eq) and Phe-OEt (289.5 mg, 1.498 mmol) in it. The mixture was stirred at 37 o C for 1h, then cooled to room temperature. The solvent was evaporated under reduced pressure, and the residue dissolved in ethyl acetate then washed successively with 1M aqueous HCl, 2M aqueous NaOH and brine. The organic layer was dried with Na2SO4, filtered, and concentrated to dryness. The crude product was purified by flash chromatography (first use 25% ethyl acetate in petroleum ether, then use 10% MeOH in DCM wash the chromatography to get the product). The title compound was prepared as a white solid (327.0 mg, 80 %). 1 H NMR (400 MHz, MeOD) δ = 7.32-7.23 (m, 5H), 4.69-4.64 (m, 1H), 4.52-4.49 (m, 1H), 4.30-4.27 (m, 1H), 4.19-4.12 (m, 2H),3.16-3.15 (m, 2H), 2.97-2.92 (m, 2H), 2.74 (d, J=12.0, 1H), 2.21-2.18 (m, 2H), 1.60-1.57 (m, 4H), 1.24 (t, J=8.0, 4H),0.94-0.90 (m, 2H), 13 C NMR (101 MHz, MeOD) δ = 174.5, 171.9, 136.8, 128.8, 128.1, 126.4, 61.8, 61.0, 60.2, 55.5, 53.8, 39.6, 37.0, 34.8, 28.0, 27.9, 25.3, 13.0; HRMS (ESI) Calcd for: C21H30N3O4S: 420.1952. Found: 420.1945 ([M+H] + ). Synthesis of 3d: Cbz-Phe-p-toluene thiol ester (405.0 mg, 998.8 µmol) was added to DMF/PBS (2000 µl, 1:1) in round-bottom flask, then added Cu(OAc)2/HOBt (1 eq) and Ser-OEt (199.5 mg, 1.499 mmol) in it. The mixture was stirred at 37 o C for 1h, then cooled to room temperature. The solvent was evaporated under reduced pressure, and the residue dissolved in ethyl acetate then washed successively with 1M aqueous HCl, 2M aqueous NaOH and brine. The organic layer was dried with Na2SO4, filtered, and concentrated to dryness. The crude product was purified by flash chromatography (first use 25% ethyl acetate in petroleum ether, then use 10% MeOH in DCM wash the chromatography to get the product). The title compound was prepared as a white solid (352.0 mg, 85%). 1 H NMR (400 MHz, CDCl3) δ = 7.27-7.06
(m, 11H), 5.58 (d, J=8.0, 1H), 5.00-4.89 (m, 2H), 4.52-4.50 (m, 2H), 4.13-4.10 (m, 2H), 3.82 (s, 2H), 3.08-2.67 (m, 2H), 1.20 (t, J=8.0, 3H), 13 C NMR (101 MHz, CDCl3) δ = 171.5, 170.1, 136.2, 136.0, 129.3, 128.6, 128.5, 128.2, 128.0, 127.0, 67.1, 62.8, 61.9, 56.2, 54.9, 38.4, 14.1; HRMS (ESI) Calcd for: C22H27N2O3: 415.1864. Found: 415.1856 ([M+H] + ). Synthesis of 3e: Boc-L-Phe-Gly-p-toluene thiol ester (428.0 mg, 998.9 µmol) was added to DMF/PBS (2000 µl, 1:1) in round-bottom flask, then added Cu(OAc)2/HOBt (1 eq) and L-Phe-Gly-OEt (375.0 mg, 1.498 mmol) in it. The mixture was stirred at 37 o C for 2 h, then cooled to room temperature. The reaction mixture was purified by HPLC to get desire product as a white solid (415.0 mg, 75 %). 1 H NMR (400 MHz, MeOD) δ = 7.31-7.21 (m, 10H), 4.65 (s, 3H), 4.29-4.19 (m, 2H), 3.95-3.94 (m, 3H), 3.67-3.62 (m, 2H), 3.27-3.23 (m, 1H), 3.16-3.11 (m, 1H), 2.98-2.95 (m, 1H), 2.87-2.85 (m, 1H), 1.37-1.18 (m, 12H), 13 C NMR (101 MHz, CDCl3) δ = 171.5, 170.1, 136.2, 136.0, 129.3, 128.6, 128.5, 128.2, 128.0, 127.0, 67.1, 62.8, 61.9, 56.2, 54.9, 38.4, 14.1; HRMS (ESI) Calcd for: C29H39N4O7: 555.2813. Found: 555.2816 ([M+H] + ). Synthesis of 3f: Cbz-L-Phe-Gly-p-toluene thiol ester (462.0 mg, 998.7 µmol) was added to DMF/PBS (2000 µl, 1:1) in round-bottom flask, then added Cu(OAc)2/HOBt (1 eq) and L-Thr-Gly-L-Ala-OEt (412.0 mg, 1.497 mmol) in it. The mixture was stirred at 37 o C for 4 h, then cooled to room temperature. The reaction mixture was purified by HPLC to get desire product as a white solid (348.0 mg, 60.0 %). 1 H NMR (400 MHz, MeOD) δ = 8.34-8.31 (m, 1H), 8.16-8.08 (m, 2H), 7.84 (d, J=8.0, 1H), 7.56 (d, J=8.0, 1H), 7.35-7.20 (m, 10H), 4.95-4.93 (m, 2H), 4.28-4.21
(m, 3H), 4.10-4.00 (m, 3H), 3.87(t, J=4.0, 2H), 3.76(t, J=4.0, 2H), 3.07-3.02 (m, 2H), 2.79-2.73 (m, 1H), 1.74 (br, 1H), 1.28(d, J=8.0, 3H), 1.19(d, J=8.0, 3H), 1.09(d, J=8.0, 3H), 13 C NMR (101 MHz, CDCl3) δ = 172.8, 170.7, 169.1, 137.4, 129.6, 128.7, 128.5, 127.9, 126.7, 67.0, 60.9, 48.1, 20.0, 17.4, 14.5; HRMS (ESI) Calcd for: C27H42N5O9: 580.2977. Found: 580.2971 ([M+H] + ). Synthesis of 3g: Thiol acid (76.00 mg, 998.4 µmol) was added to DMF/PBS (2000 µl, 1:1) in round-bottom flask, then added Cu(OAc)2/HOBt (1 eq) and Phe-OEt (289.5 mg, 1.498 mmol) in it. The mixture was stirred at 37 o C for 0.1 h, then cooled to room temperature. The solvent was evaporated under reduced pressure, and the residue dissolved in ethyl acetate then washed successively with 1M aqueous HCl, 2M aqueous NaOH and brine. The organic layer was dried with Na2SO4, filtered, and concentrated to dryness. The crude product was purified by flash chromatography (first use 25% ethyl acetate in petroleum ether, then use 10% MeOH in DCM wash the chromatography to get the product). The title compound was prepared as a white solid (211.0 mg, 90 %). 1 H NMR (400 MHz, CDCl3) δ 7.46 7.19 (m, 3H), 7.16 7.05 (m, 2H), 6.11 (d, J = 7.4 Hz, 1H), 4.87 (dt, J = 7.8, 5.9 Hz, 1H), 4.18 (q, J = 7.1 Hz, 2H), 3.46 2.93 (m, 2H), 1.99 (s, 3H), 1.25 (t, J = 7.1 Hz, 3H), 13 C NMR (101 MHz, CDCl3) δ 171.74, 169.67, 135.95, 129.32, 128.52, 127.08, 61.52, 53.17, 37.92, 23.14, 14.12; ESI-MS Calcd for: C13H18NO3: 236.13. Found: 235.88 ([M+H] + ). Synthesis of 3h: Cy5.5 thiol acid (635.0 µg, 999.5 µmol) was added to DMF/PBS (100.0 µl, 1:1) in EP tube, then added Cu(OAc)2/HOBt (1 eq) and c(rgdfk) (900.0
µg, 1.542 µmol) in it. The mixture was stirred at 37 o C for 0.5 h, then cooled to room temperature. The reaction mixture was purified by HPLC to get the product as a blue solid (759.4 µg, 60 %). ESI-MS Calcd for: C67H82N11O8 + : 1168.6. Found: 1168.9 ([M+H] + ). Synthesis of 3i: Cy5.5 thiol acid (635.0 µg, 0.9995 µmol) was added to DMF/PBS (100.0 µl, 1:1) in EP tube, then added Cu(OAc)2/HOBt (1 eq) and JMV594 (1600 µg, 1.438 µmol) in it. The mixture was stirred at 37 o C for 0.5 h, then cooled to room temperature. The reaction mixture was purified by HPLC to get the product as a blue solid (1.057 mg, 63 %). ESI-MS Calcd for: C95H121N16O10S + : 1677.9. Found: 1679.4 ([M+H] + ). Synthesis of 3j: Cy5.5 thiol acid (635.0 µg, 0.9995 µmol) was added to DMF/PBS (100.0 µl, 1:1) in EP tube, then added Cu(OAc)2/HOBt (1 eq) and AE105 (2100 µg, 1.453 µmol) in it. The mixture was stirred at 37 o C for 0.5 h, then cooled to room temperature. The reaction mixture was purified by HPLC to get the product as a blue solid (1170 µg, 58 %). ESI-MS Calcd for: C110H141N18O19: 2018.0, Found: 2017.5 ([M+H] + ). Synthesis of 3k: Cy5.5 thiol acid (635.0 µg, 0.9995 µmol) was added to DMF/PBS (100.0 µl, 1:1) in EP tube, then added Cu(OAc)2/HOBt (1 eq) and E[c(RGDyK)2] (2.020 mg, 1.496 µmol) in it. The mixture was stirred at 37 o C for 0.5 h, then cooled
to room temperature. The reaction mixture was purified by HPLC to get the product as a blue solid (1.111 mg, 58 %). ESI-MS Calcd for: C99H129N21O19 + : 1915.9. Found: 1915.1 ([M+H] - ).
NMR Spectra 1 H NMR and 13 C NMR for 1a
1 H NMR and 13 C NMR for 1b
1 H NMR and 13 C NMR for 1c
1 H NMR and 13 C NMR for 1d
1 H NMR and 13 C NMR for 3a
1 H NMR and 13 C NMR for 3b
1 H NMR and 13 C NMR for 3c
1 H NMR and 13 C NMR for 3d
1 H NMR and 13 C NMR for 3e
1 H NMR and 13 C NMR for 3f
1 H NMR and 13 C NMR for 3g