13 C Chemical Shift Constrained Crystal Structure Refinement of Cellulose I R and Its Verification by NMR Anisotropy Experiments Raiker Witter,* ,† Ulrich Sternberg, † Stephanie Hesse, ‡,|,# Tetsuo Kondo, |,⊥ Frank-Th. Koch, ‡ and Anne S. Ulrich †,§ Institute of Biological Interfaces, Forschungszentrum Karlsruhe, POB 3640, 76021 Karlsruhe, Germany; Institute of Optics and Quantum Electronics, Friedrich-Schiller-UniVersita ¨t Jena, Max-Wien-Platz 1, Jena 07743, Germany, and Forestry and Forest Products Research Institute, Matusnosato 1, Tsukuba, Ibaraki 305-8687, Japan; and Institute of Organic Chemistry, UniVersity of Karlsruhe, Fritz-Haber-Weg 6, 76131 Karlsruhe, Germany ReceiVed NoVember 15, 2005; ReVised Manuscript ReceiVed June 27, 2006 ABSTRACT: The solid-state NMR assignments of the 13 C resonances of bacterial cellulose I R were reinvestigated by INADEQUATE experiments on uniformly 13 C-enriched samples from Acetobacter xylinum. Additionally, we determined the principal chemical shift tensor components of each 13 C 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 I R was refined using the 13 C 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 13 C chemical shift structure refinement for molecular dynamics simulations and Newton-Raphson geometry optimization. Introduction Natural cellulose is a partially crystalline polymer of 1-4 linked -D-glucose residues. Its structure and NMR investiga- tions thereof are discussed by Sternberg et al. 1 VanderHart and Atalla 2 revealed the presence of two allomorphs, I R and I  , by CP-MAS 13 C NMR studies of highly crystalline native cellulose I. They published a first assignment for the resonances of C1, C4, and C6. The cluster of signals between 70 and 80 ppm was attributed to carbons C2, C3, and C5. These results were confirmed later using selectively 13 C-labeled cellulose 3,4 and by solid-state INADEQUATE NMR. 5 In both cases the C2, C3, and C5 chemical shifts were resolved and assigned. Recently, Kono et al. 6 assigned all 13 C signals to the respec- tive carbon sites in the two different anhydroglucose rings of purified Cladophora cellulose (I R ) and tunicate cellulose (I  ). Additionally, Jaeger et al. 7 assigned all carbon sites in uniformly 13 C-enriched bacterial cellulose, for which slightly different isotropic chemical shift values were found. New results on cellulose based on correlation spectroscopy are given by Cadars et al. 8 and Sakellariou et al. 9 In diffraction studies of cellulose fibers, the amorphous character of this microcrystalline material tends to produce poorly resolved diffraction patterns. Nevertheless, Reiling and Brickmann 10 constructed computer models of cellulose I R from X-ray and electron diffraction studies, 11,12 and they performed force field refinements with periodic boundary conditions. First, precise atomic coordinates based on 13 C NMR chemical shift refinements were derived by Sternberg et al. 1 At the same time, the native cellulose structures were reinvestigated by Nishiyama et al. 13 with X-ray and neutron diffraction, which yielded information about hydrogen-bond networks. Hence, it is now of interest to compare the diffraction results with the newly refined NMR structures. In this work, the recent crystal structure of Nishiyama et al. 13 is used as a starting model for the 13 C NMR structure refinement of cellulose I R . The unit cell contains one chain consisting of two crystallographically different anhydroglucose units (see Figure 1). Therefore, 12 resonances should be observable by NMR. Their isotropic chemical shifts are used for direct structure refinement, based on a newly developed NMR force field which utilizes the chemical shift target functions introduced by Witter et al. 14,15,16 Preparation of Bacterial Cellulose Bacterial cellulose was produced by Acetobacter xylinum NQ-5 (strain ATCC 53582, from the collection of T. Kondo), and relevant details about their biosynthesis behavior in standard media can be found in Brown et al. 17 and Kondo et al. 18 This microbial strain is also quite robust for cultivation in 13 C-enriched media, as inves- tigated by Hesse and Kondo. 19 * Corresponding author. E-mail: Raiker.Witter@ibg.fzk.de. † Forschungszentrum Karlsruhe. ‡ Friedrich-Schiller-Universita ¨t Jena. | Forest Products Research Institute. § University of Karlsruhe. ⊥ Current address. Biomaterial Design Lab, Bioarchitecture Center(BAC) & Graduate school of Bioresource and Bioenvironmental Sciences, Kyushu University, 6-10-1, Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan. # Current address. Centre of Excellence for Polysaccharide Research, Friedrich-Schiller-Universita ¨t Jena, Humboldtstrasse 10, Jena 07743, Ger- many. 6125 Macromolecules 2006, 39, 6125-6132 10.1021/ma052439n CCC: $33.50 © 2006 American Chemical Society Published on Web 08/09/2006