HiFSA fingerprinting applied to isomers with near-identical NMR spectra: the silybin/isosilybin case

Abstract

This study demonstrates how regio- and diastereo-isomers with near-identical NMR spectra can be distinguished and unambiguously assigned using quantum mechanical driven (1)H iterative Full Spin Analysis (HiFSA). The method is illustrated with four natural products, the flavonolignans silybin A, silybin B, isosilybin A, and isosilybin B, which exhibit extremely similar coupling patterns and chemical shift differences well below the commonly reported level of accuracy of 0.01 ppm. The HiFSA approach generated highly reproducible (1)H NMR fingerprints that enable distinction of all four isomers at (1)H frequencies from 300 to 900 MHz. Furthermore, it is demonstrated that the underlying numeric (1)H NMR profiles, combined with iterative computational analysis, allow parallel quantification of all four isomers, even in difficult to characterize reference materials and mixtures. The results shed new light on the historical challenges to the qualitative and quantitative analysis of these therapeutically relevant flavonolignans and open new opportunities to explore hidden diversity in the chemical space of organic molecules.

Figure 1

Stacked plots showing the similarities between the 1D ¹H NMR spectra of silybin A (1), silybin B (2), isosilybin A (3), and isosilybin B (4) (DMSO-d₆, 900 MHz, 298 K). Labels A-G indicate the positions of extended regions displayed in the lower panel, where small differences in ¹H chemical shifts between the four closely related compounds are shown.

Figure 2

Stacked plots showing the similarities between the 1D ¹³C–DEPTQ spectra of silybin A (1), silybin B (2), isosilybin A (3), and isosilybin B (4) (DMSO-d₆, 225 MHz, 298 K). Primary and tertiary carbons (CH, CH₃) are positive signals, quaternary and secondary carbons (C, CH₂) are negative signals. The artifact (*) and the DMSO signal are symmetric with respect to the center of the spectrum (i.e., transmitter offset). Labels A-E indicate the positions of extended regions shown in the lower panel, where small differences in ¹³C chemical shifts between the four isomers are highlighted.

Figure 3

The identification of key HMBC connectivities to distinguish between the regioisomers silybin B (A) and isosilybin B (B) required the acquisition of 2D semi-selective HMBC experiments (DMSO-d₆, 600 MHz, 298 K). In both cases, conventional HMBC experiments acquired with a 220 ppm window in the ¹³C dimension (grey) are compared to ¹³C resolution-enhanced, semi-selective HMBC experiments acquired with a 20 ppm ¹³C window centered at 145 ppm (red).

Figure 4

The ¹H NMR fingerprint of silybin A (1) generated by HiFSA (Calculated, red) represents a detailed replica of the experimental 1D ¹H NMR spectrum (Observed, blue, obtained in DMSO-d₆ at 900 MHz and 298 K). Complete ¹H assignments and simplified J-coupling trees are included, showing that the ¹H NMR spectrum of 1 can be largely interpreted under first order assumptions.

Figure 5

Graphical representation of the small chemical shift differences (Δδ, in ppb) between isomers 1–4 (DMSO-d₆, 298 K, 900 MHz for ¹H, 225 MHz for ¹³C). (*) denote diagnostic Δδ values to differentiate between diastereomeric pairs and regioisomers.

Figure 6

Simultaneous qualitative and quantitative ¹H NMR analysis of Silybin USP Reference Standard performed at both 400 and 600 MHz (DMSO-d₆, 298 K). The intensity adjusted fingerprints of silybin A (1) and silybin B (2) were generated by Total Line Shape (TLS) iteration of the corresponding ¹H NMR profiles (see Table 1). The arithmetic addition of the two fingerprints (Sum, red) matches the experimental 1D ¹H NMR spectrum (Silybin USP, blue).

Figure 7

Simultaneous identification of silybin A (1), silybin B (2), isosilybin A (3), and isosilybin B (4) in a flavonolignan-enriched fraction obtained by HSCCC. The arithmetic addition of the four intensity adjusted fingerprints (Sum, red) is in excellent agreement with the experimental ¹H NMR spectrum of the fraction (Observed, blue, obtained in DMSO-d₆ at 600 MHz and 298 K).

Scheme 1

Generation of the NMR fingerprints by ¹H iterative Full Spin Analysis (HiFSA), exemplified with the characteristic resonance of H-8″ in isomers 1–4.

Chart 1

Structures and numbering of the diastereomeric pairs of regioisomers silybins A-B (1, 2) and isosilybins A-B (3, 4).

Chart 2

Additional examples of isomeric organic molecules with near-identical NMR spectra. (A) The hydnocarpin/hydnocarpin D pair. (B) Murisolin isomers, exemplified with group 1. (C) The four diastereomers of the C-1/C-10 fragment of amphidinol 3. (D) Two macrocyclic dioxatetralactones with C_s and C₂ symmetry, respectively.