Arka Som, Marion V. F. Quijano Rincon, Nikolai Hempel, Lisa Johannknecht, Moritz Obenauer, Paul Czodrowski, Tanja Weil, Pol Besenius. “Cucurbit[8]uril couples enzymatic and supramolecular reaction networks for peptide isomerization and recognition in gain-of-function hydrogels.” ChemRxiv (). https://doi.org/10.26434/chemrxiv.15007664/v1

L–Phe–GLY–L–Ser–GLY–L–Ala (FGSGA)
Identifier 1
HRMS (ESI–MS) Calculated m/z 438.1462 [M+H]⁺; found 438.1463 [M+H]⁺
¹H NMR (400 MHz, DMSO-d₆) δ [ppm] = 8.45 (t, ³J = 6.0 Hz, 1H, –NHGly), 8.40 – 8.27 (m, 1H, –NHGly), 8.23 (d, ³J = 7.5 Hz, 1H, –NHSer), 7.52 (d, ³J = 6.7 Hz, 1H, –NHAla), 7.33 – 7.13 (m, 5H, –HAr), 4.27 (dt, ³J = 6.1 Hz, 6.0 Hz, 1H, –CHSer), 3.91 – 3.80 (m, 1H, –CHAla), 3.80 – 3.75 (m, 2H, –CH₂Ser), 3.72 – 3.54 (m, 4H, –CH₂Gly, –CH₂Gly), 3.46 (dd, ³J = 8.7 Hz, 4.5 Hz, 1H, –CHPhe), 2.99 (dd, ²J = 13.5 Hz, ³J = 4.5 Hz, 1H, –CHPhe), 2.60 (dd, ²J = 13.5 Hz, ³J = 8.8 Hz, 1H, –CH₂Phe), 1.16 (t, ³J = 6.5 Hz, 3H, –CH₃Ala)
SI, pp. 3–4
L–Phe(tBoc)–GLY
HRMS (ESI–MS) Calculated m/z 321.3535 [M+H]⁺; found 321.1456 [M+H]⁺
¹H NMR (400 MHz, DMSO-d₆) δ [ppm] = 8.23 (t,²J = 5.8 Hz, 1H, –NHGly), 7.35–7.22 (m, 4H, –HAr), 7.21 – 7.15 (m, 1H, –HAr), 6.92 (d, ³J = 8.8 Hz, 1H, –NHPhe), 4.34 – 4.11 (m, 1H, –NH–CH–CH₂), 3.85 – 3.73 (m ²J = 17.6 Hz, ³J = 5.7 Hz, 2H, –NH–CH₂), 3.05 – 2.92 (m, 1H, –CH–CH₂–), 2.71 (dd, ²J = 13.9 Hz, ³J = 10.8 Hz, 1H, CH–CH₂–), 1.28 (s, 9H, –O–C–(CH₃)₃)
SI, p. 4
Ac–L–Tyr–L–Arg–L–Ser–(L–Phe–GLY)–GLY–L–Ala (YRSFGGA)
Identifier 2
HRMS (ESI–MS) Calculated m/z 799.3734 [M+H]⁺; found 799.3733 [M+H]⁺
¹H NMR (400 MHz, DMSO-d₆) δ [ppm] = 9.07 (t, ³J = 5.8 Hz, 1H, –NHGly), 8.55 (t, ³J = 6.0 Hz, 1H, –NH–NHPhe), 8.42 (d, ³J = 7.8 Hz, 1H, –NHTyr), 8.17 (d, ³J = 7.4 Hz, 1H, –NHArg), 8.12 (d, J = 8.3 Hz, 1H, –NHSer), 7.80 (d, ³J = 6.5 Hz, 1H, –NHAla), 7.43 – 7.34 (s, 1H, –C=NH), 7.33 – 7.29 (m, 6H, –HAr, –HN=C–NH–), 7.28 – 7.22 (m, 1H, –HAr), 7.09 – 7.00 (m, 2H, –HAr), 6.68 – 6.60 (m, 2H, –HAr), 4.60 (dt, ³J = 7.9 Hz, 6.1 Hz, 1H, –CHTyr), 4.41 (dd, ³J = 4.1 Hz, 2.1 Hz, 1H, –CHSer), 4.32 (d, ³J = 6.8 Hz, 1H, –CHArg), 4.25 dd, (²J = 10.9 Hz, ³J = 5.9 Hz, –CH₂Tyr), 1H, 4.18 (dd, ²J = 10.9, ³J = 6.2 Hz, 1H, –CH₂Tyr), 4.04 (dd, ³J = 5.5 Hz, 2H, –CH₂Gly′), 3.98 (m, ³J = 6.9 Hz, 1H, –CHAla), 3.93 (d, ³J = 5.6 Hz, 2H, –CH₂Gly), 3.60 (dd, ³J = 5.5 Hz, 5.4 Hz, 1H, –CHPhe), 3.15 (dd, ²J = 14.0 Hz, ³J = 5.4 Hz, 1H, –O–CH₂), 3.08 (m, 2H, –CH₂–CH₂–CH₂–NH), 2.95 (dd, ²J = 14.0 Hz, 7.8 Hz, 1H, –O–CH₂), 2.88 (dd, ²J = 14.0 Hz, ³J = 3.8 Hz, 1H, –CH₂Phe), 2.62 (dd, ²J = 14.0 Hz, ³J = 4.8 Hz, 1H, –CH₂Phe), 1.75 (s, 3H, O=C–CH₃), 1.66 – 1.36 (m, 4H, CH₂–CH₂–CH₂), 1.24 (d, ³J = 7.1 Hz, 3H, –CH₃Ala)
SI, p. 5
Ac–L–Glu–L–Arg–L–Ser–(L–Phe–GLY)–GLY–L–Ala (ERSFGGA)
Identifier 3
HRMS (ESI–MS) Calculated m/z 765.3526 [M+H]⁺; found 765.3530 [M+H]⁺
¹H NMR (400 MHz, DMSO-d₆) δ [ppm] = 8.91 (t, ³J = 5.8 Hz, 1H, –NHGly), 8.81 (t, ³J = 5.7 Hz, 1H, –NHGly), 8.49 – 8.24 (m, 3H, –C=NHArg, –NH₂Phe), 8.11 – 7.98 (m, 2H, HN=C–NH₂Arg), 7.91 (d, ³J = 6.9 Hz, 1H, –HN=C–NHArg), 7.42 – 7.06 (m, 9H, –HAr, –NHAla, Ser, Glu, Arg), 4.58 (dd, ³J = 7.9 Hz, 5.8 Hz, 1H, –NH–CHSer), 4.38 – 4.13 (m, 4H, –NH–CHGlu, Arg, –CH₂Ser–O), 4.13 – 4.00 (m, 3H, –NH–CHAla, –NH–CH₂Gly), 3.99 – 3.88 (m, 1H, –CHPhe–NH₂), 3.85 (d, ³J = 5.6 Hz, 2H, –NH–CH₂Gly), 3.83 – 3.72 (m, 2H, NH–CH₂Arg, TFA salt), 3.71 – 3.61 (m, 2H, NH–CH₂Arg), 3.18 – 3.00 (m, 2H, –CH–CH₂Phe), 2.99 – 2.86 (m, 2H, HOOC–CH₂Glu), 2.30 – 2.17 (m, 2H, HOOC–CH₂–CH₂Glu), 1.98 (s, 1H, –NHArg), 1.84 (s, 3H, O=C–CH₃), 1.79 – 1.63 (m, 2H, –CH–CH₂–CH₂Arg), 1.60 – 1.36 (m, 2H, NH–CH₂–CH₂Arg–), 1.24 (d, 3H, –CH₃Ala)
SI, p. 6
Ac–L–His–L–Arg–L–Ser–(L–Phe–GLY)–GLY–L–Ala (HRSFGGA)
Identifier 4
HRMS (ESI–MS) Calculated m/z 773.3689 [M+H]⁺; found 773.3676 [M+H]⁺
¹H NMR (400 MHz, DMSO-d₆) δ [ppm] = 8.94 (t, ³J = 5.8 Hz, 5.8 Hz, 1H, –NHGly), 8.30 (d, ³J = 7.8 Hz, 1H, –NHHis), 8.26 – 8.20 (m, 2H, –NHArg, Ser), 8.19 – 8.15 (t, ³J = 4.5 Hz, 5.9 Hz 1H, –NHPhe), 8.13 (s, 1H, HN=C–NH₂), 8.05 (d, ³J = 7.1 Hz, 1H, –NHAla), 7.91 – 7.83 (m, 1H, –C=NH–), 7.75 (t, ³J = 5.8 Hz, 5.8 Hz, 1H, –HN=C–NH–), 7.33 (d, ³J = 1.3 Hz, 1H, –HAr), 7.28 (s, 1H, HN=C–NH₂), 7.27–7.21 (m, 3H, –HAr), 7.12 (dd, ³J = 7.5 Hz, 2.0 Hz, 2H, –HAr), 4.57 (d, ³J = 5.2 Hz, 1H, –CHHis), 4.28 (td, ³J = 7.9 Hz, 7.9 Hz, 5.0 Hz, 1H, –CHArg), 4.24 – 4.16 (m, 1H, –CHAla), 4.14 (t, ³J = 7.2 Hz, 7.2 Hz, 1H, –CHPhe), 4.05 – 4.00 (m, 1H, –CHSer), 3.80 (dd, ³J = 5.8 Hz, 1H, –CH₂Gly–), 3.67 (dd, 2H, –O–CH₂–),3.64 – 3.47 (m, 2H, –CH₂Gly′), 3.31 (dd, ²J = 7.4 Hz, ³J = 2.9 Hz, 1H, –CH₂His), 3.06 (dq, ²J = 13.4 Hz, ³J = 4.8 Hz, 3H, –CH₂Gly, –CH₂–CH₂–CH₂–NH–), 2.95 (ddd, ³J = 7.9 Hz, ³J = 5.8 Hz, 1H, –CH–NH=CH–), 2.85 (dd, ²J = 13.5 Hz, ³J = 5.0 Hz, 1H, –CH₂Phe), 2.74 – 2.67 (m, 1H, –CH₂Phe), 1.81 (s, 3H, O=C–CH₃), 1.75 – 1.42 (m, 4H, –CH₂–CH₂–CH₂–), 1.22 (d, ³J = 7.3 Hz, 3H, –CH₃Ala)
SI, p. 7
Ac–L–Trp–L–Arg–L–Ser–(L–Phe–GLY)–GLY–L–Ala (WRSFGGA)
Identifier 5
HRMS (ESI–MS) Calculated m/z 822.3893 [M+H]⁺; found 822.3870 [M+H]⁺
¹H NMR (400 MHz, DMSO-d₆) δ [ppm] = 10.86 (d, ³J = 2.4 Hz, 1H, NH=C–NH–), 9.15 (t, ³J = 5.8 Hz, 1H, –NHAla), 8.38 (t, ³J = 5.9 Hz, 1H, –NHPhe), 8.32 (d, 1H, –NHSer), 8.30 (s, 3H HN=C–NH), 8.24 (d, ³J = 7.7 Hz, 1H, –NHGly), 8.19 (dd, ³J = 7.5 Hz, 2.8 Hz, 2H, –NHArg, –NHTrp), 7.82 (t, ³J = 5.8 Hz, 1H, HN=C–NH–) 7.62 (d, 1H, –NH–CH=C–), 7.45 (s, 1H, –C=NH), 7.38 – 7.28 (m, 6H, –HAr, HN=C–NH–), 7.19 – 7.15 (m, 1H, –HAr), 7.06 – 6.92 (m, 3H, –HAr), 4.62 – 4.55 (m, 1H, –CHSer), 4.55 – 4.49 (m, 1H, –CHTrp), 4.30 (ddd, ³J = 11.8 Hz, ³J = 6.6 Hz, 1H, –CHArg), 4.23 – 4.19 (m, 1H, –CHAla), 4.14 (m, 1H, –CHPhe), 3.96 (d, ³J = 5.7 Hz, 2H, –CH₂Gly, –O–CH₂), 3.77 (dd, ²J = 15.1 Hz, ³J = 5.8 Hz, 2H, –CH₂Gly′), 3.23 – 3.13 (m, 1H, –CH=C–CH₂–), 3.10 (q, ³J = 4.9 Hz, 2H, –CH₂–CH₂–CH₂–NH–), 3.05 – 2.87 (m, 2H, –CH₂Phe, –CH=C–CH₂–), 1.78 (s, 3H, O=C–CH₃), 1.73 – 1.34 (m, 4H, –CH₂–CH₂–CH₂–), 1.28 (d, ³J = 7.3 Hz, 3H, –CH₃Ala)
SI, p. 8
L–Phe(1(2–nitrophenyl)ethyl–N–succinimidyl carbonate)–GLY (O–NB–FG)
HRMS (ESI–MS) Calculated m/z 416.1452 [M+H]⁺; found 416.1453 [M+H]⁺
¹H NMR (400 MHz, DMSO-d₆) δ [ppm] = 8.02 – 7.91 (m, 1H, –NHGly), 7.82 – 7.39 (m, 5H, –HAr), 7.30 – 7.11 (m, 4H, –HAr), 5.86 (dd, ³J = 6.2 Hz 6.2 Hz. 1H, –O–CH–), 5.21 – 5.07 (m, 1H, –NHPhe), 4.20 – 3.88 (m, 1H, –CHPhe), 3.64 – 3.42 (m, 2H, –CH₂Gly), 3.18 – 2.89 (m, 1H, –CH₂Phe), 2.76 (d, ³J = 7.5 Hz, 1H, –CH₂Phe), 1.43 (ddd, ²J = 24.40 Hz, 19.12 Hz, ³J = 6.5 Hz, 3H, –O–CH–CH₃)
SI, p. 9
Ac–L–Tyr(OtBu)–L–Arg(Pbf)–L–Ser–GLY–L–Ala–propargyl
HRMS (ESI–MS) Calculated m/z 941.4627 [M+H]⁺; found 941.4611 [M+H]⁺
¹H NMR (400 MHz, DMSO-d₆) δ [ppm] = 8.34 – 8.21 (m, 1H, –NHGly), 8.19 – 8.15 (m, 1H, HC≡C–CH₂–NH–), 8.15 – 8.07 (m, 2H, –NHSer, –NHTyr), 8.05 – 7.92 (m, 2H, –NHArg, –NHAla), 7.18 – 7.10 (m, 2H. –HAr), 6.88 – 6.80 (m, 2H, –HAr), 6.40 (s, 1H, –C=NH), 5.14 – 5.01 (m, 1H, –HN=C–NH–CH₂–), 4.51 – 4.42 (m, 1H, –CHTyr), 4.34 – 4.18 (m, 2H, –CHArg, –CHSer), 3.83 (dd, ³J = 5.6 Hz, 2.45 Hz, 2H, –CH₂Gly), 3.80 – 3.65 (m, 2H, CH≡C–CH₂–), 3.65 – 3.51 (m, 2H, –CH₂Ser), 3.09 (s, 1H, –NH–S–), 3.07 – 2.99 (m, 2H, –CH₂–CH₂–CH₂–NH), 2.96 (s, 2H, –O–C(CH₃)₂–CH₂–), 2.93 (d, ³J = 4.0 Hz, 1H, –CH₂Tyr), 2.67 (dd, ²J = 14.1 Hz, ³J = 10.24 Hz, 1H, –CH₂Tyr), 2.48 (s, 3H, –CAr(CH₃)), 2.42 (s, 3H, –CAr(CH₃)), 2.00 (s, 3H, –CAr(CH₃)), 1.86 (s, 1H, –C≡CH), 1.74 (s, 3H, O=C–CH₃), 1.74 – 1.44 (m, 4H, –CH₂–CH₂–CH₂–), 1.40 (s, 6H, –O–C(CH₃)₂–CH₂–), 1.25 (s, 9H, –O–C(CH₃)₃), 1.19 (d, ³J = 7.1 Hz, 3H, –CH₃Ala)
SI, p. 11
Ac–L–Tyr(OtBu)–L–Arg(Pbf)–L–Ser(L–Phe(1(2–nitrophenyl)ethyl–N–succinimidyl carbonate)–GLY)–GLY–L–Ala–propargyl
LC–MS 1337.8 [M+H]⁺; calculated 1337.6
SI, p. 11
Ac–L–Tyr–L–Arg–L–Ser(L–Phe(1(2–nitrophenyl)ethyl–N–succinimidyl carbonate)–GLY)–GLY–L–Ala–propargyl (YRSo–NBFGGA–propargyl)
Identifier 7
HRMS (ESI–MS) Calculated m/z 1029.4425 [M+H]⁺; found 1029.4435 [M+H]⁺
¹H NMR (400 MHz, DMSO-d₆) δ [ppm] = 8.55 – 8.45 (m, 1H, –NHGly), 8.42 – 8.24 (m, 4H, –NHGly′, –NHPhe, –NHSer, HC≡C–CH₂–NH–), 8.12 (t, ³J = 7.4 Hz, 2H, –NHAla, –NHArg), 7.97 – 7.89 (m, 1H, –HAr), 7.81 – 7.65 (m, 4H, HC≡C–CH₂–NH–, HN=C–NH–CH₂–,–HAr,), 7.61 – 7.44 (m, 1H, –HAr), 7.43 – 7.38 (m, 1H, –HAr), 7.27 (d, ³J = 4.4 Hz, 2H, –HAr), 7.24 – 7.11 (m, 2H, –HAr), 7.06 – 6.97 (m, 2H, –HAr), 6.63 (d, ³J = 8.2 Hz, 2H, –HAr), 5.94 –5.74 (m, 1H, , –O–CH–), 4.56 (d, ³J = 8.7 Hz, 1H, –CHSer), 4.43 (td, ³J = 8.8 Hz, ³J = 3.9 Hz, 1H, –CHTyr), 4.31 – 4.08 (m, 5H, –CHAla, –CHArg, –CHPhe, –CH₂Ser), 3.84 (dt, ³J = 5.5 Hz, ³J = 2.1 Hz, 4H, –CH₂Gly, CH≡C–CH₂–), 3.76 (q, ³J = 5.4 Hz, 2H, –CH₂Gly′), 3.14 – 3.07 (m, 1H, –CH₂Tyr), 3.07 – 3.00 (m, 2H, –CH₂Tyr, –CH₂–CH₂–CH₂–NH–), 2.89 (d, ²J = 13.7 Hz, 1H, –CH₂Phe), 2.80 –2.69 (m, 1H, –CH₂–CH₂–CH₂–NH–, –CH₂Phe), 2.69 – 2.59 (m, 1H, –CH₂Tyr), 2.54 (s, 1H, –C≡CH), 1.75 (s, 3H, O=C–CH₃), 1.72 – 1.46 (m, 2H, –CH₂–CH₂–CH₂–), 1.48 (d, ³J = 6.5 Hz, 3H, –O–CH–CH₃), 1.41 – 1.39 (m, 2H, –CH₂–CH₂–CH₂–), 1.19 (dd, ³J = 7.2 Hz, 2.41 Hz 3H, –CH₃Ala)
SI, pp. 11–12
Ac–L–Tyr–L–Arg–L–Ser(L–Phe–Gly)–Gly–L–Ala–propargyl
LC–MS (ESI–MS) Calculated m/z 835.4 [M+H]⁺; found 835.6 [M+H]⁺
¹H NMR (400 MHz, DMSO-d₆) δ [ppm] = 8.54 – 8.44 (m, 1H, –NHGly), 8.40 – 8.23 (m, 3H, –NHGly′, –NHPhe, –NHSer), 8.23 – 8.13 (m, 2H, HC≡C–CH₂–NH–, –NHAla), 8.13 – 8.06 (m, 2H, –NH₂Phe), 8.06 – 7.96 (m, 1H, –NHTyr), 7.89 (s, 1H, –NHArg), 7.71 – 7.42 (m, 4H, HC≡C–CH₂–NH–, HN=C–NH–CH₂–, –NH₂Arg), 7.32 – 7.24 (m, 2H, –HAr), 7.22 (d, ³J = 8.2 Hz, 1H, –HAr), 7.17 (dd, J = 7.5, 2.0 Hz, 2H, –HAr), 7.03 (d, ³J = 8.6 Hz, 2H, –HAr), 6.63 (d, ³J = 8.6 Hz, 2H, –HAr), 4.48 – 4.37 (m, 1H, –CH₂Ser), 4.31 (d, ³J = 5.0 Hz, 1H, –CHTyr), 4.29 – 4.18 (m, 2H, –CHArg, –CHAla), 4.08 – 4.01 (m, 1H, –CHPhe), 3.91 – 3.79 (m, 2H, CH≡C–CH₂–), 3.79 – 3.69 (m, 2H), 3.13 – 2.95 (m, 6H, –CH₂Gly′, –CH₂Gly, –CH₂Ser), 2.90 - 2.86 (m, 2H, –CH₂Phe), 2.69 – 2.56 (m, 2H, –CH₂Tyr), 2.54 (s, 1H, –C≡CH), 1.75 (s, 3H, O=C–CH₃), 1.64 – 1.53 (m, 2H, –CH₂–CH₂–CH₂–), 1.47 (b, 3H, –O–CH–CH₃), 1.23 (m, 2H, –CH₂–CH₂–CH₂–), 1.19 (d, ³J = 7.2 Hz, 3H, –CH₃Ala)
SI, p. 12
Ac–L–Glu(OtBu)–L–Arg(Pbf)–L–Ser–GLY–L–Ala–propargyl
HRMS (ESI–MS) Calculated m/z 906.4390 [M+H]⁺; found 906.4381 [M+H]⁺
¹H NMR (400 MHz, DMSO-d₆) δ [ppm] = 8.30 -8.23 (m, 1H, –NHGly), 8.18 – 8.11 (m, 1H, –C=NHArg), 8.01 – 7.91 (m, 5H, –NHAla,–NHSer, –NHGlu,–NH Arg, HC≡C–CH₂–NH–), 5.17 – 4.90 (m, 1H, –CH₂–OH), 4.33 – 4.18 (m, 5H, –CHGlu, –CH Arg, –CH Ser, –CH Ala), 3.89 – 3.48 (m, 8H, –CH₂Gly, –CH₂Arg–, –CH₂–C≡C–, –CH₂Ser), 3.09 (s, 1H, –C≡CH), 3.07 – 2.92 (m, 2H, -CHOOC–CH₂), 2.54 (s, 2H, CH≡C–CH₂–), 2.47 (s, 3H, –CAr(CH₃)), 2.42 (s, 3H, –CAr(CH₃)), 2.26 – 2.15 (m, 2H, –CH₂–CH₂Glu–), 2.00 (s, 3H, –CAr(CH₃)–), 1.89 (s, 1H, –SO₂–NH–), 1.84 (s, 3H, O=C–CH₃), 1.75 – 1.62 (m, 2H, –CH₂–CH₂–CH₂Arg), 1.44 – 1.35 (m, 17H, –CH₃Pbf, Boc, NH–CH₂–CH₂Arg), 1.19 (d, ³J = 7.2 Hz, 3H, –CH₃Ala)
SI, p. 13
Ac–L–Glu(OtBu)–L–Arg(Pbf)–L–Ser(L–Phe(1(2–nitrophenyl)ethyl–N–succinimidyl carbonate)–GLY)–GLY–L–Ala–propargyl
HRMS (ESI–MS) Calculated m/z 995.4217 [M+H]⁺; found 995.4218 [M+H]⁺
¹H NMR (400 MHz, DMSO-d₆) δ [ppm] = 8.60 – 8.53 (m, 1H, –NHGly), 8.44 – 8.38 (m, 1H, –NHGly′), 8.30 – 8.24 (m, 3H, –NHAla,–NHSer, –NHGlu), 8.08 – 7.98 (m, 3H, –NHArg,HC≡C–CH₂–NH–, –NHPhe), 7.97 - 7.88 (m, 1H, –C=NHArg), 7.77 – 7.63 (m, 2H,-HAr), 7.60 – 7.47 (m, 2H, -HAr), 7.35 – 7.07 (m, 5H, -HAr), 5.93 – 5.76 (m, 1H, –CHPhe–), 4.62 – 4.47 (m, 1H, –CH Ser), 4.32 – 4.06 (m, 6H, –CHGlu, –CH Arg, –CH Phe, –CH Ala, –CH₂Gly′), 3.98 – 3.62 (m, 6H, –CH₂Ser–O–, –CH₂–C≡C–, –CH₂Gly), 3.13 – 2.90 (m, 6H, –C≡CH, –CH₂–CH₂–CH₂Arg, –NHArg–, –CH₂Ser), 2.80 – 2.63 (m, 1H, –NH–SO₂–), 2.47 (s, 3H, –CH₃Arg, Pbf), 2.42 (s, 3H, –CH₃Arg, Pbf), 2.26 – 2.12 (m, 2H, –CH₂Arg–), 2.00 (s, 3H, –CH₃Arg, Pbf), 1.90 – 1.77 (m, 5H, O=C–CH₃, –CH₂–CH₂–CH₂Arg), 1.75 – 1.60 (m, 2H, –CH₂–CH₂–CH₂Arg), 1.54 – 1.41 (m, 3H, –CH₃o-NB)1.46 – 1.31 (m, 15H, –CH₃Arg, –CH₃Glu), 1.19 (dd, 3H, ³J = 7.2 Hz, 2.7 Hz,–CH₃Ala)
SI, p. 14
Ac–L–Glu–L–Arg–L–Ser(L–Phe(1(2–nitrophenyl)ethyl–N–succinimidyl carbonate)–GLY)–GLY–L–Ala–propargyl (ERSo–NBFGGA–propargyl)
Identifier 8
HRMS (ESI–MS) Calculated m/z 906.4390 [M+H]⁺; found 906.4381 [M+H]⁺
¹H NMR (400 MHz, DMSO-d₆) δ [ppm] = 9.20 (b, 1H, -NHGly), 8.781 – 8.71 (m, 1H, -NHGly), 8.63 – 8.50 (m, 1H, -C=NHArg), 8.43 – 8.27 (m, 4H, -NH₂Phe, HN=C-NH₂Arg), 8.13 – 8.04 (m, 1H, -NHPropargyl), 8.00 – 7.87 - (d, 1H, -NHArg), 7.82 – 7.04 (m, 12H, -HAr, NHAla, Ser, Glu), 5.91 – 5.80 (m, 1H, -CH-O-), 4.65 – 4.44 (m, 1H, -NH-CHSer), 4.41 – 4.03 (m, 7H, -NH-CHGlu, Arg, -CH₂Ser-O-, -NH-CH₂Gly, -NH-CHAla), 4.00 – 3.63 (m, 7H, -CH₂-C≡C-, -CHPhe-NH₂, -NH-CH₂Gly, NH-CH₂Arg-), 3.14 – 2.91 (m, 5H, -C≡CH, NH-CH₂Arg-, -CH-CH₂Phe-), 2.82 – 2.61 m, 2H, HOOC-CH₂Glu-), 2.27 – 2.05 (m, 2H, HOOC-CH₂-CH₂Glu-), 1.96 (s, 1H, -NHArg), 1.89 – 1.65 (m, 5H, O=C-CH₃, -CH-CH₂-CH₂Arg-), 1.66 – 1.33 (m, 5H, -CH₃, NH-CH₂-CH₂Arg-), 1.32 – 1.13 (m, 3H, -CH₃Ala)
SI, pp. 14–15
Ac–L–His(Trt)–L–Arg(Pbf)–L–Ser–GLY–L–Ala–propargyl
HRMS (ESI–MS) Calculated m/z 1101.5052 [M+H]⁺; found 1101.5037 [M+H]⁺
¹H NMR (400 MHz, DMSO-d₆) δ [ppm] = 8.32 – 8.27 (m, 1H, –NHGly), 8.25 – 8.18 (m, 1H, –NHArg), 8.15 (d, ³J = 7.6 Hz, 1H, HC≡C–CH₂–NH–), 8.12 – 8.06 (m, 1H, –NHAla), 7.99 (d, ³J = 8.4 Hz, 1H, –NHHis), 7.96 – 7.90 (m, 1H, –NHSer), 7.47 – 7.35 (m, 9H, –HAr), 7.32 – 7.27 (m, 1H, –N=CH–), 7.18 – 7.00 (m, 6H, –HAr), 6.79 – 6.62 (m, 1H, –N=CH–N–), 6.44 (s, 1H,-C=NH), 5.32 – 4.86 (m, 1H, –HN=C–NH–CH₂–), 4.52 – 4.42 (m, 1H, –CHHis), 4.29 – 4.18 (m, 3H, –CHAla, –CHArg, –CHSer), 3.87 – 3.80 (m, 2H, –CH₂Gly), 3.77 –3.51 (m, 4H, –CH₂Ser, CH≡C–CH₂–), 3.08 (s, 1H, –NH–S–), 3.03 – 2.99 (m, 2H, –CH₂–CH₂–CH₂–NH–), 2.95 (s, 2H, –O–C(CH₃)₂–CH₂–), 2.87 (dd, ²J = 14.7 Hz, ³J = 4.5 Hz, 1H, CH₂His), 2.74 – 2.63 (m, 1H, –CH₂His), 2.47 (s, 3H, –CAr(CH₃)), 2.42 (s, 3H, –CAr(CH₃)), 2.00 (s, 3H, –CAr(CH₃)), 1.89 (s, 1H, –C≡CH), 1.76 (s, 3H, O=C–CH₃), 1.74 – 1.44 (m, 4H, –CH₂–CH₂–CH₂–), 1.40 (s, 6H, –O–C(CH₃)₂–CH₂–), 1.18 (d, ³J = 4.0 Hz, 3H, –CH₃Ala)
SI, p. 15
Ac–L–His(Trt)–L–Arg(Pbf)–L–Ser(L–Phe(1(2–nitrophenyl)ethyl–N–succinimidyl carbonate)–GLY)–GLY–L–Ala–propargyl
LC–MS 1497.9 [M+H]⁺; calculated 1497.6
SI, p. 16
HRSo–NBFGGA–propargyl
Identifier 9
HRMS (ESI–MS) Calculated m/z 1003.4381 [M+H]⁺; found 1003.4378 [M+H]⁺
¹H NMR (400 MHz, DMSO-d₆) δ [ppm] = 9.15 – 8.79 (m, 1H, –NHSer), 8.63 – 8.47 (m, 1H, –NHGly), 8.41 – 8.31 (m, 2H, –NHArg, –NHPhe), 8.29 – 8.21 (m, 1H, –NHGly′), 8.12 – 8.06 (m, 2H, –NHAla, –NHHis), 7.94 (ddd, ³J = 12.2 Hz, 8.2 Hz, ⁴J = 1.28, 1H, –HAr), 7.81 – 7.63 (m, 4H, HN=C–NH–CH₂–, HC≡C–CH₂–NH– –HAr), 7.60 – 7.50 (m, 2H, –HAr), 7.42 (dd, ³J = 7.9 Hz, ⁴J = 1.42 Hz, 1H, –HAr), 7.35 – 7.25 (m, 2H, –HAr), 7.23 – 7.14 (m, 3H, –HAr), 6.82 (s, 1H, HN=C–NH–CH₂–), 5.92 – 5.78 (m, 1H, –O–CH–), 4.70 – 4.53 (m, 1H, –NHSer), 4.47 (d, ³J = 7.7 Hz, 1H, –NHHis), 4.33 (d, ³J = 6.0 Hz, 1H, –CHPhe), 4.30 – 4.15 (m, 4H, –CHAla, –CHArg, –CH₂Ser), 3.99 – 3.81 (m, 4H, –CHGly, CH≡C–CH₂–), 3.75 (m, 2H, –CH₂Gly), 3.09 – 3.07 (m, 1H, =CH–NH–CH=), 3.01 (d, ³J = 7.7 Hz, 3H, –CH₂His, –CH₂–CH₂–CH₂–NH–), 2.94 – 2.79 (m, 2H, –CH₂Phe), 2.7 - 2.62 (m, 1H, –CH₂–CH₂–CH₂–NH–), 2.54 (s, 1H, –C≡CH), 1.84 (d, ³J = 3.3 Hz, 3H, –O–CH–CH₃), 1.80 (s, 3H, O=C–CH₃ ), 1.78 – 1.54 (m, 2H, –CH₂–CH₂–CH₂–), 1.45 (d, ³J = 6.5 Hz, 1H, –CH–CH₂–CH₂–), 1.20 (d, ³J = 7.2 Hz, 1H, –CH₃)
SI, p. 16
Ac–L–Trp(Boc)–L–Arg(Pbf)–L–Ser–GLY–L–Ala–propargyl
HRMS (ESI–MS) Calculated m/z 1008.4685 [M+H]⁺; found 1008.4685 [M+H]⁺
¹H NMR (400 MHz, DMSO-d₆) δ [ppm] = 8.30 – 8.11 (m, 4H, –NHArg, –NHGly, –NHHis, –NHSer), 7.99 – 7.95 (m, 3H, –NHAla, HC≡C–CH₂–NH–, –HAr), 7.71 (d, ³J = 7.7 Hz, 1H, –HAr), 7.51 (s, 1H, –C=CH–N), 7.27 (m, 2H, –HAr), 6.38 (s, 1H, –C=NH), 5.14 – 5.01 (m, 1H, –HN=C–NH–CH₂–), 4.65 – 4.56 (m, 1H, –CHHis), 4.33 – 4.16 (m, 3H, –CHAla, –CHArg, –CHSer), 3.82 (dd, ³J = 5.6 Hz, 2.5 Hz, 2H, –CH₂Gly), 3.74 (m, 2H, CH≡C–CH₂–), 3.65 (dt, ²J = 11.7 Hz, ³J = 5.5 Hz, 1H, –CH₂Ser), 3.55 (dd, ²J = 11.7 Hz, ³J = 5.0 Hz, 1H, –CH₂Ser), 3.07 (s, 1H, –NH–S–), 3.06 – 3.01 (m, 1H, –CH₂–CH₂–CH₂–NH–), 2.95 (s, 1H, –O–C(CH₃)₂–CH₂–), 2.86 (dd, ²J = 14.9 Hz, ³J = 5.1 Hz, 2H, –CH₂His), 2.48 (s, 3H, –CAr(CH₃)), 2.42 (s, 3H, –CAr(CH₃)), 2.00 (s, 3H, –CAr(CH₃)), 1.89 (s, 1H, –C≡CH), 1.76 (s, 3H; O=C–CH₃), 1.72 – 1.65 (m, 2H; –CH–CH₂–CH₂–), 1.61 (s, 9H, –O–C(CH₃)₃), 1.55 –1.47 (m, 2H; –CH₂–CH₂–CH₂–), 1.39 (s, 6H, –O–C(CH₃)₂–CH₂–), 1.18 (dd, ³J = 7.2 Hz, 2.9 Hz, 3H, –CH₃Ala)
SI, p. 17
Ac–L–Trp(Boc)–L–Arg(Pbf)–L–Ser(L–Phe(1(2–nitrophenyl)ethyl–N–succinimidyl carbonate)–GLY)–GLY–L–Ala–propargyl
LC–MS 1404.8 [M+H]⁺; calculated m/z 1404.6 [M+H]⁺
SI, p. 18
Ac–L–Trp–L–Arg–L–Ser(L–Phe(1(2–nitrophenyl)ethyl–N–succinimidyl carbonate)–GLY)–GLY–L–Ala–propargyl (WRSo–NBFGGA–propargyl)
Identifier 10
HRMS (ESI–MS) Calculated m/z 1052.4585 [M+H]⁺; found 1052.4590 [M+H]⁺
¹H NMR (400 MHz, DMSO-d₆) δ [ppm] = 10.86 (s, 1H, =CH–NH–C=), 8.97 – 8.77 (m, 1H, –NHGly), 8.55 – 8.44 (m,1H, –NHTrp), 8.44 – 8.31 (m, 4H, –NHAla, –NHArg, –NHGly′, –NHSer), 8.14 (m, 2H, –NHPhe, HN=C–NH–CH₂), 7.94 (ddd, ³J = 9.7 Hz, 8.2 Hz, ⁴J = 1.3 Hz 1H, –HAr), 7.82 – 7.59 (m, 5H, HC≡C–CH₂–NH–, –HAr), 7.56 – 7.49 (m, 6.6 Hz, 1H, –HAr), 7.35 – 7.23 (m, 3H, HN=C–NH–CH₂, –HAr), 7.22 – 7.13 (m, 3H, –HAr), 7.08 – 6.99 (m, 1H, –HAr), 6.97(td, ³J = 7.4 Hz, 1H, –HAr), 5.92 – 5.80 (m, 1H, –O–CH–), 4.63 – 4.52 (m, 2H, –CHSer, –CHTrp), 4.39 – 4.11 (m, 5H, –CHAla, –CHArg, –CHPhe, –CH₂Ser), 3.92 – 3.80 (m, 4H, CH≡C–CH₂–, –CH₂Gy), 3.78 (d, ³J = 5.8 Hz, 2H, –CH₂Gly′), 3.16 – 3.10 (m, 1H, –CH₂Trp), 3.10 – 3.08 (m, 1H, –CH₂Trp), 3.06 – 3.00 (m, 2H, –CH₂Phe, –CH₂–CH₂–CH₂–NH–), 2.99 – 2.87 (m, 1H, –CHPhe), 2.80 – 2.66 (m, 2H, –CH₂–CH₂–CH₂–NH–), 2.55 (s, 2H, –C≡CH), 1.77 (s, 3H, O=C–CH₃), 1.75 – 1.54 (m, 2H, –CH₂–CH₂–CH₂–NH–), 1.48 (d, ³J = 6.5 Hz, 3H, –O–CH–CH₃), 1.46 – 1.40 (m, 2H, –CH₂–CH₂–CH₂–NH–), 1.20 (d, ³J = 7.2 Hz, 3H, –CH₃Ala)
LC–MS, hydrolysis conditions, Main report Phosphate buffer, pH 7.0; 25 °C
LC–MS, hydrolysis conditions, SI report 5 mg mL⁻¹ in 10 mM phosphate buffer, pH 7.0; room temperature in a thermoshaker; aliquots diluted to 1 mg mL⁻¹ before analysis; peak areas of the depsipeptide and hydrolyzed product oNB–FG–COOH integrated and relative percentages calculated
LC–MS, gradient, time/A/B (%) 0.00/95.00/5.00; 0.50/95.00/5.00; 1.50/80.00/20.00; 10.00/50.00/50.00; 12.00/1.00/99.00; 13.00/1.00/99.00; 13.50/95.00/5.00
LC–MS, hydrolysis after 15 min ~18 %
LC–MS, hydrolysis after 24 h, Main report 40 %
LC–MS, hydrolysis after 24 h, SI report 40.13 %
Main, p. 9; SI, pp. 18, 33, 35
o-NB–FGSGA
LC–MS Calculated m/z 631.2 [M+H]⁺; found 631.4 [M+H]⁺
SI, p. 19
L–Phe(1(2–nitrophenyl)ethyl–N–succinimidyl carbonate)–GLY–L–Arg–L–Ser–GLY–L–Ala–propargyl (O–NB–FGSGA–propargyl)
Identifier 6
LC–MS Calculated 668.2675 [M+H]⁺; found 668.2674 [M+H]⁺
¹H NMR (400 MHz, DMSO-d₆) δ [ppm] = 8.42 – 8.02 (m, 4H, –NHAla, –NHGly, –NHGly, HC≡C–CH₂–NH–), 8.01 – 7.85 (m, 1H, –NHSer), 7.83 – 7.44 (m, 4H, –HAr), 7.30 – 7.22 (m, 2H, –HAr), 7.20 – 7.15 (m, 3H, –HAr), 5.92 – 5.78 (m, 1H, –O–CH–), 4.25 (qd, ³J = 7.4 Hz, 5.3 Hz, 1H, –CHAla), 4.20 – 4.08 (m, 1H, –CHSer), 3.88 – 3.78 (m, 1H, –CHGly, –CH₂–C≡CH), 3.78 – 3.69 (m, 3H, –CHGly), 3.65 – 3.55 (m, 2H, –CH₂Phe), 3.10 (d, ³J = 2.6 Hz, 1H, –CHPhe), 3.04 – 2.95 (m, 1H, –CH₂Ser), 2.71 (d, ²J = 14.2 Hz, 1H, –CH₂Phe), 1.89 (s, 1H, –C≡CH), 1.45 (dd, ²J = 25.3 Hz, ²J = 6.5 Hz, 3H, –O–CH–CH₃), 1.19 (dt, ³J = 7.3 Hz, 1H, –CH₃Ala)
SI, p. 19
Ac–L–Tyr(OtBu)–L–Arg(Pbf)–L–Ser–GLY–L–Ala–EDANHBoc
HRMS (ESI–MS) Calculated m/z 1045.5387 [M+H]⁺; found 1045.5369 [M+H]⁺
¹H NMR (400 MHz, DMSO-d₆) δ [ppm] = 8.32 – 8.13 (m, 2H, –NHGly, HC≡C–CH₂–NH–), 8.10 (d, ³J = 8.3 Hz, 1H, –NHSer), 8.01 (d, ³J = 7.1 Hz, 1H, –NHTyr), 7.95 (d, ³J = 7.5 Hz, 1H, –NHArg), 7.87 – 7.80 (m, 1H, –NHAla), 7.14 (d, ³J = 8.6 Hz, 2H, –HAr), 6.84 (d, ³J = 8.4 Hz, 2H, –HAr), 6.78 (t, ³J = 5.4 Hz, 1H, –C₂H₄–NH–), 6.43 (s, 1H, –C=NH), 4.54 – 4.41 (m, 1H, –CHTyr), 4.34 – 4.11 (m, 3H, –HN=C–NH–CH₂–, –CHArg, –CHSer), 3.74 (dd, ²J = 11.0 Hz, ³J = 7.8 Hz, 2H, –CH₂Gly), 3.69 – 3.51 (m, 4H, –CH₂–CH₂–), 3.03 (d, ³J = 6.7 Hz, 4H, –CH₂Ser, –CH₂–CH₂–CH₂–NH), 2.96 (s, 5H, –CH₂Tyr, –O–C(CH₃)₂–CH₂–, –NH–S–), 2.67 (dd, ²J = 14.1 Hz, 1H, –CH₂Tyr), 2.48 (s, 3H, –CAr(CH₃)), 2.42 (s, 3H, –CAr(CH₃)), 2.00 (s, 3H, –CAr(CH₃)), 1.87 (s, 1H, –NH–), 1.74 (s, 3H, O=C–CH₃), 1.54 – 1.44 (m, 1H), 1.40 (s, 6H, –O–C(CH₃)₂–CH₂Pbf–), 1.36 (s, 9H, –O–C(CH₃)₃Boc), 1.25 (s, 9H, –O–C(CH₃)₃Tyr), 1.18 (d, ³J = 7.1 Hz, 3H, –CH₃Ala)
SI, p. 21
Ac–L–Tyr(OtBu)–L–Arg(Pbf)–L–Ser(L–Phe(1(2–nitrophenyl)ethyl–N–succinimidyl carbonate)–GLY)–GLY–L–Ala–EDANHBoc
HRMS (ESI–MS) Calculated m/z 1442.6661 [M+H]⁺; found 1442.66 [M+H]⁺
¹H NMR (400 MHz, DMSO-d₆) δ [ppm] = 8.43 (t, ³J = 6.1 Hz, 1H, –NHGly), 8.27 (d, ³J = 7.8 Hz, 1H, –NHGly), 8.24 – 8.19 (m, 1H, HC≡C–CH₂–NH–), 8.15 (d, ³J = 4.4 Hz, 1H, –NHPhe), 8.08 (d, ³J = 8.2 Hz, 1H, –NHSer), 8.02 (d, ³J = 5.0 Hz, 1H, –NHTyr), 7.97 – 7.84 (m, 2H, –NHArg, –NHArg), 7.79 – 7.62 (m, 2H, –HAr), 7.62 – 7.47 (m, 1H, –HAr), 7.27 (d, ³J = 4.5 Hz, 2H, –HAr), 7.24 – 7.06 (m, 6H, –HAr), 6.83 (d, ³J = 8.4 Hz, 2H, –HAr), 6.78 (s, 1H, –C₂H₄–NH–), 6.64 (s, 1H, –C=NH), 6.40 (b, 1H, –CHPhe), 5.94 – 5.78 (m, 1H, –CH(CH₃)–), 4.64 – 4.53 (m, 1H, –CH₂Ser–O–), 4.53 – 4.43 (m, 1H, CH₂Ser–O–), 4.33 – 4.06 (m, 6H, –CHTyr, –HN=C–NH–CH₂–, –CHArg, –CHSer, –CH₂Gly), 3.99 – 3.81 (m, 2H, –CH₂–CArPhe–), 3.76 (s, 2H, –CH₂Gly), 3.03 (s, 5H, –CH₂–CH₂–CH₂–NH, –CH₂–CH₂–), 2.95 (s, 7H, –CH₂Tyr, –NH–S–, –CH₂–CH₂–), 2.80 – 2.59 (m, 2H, –O–C(CH₃)₂–CH₂–), 2.48 (s, 3H, –CAr(CH₃)), 2.42 (s, 3H, –CAr(CH₃)), 2.07 (s, 1H, –NH–), 2.00 (s, 3H, –CAr(CH₃), 1.74 (d, ³J = 4.4 Hz, 4H, O=C–CH₃), 1.48 (d, ³J = 6.6 Hz, 3H, –CH₃), 1.40 (s, 9H, –O–C(CH₃)₂–CH₂Pbf–, –CH₃), 1.36 (s, 9H, –O–C(CH₃)₃Boc), 1.24 (d, J = 2.0 Hz, 9H, –O–C(CH₃)₃Tyr), 1.19 (d, J = 7.1 Hz, 3H, –CH₃Ala)
SI, pp. 21–22
Ac–L–Tyr–L–Arg–L–Ser(L–Phe(1-(2–nitrophenyl)ethyl–N succinimidyl carbonate)–Gly)–Gly–L–Ala–EDANH₂
Identifier X
HRMS (ESI–MS) Calculated m/z 1034.4691 [M+H]⁺; found 1034.4681 [M+H]⁺
¹H NMR (400 MHz, DMSO-d₆) δ [ppm] = 10.24 (s, 1H, –OH), 8.70 (t, ³J = 6.8 Hz, 1H, –NHGly), 8.53 (t, ³J = 8.6 Hz, 1H, –NHGly′), 8.49 – 8.35 (m, 1H, – HC≡C–CH₂–NH–), 8.07 (d, ³J = 6.2 Hz, 1H, –NHPhe), 8.03 – 7.85 (m, 3H, –NHSer, –NHTyr, –NHArg), 7.81 – 7.58 (m, 3H, –NHAla, –NH₂Arg), 7.58 – 7.46 (m, 1H, –HAr), 7.42 (d, ³J = 7.9 Hz, 1H, –HAr), 7.37 – 7.32 (m, 1H, –HAr), 7.32 – 6.94 (m, 11H, –NH₂, –NHArg–, –HAr), 6.63 (d, ³J = 7.3 Hz, 2H, –HAr), 5.87 (q, ³J = 7.0 Hz, 1H, –CH(CH₃)–), 4.70 – 4.54 (m, 1H, –CHSer), 4.50 – 4.29 (m, 3H, –CHTyr, –CHAla, –CHArg), 4.27 – 4.15 (m, 3H, –CHPhe, –CH₂Ser–O–), 4.15 – 4.04 (m, 2H, –CH₂Gly′), 4.02 – 3.89 (m, 2H, –NH–CH₂–CH₂–), 3.88 – 3.79 (m, 2H, –CH₂Gly), 3.79 – 3.68 (m, 2H, –CH₂Tyr), 3.68 – 3.48 (m, 2H, –CH₂Phe), 3.15 – 2.93 (m, 2H, –CH₂–CH₂–CH₂–), 2.79 – 2.64 (m, 2H, –NH–CH₂–CH₂–NH₂), 1.75 – 1.71 (m, 3H, O=C–CH₃), 1.63 – 1.51 (m, 2H, –CH₂–CH₂–CH₂–), 1.48 (d, ³J = 6.6 Hz, 3H, –CH–CH₃), 1.46 – 1.39 (m, 2H, –CH–CH₂–CH₂–), 1.21 (d, ³J = 7.0 Hz, 3H, –CH₃Ala)
SI, p. 22
Hyaluronic acid–YRSFGGA–EDA conjugate, Batch 1
¹H NMR (400 MHz, D₂O) δ [ppm] = 7.50 – 7.18 (m, 0.30 H, –HArPhe), 7.12 (d, J = 8.1 Hz, 0.13 H, –HArTyr), 6.82 (d, J = 8.0 Hz, 0.13 H, –HArTyr), 4.68 – 4.32 (m, 1H, –CHHA–CO–), 3.91 – 3.62 (m, 4H, –CHHA–), 3.64 – 3.39 (m, 4H, –CHHA–), 3.39 – 3.19 (m, 2H, –NH–CH₂HA–), 1.99 (s, 3H, –NH–CO–CH₃HA), 1.42 – 1.27 (m, 0.24H, –CH₃Ala)
¹H NMR, peptide functionalization, SI report Approximately 6.4 %
Average functionalization degree, Main report 6.4 mol %
¹H NMR, peptide functionalization basis Calculated by normalizing the CH₃ (acetyl) peak of hyaluronic acid to 3 and comparing it to the aromatic proton signals of phenylalanine
Main, p. 14; SI, p. 23
Hyaluronic acid–YRSFGGA–EDA conjugate, Batch 2
¹H NMR, peptide functionalization 15.8 %
SI, p. 23
CB[8] complexation with peptides
Mass spectrometry (ESI), protocol conditions Peptide 0.5 mM and CB[8] 0.25 mM in Milli–Q water; 25 °C; 18 h with gentle shaking; 5 µL injection in bypass mode without a column; 70 % A (20 mM NH₄OAc with 0.1 % HOAc) and 30 % B (acetonitrile) for 0.5 min, then 1 % A and 99 % B over 1.0 min; total 1.5 min
Mass spectrometry (ESI), incubation ratio stated in the analysis Peptide:CB[8] molar ratio of 1:2 in deionised water overnight
YRSFGGA (2), binary complex m/z 1064.8 [2·CB[8]+2H]²⁺; 1:1 complex
YRSFGGA (2), ternary complex No ternary (2:1) complex peaks were observed
YRSFGGA (2), linear peptide YRSGA m/z 595.5
ERSFGGA (3), binary complex m/z 1047.1 [3·CB[8]+2H]²⁺
ERSFGGA (3), ternary complex No ternary (2:1) complex was detected
HRSFGGA (4), binary and ternary complexes Mass signals corresponding to binary or ternary CB[8] complexes were not observed
HRSFGGA (4), linear peptide HRSGA m/z 569.4
WRSFGGA (5), binary and ternary complexes Mass signals corresponding to binary or ternary CB[8] complexes were not observed
WRSFGGA (5), linear peptide WRSGA m/z 618.5
FGSGA (1), ternary complex m/z 1102.4 [(1)₂·CB[8]+2H]²⁺
FGSGA (1), binary complex No signal was detected at m/z 883.8 [1·CB[8]+2H]²⁺
FGSGA (1), hydrolysis products No hydrolysis products were observed
FGSGA (1), mass-spectrum annotations m/z 438.1 [1+H]⁺, 726.3, 875.2 [(1)₂+H]⁺
ERSFGGA (3), mass-spectrum annotations m/z 484.1, 603.2, 765.3 [3+H]⁺
YRSFGGA (2), mass-spectrum annotation m/z 799.6 [2+H]⁺
WRSFGGA (5), mass-spectrum annotation m/z 822.6 [5+H]⁺
Main, p. 5; SI, pp. 24–25
Peptide–CB[8] isothermal titration calorimetry series
ITC, conditions 27 °C; 10 mM phosphate buffer, pH 7.0; peptide 1.35 mM; CB[8] 54 μM; MicroCal PEAQ–ITC; reference power 5 μcal·s⁻¹; 500 rpm; initial injection 0.4 μL over 0.8 s, then 38 injections of 1 μL over 2 s with 150 s spacing; first injection excluded; duplicate titrations
YRSFGGA (2), single-site fit with N fixed at 1, Kᴅ 3.920 ± 0.002 µM
ERSFGGA (3), single-site fit with N fixed at 1, Kᴅ 6.7 ± 0.4 µM
HRSFGGA (4), single-site fit with N fixed at 1, Kᴅ 13.8 ± 1.38 µM
WRSFGGA (5), single-site fit with N fixed at 1, Kᴅ 11.0 ± 0.2 µM
FGSGA (1), sequential-binding fit, overall Kᴅ 1.41 × 10⁻¹¹ M²
FGSGA (1), binding stoichiometry 2
Main, p. 6; SI, p. 26
Peptide–CB[8] ¹H NMR complexation series
¹H NMR, conditions Peptide 1.0 mM; CB[8] 0.5 mM; D₂O; 25 °C; FGSGA pD 7.2–7.3
FGSGA (1), phenylalanine aromatic resonances 7.2 –7.5 ppm (a–c); with CB[8], 6.2 – 6.6 ppm (b′, c′; Δδ ≈ 1.1 ppm) and 5.9 ppm (a′; Δδ ≈ 1.4 ppm)
FGSGA (1), phenylalanine-adjacent benzylic methylene signal d Shifted from 3.2 ppm to 2.2 ppm; Δδ ≈ 1.0 ppm
FGSGA (1), degree of CB[8] complexation Approximately 93 %
YRSFGGA (2), degree of CB[8] complexation 47 %
ERSFGGA (3), degree of CB[8] complexation 49 %
HRSFGGA (4), degree of CB[8] complexation 44 %
WRSFGGA (5), degree of CB[8] complexation 43 %
YRSFGGA (2) and ERSFGGA (3), aromatic resonances Sharp resonances; slow exchange behavior
HRSFGGA (4) and WRSFGGA (5), aromatic resonances Broader peaks; intermediate-to-fast exchange behavior
Switch peptides, benzylic methylene peaks at 3.20 ppm Could not be resolved because of overlap with neighboring peptide signals
Main, pp. 6–8; SI, pp. 27–30, 56
WRSFGGA (5) stability in the absence and presence of CB[8]
LC–MS, conditions 10 mM phosphate buffer, pH 7.0; room temperature; incubated in a thermoshaker; WRSFGGA 0.5 mM; LC–MS results in % for WRSFGGA (5) and WRSGA (12)
LC–MS, gradient, time/A/B (%) 0.00/95.00/5.00; 0.50/95.00/5.00; 2.00/1.00/99.00; 3.00/1.00/99.00; 3.50/95.00/5.00
Without CB[8], 15 min 5: 92.1 %; 12: 7.9 %
Without CB[8], 24 h 5: 12.7 %; 12: 87.3 %
CB[8] 0.25 mM, 15 min 5: 72.5 %; 12: 27.5 %
CB[8] 0.25 mM, 24 h 5: 38.0 %; 12: 62.0 %
CB[8] 0.5 mM, 15 min 5: 76.4 %; 12: 23.6 %
CB[8] 0.5 mM, 24 h 5: 62.4 %; 12: 37.6 %
SI, pp. 30–31
YRSFGGA (2) stability in the absence and presence of CB[8]
LC–MS, conditions 10 mM phosphate buffer, pH 7.0; room temperature; incubated in a thermoshaker; YRSFGGA 0.5 mM; LC–MS results in % for YRSFGGA (2) and YRSGA (13)
LC–MS, gradient, time/A/B (%) 0.00/95.00/5.00; 0.50/95.00/5.00; 2.00/1.00/99.00; 3.00/1.00/99.00; 3.50/95.00/5.00
Without CB[8], 15 min 2: 75.4 %; 13: 24.6 %
Without CB[8], 24 h 2: 34.3 %; 13: 65.7 %
CB[8] 0.25 mM, 15 min 2: 72.1 %; 13: 27.9 %
CB[8] 0.25 mM, 24 h 2: 46.6 %; 13: 53.4 %
CB[8] 0.5 mM, 15 min 2: 72.1 %; 13: 27.9 %
CB[8] 0.5 mM, 24 h 2: 58.5 %; 13: 41.5 %
SI, pp. 30, 32
FG and cyclicFG detected in the depsipeptide stability study
Conditions 10 mM phosphate buffer, pH 7.0; room temperature
FG, assigned mass signal m/z 223.1; tᵣ = 0.73 min
FG sample, other mass-spectrum annotations m/z 245.1, 445.3, 467.3
cyclicFG (3-benzylpiperazine-2,5-dione), assigned mass signal m/z 205.2; tᵣ = 1.37 min
cyclicFG sample, other mass-spectrum annotation m/z 409.3
Main, p. 7; SI, p. 33
YRSFGGA (2) ¹H NMR sampled states
Conditions YRSFGGA 1 mM; D₂O; pD 7.4
YRSFGGA (2), phenylalanine signals a–c δ = 7.2–7.5 ppm
YRSFGGA (2), tyrosine signals d and e δ = 6.8–7.2 ppm
YRSFGGA (2) 1 mM with CB[8] 0.5 mM, incubated with gentle agitation for 10 min and briefly sonicated, phenylalanine signals a′–c′ δ = 5.9–6.6 ppm; approximately half of the phenylalanine aromatic signal intensity shifted
YRSFGGA (2) 1 mM with CB[8] 0.5 mM, incubated with gentle agitation for 10 min and briefly sonicated, tyrosine signals d and e Remained essentially unchanged
YRSFGGA (2) 1 mM with CB[8] 0.5 mM, incubated with gentle agitation for 10 min and briefly sonicated, sample state Faintly turbid
YRSFGGA (2) 1 mM with 100 µL immobilized trypsin (TPCK-treated agarose resin) at 37 °C for 12–16 h, filtration, and CB[8] 0.5 mM, phenylalanine signals a′–c′ Complete upfield shift to δ = 5.9–6.6 ppm
YRSFGGA (2) 1 mM with 100 µL immobilized trypsin (TPCK-treated agarose resin) at 37 °C for 12–16 h, filtration, and CB[8] 0.5 mM, integration Approximate peptide:CB[8] stoichiometry of 2:1
YRSFGGA (2) 1 mM with 100 µL immobilized trypsin (TPCK-treated agarose resin) at 37 °C for 12–16 h, filtration, and CB[8] 0.5 mM, sample state Complete clearing of the previously turbid host solution
Main, pp. 12–13; SI, p. 27
YRSFGGAR′ (2′) LC–MS sampled states
Conditions Phosphate buffer, pH 7.0
After 3 h at room temperature, YRSGAR′ tᵣ = 1.33 and 1.36 min; m/z = 632.4 for both peaks; potentially arising from conformational isomers
After 3 h at room temperature, residual YRSFGGAR′ (2′) tᵣ = 1.40 min; m/z = 836.6
YRSFGGAR′ (2′) 0.50 mM with CB[8] 0.25 mM, vortexed and sonicated for 5 min, immediate LC–MS Parent tᵣ = 1.40 min remained unchanged; new signal at m/z = 1083.3 corresponding to the binary complex 2′·CB[8]; no ternary complex was observed
After adding trypsin to the pre-formed 2′·CB[8] complex at 37 °C for 3 h, YR tᵣ = 0.88 min; m/z = 380.2
After adding trypsin to the pre-formed 2′·CB[8] complex at 37 °C for 3 h, FGSGAR′ (6′) tᵣ = 1.33 min; m/z = 475.3
After adding trypsin to the pre-formed 2′·CB[8] complex at 37 °C for 3 h, FGSGAR′/CB[8] complex m/z = 1140.3; 2:1 ternary complex
Main, pp. 11, 13
HA–YRSFGGA inverted-vial states
Conditions Following overnight incubation at 37 °C
HA–YRSFGGA + trypsin + CB[8] (A) Stable, self-supporting gel
HA–YRSFGGA + CB[8] (B) Modest increase in viscosity
HA–YRSFGGA (C) Remained fluid
Main, pp. 14–15
HA–YRSFGGA, Batch 1, rheology systems A and B
Samples A = HA–YRSFGGA 1.25 wt % + CB[8] 0.3 wt % + trypsin at 1:20 of the peptide-unit molar concentration, stirred at 37 °C for 3 h and then at room temperature overnight; B = HA–YRSFGGA 1.25 wt % + CB[8] 0.3 wt %, stirred at room temperature overnight
Rheology, general conditions Anton Paar MCR 302e; aluminium plate with PP25 geometry; 1 mm gap; PBS; 25 °C
Storage modulus B: 33 ± 2 Pa; A: 70 ± 4 Pa
Damping factor, tan(δ) B: 67 ± 3 %; A: 43 ± 1 %
Shear-rate-dependent rheology A and B exhibited similar shear thinning behavior at shear rates higher than 0.5 s⁻¹
Viscosity A was 32.4 ± 0.8 % higher than B for shear rates up to 16 s⁻¹
Characteristic relaxation time B: 26.0 s; A: 42.8 s; 64 % increase
Stress relaxation, B Initial strain 10 %; single relaxation mode; exponent = −0.59
Stress relaxation, A Initial strain 10 %; bicomponent relaxation system; exponents = −0.36 and −0.53
Stress relaxation after 1 min Initial strain 10 %; B: (1.9 ± 0.1) % residual stress; A: (7 ± 1) % residual stress
Main, pp. 15–16; SI, pp. 2, 39, 41, 56
HA–YRSFGGA, Batch 2, in situ rheology
HA–YRSFGGA 1 wt % conditions HA–YRSFGGA 1 wt %; CB[8] 0.25 equiv; trypsin 0.01 equiv; PBS, pH 7.4; 25 °C
Initial moduli G′ ≈ G″
Storage modulus G′ began increasing after 60 min
Damping factor, tan(δ) Initially 98 %; 25.3 ± 0.9 % after 12 h
Main, pp. 15–16; SI, p. 23
HA–YRSFGGA 2 wt % with CB[8] and trypsin
Amplitude-sweep rheology, storage modulus 195.3 ± 0.3 Pa
Main, p. 16; SI, p. 42
HA–YRSFGGA, Batch 1, with CB[8], ¹H NMR sampled states
Sample 1, conditions HA–YRSFGGA 1.25 wt %; trypsin 9.1 mg; 37 °C for 3 h, then overnight; recovered after rheology; dialyzed against NaCl-containing distilled water for 2 days; lyophilized; redissolved in D₂O
Sample 1, signals Characteristic CB[8] resonances and upfield-shifted phenylalanine aromatic signals were observed; tyrosine signals were absent
Sample 2, conditions HA–YRSFGGA 1.25 wt %; no trypsin; stirred for 1 h; recovered after rheology; dialyzed against NaCl-containing distilled water for 2 days; lyophilized; redissolved in D₂O
Sample 2, signals Only tyrosine aromatic peaks were observed; phenylalanine signals were absent
Peak assignments a′ corresponds to the phenylalanine residue complexed within CB[8]; b corresponds to tyrosine residues