¹³C Chemical Shift Constrained Crystal Structure Refinement of Cellulose I_α and Its Verification by NMR Anisotropy Experiments

Abstract

The solid-state NMR assignments of the 13C resonances of bacterial cellulose Iα were reinvestigated by INADEQUATE experiments on uniformly 13C-enriched samples from Acetobacter xylinum. Additionally, we determined the principal chemical shift tensor components of each 13C labeled site from a 2D iso-aniso RAI (recoupling of anisotropy information) spectrum acquired at magic angle spinning speed of 10 kHz. On the basis of these NMR data, the crystal structure of cellulose Iα was refined using the 13C chemical shifts for target functions. Starting off with coordinates derived from neutron scattering, our molecular dynamics simulations yielded four ensembles of 200 structures, two ensembles for hydrogen bond scheme A and B and two ensembles for different chemical shift assignments I and II, giving 800 structures in total. These were subsequently geometry-optimized with the given isotropic chemical shift constraints applying crystallographic boundary conditions, to identify a structure for every ensemble that fit best to the experimental NMR data. The resulting four model structures were then assessed by simulating the chemical shift tensors (using the bond polarization theory) and comparing these values with the experimental chemical shift anisotropy information (obtained by RAI). The earlier neutron diffraction study had reported two possible occupation schemes for the hydrogen-bonded hydroxyl-groups (A, B) which connect the cellulose chains. From these two possibilities, our NMR results single out pattern A as the most probable structure. In this work, the first time crystallographic boundary conditions were applied for 13C chemical shift structure refinement for molecular dynamics simulations and Newton−Raphson geometry optimization.

Spectral assignments and anisotropy data of cellulose Iα: 13C‐NMR chemical shift data of cellulose Iα determined by INADEQUATE and RAI techniques applied to uniformly 13C‐labeled bacterial celluloses of different Gluconacetobacter xylinus strains

“…The experimental values were generally much smaller than their theoretical predictions based on neutron diffraction crystal structures. [10] A crystal structure refinement utilizing NMR chemical shift constraints was finally able to yield a good agreement between the experimental and theoretical chemical shift tensor values. It turned out that the intrachain hydrogen bonds are responsible for the special glycosidic linkage.…”

“…[21,27] The data also show that the 13 Cenrichment of samples 1 and 2 was successful with about 94% yield, virtually uniformly at all carbon positions. In view of their rather well-resolved 13 C-NMR lines compared to BC DSM 13 368, [10] with slight modifications (Avance 500, 4 mm, ν R = 12.5 kHz, ns = 256, t w = 1 s, t CP = 5 ms, TPPM: ±20 • , 10 µs; echo time for J-couplings = 3.04 ms) of uniformly 13 C-labeled bacterial cellulose from Gluconacetobacter xylinus NQ-5 (1). samples 1 and 2 provide excellent structural characteristics for the analysis of crystalline properties.…”

“…[1] These conclusions were subsequently confirmed by solidstate INADEQUATE NMR. [6] Further details based on correlation spectroscopy were presented by Sakellariou et al [7] and Cadars et al, [8] and the three-dimensional cellulose structure and NMR investigations thereof were discussed by Sternberg et al [9] and Witter et al [10] The complete assignment of all carbon sites in the crystalline structure was finally possible using specifically 13 Clabeled D-glucose and D-glycerol for biosynthesis, [11,12] allowing to resolve the individual chemical shifts of C2, C3, and C5. Next, the structural assignment of all 13 C-NMR signals in the two different anhydroglucose rings were succeeded by solid-state INADEQUATE NMR of specifically prepared allomorphs I α (purified Cladophora) and I β (purified tunicate cellulose).…”

“…This effect also explains the characteristic isotropic chemical shift of C4 (90 ppm) that is observed in crystalline cellulose. [10] In the present study, uniformly 13 C-labeled bacterial cellulose samples from two different strains of G. xylinus (ATCC 53 582 and ATCC 23 769) have been investigated. These strains were chosen because of their slightly different behavior in cellulose production.…”

“…Accurate principal tensor components were obtained using an optimized version of the 2D RAI sequence. [10,22] Here, the influence of finite pulse length was taken into account, and proton decoupling with a ratio of ω 1H /ω 13C ≥ 2 was achieved, which significantly improved the quality of the quasi-static powder patterns.…”