NMR spectroscopic detection of chirality and enantiopurity in referenced systems without formation of diastereomers

Abstract

Enantiomeric excess of chiral compounds is a key parameter that determines their activity or therapeutic action. The current paradigm for rapid measurement of enantiomeric excess using NMR is based on the formation of diastereomeric complexes between the chiral analyte and a chiral resolving agent, leading to (at least) two species with no symmetry relationship. Here we report an effective method of enantiomeric excess determination using a symmetrical achiral molecule as the resolving agent, which is based on the complexation with analyte (in the fast exchange regime) without the formation of diastereomers. The use of N,N'-disubstituted oxoporphyrinogen as the resolving agent makes this novel method extremely versatile, and appropriate for various chiral analytes including carboxylic acids, esters, alcohols and protected amino acids using the same achiral molecule. The model of sensing mechanism exhibits a fundamental linear response between enantiomeric excess and the observed magnitude of induced chemical shift non-equivalence in the (1)H NMR spectra.

Figure 1. Structures of host and guests and their NMR properties.

(a) Structure of achiral host N₂₁,N₂₃-bis(4-bromobenzyl)-5,10,15,20-tetrakis(3,5-di-t-butyl-4-oxocyclohexadien-2,5-ylidene)porphyrinogen, H. Symbols denote the peak assignments in all ¹H NMR spectra in this study. (b) Structure of chiral guests used: ibuprofen 1a, 2-phenylpropionic acid 1b, N-boc-2-phenylglycine 1c, N-boc-phenylalanine 1d, leucic acid 1e, 2-phenoxypropionic acid methyl ester 2a, valine methyl ester 2b, atropine (hyoscyamine) 2c, menthol 3 and camphor 4 (boc=tert-butoxycarbonyl). (c) Typical ¹H NMR spectra of chloroform-d solution of neat H (ca. 0.7 mM) and H (ca. 0.7 mM) with ca. 400 equiv. of selected chiral guests. Key: Green square (pyrrolic NH); enantiotopic CH reporter groups: orange square and circle (hemiquinonoid ortho-H), green circle (N-H pyrrolic β-H) and red circle (N-alkylated pyrrolic β-H). Hash symbol denotes residual chloroform (CHCl₃). Full H spectrum assignment is provided in Supplementary Figs S1–S3.

Figure 2. Effect of guest complexation on symmetry of the host molecule.

(a) Scheme of symmetry of host molecule and corresponding shape of NMR spectra. Dashed lines denote axes of S₁ symmetry elements (mirror planes) also identical to prochiral planes. (b) Disruption of host’s symmetry upon complexation with chiral guest and resulting splitting of NMR spectral pattern. (c) Schematic plot of reference system connected with host and orientation of guest within it using φ and θ coordinates used in the MD simulations. (d) Plot of probability density function ρ(φ) as obtained from MD simulations (for H·(R)–1a and H·(S)–1a complexes) for phenyl centroid (PhC). Solid red or blue lines show average ρ(φ) over 5°. Light red or blue background denotes standard variance of ρ(φ). Supplementary Movie 1 depicts MD of the spontaneous release of (S)-1a from the binding site with subsequent binding of (R)-1a (video length in real time is 4.4 ns).

Figure 3. ¹H NMR data showing dependency of chemical shift non-equivalency on e.e.

(a,b,c) Spectra of H (0.68 mM, chloroform-d) in the presence of various e.e. of 1a (460 equiv.), 2a (370 equiv.) and 4 (490 equiv.) at 25 °C, respectively. Hash symbol denotes carbon-13 satellite from chloroform-d solvent. All spectra were fitted (grey=original spectrum, red=fit) and true resonance frequencies as obtained by fit are indicated by lines under each spectrum. Spectra for 1a and 2a were taken as AB second-order strongly coupled quartet. Left part of spectrum (orange circle and square) for guest 4 was fitted as two independent EF and E′F′ second-order spin systems and right part of spectrum (green circle) as CDX strongly coupled quartet of doublets (for details see ¹H NMR spectra fitting in Supplementary Methods). (d) Plot of dependency of Δδ on the e.e. value of host’s splitted resonance (denoted by arrows in a–c) showing linear relationship (for all R²>0.997). (e) Homonuclear ¹H–¹H COSY spectrum of N-alkylated pyrrolic β-H resonances of H (1.42 mM, chloroform-d) with (S)-1a (200 equiv.) at 25 °C.

Figure 4. ¹H NMR titration experiments supporting the competitive binding model.

(a) Titration of H (0.41 mM, chloroform-d) with water W at 25 °C. (b) Titration of H (0.65 mM, chloroform-d) with (–)−3 at 25 °C. (c) Titration of H (0.74 mM, chloroform-d) with (–)−3 at 25 °C. Before titration (with (–)−3) 150 equiv. of (+)−3 was added to the solution. Bottom panel shows dependency of Δδ on e.e. during the titration. Grey curves were fitted according to a competitive binding model (see Fig. 5) using isotherms expressed in equations 2 and 3. During titrations chiral guest and water concentrations were evaluated although water concentration did not vary significantly from initial value during titration. For binding constants and other parameters see Table 1. (d,e) Spectra taken during titration shown in b,c, respectively (grey=original spectrum, red=fit). Double dagger (‡) in c,e denotes combined concentration (in equiv.) of both enantiomers.

Figure 5. Competitive binding model and its schematic ¹H NMR spectral manifestation.

(a) Partial (meso-substituents removed) structural model of host, H, showing its three forms and (at right) an image illustrating the situation in solution. Equilibrium binding constants and populations are indicated. Protons of special interest (H_A, H_B and H_X) are also highlighted. (b) Schematic description of pyrrolic NH resonance (H_X) behaviour. (c) Schematic description of N-alkylated pyrrolic β-H resonances (H_A and H_B). Arrows indicate the true resonance frequency of spectral patterns.