Understanding the Solid-State Structure of Riboflavin through a Multitechnique Approach

Abstract

Crystalline riboflavin (vitamin B₂) performs an important biological role as an optically functional material in the tapetum lucidum of certain animals, notably lemurs and cats. The tapetum lucidum is a reflecting layer behind the retina, which serves to enhance photon capture and vision in low-light settings. Motivated by the aim of rationalizing its biological role, and given that the structure of biogenic solid-state riboflavin remains unknown, we have used a range of experimental and computational techniques to determine the solid-state structure of synthetic riboflavin. Our multitechnique approach included microcrystal XRD, powder XRD, three-dimensional electron diffraction (3D-ED), high-resolution solid-state ¹³C NMR spectroscopy, and dispersion-augmented density functional theory (DFT-D) calculations. Although an independent report of the crystal structure of riboflavin was published recently, our structural investigations reported herein provide a different interpretation of the intermolecular hydrogen-bonding arrangement in this material, supported by all the experimental and computational approaches utilized in our study. We also discuss, more generally, potential pitfalls that may arise in applying DFT-D geometry optimization as a bridging step between structure solution and Rietveld refinement in the structure determination of hydrogen-bonded materials from powder XRD data. Finally, we report experimental and computational values for the refractive index of riboflavin, with implications for its optical function.

Figure 1

Riboflavin molecule with chirality {S, S, R} for the chiral centers C20, C22, and C24, respectively. The atom-numbering scheme shown is used throughout this article.

Figure 2

Comparison of the solid-state structures of riboflavin determined at 293 K in the present work from microcrystal XRD (structure A) and in ref from powder XRD (structure B). (a) Overlay of structure A (cyan) and structure B (magenta). (b) Overlay of the side chain of the riboflavin molecule (also including the C2–N1–C18 portion of the aromatic ring system) in structure A (cyan) and structure B (magenta), highlighting the conformational differences, particularly regarding the orientation of the terminal OH bond (right side of figure). (c, d) The intermolecular hydrogen-bonding arrangement between a given molecule (the central molecule shown) and two neighboring molecules in (c) structure A and (d) structure B, highlighting the different intermolecular hydrogen-bonding of the terminal CH₂OH group (containing O27). (e, f) Expanded view of the hydrogen-bonding involving the terminal CH₂OH group of the side chain of the riboflavin molecule in (e) structure A and (f) structure B. Hydrogen bonds are represented by green dashed lines.

Figure 3

Final Rietveld refinements of the powder XRD data (background subtracted) for riboflavin using (a) structure A and (b) structure B as the structural model (red plus marks, experimental powder XRD data; green line, calculated powder XRD data; magenta line, difference between experimental and calculated powder XRD data; black tick marks, peak positions).

Figure 4

(a) Bright field TEM overview of the needle-like riboflavin crystals. (b) μ-STEM image of a single crystal selected for FAST-ADT acquisition (the red circle indicates the position of the electron beam in the diffraction experiment). (c) The (h0l) reciprocal lattice plane in the 3D-ED data with extinctions due to 2₁ screw axes indicated by yellow circles. Projections of the 3D reconstruction are shown in Figure S3.

Figure 5

(a) The experimental high-resolution solid-state ¹³C NMR spectrum for riboflavin recorded at 293 K and (b, c) values of the isotropic ¹³C NMR chemical shifts calculated using DFT-GIPAW methodology for (b) structure A and (c) structure B. The red arrow in each spectrum indicates the isotropic peak for the ¹³C environment (C26) in the terminal CH₂OH group of the side chain.

Figure 6

Local environment of the terminal OH group (containing O27) of the side chain of the riboflavin molecule in (a) the structure solution obtained directly from powder XRD data, (b) the structure obtained after applying DFT-D geometry optimization to the structure shown in (a), and (c) the correct hydrogen-bonding arrangement in structure A, determined from microcrystal XRD data. The O27···O25 and O27···O8 distances in each structure are indicated, and hydrogen bonds are represented by green dashed lines.