Tianyi Yang, Yujia Cui, Daria K. Wanic, Linwei Zeng, Mark R. Howarth, David R. Spring. “Divinyl-s-tetrazine as a minimal linchpin for peptide stapling, protein disulfide re-bridging and click functionalisation.” ChemRxiv (). https://doi.org/10.26434/chemrxiv.15007631/v1

2,2′-(1,2,4,5-tetrazine-3,6-diyl)bis(ethan-1-ol)
Identifier Int-1
TLC Rf = 0.32 (SiO₂, 3 % MeOH in EtOAc)
¹H NMR (700 MHz, CDCl₃) δ 4.27 (t, J = 5.8 Hz, 4H), 3.59 (t, J = 5.8 Hz, 4H), 2.37 (s, 2H)
¹³C NMR (176 MHz, CDCl₃) δ 169.0, 60.1, 37.6
SI, p. 4
3,6-divinyl-1,2,4,5-tetrazine
Identifier DvTz
TLC Rf = 0.48 (20 % Et₂O in PE)
¹H NMR (700 MHz, DMSO-d₆) δ 7.19 (2H, dd, J = 17.5, 10.9 Hz, H3), 6.87 (2H, dd, J = 17.5, 1.1 Hz, H1), 6.12 (2H, dd, J = 10.9, 1.1 Hz, H2)
¹³C NMR (176 MHz, DMSO-d₆) δ 163.1 (Cc), 130.7 (Cb), 127.5 (Ca)
SI, p. 4
Peptide 1
Identifier 1
Analytical HPLC (Agilent 1260 Infinity II; Eclipse Plus C18, 150 × 4.6 mm, 3.5 µm; 1 mL min⁻¹; 220 nm; A = 0.05 % TFA in water (v/v), B = 0.05 % TFA in MeCN (v/v)) Rₜ 5.19 min (5–95 % B over 15 min)
Analytical HPLC injection results (5–95 % B over 15 min; DAD1A, Sig=220,4 Ref=360,100) Peak 1: RT 5.836 min, area 1994.862 mAU·s, area 100.000 %, height 692.311 mAU, height 100.00 %, start 5.680 min
Analytical HPLC injection results (10–30 % B over 15 min; DAD1A, Sig=220,4 Ref=360,100) Peak 1: RT 7.944 min, area 1923.970 mAU·s, area 100.000 %, height 384.043 mAU, height 100.00 %, start 7.797 min
HRMS (ESI) m/z calcd for C₃₃H₄₈N₁₀O₁₄S₂ [M+H]⁺ 873.2866, found 873.2825
SI, pp. 2–3, 14, 22
Peptide 1a
Identifier 1a
Analytical HPLC (Agilent 1260 Infinity II; Eclipse Plus C18, 150 × 4.6 mm, 3.5 µm; 1 mL min⁻¹; 220 nm; A = 0.05 % TFA in water (v/v), B = 0.05 % TFA in MeCN (v/v)) Rₜ 5.84 min (5–95 % B over 15 min); 7.10 min (5–60 % B over 15 min); 8.76 min (5–40 % B over 15 min); 7.94 min (10–30 % B over 15 min)
HRMS (ESI) m/z calcd for C₃₉H₅₄N₁₄O₁₄S₂ [M+H]⁺ 1007.3459, found 1007.3478
SI, pp. 2–3, 14
Peptide 3
Identifier 3
Analytical HPLC (Agilent 1260 Infinity II; Eclipse Plus C18, 150 × 4.6 mm, 3.5 µm; 1 mL min⁻¹; 220 nm; A = 0.05 % TFA in water (v/v), B = 0.05 % TFA in MeCN (v/v)) Rₜ 5.57 min (1–99 % B over 15 min); 5.01 min (5–95 % B over 15 min); 5.60 min (5–60 % B over 15 min); 6.29 min (5–40 % B over 15 min); 7.87 min (5–20 % B over 15 min)
Analytical HPLC injection results (1–99 % B over 15 min; DAD1A, Sig=220,4 Ref=360,100) Peak 1: RT 5.569 min, area 2193.764 mAU·s, area 100.000 %, height 874.820 mAU, height 100.00 %, start 5.509 min
HRMS (ESI) m/z calcd for C₁₈H₂₇N₅O₅S₂ [M+H]⁺ 458.1527, found 458.1501
SI, pp. 2–3, 15, 22
Peptide 3a
Identifier 3a
LC–MS (ESI; Waters ACQUITY HClass UPLC with SQ Detector 2; ACQUITY UPLC CSH C18, 2.1 × 50 mm, 1.7 µm, 130 Å, 40 °C; 0.6 mL min⁻¹; A = 2 mM NH₄OAc in H₂O/MeCN 95:5, B = MeCN, C = 2 % aqueous formic acid; PDA 220–800 nm, 1.2 nm interval) Rₜ 1.24 min (5–95 % B over 3 min, constant 5 % C over 1 min); m/z calcd for C₂₄H₃₃N₉O₅S₂ [M+H]⁺ 592.2, found 592.3
Analytical HPLC (Agilent 1260 Infinity II; Eclipse Plus C18, 150 × 4.6 mm, 3.5 µm; 1 mL min⁻¹; 220 nm; A = 0.05 % TFA in water (v/v), B = 0.05 % TFA in MeCN (v/v)) Rₜ 6.36 min (1–99 % B over 15 min)
Visible analytical HPLC injection-results rows (1–99 % B over 15 min; DAD1A, Sig=220,4 Ref=360,100) Peak 1: RT 5.421 min, area 145.434 mAU·s, area 4.745 %, height 61.537 mAU, height 5.52 %, start 5.375 min; peak 2: RT 5.764 min, area 22.307 mAU·s, area 0.728 %, height 9.684 mAU, height 0.87 %, start 5.728 min; peak 3: RT 5.927 min, area 27.748 mAU·s, area 0.905 %, height 12.032 mAU, height 1.08 %, start 5.896 min; peak 4: RT 6.224 min, area 59.198 mAU·s, area 1.931 %, height 15.493 mAU, height 1.39 %, start 6.159 min; peak 5: RT 6.361 min, area 2230.581 mAU·s, area 72.776 %, height 922.474 mAU, height 82.68 %, start 6.307 min
HRMS (ESI) m/z calcd for C₂₄H₃₃N₉O₅S₂ [M+H]⁺ 592.2119, found 592.2117
SI, pp. 2–3, 15, 23
Peptide 4
Identifier 4
Analytical HPLC (Agilent 1260 Infinity II; Eclipse Plus C18, 150 × 4.6 mm, 3.5 µm; 1 mL min⁻¹; 220 nm; A = 0.05 % TFA in water (v/v), B = 0.05 % TFA in MeCN (v/v)) Rₜ 7.13 min (1–99 % B over 15 min); 6.75 min (5–95 % B over 15 min); 8.47 min (5–60 % B over 15 min); 10.47 min (5–40 % B over 15 min)
Analytical HPLC injection results (1–99 % B over 15 min; DAD1A, Sig=220,4 Ref=360,100) Peak 1: RT 7.128 min, area 7786.510 mAU·s, area 100.000 %, height 2567.204 mAU, height 100.00 %, start 7.071 min
Analytical HPLC injection results (5–40 % B over 15 min; DAD1A, Sig=220,4 Ref=360,100) Peak 1: RT 10.735 min, area 20266.140 mAU·s, area 100.000 %, height 2871.551 mAU, height 100.00 %, start 10.598 min
HRMS (ESI) m/z calcd for C₂₇H₄₀N₈O₆S₂ [M+H]⁺ 637.2585, found 637.2621
SI, pp. 2–3, 15, 23–24
Peptide 4a
Identifier 4a
LC–MS (ESI; Waters ACQUITY HClass UPLC with SQ Detector 2; ACQUITY UPLC CSH C18, 2.1 × 50 mm, 1.7 µm, 130 Å, 40 °C; 0.6 mL min⁻¹; A = 2 mM NH₄OAc in H₂O/MeCN 95:5, B = MeCN, C = 2 % aqueous formic acid; PDA 220–800 nm, 1.2 nm interval) Rₜ 1.39 min (5–95 % B over 3 min, constant 5 % C over 1 min); m/z calcd for C₃₃H₄₆N₁₂O₆S₂ [M+H]⁺ 771.3, found 771.6
Analytical HPLC (Agilent 1260 Infinity II; Eclipse Plus C18, 150 × 4.6 mm, 3.5 µm; 1 mL min⁻¹; 220 nm; A = 0.05 % TFA in water (v/v), B = 0.05 % TFA in MeCN (v/v)) Rₜ 7.36 min (1–99 % B over 15 min)
Analytical HPLC injection results (1–99 % B over 15 min; DAD1A, Sig=220,4 Ref=360,100) Peak 1: RT 6.714 min, area 426.612 mAU·s, area 4.449 %, height 177.446 mAU, height 6.14 %, start 6.663 min; peak 2: RT 7.241 min, area 908.326 mAU·s, area 9.473 %, height 189.473 mAU, height 6.55 %, start 7.101 min; peak 3: RT 7.364 min, area 7812.437 mAU·s, area 81.479 %, height 2394.323 mAU, height 82.82 %, start 7.293 min; peak 4: RT 7.724 min, area 327.265 mAU·s, area 3.413 %, height 98.818 mAU, height 3.42 %, start 7.574 min; peak 5: RT 8.384 min, area 113.620 mAU·s, area 1.185 %, height 31.016 mAU, height 1.07 %, start 8.357 min
HRMS (ESI) m/z calcd for C₃₃H₄₆N₁₂O₆S₂ [M+H]⁺ 771.3178, found 771.3179
SI, pp. 2–3, 15, 24
Peptide 5
Identifier 5
Analytical HPLC (Agilent 1260 Infinity II; Eclipse Plus C18, 150 × 4.6 mm, 3.5 µm; 1 mL min⁻¹; 220 nm; A = 0.05 % TFA in water (v/v), B = 0.05 % TFA in MeCN (v/v)) Rₜ 6.72 min (5–95 % B over 15 min)
Analytical HPLC injection results (5–95 % B over 15 min; DAD1A, Sig=220,4 Ref=360,100) Peak 1: RT 6.721 min, area 2374.356 mAU·s, area 100.000 %, height 885.277 mAU, height 100.00 %, start 6.634 min
HRMS (ESI) m/z calcd for C₂₅H₃₈N₆O₇S₃ [M+H]⁺ 631.2037, found 631.2015
SI, pp. 2–3, 16, 25
Peptide 5a
Identifier 5a
LC–MS (ESI; Waters ACQUITY HClass UPLC with SQ Detector 2; ACQUITY UPLC CSH C18, 2.1 × 50 mm, 1.7 µm, 130 Å, 40 °C; 0.6 mL min⁻¹; A = 2 mM NH₄OAc in H₂O/MeCN 95:5, B = MeCN, C = 2 % aqueous formic acid; PDA 220–800 nm, 1.2 nm interval) Rₜ 1.38 min (5–95 % B over 3 min, constant 5 % C over 1 min); m/z calcd for C₃₁H₄₄N₁₀O₇S₃ [M+H]⁺ 765.3, found 765.5
Analytical HPLC (Agilent 1260 Infinity II; Eclipse Plus C18, 150 × 4.6 mm, 3.5 µm; 1 mL min⁻¹; 220 nm; A = 0.05 % TFA in water (v/v), B = 0.05 % TFA in MeCN (v/v)) Rₜ 7.46 min (5–95 % B over 15 min)
Visible analytical HPLC injection-results rows (5–95 % B over 15 min; DAD1A, Sig=220,4 Ref=360,100) Peak 1: RT 5.271 min, area 35.667 mAU·s, area 1.494 %, height 12.576 mAU, height 1.43 %, start 5.233 min; peak 2: RT 6.698 min, area 588.927 mAU·s, area 24.667 %, height 213.706 mAU, height 24.37 %, start 6.611 min; peak 3: RT 6.894 min, area 23.945 mAU·s, area 1.003 %, height 11.247 mAU, height 1.28 %, start 6.862 min; peak 4: RT 7.458 min, area 1598.696 mAU·s, area 66.960 %, height 583.686 mAU, height 66.55 %, start 7.399 min
HRMS (ESI) m/z calcd for C₃₁H₄₄N₁₀O₇S₃ [M+H]⁺ 765.2630, found 765.2628
SI, pp. 2–3, 16, 25
Peptide 6
Identifier 6
Analytical HPLC (Agilent 1260 Infinity II; Eclipse Plus C18, 150 × 4.6 mm, 3.5 µm; 1 mL min⁻¹; 220 nm; A = 0.05 % TFA in water (v/v), B = 0.05 % TFA in MeCN (v/v)) Rₜ 6.32 min (1–99 % B over 15 min); 5.85 min (5–95 % B over 15 min); 8.73 min (5–40 % B over 15 min); 13.53 min (5–20 % B over 15 min)
Analytical HPLC injection results (1–99 % B over 15 min; DAD1A, Sig=220,4 Ref=360,100) Peak 1: RT 6.319 min, area 1012.453 mAU·s, area 100.000 %, height 404.624 mAU, height 100.00 %, start 6.269 min
Analytical HPLC injection results (5–40 % B over 15 min; DAD1A, Sig=220,4 Ref=360,100) Peak 1: RT 8.726 min, area 5258.813 mAU·s, area 100.000 %, height 941.227 mAU, height 100.00 %, start 8.649 min
HRMS (ESI) m/z calcd for C₂₇H₅₀N₈O₈S₂ [M+H]⁺ 679.3266, found 679.3229
SI, pp. 2–3, 16, 26
Peptide 6a
Identifier 6a
LC–MS (ESI; Waters ACQUITY HClass UPLC with SQ Detector 2; ACQUITY UPLC CSH C18, 2.1 × 50 mm, 1.7 µm, 130 Å, 40 °C; 0.6 mL min⁻¹; A = 2 mM NH₄OAc in H₂O/MeCN 95:5, B = MeCN, C = 2 % aqueous formic acid; PDA 220–800 nm, 1.2 nm interval) Rₜ 1.25 min (5–95 % B over 3 min, constant 5 % C over 1 min); m/z calcd for C₃₃H₅₆N₁₂O₈S₂ [M+H]⁺ 813.4, found 813.7
Analytical HPLC (Agilent 1260 Infinity II; Eclipse Plus C18, 150 × 4.6 mm, 3.5 µm; 1 mL min⁻¹; 220 nm; A = 0.05 % TFA in water (v/v), B = 0.05 % TFA in MeCN (v/v)) Rₜ 6.71 min (1–99 % B over 15 min)
Visible analytical HPLC injection-results rows (1–99 % B over 15 min; DAD1A, Sig=220,4 Ref=360,100) Peak 1: RT 5.934 min, area 137.403 mAU·s, area 8.421 %, height 47.508 mAU, height 8.41 %, start 5.859 min; peak 2: RT 6.254 min, area 12.119 mAU·s, area 0.743 %, height 5.523 mAU, height 0.98 %, start 6.198 min; peak 3: RT 6.708 min, area 1154.337 mAU·s, area 70.744 %, height 440.971 mAU, height 78.04 %, start 6.608 min
HRMS (ESI) m/z calcd for C₃₃H₅₆N₁₂O₈S₂ [M+H]⁺ 813.3859, found 813.3803
SI, pp. 2–3, 16, 26
Peptide 7
Identifier 7
Analytical HPLC (Agilent 1260 Infinity II; Eclipse Plus C18, 150 × 4.6 mm, 3.5 µm; 1 mL min⁻¹; 220 nm; A = 0.05 % TFA in water (v/v), B = 0.05 % TFA in MeCN (v/v)) Rₜ 6.38 min (5–95 % B over 15 min); 8.01 min (5–60 % B over 15 min); 10.15 min (5–40 % B over 15 min)
Analytical HPLC injection results (5–95 % B over 15 min; DAD1A, Sig=220,4 Ref=360,100) Peak 1: RT 6.383 min, area 12119.705 mAU·s, area 100.000 %, height 2693.548 mAU, height 100.00 %, start 6.320 min
Analytical HPLC injection results (5–40 % B over 15 min; DAD1A, Sig=220,4 Ref=360,100) Peak 1: RT 10.154 min, area 15553.475 mAU·s, area 100.000 %, height 2418.165 mAU, height 100.00 %, start 9.964 min
HRMS (ESI) m/z calcd for C₃₅H₅₇N₉O₉S₂ [M+H]⁺ 812.3794, found 812.3772
SI, pp. 2–3, 16, 27
Peptide 7a
Identifier 7a
LC–MS (ESI; Waters ACQUITY HClass UPLC with SQ Detector 2; ACQUITY UPLC CSH C18, 2.1 × 50 mm, 1.7 µm, 130 Å, 40 °C; 0.6 mL min⁻¹; A = 2 mM NH₄OAc in H₂O/MeCN 95:5, B = MeCN, C = 2 % aqueous formic acid; PDA 220–800 nm, 1.2 nm interval) Rₜ 1.30 min (5–95 % B over 5 min, constant 5 % C over 1 min); m/z calcd for C₄₁H₆₃N₁₃O₉S₂ [M+H]⁺ 946.4, found 946.6
Analytical HPLC (Agilent 1260 Infinity II; Eclipse Plus C18, 150 × 4.6 mm, 3.5 µm; 1 mL min⁻¹; 220 nm; A = 0.05 % TFA in water (v/v), B = 0.05 % TFA in MeCN (v/v)) Rₜ 6.71 min (5–95 % B over 15 min)
Visible analytical HPLC injection-results rows (5–95 % B over 15 min; DAD1A, Sig=220,4 Ref=360,100) Peak 1: RT 6.110 min, area 77.560 mAU·s, area 1.774 %, height 17.651 mAU, height 2.09 %, start 6.064 min; peak 2: RT 6.713 min, area 3634.244 mAU·s, area 83.118 %, height 697.247 mAU, height 82.74 %, start 6.466 min
HRMS (ESI) m/z calcd for C₄₁H₆₃N₁₃O₉S₂ [M+H]⁺ 946.4386, found 946.4434
SI, pp. 2–3, 17, 28
Peptide 8
Identifier 8
Analytical HPLC (Agilent 1260 Infinity II; Eclipse Plus C18, 150 × 4.6 mm, 3.5 µm; 1 mL min⁻¹; 220 nm; A = 0.05 % TFA in water (v/v), B = 0.05 % TFA in MeCN (v/v)) Rₜ 6.75 min (5–95 % B over 15 min); 8.49 min (5–60 % B over 15 min); 10.79 min (5–40 % B over 15 min)
Analytical HPLC injection results (5–95 % B over 15 min; DAD1A, Sig=220,4 Ref=360,100) Peak 1: RT 6.647 min, area 600.088 mAU·s, area 4.272 %, height 211.847 mAU, height 6.66 %, start 6.501 min; peak 2: RT 6.747 min, area 13446.197 mAU·s, area 95.728 %, height 2970.691 mAU, height 93.34 %, start 6.694 min
Analytical HPLC injection results (5–40 % B over 15 min; DAD1A, Sig=220,4 Ref=360,100) Peak 1: RT 10.785 min, area 19048.965 mAU·s, area 100.000 %, height 2902.766 mAU, height 100.00 %, start 10.652 min
HRMS (ESI) m/z calcd for C₃₁H₄₆N₁₀O₈S₃ [M+H]⁺ 783.2735, found 783.2709
SI, pp. 2–3, 17, 28–29
Peptide 8a
Identifier 8a
LC–MS (ESI; Waters ACQUITY HClass UPLC with SQ Detector 2; ACQUITY UPLC CSH C18, 2.1 × 50 mm, 1.7 µm, 130 Å, 40 °C; 0.6 mL min⁻¹; A = 2 mM NH₄OAc in H₂O/MeCN 95:5, B = MeCN, C = 2 % aqueous formic acid; PDA 220–800 nm, 1.2 nm interval) Rₜ 1.28 min (5–95 % B over 3 min, constant 5 % C over 1 min); m/z calcd for C₃₇H₅₂N₁₄O₈S₃ [M+H]⁺ 917.3, found 917.7
Analytical HPLC (Agilent 1260 Infinity II; Eclipse Plus C18, 150 × 4.6 mm, 3.5 µm; 1 mL min⁻¹; 220 nm; A = 0.05 % TFA in water (v/v), B = 0.05 % TFA in MeCN (v/v)) Rₜ 7.07 min (5–95 % B over 15 min)
Visible analytical HPLC injection-results rows (5–95 % B over 15 min; DAD1A, Sig=220,4 Ref=360,100) Peak 1: RT 6.597 min, area 576.726 mAU·s, area 5.954 %, height 179.065 mAU, height 5.51 %, start 6.447 min; peak 2: RT 6.977 min, area 113.673 mAU·s, area 1.174 %, height 53.233 mAU, height 1.64 %, start 6.903 min; peak 3: RT 7.071 min, area 7878.934 mAU·s, area 81.344 %, height 2637.415 mAU, height 81.21 %, start 7.018 min
HRMS (ESI) m/z calcd for C₃₇H₅₂N₁₄O₈S₃ [M+H]⁺ 917.3328, found 917.3339
SI, pp. 2–3, 17, 29
Peptide 9
Identifier 9
Analytical HPLC (Agilent 1260 Infinity II; Eclipse Plus C18, 150 × 4.6 mm, 3.5 µm; 1 mL min⁻¹; 220 nm; A = 0.05 % TFA in water (v/v), B = 0.05 % TFA in MeCN (v/v)) Rₜ 6.95 min (5–95 % B over 15 min); 8.81 min (5–60 % B over 15 min); 11.24 min (5–40 % B over 15 min)
Analytical HPLC injection results (5–95 % B over 15 min; DAD1A, Sig=220,4 Ref=360,100) Peak 1: RT 6.951 min, area 6679.809 mAU·s, area 100.000 %, height 2041.459 mAU, height 100.00 %, start 6.868 min
Analytical HPLC injection results (5–40 % B over 15 min; DAD1A, Sig=220,4 Ref=360,100) Peak 1: RT 11.237 min, area 7163.162 mAU·s, area 100.000 %, height 1488.624 mAU, height 100.00 %, start 11.154 min
HRMS (ESI) m/z calcd for C₃₁H₄₉N₁₃O₁₀S₂ [M+H]⁺ 844.3189, found 844.3190
SI, pp. 2–3, 17, 30
Peptide 9a
Identifier 9a
LC–MS (ESI; Waters ACQUITY HClass UPLC with SQ Detector 2; ACQUITY UPLC CSH C18, 2.1 × 50 mm, 1.7 µm, 130 Å, 40 °C; 0.6 mL min⁻¹; A = 2 mM NH₄OAc in H₂O/MeCN 95:5, B = MeCN, C = 2 % aqueous formic acid; PDA 220–800 nm, 1.2 nm interval) Rₜ 1.44 min (5–95 % B over 5 min, constant 5 % C over 1 min); m/z calcd for C₃₇H₅₅N₁₇O₁₁S₂ [M+H]⁺ 978.4, found 978.5
Analytical HPLC (Agilent 1260 Infinity II; Eclipse Plus C18, 150 × 4.6 mm, 3.5 µm; 1 mL min⁻¹; 220 nm; A = 0.05 % TFA in water (v/v), B = 0.05 % TFA in MeCN (v/v)) Rₜ 7.58 min (5–95 % B over 15 min)
Visible analytical HPLC injection-results rows (5–95 % B over 15 min; DAD1A, Sig=220,4 Ref=360,100) Peak 1: RT 5.284 min, area 39.944 mAU·s, area 1.069 %, height 11.845 mAU, height 1.09 %, start 5.227 min; peak 2: RT 6.080 min, area 13.907 mAU·s, area 0.372 %, height 6.021 mAU, height 0.55 %, start 6.041 min; peak 3: RT 6.784 min, area 163.839 mAU·s, area 4.385 %, height 36.992 mAU, height 3.39 %, start 6.720 min; peak 4: RT 7.334 min, area 43.415 mAU·s, area 1.162 %, height 12.936 mAU, height 1.19 %, start 7.263 min; peak 5: RT 7.510 min, area 376.151 mAU·s, area 10.066 %, height 85.728 mAU, height 7.86 %, start 7.377 min; peak 6: RT 7.584 min, area 2486.620 mAU·s, area 66.546 %, height 824.637 mAU, height 75.59 %, start 7.534 min
HRMS (ESI) m/z calcd for C₃₇H₅₅N₁₇O₁₁S₂ [M+H]⁺ 978.3782, found 978.3734
SI, pp. 2–3, 17, 30
Peptide 10
Identifier 10
Analytical HPLC (Agilent 1260 Infinity II; Eclipse Plus C18, 150 × 4.6 mm, 3.5 µm; 1 mL min⁻¹; 220 nm; A = 0.05 % TFA in water (v/v), B = 0.05 % TFA in MeCN (v/v)) Rₜ 4.96 min (1–99 % B over 15 min); 4.37 min (5–95 % B over 15 min); 5.10 min (5–40 % B over 15 min); 5.97 min (5–20 % B over 15 min)
Analytical HPLC injection results (1–99 % B over 15 min; DAD1A, Sig=220,4 Ref=360,100) Peak 1: RT 4.959 min, area 9137.523 mAU·s, area 100.000 %, height 2653.484 mAU, height 100.00 %, start 4.916 min
Analytical HPLC injection results (5–20 % B over 15 min; DAD1A, Sig=220,4 Ref=360,100) Peak 1: RT 5.970 min, area 7574.108 mAU·s, area 100.000 %, height 1193.046 mAU, height 100.00 %, start 5.886 min
LC–MS (ESI; Waters ACQUITY HClass UPLC with SQ Detector 2; ACQUITY UPLC CSH C18, 2.1 × 50 mm, 1.7 µm, 130 Å, 40 °C; 0.6 mL min⁻¹; A = 2 mM NH₄OAc in H₂O/MeCN 95:5, B = MeCN, C = 2 % aqueous formic acid; PDA 220–800 nm, 1.2 nm interval) Rₜ 0.32 min (5–95 % B over 3 min, constant 5 % C over 1 min); m/z [M+2H]²⁺ calcd for C₃₃H₅₄N₁₂O₁₀S₂ 422.2, found 422.4
SI, pp. 2, 18, 31
Peptide 10a
Identifier 10a
LC–MS (ESI; Waters ACQUITY HClass UPLC with SQ Detector 2; ACQUITY UPLC CSH C18, 2.1 × 50 mm, 1.7 µm, 130 Å, 40 °C; 0.6 mL min⁻¹; A = 2 mM NH₄OAc in H₂O/MeCN 95:5, B = MeCN, C = 2 % aqueous formic acid; PDA 220–800 nm, 1.2 nm interval) Rₜ 1.00 min (5–95 % B over 3 min, constant 5 % C over 1 min); m/z [M+2H]²⁺ calcd for C₃₉H₆₀N₁₆O₁₀S₂ 489.2, found 489.5
Analytical HPLC (Agilent 1260 Infinity II; Eclipse Plus C18, 150 × 4.6 mm, 3.5 µm; 1 mL min⁻¹; 220 nm; A = 0.05 % TFA in water (v/v), B = 0.05 % TFA in MeCN (v/v)) Rₜ 4.90 min (1–99 % B over 15 min)
Visible analytical HPLC injection-results rows (1–99 % B over 15 min; DAD1A, Sig=220,4 Ref=360,100) Peak 1: RT 4.112 min, area 8.172 mAU·s, area 0.203 %, height 6.974 mAU, height 0.54 %, start 4.060 min; peak 2: RT 4.702 min, area 40.288 mAU·s, area 1.001 %, height 10.472 mAU, height 0.82 %, start 4.649 min; peak 3: RT 4.895 min, area 3194.994 mAU·s, area 79.355 %, height 1034.917 mAU, height 80.62 %, start 4.802 min
SI, pp. 2, 18, 32
Peptide 11
Identifier 11
Analytical HPLC (Agilent 1260 Infinity II; Eclipse Plus C18, 150 × 4.6 mm, 3.5 µm; 1 mL min⁻¹; 220 nm; A = 0.05 % TFA in water (v/v), B = 0.05 % TFA in MeCN (v/v)) Rₜ 7.01 min (5–95 % B over 15 min); 8.54 min (15–40 % B over 15 min)
Analytical HPLC injection results (5–95 % B over 15 min; DAD1A, Sig=220,4 Ref=360,100) Peak 1: RT 6.880 min, area 875.022 mAU·s, area 5.541 %, height 280.270 mAU, height 9.05 %, start 6.770 min; peak 2: RT 7.007 min, area 14915.876 mAU·s, area 94.459 %, height 2816.170 mAU, height 90.95 %, start 6.944 min
Analytical HPLC injection results (15–40 % B over 15 min; DAD1A, Sig=220,4 Ref=360,100) Peak 1: RT 8.188 min, area 848.846 mAU·s, area 3.234 %, height 121.191 mAU, height 4.17 %, start 7.965 min; peak 2: RT 8.538 min, area 25402.583 mAU·s, area 96.766 %, height 2782.364 mAU, height 95.83 %, start 8.451 min
HRMS (ESI) m/z calcd for C₄₃H₆₈N₁₂O₁₂S₂ [M+H]⁺ 1009.4594, found 1009.4549
SI, pp. 2–3, 18, 32–33
Peptide 11a
Identifier 11a
LC–MS (ESI; Waters ACQUITY HClass UPLC with SQ Detector 2; ACQUITY UPLC CSH C18, 2.1 × 50 mm, 1.7 µm, 130 Å, 40 °C; 0.6 mL min⁻¹; A = 2 mM NH₄OAc in H₂O/MeCN 95:5, B = MeCN, C = 2 % aqueous formic acid; PDA 220–800 nm, 1.2 nm interval) Rₜ 6.46 min (5–95 % B over 17.5 min, constant 5 % C over 1 min); m/z calcd for C₄₉H₇₄N₁₆O₁₂S₂ [M+H]⁺ 1143.52, found 1143.66
Analytical HPLC (Agilent 1260 Infinity II; Eclipse Plus C18, 150 × 4.6 mm, 3.5 µm; 1 mL min⁻¹; 220 nm; A = 0.05 % TFA in water (v/v), B = 0.05 % TFA in MeCN (v/v)) Rₜ 7.28 min (5–95 % B over 15 min)
Visible analytical HPLC injection-results rows (5–95 % B over 15 min; DAD1A, Sig=220,4 Ref=360,100) Peak 1: RT 6.847 min, area 464.496 mAU·s, area 8.505 %, height 169.696 mAU, height 9.37 %, start 6.811 min; peak 2: RT 7.281 min, area 4128.280 mAU·s, area 75.590 %, height 1493.058 mAU, height 82.40 %, start 7.237 min
HRMS (ESI) m/z calcd for C₄₉H₇₄N₁₆O₁₂S₂ [M+H]⁺ 1143.5187, found 1143.5214
SI, pp. 2–3, 18, 33
Peptide 1b
Identifier 1b
Analytical HPLC (Agilent 1260 Infinity II; Eclipse Plus C18, 150 × 4.6 mm, 3.5 µm; 1 mL min⁻¹; 220 nm; A = 0.05 % TFA in water (v/v), B = 0.05 % TFA in MeCN (v/v)) Rₜ 5.65 min (5–95 % B over 15 min); 6.83 min (5–60 % B over 15 min); 8.41 min (5–40 % B over 15 min)
HRMS (ESI) m/z calcd for C₄₉H₆₈N₁₂O₁₅S₂ [M+H]⁺ 1129.4442, found 1129.4485
SI, pp. 2–3, 20
IEDDA of peptide 1a with BCN derivative 2
HPLC reaction-monitoring conditions 2.0 equivalents of 2; 5 % DMSO in 20 mM NaPi, pH 8.0; peptide 0.1 mM; 37 °C; 400 rpm; Agilent 1260 Infinity II; Eclipse Plus C18 column, 150 × 4.6 mm, 3.5 µm; 1 mL min⁻¹; HPLC-UV at 220 nm; 5–95 % MeCN/water with constant 0.05 % TFA
After 30 min 1b, Rₜ = 5.627 min; 1a, Rₜ = 5.817 min
After 2 h 1b, Rₜ = 5.628 min; reaction reached completion
SI, pp. 2, 19
One-pot stapling and IEDDA functionalisation of peptide 1
HPLC reaction-monitoring conditions 2.0 equivalents of DvTz; 5 % DMSO in 20 mM NaPi, pH 8.0; peptide 1 at 0.1 mM; 37 °C for 15 min; then 2.0 equivalents of BCN derivative 2 at 37 °C for 2 h; Agilent 1260 Infinity II; Eclipse Plus C18 column, 150 × 4.6 mm, 3.5 µm; 1 mL min⁻¹; HPLC-UV at 220 nm; 5–95 % MeCN/water with constant 0.05 % TFA
Peptide 1 (reported in Main) Rₜ = 5.19 min
After 15 min (reported in Main) 1a, Rₜ = 5.86 min
After 15 min (reported in SI) 1a, Rₜ = 5.857 min
After 2 h (reported in Main) 1b, Rₜ = 5.63 min
After 2 h (reported in SI) 1b, Rₜ = 5.630 min; side product from excess DvTz with 2, Rₜ = 6.347 min
Main, p. 6; SI, pp. 2, 20–21
αHER2 Fab conjugate series
Protein LC–MS acquisition Xevo G2-S TOF mass spectrometer coupled to an Acquity UPLC system; Acquity UPLC BEH300 C4 column, 1.7 µm, 2.1 × 50 mm; A = 0.1 % aqueous formic acid, B = 95 % MeCN with 5 % 0.1 % aqueous formic acid; 0.2 mL min⁻¹; 95 % A for 0.93 min, linear gradient to 100 % B over 4.28 min, 100 % B for 1.04 min, then linear gradient to 95 % A over 1.04 min; electrospray source S-5, capillary voltage 2.0 kV, cone voltage 190 V; N₂ desolvation gas 850 L h⁻¹; total mass spectra reconstructed from the ion series using MaxEnt 1 in MassLynx 4.2; only the total ion chromatogram region containing protein signals was analysed
Deconvoluted MS reporting qualifier Peak height referenced to most intense signal
Native αHER2 Fab 12, deconvoluted protein LC–MS (1500–4000 m/z) Expected mass 47638 Da; observed mass 47636 Da
DvTz-re-bridged αHER2 Fab 12a, deconvoluted protein LC–MS (1500–4000 m/z) Expected mass 47772 Da; observed mass 47774 Da
BCN-functionalised DvTz-re-bridged αHER2 Fab 12b, deconvoluted protein LC–MS (1500–4000 m/z) Expected mass 47894 Da; observed mass 47896 Da
MS assignment for 12a Clean, single addition of DvTz; no other conjugated species were detected
Reducing SDS-PAGE (12 % polyacrylamide running gel, 4 % stacking gel; 15 µL sample, 0.67 µM; Coomassie brilliant blue loading dye with 2-mercaptoethanol; 90 °C for 5 min; 5 µL 10–200 kDa marker, New England BioLabs; 200 V for 65 min in Laemmli running buffer; Coomassie Blue staining; Syngene imaging) The native interchain disulfide bond in αHER2 Fab 12 was cleaved into heavy and light chains, while both 12a and 12b remained intact
Main, pp. 8–9; SI, pp. 3, 40
αCD45 Fab conjugate series
Protein LC–MS acquisition Xevo G2-S TOF mass spectrometer coupled to an Acquity UPLC system; Acquity UPLC BEH300 C4 column, 1.7 µm, 2.1 × 50 mm; A = 0.1 % aqueous formic acid, B = 95 % MeCN with 5 % 0.1 % aqueous formic acid; 0.2 mL min⁻¹; 95 % A for 0.93 min, linear gradient to 100 % B over 4.28 min, 100 % B for 1.04 min, then linear gradient to 95 % A over 1.04 min; electrospray source S-5, capillary voltage 2.0 kV, cone voltage 190 V; N₂ desolvation gas 850 L h⁻¹; total mass spectra reconstructed from the ion series using MaxEnt 1 in MassLynx 4.2; only the total ion chromatogram region containing protein signals was analysed
Deconvoluted MS reporting qualifier Peak height referenced to most intense signal
Native αCD45 Fab 13, deconvoluted protein LC–MS (1500–4000 m/z) Expected mass 51016 Da; observed mass 51016 Da
DvTz-re-bridged αCD45 Fab 13a, deconvoluted protein LC–MS (1500–4000 m/z) Expected mass 51152 Da; observed mass 51153 Da
BCN-functionalised DvTz-re-bridged αCD45 Fab 13b, deconvoluted protein LC–MS (1500–4000 m/z) Expected mass 51274 Da; observed mass 51275 Da
SI, pp. 3, 42
αSdrF Fab conjugate series
Protein LC–MS acquisition Xevo G2-S TOF mass spectrometer coupled to an Acquity UPLC system; Acquity UPLC BEH300 C4 column, 1.7 µm, 2.1 × 50 mm; A = 0.1 % aqueous formic acid, B = 95 % MeCN with 5 % 0.1 % aqueous formic acid; 0.2 mL min⁻¹; 95 % A for 0.93 min, linear gradient to 100 % B over 4.28 min, 100 % B for 1.04 min, then linear gradient to 95 % A over 1.04 min; electrospray source S-5, capillary voltage 2.0 kV, cone voltage 190 V; N₂ desolvation gas 850 L h⁻¹; total mass spectra reconstructed from the ion series using MaxEnt 1 in MassLynx 4.2; only the total ion chromatogram region containing protein signals was analysed
Deconvoluted MS reporting qualifier Peak height referenced to most intense signal
Native αSdrF Fab 14, deconvoluted protein LC–MS (1500–4000 m/z) Expected mass 49862 Da; observed mass 49862 Da
DvTz-re-bridged αSdrF Fab 14a, deconvoluted protein LC–MS (1500–4000 m/z) Expected mass 49998 Da; observed mass 49999 Da
BCN-functionalised DvTz-re-bridged αSdrF Fab 14b, deconvoluted protein LC–MS (1500–4000 m/z) Expected mass 50120 Da; observed mass 50121 Da
SI, pp. 3, 44
Native trastuzumab
Identifier 15
Protein LC–MS sample state Deglycosylated with PNGase F before analysis
Protein LC–MS acquisition Xevo G2-S TOF mass spectrometer coupled to an Acquity UPLC system; Acquity UPLC BEH300 C4 column, 1.7 µm, 2.1 × 50 mm; A = 0.1 % aqueous formic acid, B = 95 % MeCN with 5 % 0.1 % aqueous formic acid; 0.2 mL min⁻¹; 95 % A for 0.93 min, linear gradient to 100 % B over 4.28 min, 100 % B for 1.04 min, then linear gradient to 95 % A over 1.04 min; electrospray source S-5, capillary voltage 2.0 kV, cone voltage 190 V; N₂ desolvation gas 850 L h⁻¹; total mass spectra reconstructed from the ion series using MaxEnt 1 in MassLynx 4.2; only the total ion chromatogram region containing protein signals was analysed
Deconvoluted protein LC–MS Observed mass 145,171 Da
SI, p. 3
DvTz-re-bridged trastuzumab
Identifier 15a
Protein LC–MS sample state Deglycosylated with PNGase F before analysis; formed with 60 equivalents of DvTz for 2 h
Protein LC–MS acquisition Xevo G2-S TOF mass spectrometer coupled to an Acquity UPLC system; Acquity UPLC BEH300 C4 column, 1.7 µm, 2.1 × 50 mm; A = 0.1 % aqueous formic acid, B = 95 % MeCN with 5 % 0.1 % aqueous formic acid; 0.2 mL min⁻¹; 95 % A for 0.93 min, linear gradient to 100 % B over 4.28 min, 100 % B for 1.04 min, then linear gradient to 95 % A over 1.04 min; electrospray source S-5, capillary voltage 2.0 kV, cone voltage 190 V; N₂ desolvation gas 850 L h⁻¹; total mass spectra reconstructed from the ion series using MaxEnt 1 in MassLynx 4.2; only the total ion chromatogram region containing protein signals was analysed
Deconvoluted MS reporting qualifier Peak height referenced to most intense signal
Protein LC–MS reporting basis Expected masses are based on the observed mass ion of native trastuzumab under the same preparation and ionisation conditions (145,171 Da)
Half antibody + 1 DvTz, deconvoluted protein LC–MS (1500–4000 m/z) Expected mass 72719 Da; observed mass 72720 Da
Half antibody + 2 DvTz, deconvoluted protein LC–MS (1500–4000 m/z) Expected mass 72853 Da; observed mass 72856 Da
Full antibody + 2 DvTz, deconvoluted protein LC–MS (1500–4000 m/z) Expected mass 145438 Da; observed mass 145441 Da
Full antibody + 3 DvTz, deconvoluted protein LC–MS (1500–4000 m/z) Expected mass 145572 Da; observed mass 145578 Da
Full antibody + 4 DvTz, deconvoluted protein LC–MS (1500–4000 m/z) Expected mass 145706 Da; observed mass 145713 Da
Reducing SDS-PAGE (12 % polyacrylamide running gel, 4 % stacking gel; 15 µL sample, 0.67 µM; Coomassie brilliant blue loading dye with 2-mercaptoethanol; 90 °C for 5 min; 5 µL 10–200 kDa marker, New England BioLabs; 200 V for 65 min in Laemmli running buffer; Coomassie Blue staining; Syngene imaging) Formation of half antibodies was observed
Main, p. 11; SI, pp. 3, 47–48
BCN-functionalised DvTz-re-bridged trastuzumab
Identifier 15b
Protein LC–MS sample state Deglycosylated with PNGase F before analysis; formed with 100 equivalents of BCN for 16 h
Protein LC–MS acquisition Xevo G2-S TOF mass spectrometer coupled to an Acquity UPLC system; Acquity UPLC BEH300 C4 column, 1.7 µm, 2.1 × 50 mm; A = 0.1 % aqueous formic acid, B = 95 % MeCN with 5 % 0.1 % aqueous formic acid; 0.2 mL min⁻¹; 95 % A for 0.93 min, linear gradient to 100 % B over 4.28 min, 100 % B for 1.04 min, then linear gradient to 95 % A over 1.04 min; electrospray source S-5, capillary voltage 2.0 kV, cone voltage 190 V; N₂ desolvation gas 850 L h⁻¹; total mass spectra reconstructed from the ion series using MaxEnt 1 in MassLynx 4.2; only the total ion chromatogram region containing protein signals was analysed
Deconvoluted MS reporting qualifier Peak height referenced to most intense signal
Protein LC–MS reporting basis Expected masses are based on the observed mass ion of native trastuzumab under the same preparation and ionisation conditions (145,171 Da)
Half antibody + 1 DvTz-BCN, deconvoluted protein LC–MS (1500–4000 m/z) Expected mass 72841 Da; observed mass 72842 Da
Half antibody + 1 DvTz-BCN + 1 DvTz, deconvoluted protein LC–MS (1500–4000 m/z) Expected mass 72975 Da; observed mass 72978 Da
Half antibody + 2 DvTz-BCN, deconvoluted protein LC–MS (1500–4000 m/z) Expected mass 73097 Da; observed mass 73101 Da
Full antibody + 2 DvTz-BCN, deconvoluted protein LC–MS (1500–4000 m/z) Expected mass 145682 Da; observed mass 145683 Da
Full antibody + 2 DvTz-BCN + 2 DvTz, deconvoluted protein LC–MS (1500–4000 m/z) Expected mass 145950 Da; observed mass 145955 Da
Full antibody + 4 DvTz-BCN, deconvoluted protein LC–MS (1500–4000 m/z) Expected mass 146194 Da; observed mass 146201 Da
MS assignment MS revealed a mixture of species, with the fully ‘clicked’ tetra-functionalised antibody (bearing four BCN groups) as the major product, alongside partially converted double-‘clicked’ species