TA-AA synthesis
TA-AA synthesis protocol and TA-Arg and TA-Lys structural characterization by proton and carbon nuclear magnetic resonance (NMR) and mass spectrometric (HRMS) analysis were previously reported [21].
TA-Ala, TA-Asn, TA-Gln, TA-Gly, TA-His, TA-Ile, TA-Met and TA-Val carbon and hydrogen NMR analysis as well as HRMS are listed below:
TA-Ala : 1H NMR (300 MHz, DMSO-d6) δ 8.37 (s, 3H), 7.30 (d, J = 9 Hz, 1H), 6.24 (d, J= 9 Hz, 1H), 6.02 (s, 1H), 5.56 (d, J = 6 Hz, 1H), 5.31 (d, J = 18 Hz, 1H), 4,94 (d, J = 18 Hz, 1H), 4.88, (d, J= 6 Hz, 1H), 4.30 (q, J= 3, 1H), 4.20 (bs, 2H), 2.63 (m, 1H), 2.35 (m, 2H), 2.04-1.54 (m, 6H), 1.50 (t, J= 3, 3H), 1.49 (s, 3H), 1.36 (s, 3H), 1.32 (m, 1H), 1.16 (s, 3H), 0.84 (s, 3H); 13C NMR (300 MHz, DMSO-d6) δ 203.55, 185.85, 170.56, 167.24, 153.09, 129.75, 124.95, 111.71, 102.85-100.52, 97.70, 81.90, 71.03-70.55, 69.06, 48.55-48.25, 48.34, 45.98, 43.52, 36.70, 33.71, 33.19-32.94, 30.76, 28.19, 26.96, 25.99, 23.53, 16.71, 16.75; HRMS (ESI) calculated for C27H37FNO7 [M+H]+: 506.2549, found: 506.2549.
TA-Asn Yield: 92 %; 1H NMR (600MHz, DMSO-d6) δ 8.39 (s, 3H), 7.71 (s, 1H), 7.30 (d, J= 12 Hz, 1H), 7.28 (s, 1H), 6.24 (d, J= 12Hz, 1H), 6.02 (s,1H), 5.51 (d, J= 6Hz, 1H), 5.32 (d, J= 18Hz, 1H), 4.88 (d, J=
6Hz, 1H), 4.87 (d, J= 18Hz, 1H), 4.46 (bs, 1H), 4.21 (bs, 1H), 2.82 (m,2H) 2.62-2.35 (m, 3H), 2.06-1.55 (m, 6H), 1.49 (s,3H), 1.36 (s,3H), 1.34 (m,1H), 1.17 (s,3H), 0.84 (s,3H); 13C NMR (600 MHz, DMSO-d6) δ 203.61, 186.08, 170.91, 169.63, 167.44, 153.32, 129.99, 125.21, 111.98, 102.48-101.31, 97.95, 82.12, 71.21-70.97, 65.84, 49.41, 48.72-48.57, 46.17, 43.77, 36.89, 35.36, 33.95, 33.40-33.27, 31.00, 28.42, 27.22, 26.30, 23.71, 17.03; MS (ESI) calculated for C28H38FN2O8 [M+H]+: 549.3, found: 549.2.
TA-Gln Yield: 91%; 1H NMR (600MHz, DMSO-d6) δ 8.47 (s, 3H), 7.43 (s, 1H), 7.30 (d, J= 12Hz, 1H), 6.98 (s, 1H), 6.25 (d, J= 12Hz, 1H), 6.03 (s, 1H), 5.53 (d, J= 5Hz, 1H), 5.34 (d, J= 18Hz, 1H), 4.90 (d, J=
18Hz, 1H), 4.88 (d, J= 5Hz, 1H), 4.28 (bs, 1H), 4.19 (bs, 1H), 2.60 (m,1H), 2.37 (m, 4H), 2.11-1.55 (m,
8H), 1.49 (s, 3H), 1.36 (s,3H), 1.34 (m, 1H), 1.17 (s, 3H), 0.84 (s, 3H); 13C NMR (600 MHz, DMSO-d6) δ 203.79, 186.08, 174.01, 170.12, 167.45, 153.30, 130.00, 125.20, 111.99, 102.50-101.34, 98.00, 82.17, 70.47- 69.43, 65.85, 52.34, 48.72-48.57, 46.23, 43.76, 36.97, 33.96, 33.39-33.26, 31.00, 30.86, 28.38, 27.23, 26.90, 26.33, 23.76-23.72, 17.02; MS (ESI) calculated for C29H40FN2O8 [M+H]+: 563.3, found:
563.2.
TA-Gly: 1H NMR (300 MHz, DMSO-d6) δ 8.33 (s, 3H), 7.30 (d, J = 9 Hz, 1H), 6.24 (d, J= 9 Hz, 1H), 6.02 (s, 1H), 5.55 (d, J = 6 Hz, 1H), 5.35 (d, J = 18 Hz, 1H), 4,90 (d, J = 18 Hz, 1H), 4.87, (d, J= 6 Hz, 1H) 4.21 (bs, 1H), 4.03 (s, 2H), 2.64 (m, 1H), 2.34 (m, 2H), 2.08-1.57 (m, 6H), 1.49 (s, 3H), 1.36 (s, 3H), 1.32 (m, 1H), 1.16 (s, 3H), 0.82 (s, 3H); 13C NMR (300 MHz, DMSO-d6) δ 203.53, 185.86, 168.10, 167.25, 153.14, 129.76, 124.96, 111.71, 102.84-100.51, 97.71, 81.90, 71.04-70.58, 69.02, 48.55-48.25, 45.94, 43.53, 40.56, 36.67, 33.69, 33.21-32.95, 30.76, 28.17, 26.96, 25.99, 23.45, 16.83; HRMS (ESI) calculated for C26H35FNO7 [M+H]+: 492.2393, found: 492.2392.
TA-His Yield: 42%; 1H NMR (600MHz, DMSO-d6) δ 14.17 (bs, 1H), 8.56 (bs, 3H), 7.40 (s, 1H), 7.39 (s, 1H), 7.31 (d, J= 12Hz, 1H), 6.25 (d, J= 12Hz, 1H), 6.03 (s, 1H), 5.55 (d, J= 6Hz, 1H), 5.35 (d, J= 18, 1H), 4.93 (d, J= 18, 1H), 4.88 (d, J= 6H, 1H), 4.55 (bs, 1H), 4.22 (bs, 1H), 3.23 (m, 2H), 2.61 (m, 1H), 2.34 (m 2H) 2.07-1.56 (m, 6H), 1.49 (s, 3H), 1.36 (s, 3H), 1.35 (m, 1H), 1.14 (s, 3H), 0.83 (s, 3H); 13C NMR (600 MHz, DMSO-d6) δ 203.70, 186.08, 169.13, 167.40, 158.70, 153.28, 135.75, 134.96, 130.00, 125.22, 112.00, 102.49-101.32, 97.93, 82.15, 71.14-70.90, 65.84, 55.63, 48.71, 46.20, 43.77, 36.95, 33.94, 33.38-33.25, 31.29, 28.42, 27.20, 26.23, 24.64, 23.71, 17.03; MS (ESI) calculated for C30H39FN3O7 [M+H]+: 572.3, found: 572.2.
TA-Ile: 1H NMR (300 MHz, DMSO-d6) δ 8.38 (s, 3H), 7.31 (d, J = 12 Hz, 1H), 6.25 (d, J= 12 Hz, 1H), 6.03 (s, 1H), 5.52 (d, J = 6 Hz, 1H), 5.32 (d, J = 18 Hz, 1H), 4,93 (d, J = 18 Hz, 1H), 4.88, (d, J= 6 Hz, 1H) 4.20 (bs, 2H), 2.59 (m, 1H), 2.36(m, 2H), 2.08-1.53 (m, 8H), 1.49 (s, 3H), 1.36 (s, 3H), 1.32 (m, 2H), 1.16 (s, 3H), 1,01 (d, J = 6 Hz, 3H), 0.92 (t, J = 9 Hz, 3H), 0.82 (s, 3H); 13C NMR (300 MHz, DMSO-d6) δ
203.63, 185.84, 169.78, 167.24, 153.09, 129.73, 124.95, 111.70, 102.86-100.53, 97.79, 81.88, 71.02-70.53, 69.00, 56.94, 48.56-48.26, 45.92, 43.49, 37.16, 36.68, 33.71, 33.20-32.94, 30.76, 28.21, 26.98, 26.05, 25.47, 23.46, 16.69, 14.71, 12.36; HRMS (ESI) calculated for C30H42FNO7 [M+H]+: 548.3018, found:
548.3017.
TA-Met Yield: 52%; 1H NMR (600MHz, DMSO-d6) δ 8.47 (s, 3H), 7.30 (d, J= 12Hz, 1H), 6.24 (d, J=
12Hz, 1H), 6.02 (s, 1H), 5.54 (d, J= 6Hz, 1H), 5.34 (d, J= 18Hz, 1H), 4.95 (d, J= 18Hz, 1H), 4.89 (d, J=
6Hz, 1H), 4.33 (bs, 1H), 4.21 (bs, 1H), 2.67 (m,2H), 2.62 (m,1H), 2.45 (m, 1H), 2.35 (m,1H), 2.14 (m, 2H), 2.07 (s, 3H), 2.05-1.54 (m, 6H), 1.49 (s, 3H), 1.36 (s, 3H), 1.34 (m, 1H), 1.16 (s, 3H), 0.84 (s, 3H); 13C NMR (600 MHz, DMSO-d6) δ 203.98, 186.08, 169.99, 167.45, 153.31, 129.99, 125.20, 111.99, 102.50-101.34, 97.99, 82.16, 71.15-70.91, 69.47, 51.72, 48.72-48.57, 46.22, 43.77, 36.94, 33.95, 33.39-33.26, 31.01, 30.77, 29.02, 28.43, 27.22, 26.26, 23.76-23.72, 16.97, 15.16; MS (ESI) calculated for C29H41FNO7S [M+H]+: 566.3, found: 566.2.
TA-Val Yield: 81 %; 1H NMR (300 MHz, DMSO-d6) δ 8.39 (s, 3H), 7.30 (d, J = 9 Hz, 1H), 6.24 (d, J= 9 Hz, 1H), 6.02 (s, 1H), 5.54 (d, J= 6 Hz, 1H), 5.33 (d, J= 18 Hz, 1H), 4,94 (d, J= 18 Hz, 1H), 4.88, (d, J= 6 Hz, 1H) 4.20 (bs, 2H), 2.63 (m, 1H), 2.34 (m, 3H), 2.08-1.57 (m, 6H), 1.49 (s, 3H), 1.36 (s, 3H), 1.32 (m, 1H), 1.16 (s, 3H), 1.06 (t, 6H), 0.82 (s, 3H); 13C NMR (300 MHz, DMSO-d6) δ 203.63, 185.85, 169.40, 167.24, 153.09, 129.74, 124.96, 111.70, 102.87-100.54, 97.75, 81.89, 71.00-70.51, 69.15, 57.65, 48.55-48.25, 45.93, 43.51, 36.68, 33.70, 33.19-32.93, 30.76, 30.25, 28.20, 26.97, 26.00, 23.46, 18.47, 18.09, 16.69; HRMS (ESI) calculated for C29H41FNO7 [M+H]+: 534.3, found: 534.3.
Validation of HPLC-UV method for the quantification of TA and TA-AA
Specificity, linearity, LOD and LOQ
The method specificity and linearity were established in a six point calibration curve in a concentration range from 1.41 to 230.15 nmol/ml; R2 was found consistently in between 0.98-1. The limit of detection (LOD) and quantification (LOQ) were established according to ICH Q2 (R1) guidelines [33].
Table S1. Retention time and calculated LOQ and LOD for TA and TA-AA prodrugs Retention solutions. Results of accuracy and precision are listed in Table S2 and were found to be included within the acceptance limits.
Table S2. Intra-and inter-day precision and accuracy for TA and TA-AA quantification method
Intra-day Inter-day
TA-Val
230.15 231.84 ± 1.30 0.56 100.7 228.59 ± 7.66 3.23 99.3
46.03 46.01 ± 0.26 0.56 100 45.62 ± 1.48 3.58 99.1
11.51 11.71 ±0.13 1.11 101.7 11.09 ± 0.40 4.03 96.3
a) RSD= (SD/mean) *100
b) Accuracy = (obtained concentration/theoretical concentration) *100
Acknowledgments
We would like to thank very warmly our colleagues, Prof. S. Rudaz, Dr. J. Schappler and J. Jacquat of the School of Pharmaceutical Sciences (University of Geneva) for the help with performing the capillary zone electrophoresis studies. V. Santer would like to thank F. Tessaro for the constructive exchange and help with the in silico modelling software. We are extremely grateful to SOOFT Italia S.p.A. company and in specific Dr. D. Rusciano for providing us with their iontophoretic device (power supply (I-ON CXL) and corneal application system (Iontofor CXL)).
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