Extended Data Fig. 4: NMR signal assignments support P1 identity as 5-(1-[2,3,4-trihydroxybutyl])-2′-deoxycytidine.

a, ¹H-NMR spectrum of P1 with signal assignments. The spectrum shows all the non-exchangeable proton signals with their chemical shifts and J-coupling constants for P1 (Extended Data Table 1). b, ¹H-¹H 2D COSY spectrum for P1 with assignments. The sequential positions of protons showed in two spin-coupling systems as δ_H 6.299–2.320/3.437–4.455–4.062–3.773/3.860 in a deoxyribosyl moiety and δ_H 3.813/3.664–3.615–3.811–2.793/2.505. c, ¹H-¹H 2D TOCSY spectrum for P1 with assignments. Three coupling systems were observed in this TOCSY spectrum. The first coupling system showed a typical signal pattern for a deoxyriboside moiety, here with seven protons at δ_H 6.299 (1H, t, H1′), 4.455 (1H, m, H3′), 4.062 (1H, m, H4′), 3.860 (1H, dd, H5′b), 3.773 (1H, dd, H5′a), 2.437 (1H, ddd, H2′b) and 2.320 (1H, dt, H2′a). The second one was observed for six protons at δ_H 3.813 (1H, H10b), 3.811 (1H, ddd, H8), 3.664 (1H, dd, H10a), 3.615 (1H, ddd, H9), 2.793 (1H, ddd, H7b), 2.505 (1H, ddd, H7a) and 2.320 (1H, dt, H2′a). A third coupling system was observed as a weak correlation between δ_H 7.759 (1H, t, H6) and a CH₂ moiety (H7a and H7b, δ_H 2.793 and 2.505). d, ¹H-¹H JRES spectrum for P1 showing J-coupling patterns from all protons (Extended Data Table 1). The F₁ dimension gives coupling constants (Hz) and the F₂ dimension gives chemical shift information. e, ¹H-¹³C 2D HSQC spectrum for P1 with assignments. The direct H–C linkages were detected by the one-bond ¹H–¹³C correlations in this HSQC spectrum. f, ¹H-¹³C 2D HMBC spectrum for P1 with assignments. The long-range ¹H–¹³C correlations were detected in the HMBC spectrum. The proton at δ_H 7.759 showed long-range correlations with C2, C4 and C5 (δ_C 159.98, 168.53 and 107.64, respectively) of a cytosine residue, with C7 of the THB moiety (δ_C 33.64), and with the deoxyribosyl C1′ (δ_C 88.95). This indicates that C7 (CH₂) of the THB moiety was attached to C6 of a cytosine ring. This was further confirmed with long-range correlations between H7 (δ_H 2.793, 2.505) and C4, C5, C6, C8 and C9 (δ_C 168.53, 107.64, 143.83, 72.56 and 76.94). The long correlations between H1′ (δ_H 6.299) and C2 and C6 (δ_C 168.53 and 143.83) in the HMBC spectrum further confirmed the N1–C1′ linkage between the deoxyribosyl and cytosine moieties. Taking the above into consideration, P1 was finally determined as 5-(1-[2,3,4-trihydroxybutyl])-2′-deoxycytidine (Fig. 2c) with its ¹H and ¹³C signals unambiguously assigned and tabulated in Extended Data Table 1. Representative results are shown from two independent experiments.