Leucopterin, the white pigment in butterfly wings: structural analysis by PDF fit, FIDEL fit, Rietveld refinement, solid-state NMR and DFT-D

Abstract

Leucopterin (C₆H₅N₅O₃) is the white pigment in the wings of Pieris brassicae butterflies, and other butterflies; it can also be found in wasps and other insects. Its crystal structure and its tautomeric form in the solid state were hitherto unknown. Leucopterin turned out to be a variable hydrate, with 0.5 to about 0.1 molecules of water per leucopterin molecule. Under ambient conditions, the preferred state is the hemihydrate. Initially, all attempts to grow single crystals suitable for X-ray diffraction were to no avail. Attempts to determine the crystal structure by powder diffraction using the direct-space method failed, because the trials did not include the correct, but rare, space group P2/c. Attempts were made to solve the crystal structure by a global fit to the pair distribution function (PDF-Global-Fit), as described by Prill and co-workers [Schlesinger et al. (2021). J. Appl. Cryst. 54, 776-786]. The approach worked well, but the correct structure was not found, because again the correct space group was not included. Finally, tiny single crystals of the hemihydrate could be obtained, which allowed at least the determination of the crystal symmetry and the positions of the C, N and O atoms. The tautomeric state of the hemihydrate was assessed by multinuclear solid-state NMR spectroscopy. ¹⁵N CPMAS spectra showed the presence of one NH₂ and three NH groups, and one unprotonated N atom, which agreed with the ¹H MAS and ¹³C CPMAS spectra. Independently, the tautomeric state was investigated by lattice-energy minimizations with dispersion-corrected density functional theory (DFT-D) on 17 different possible tautomers, which also included the prediction of the corresponding ¹H, ¹³C and ¹⁵N chemical shifts in the solid. All methods showed the presence of the 2-amino-3,5,8-H tautomer. The DFT-D calculations also confirmed the crystal structure. Heating of the hemihydrate results in a slow release of water between 130 and 250 °C, as shown by differential thermal analysis and thermogravimetry (DTA-TG). Temperature-dependent powder X-ray diffraction (PXRD) showed an irreversible continuous shift of the reflections upon heating, which reveals that leucopterin is a variable hydrate. This observation was also confirmed by PXRD of samples obtained under various synthetic and drying conditions. The crystal structure of a sample with about 0.2 molecules of water per leucopterin was solved by a fit with deviating lattice parameters (FIDEL), as described by Habermehl et al. [Acta Cryst. (2022), B78, 195-213]. A local fit, starting from the structure of the hemihydrate, as well as a global fit, starting from random structures, were performed, followed by Rietveld refinements. Despite dehydration, the space group remains P2/c. In both structures (hemihydrate and variable hydrate), the leucopterin molecules are connected by 2-4 hydrogen bonds into chains, which are connected by further hydrogen bonds to neighbouring chains. The molecular packing is very efficient. The density of leucopterin hemihydrate is as high as 1.909 kg dm^-3, which is one of the highest densities for organic compounds consisting of C, H, N and O only. The high density might explain the good light-scattering and opacity properties of the wings of Pieris brassicae and other butterflies.

Keywords: DFT-D; FIDEL; PDF; Rietveld refinement; chemical shift calculation; high density; leucopterin; nonstoichiometric hydrate; organic white pigment; pair distribution function; powder data; solid-state NMR; structure determination.

Figure 1

Pieris napi (Photo: Robert Hühn, Frankfurt am Main, 23 June 2022).

Figure 2

17 of the possible tautomers of leucopterin.

Figure 3

(a) Temperature-dependent powder patterns of leucopterin; (b) magnification of the tem­per­ature-dependent powder patterns of leucopterin in the region 17.5–23.0° (2θ).

Figure 4

Powder patterns of leucopterin before (black) and after heating (blue) to 250 °C. Both patterns were measured at room tem­per­ature. The circle denotes one of the weak reflections which shifted upon dehydration.

Figure 5

Successful crystal structure determination of leucopterin hemihydrate by a global fit to the PDF, performed after the correct space group (P2/c) was known. The PDF global fit was done without the water molecule. The experimental PDF is shown in black, the calculated PDF in red and the difference curve below in blue.

Figure 6

(a) Crystal structure of leucopterin 0.2-hydrate from Rietveld refinement; the view direction is [0 0]. The red points indicate the position of the water molecules refined as an O atom with an occupancy of 0.42. (b) Rietveld plot of leucopterin 0.2-hydrate. The experimental pattern is drawn with black points, the calculated pattern as a red line and the difference curve in blue. The vertical tick marks denote the reflection positions.

Figure 7

¹³C (150.9 MHz) CPMAS spectra of leucopterin hemihydrate and anhydrate, acquired at room tem­per­ature at a spinning speed of 20 kHz.

Figure 8

Overlays of the ¹⁵N (60.8 MHz) CPMAS (12 kHz) spectra at room tem­per­ature of leucopterin hemihydrate and anhydrate acquired with different contact times to highlight signals due to protonated and non-protonated N-atom sites. Top: hemihydrate, acquired with contact times of 0.1 (red) or 4 ms (black); bottom: anhydrate, acquired with contact times of 0.1 (red) or 4 ms (black).

Figure 9

¹H (1000.4 MHz) MAS spectra of leucopterin hemihydrate (above) and anhydrate (below), acquired at room tem­per­ature at a spinning speed of 100 kHz. H3^h and H₂O^h, in the anhydrate spectrum, refer to H3 and H₂O signals originating from the amount of partially hydrated leucopterin.

Figure 10

(a) ¹H (1000.4 MHz) DQ MAS spectrum of leucopterin hemihydrate acquired at room tem­per­ature at a spinning speed of 100 kHz. Only the most relevant correlations are highlighted. (b) Scheme of the main H–H proximities observed in the ¹H DQ MAS spectrum. The H3–H3 correlation is not shown since it is an interlayer proximity.

Figure 11

¹H (1000.4 MHz) DQ MAS spectrum of leucopterin anhydrate acquired at room tem­per­ature at a spinning speed of 100 kHz. Only the most relevant correlations are highlighted. H3^h and H₂O^h, in the anhydrate spectrum, refer to H3 and H₂O signals originating from the amount of partially hydrated leucopterin.

Figure 12

Molecular chains in the crystal structure of leucopterin hemihydrate, after DFT-D optimization with fixed lattice parameters. Colour code in all drawings: C = grey, O = red, N = blue, H = white and hydrogen bonds = turquoise. The view direction is [120].

Figure 13

(a) Crystal packing (view direction [00 ]) and (b) hydrogen-bond pattern (view direction [0 0]) in the crystal structure of leucopterin hemihydrate.

Figure 14

Overlay of the 0.2-hydrate (in black) and hemihydrate structures of leucopterin.