Compound characterization data

Xiao-Cui Wan, Yue Hou, Ya-Ning Sun, Cai-Xia Zhang, Hao-Tian Hou, Jing Zhang, Ran Xie, Yu-Xuan Wang, Fan Wu, Yu-Lei Li, Ge-Min Fang. “Ribosomal synthesis of noncanonical cyclic peptides via DNA-templated, PNA-mediated tRNA aminoacylation.” ChemRxiv (2026). https://doi.org/10.26434/chemrxiv.15007903/v1

Values presented only in chromatograms, mass spectra, gel images, or other plots or images were not transcribed. The supporting information is appended to the main paper

Gly-sPNA
HPLC (2–90 % B in 40 min, 1.0 ml min⁻¹, 210 nm)	tR = 20.5 min (major)
ESI-MS	m/z = 2534.49 (C₁₀₀H₁₂₇N₅₃O₂₇S, calcd.: 2533.99 g mol⁻¹)

Tyr-sPNA
HPLC (2–90 % B in 40 min, 1.0 ml min⁻¹, 210 nm)	tR = 21.9 min (major)
ESI-MS	m/z = 2640.24 (C₁₀₇H₁₃₃N₅₃O₂₈S, calcd.: 2640.03 g mol⁻¹)

Sar-sPNA
HPLC (2–90 % B in 40 min, 1.0 ml min⁻¹, 210 nm)	tR = 20.6 min (major)
ESI-MS	m/z = 2548.41 (C₁₀₁H₁₂₉N₅₃O₂₇S, calcd.: 2548.01 g mol⁻¹)

D-Phe-sPNA
HPLC (2–90 % B in 40 min, 1.0 ml min⁻¹, 210 nm)	tR = 21.7 min (major)
ESI-MS	m/z = 2624.31 (C₁₀₇H₁₃₃N₅₃O₂₇S, calcd.: 2624.04 g mol⁻¹)

MPhe-sPNA
HPLC (2–90 % B in 40 min, 1.0 ml min⁻¹, 210 nm)	tR = 22.0 min (major)
ESI-MS	m/z = 2638.44 (C₁₀₈H₁₃₅N₅₃O₂₇S, calcd.: 2638.05 g mol⁻¹)

Achc-sPNA
HPLC (2–90 % B in 40 min, 1.0 ml min⁻¹, 210 nm)	tR = 20.0 min (major)
ESI-MS	m/z = 2602.62 (C₁₀₅H₁₃₅N₅₃O₂₇S, calcd.: 2602.05 g mol⁻¹)

His-sPNA
HPLC (2–90 % B in 40 min, 1.0 ml min⁻¹, 210 nm)	tR = 20.4 min (major)
ESI-MS	m/z = 2614.71 (C₁₀₄H₁₃₁N₅₅O₂₇S, calcd.: 2614.03 g mol⁻¹)

Lys-sPNA
HPLC (2–90 % B in 40 min, 1.0 ml min⁻¹, 210 nm)	tR = 20.5 min (major)
ESI-MS	m/z = 2605.44 (C₁₀₄H₁₃₆N₅₄O₂₇S, calcd.: 2605.06 g mol⁻¹)

D-Lys-sPNA
HPLC (2–90 % B in 40 min, 1.0 ml min⁻¹, 210 nm)	tR = 20.5 min (major)
ESI-MS	m/z = 2605.47 (C₁₀₄H₁₃₆N₅₄O₂₇S, calcd.: 2605.06 g mol⁻¹)

MVal-sPNA
HPLC (2–90 % B in 40 min, 1.0 ml min⁻¹, 210 nm)	tR = 20.9 min (major)
ESI-MS	m/z = 2590.53 (C₁₀₄H₁₃₅N₅₃O₂₇S, calcd.: 2590.05 g mol⁻¹)

H-sPNA
HPLC (2–90 % B in 40 min, 1.0 ml min⁻¹, 210 nm)	tR = 20.8 min (major)
ESI-MS	m/z = 2477.07 (C₉₈H₁₂₄N₅₂O₂₆S, calcd.: 2476.97 g mol⁻¹)

Gly-oPNA
HPLC (2–90 % B in 40 min, 1.0 ml min⁻¹, 210 nm)	tR = 21.2 min (major)
ESI-MS	m/z = 2580.04 (C₁₀₅H₁₂₉N₅₃O₂₈, calcd.: 2580.03 g mol⁻¹)

Tyr-oPNA
HPLC (2–90 % B in 40 min, 1.0 ml min⁻¹, 210 nm)	tR = 21.9 min (major)
ESI-MS	m/z = 2686.80 (C₁₁₂H₁₃₅N₅₃O₂₉, calcd.: 2686.07 g mol⁻¹)

Sar-oPNA
HPLC (2–90 % B in 40 min, 1.0 ml min⁻¹, 210 nm)	tR = 21.6 min (major)
ESI-MS	m/z = 2594.25 (C₁₀₆H₁₃₁N₅₃O₂₈, calcd.: 2594.05 g mol⁻¹)

D-Phe-oPNA
HPLC (2–90 % B in 40 min, 1.0 ml min⁻¹, 210 nm)	tR = 23.1 min (major)
ESI-MS	m/z = 2670.45 (C₁₁₂H₁₃₅N₅₃O₂₈, calcd.: 2670.08 g mol⁻¹)

MPhe-oPNA
HPLC (2–90 % B in 40 min, 1.0 ml min⁻¹, 210 nm)	tR = 21.1 min (major)
ESI-MS	m/z = 2684.67 (C₁₁₃H₁₃₇N₅₃O₂₈, calcd.: 2684.09 g mol⁻¹)

Achc-oPNA
HPLC (2–90 % B in 40 min, 1.0 ml min⁻¹, 210 nm)	tR = 23.2 min (major)
ESI-MS	m/z = 2648.54 (C₁₁₀H₁₃₇N₅₃O₂₈, calcd.: 2648.09 g mol⁻¹)

His-oPNA
HPLC (2–90 % B in 40 min, 1.0 ml min⁻¹, 210 nm)	tR = 20.8 min (major)
ESI-MS	m/z = 2660.49 (C₁₀₉H₁₃₃N₅₅O₂₈, calcd.: 2660.07 g mol⁻¹)

Lys-oPNA
HPLC (2–90 % B in 40 min, 1.0 ml min⁻¹, 210 nm)	tR = 20.9 min (major)
ESI-MS	m/z = 2651.19 (C₁₀₉H₁₃₈N₅₄O₂₈, calcd.: 2651.10 g mol⁻¹)

D-Lys-oPNA
HPLC (2–90 % B in 40 min, 1.0 ml min⁻¹, 210 nm)	tR = 20.9 min (major)
ESI-MS	m/z = 2651.28 (C₁₀₉H₁₃₈N₅₄O₂₈, calcd.: 2651.10 g mol⁻¹)

MVal-oPNA
HPLC (2–90 % B in 40 min, 1.0 ml min⁻¹, 210 nm)	tR = 21.9 min (major)
ESI-MS	m/z = 2636.52 (C₁₀₉H₁₃₈N₅₄O₂₈, calcd.: 2636.09 g mol⁻¹)

Phe-oPNA
HPLC (2–90 % B in 40 min, 1.0 ml min⁻¹, 210 nm)	tR = 25.1 min (major)
ESI-MS	m/z = 2670.30 (C₁₁₂H₁₃₅N₅₃O₂₈, calcd.: 2670.08 g mol⁻¹)

Trp-oPNA
HPLC (2–90 % B in 40 min, 1.0 ml min⁻¹, 210 nm)	tR = 22.3 min (major)
ESI-MS	m/z = 2709.51 (C₁₁₄H₁₃₆N₅₄O₂₈, calcd.: 2709.09 g mol⁻¹)

D-Trp-oPNA
HPLC (2–90 % B in 40 min, 1.0 ml min⁻¹, 210 nm)	tR = 22.3 min (major)
ESI-MS	m/z = 2709.57 (C₁₁₄H₁₃₆N₅₄O₂₈, calcd.: 2709.09 g mol⁻¹)

D-Tyr-oPNA
HPLC (2–90 % B in 40 min, 1.0 ml min⁻¹, 210 nm)	tR = 21.2 min (major)
ESI-MS	m/z = 2686.62 (C₁₁₂H₁₃₅N₅₃O₂₉, calcd.: 2686.07 g mol⁻¹)

Tyr-sPNA′
HPLC (2–90 % B in 40 min, 1.0 ml min⁻¹, 210 nm)	tR = 20.5 min (major)
ESI-MS	m/z = 2937.51 (C₁₁₈H₁₄₄N₅₆O₃₅S, calcd.: 2937.09 g mol⁻¹)

D-Phe-sPNA′
HPLC (2–90 % B in 40 min, 1.0 ml min⁻¹, 210 nm)	tR = 20.3 min (major)
ESI-MS	m/z = 2921.40 (C₁₁₈H₁₄₄N₅₆O₃₄S, calcd.: 2921.10 g mol⁻¹)

MPhe-sPNA′
HPLC (2–90 % B in 40 min, 1.0 ml min⁻¹, 210 nm)	tR = 21.7 min (major)
ESI-MS	m/z = 2935.62 (C₁₁₉H₁₄₆N₅₆O₃₄S, calcd.: 2935.11 g mol⁻¹)

Achc-sPNA′
HPLC (2–90 % B in 40 min, 1.0 ml min⁻¹, 210 nm)	tR = 25.5 min (major)
ESI-MS	m/z = 2899.83 (C₁₁₆H₁₄₆N₅₆O₃₄S, calcd.: 2899.11 g mol⁻¹)

MVal-sPNA′
HPLC (2–90 % B in 40 min, 1.0 ml min⁻¹, 210 nm)	tR = 19.5 min (major)
ESI-MS	m/z = 2887.23 (C₁₁₅H₁₄₆N₅₆O₃₄S, calcd.: 2887.11 g mol⁻¹)

Gly-sPNA′-II
HPLC (2–90 % B in 40 min, 1.0 ml min⁻¹, 210 nm)	tR = 21.0 min (major)
ESI-MS	m/z = 2388.33 (C₉₁H₁₁₆N₅₀O₂₈S, calcd.: 2388.89 g mol⁻¹)

Tyr-sPNA′-II
HPLC (2–90 % B in 40 min, 1.0 ml min⁻¹, 210 nm)	tR = 21.5 min (major)
ESI-MS	m/z = 2494.62 (C₉₈H₁₂₂N₅₀O₂₉S, calcd.: 2494.93 g mol⁻¹)

Sar-sPNA′-II
HPLC (2–90 % B in 40 min, 1.0 ml min⁻¹, 210 nm)	tR = 20.9 min (major)
ESI-MS	m/z = 2402.86 (C₉₂H₁₁₈N₅₀O₂₈S, calcd.: 2402.91 g mol⁻¹)

D-Phe-sPNA′-II
HPLC (2–90 % B in 40 min, 1.0 ml min⁻¹, 210 nm)	tR = 21.9 min (major)
ESI-MS	m/z = 2478.57 (C₉₈H₁₂₂N₅₀O₂₈S, calcd.: 2478.94 g mol⁻¹)

MPhe-sPNA′-II
HPLC (2–90 % B in 40 min, 1.0 ml min⁻¹, 210 nm)	tR = 19.9 min (major)
ESI-MS	m/z = 2492.64 (C₉₉H₁₂₄N₅₀O₂₈S, calcd.: 2492.95 g mol⁻¹)

His-sPNA′-II
HPLC (2–90 % B in 40 min, 1.0 ml min⁻¹, 210 nm)	tR = 18.6 min (major)
ESI-MS	m/z = 2468.49 (C₉₅H₁₂₀N₅₂O₂₈S, calcd.: 2468.93 g mol⁻¹)

Lys-sPNA′-II
HPLC (2–90 % B in 40 min, 1.0 ml min⁻¹, 210 nm)	tR = 20.5 min (major)
ESI-MS	m/z = 2459.37 (C₉₅H₁₂₅N₅₁O₂₈S, calcd.: 2459.96 g mol⁻¹)

D-Lys-sPNA′-II
HPLC (2–90 % B in 40 min, 1.0 ml min⁻¹, 210 nm)	tR = 20.5 min (major)
ESI-MS	m/z = 2459.31 (C₉₅H₁₂₅N₅₁O₂₈S, calcd.: 2459.96 g mol⁻¹)

MVal-sPNA′-II
HPLC (2–90 % B in 40 min, 1.0 ml min⁻¹, 210 nm)	tR = 19.1 min (major)
ESI-MS	m/z = 2444.55 (C₉₅H₁₂₄N₅₀O₂₈S, calcd.: 2444.95 g mol⁻¹)

Achc-sPNA′-II
HPLC (2–90 % B in 40 min, 1.0 ml min⁻¹, 210 nm)	tR = 22.5 min (major)
ESI-MS	m/z = 2456.91 (C₉₆H₁₂₄N₅₀O₂₈S, calcd.: 2456.95 g mol⁻¹)

Gly-sPNA′-III
HPLC (2–90 % B in 40 min, 1.0 ml min⁻¹, 210 nm)	tR = 22.5 min (major)
ESI-MS	m/z = 2597.44 (C₁₀₀H₁₂₇N₅₃O₃₁S, calcd.: 2597.97 g mol⁻¹)

Tyr-sPNA′-III
HPLC (2–90 % B in 40 min, 1.0 ml min⁻¹, 210 nm)	tR = 22.9 min (major)
ESI-MS	m/z = 2704.65 (C₁₀₇H₁₃₃N₅₃O₃₂S, calcd.: 2704.01 g mol⁻¹)

Sar-sPNA′-III
HPLC (2–90 % B in 40 min, 1.0 ml min⁻¹, 210 nm)	tR = 22.6 min (major)
ESI-MS	m/z = 2611.89 (C₁₀₁H₁₂₉N₅₃O₃₁S, calcd.: 2611.99 g mol⁻¹)

D-Phe-sPNA′-III
HPLC (2–90 % B in 40 min, 1.0 ml min⁻¹, 210 nm)	tR = 23.7 min (major)
ESI-MS	m/z = 2688.24 (C₁₀₇H₁₃₃N₅₃O₃₁S, calcd.: 2688.02 g mol⁻¹)

MPhe-sPNA′-III
HPLC (2–90 % B in 40 min, 1.0 ml min⁻¹, 210 nm)	tR = 23.8 min (major)
ESI-MS	m/z = 2702.40 (C₁₀₈H₁₃₅N₅₃O₃₁S, calcd.: 2702.03 g mol⁻¹)

His-sPNA′-III
HPLC (2–90 % B in 40 min, 1.0 ml min⁻¹, 210 nm)	tR = 23.1 min (major)
ESI-MS	m/z = 2678.76 (C₁₀₄H₁₃₁N₅₅O₃₁S, calcd.: 2678.01 g mol⁻¹)

Lys-sPNA′-III
HPLC (2–90 % B in 40 min, 1.0 ml min⁻¹, 210 nm)	tR = 22.3 min (major)
ESI-MS	m/z = 2668.83 (C₁₀₄H₁₃₆N₅₄O₃₁S, calcd.: 2669.04 g mol⁻¹)

D-Lys-sPNA′-III
HPLC (2–90 % B in 40 min, 1.0 ml min⁻¹, 210 nm)	tR = 22.3 min (major)
ESI-MS	m/z = 2668.71 (C₁₀₄H₁₃₆N₅₄O₃₁S, calcd.: 2669.04 g mol⁻¹)

MVal-sPNA′-III
HPLC (2–90 % B in 40 min, 1.0 ml min⁻¹, 210 nm)	tR = 22.8 min (major)
ESI-MS	m/z = 2654.43 (C₁₀₄H₁₃₅N₅₃O₃₁S, calcd.: 2654.03 g mol⁻¹)

Achc-sPNA′-III
HPLC (2–90 % B in 40 min, 1.0 ml min⁻¹, 210 nm)	tR = 24.2 min (major)
ESI-MS	m/z = 2666.55 (C₁₀₅H₁₃₅N₅₃O₃₁S, calcd.: 2666.03 g mol⁻¹)

Gly-sPNA′-IV
HPLC (2–90 % B in 40 min, 1.0 ml min⁻¹, 210 nm)	tR = 18.0 min (major)
ESI-MS	m/z = 2654.46 (C₁₀₂H₁₃₀N₅₄O₃₂S, calcd.: 2654.99 g mol⁻¹)

Tyr-sPNA′-IV
HPLC (2–90 % B in 40 min, 1.0 ml min⁻¹, 210 nm)	tR = 18.5 min (major)
ESI-MS	m/z = 2761.56 (C₁₀₉H₁₃₆N₅₄O₃₃S, calcd.: 2761.03 g mol⁻¹)

Sar-sPNA′-IV
HPLC (2–90 % B in 40 min, 1.0 ml min⁻¹, 210 nm)	tR = 18.1 min (major)
ESI-MS	m/z = 2669.43 (C₁₀₃H₁₃₂N₅₄O₃₂S, calcd.: 2669.01 g mol⁻¹)

D-Phe-sPNA′-IV
HPLC (2–90 % B in 40 min, 1.0 ml min⁻¹, 210 nm)	tR = 19.2 min (major)
ESI-MS	m/z = 2745.69 (C₁₀₉H₁₃₆N₅₄O₃₂S, calcd.: 2745.04 g mol⁻¹)

MPhe-sPNA′-IV
HPLC (2–90 % B in 40 min, 1.0 ml min⁻¹, 210 nm)	tR = 19.5 min (major)
ESI-MS	m/z = 2759.13 (C₁₁₀H₁₃₈N₅₄O₃₂S, calcd.: 2759.06 g mol⁻¹)

His-sPNA′-IV
HPLC (2–90 % B in 40 min, 1.0 ml min⁻¹, 210 nm)	tR = 18.3 min (major)
ESI-MS	m/z = 2735.37 (C₁₀₆H₁₃₄N₅₆O₃₂S, calcd.: 2735.03 g mol⁻¹)

Lys-sPNA′-IV
HPLC (2–90 % B in 40 min, 1.0 ml min⁻¹, 210 nm)	tR = 18.0 min (major)
ESI-MS	m/z = 2726.52 (C₁₀₆H₁₃₉N₅₅O₃₂S, calcd.: 2726.07 g mol⁻¹)

D-Lys-sPNA′-IV
HPLC (2–90 % B in 40 min, 1.0 ml min⁻¹, 210 nm)	tR = 18.0 min (major)
ESI-MS	m/z = 2726.64 (C₁₀₆H₁₃₉N₅₅O₃₂S, calcd.: 2726.07 g mol⁻¹)

MVal-sPNA′-IV
HPLC (2–90 % B in 40 min, 1.0 ml min⁻¹, 210 nm)	tR = 18.6 min (major)
ESI-MS	m/z = 2711.04 (C₁₀₆H₁₃₈N₅₄O₃₂S, calcd.: 2711.06 g mol⁻¹)

Achc-sPNA′-IV
HPLC (2–90 % B in 40 min, 1.0 ml min⁻¹, 210 nm)	tR = 19.5 min (major)
ESI-MS	m/z = 2723.67 (C₁₀₇H₁₃₈N₅₄O₃₂S, calcd.: 2723.06 g mol⁻¹)

ClAcPhe-oPNA
HPLC (2–90 % B in 40 min, 1.0 ml min⁻¹, 210 nm)	tR = 24.4 min (major)
ESI-MS	m/z = 2746.68 (C₁₁₄H₁₃₆N₅₃O₂₉Cl, calcd.: 2746.05 g mol⁻¹)

ClAcD-Phe-oPNA
HPLC (2–90 % B in 40 min, 1.0 ml min⁻¹, 210 nm)	tR = 27.3 min (major)
ESI-MS	m/z = 2746.08 (C₁₁₄H₁₃₆N₅₃O₂₉Cl, calcd.: 2746.05 g mol⁻¹)

ClAcTyr-oPNA
HPLC (2–90 % B in 40 min, 1.0 ml min⁻¹, 210 nm)	tR = 25.6 min (major)
ESI-MS	m/z = 2762.01 (C₁₁₄H₁₃₆N₅₃O₃₀Cl, calcd.: 2762.04 g mol⁻¹)

ClAcD-Tyr-oPNA
HPLC (2–90 % B in 40 min, 1.0 ml min⁻¹, 210 nm)	tR = 25.8 min (major)
ESI-MS	m/z = 2762.07 (C₁₁₄H₁₃₆N₅₃O₃₀Cl, calcd.: 2762.04 g mol⁻¹)

ClAcTrp-oPNA
HPLC (2–90 % B in 40 min, 1.0 ml min⁻¹, 210 nm)	tR = 27.2 min (major)
ESI-MS	m/z = 2785.71 (C₁₁₆H₁₃₇N₅₄O₂₉Cl, calcd.: 2785.06 g mol⁻¹)

ClAcD-Trp-oPNA
HPLC (2–90 % B in 40 min, 1.0 ml min⁻¹, 210 nm)	tR = 27.2 min (major)
ESI-MS	m/z = 2785.38 (C₁₁₆H₁₃₇N₅₄O₂₉Cl, calcd.: 2785.06 g mol⁻¹)

Aminoacyl-µhRNA products
ESI-MS	Products were detected for Gly, Tyr, Sar, D-Phe, MPhe, Achc, His, Lys, D-Lys, and MVal

Head-to-side-chain cross-linked peptides
MALDI-TOF MS	Six head-to-side-chain cross-linked peptides were detected