Hydrazine Complex: Dynamic

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Molecular Interactions in an #-chitin/Hydrazine Complex: Dynamic Hydrogen Bonds and Improvement of Polymeric Crystallinity Daisuke Sawada, Yu Ogawa, Yoshiharu Nishiyama, Eiji Togawa, Satoshi Kimura, and Paul Langan Cryst. Growth Des., Just Accepted Manuscript • DOI: 10.1021/acs.cgd.6b00315 • Publication Date (Web): 21 Apr 2016 Downloaded from http://pubs.acs.org on April 26, 2016

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Crystal Growth & Design

Title: Molecular Interactions in an α-chitin/Hydrazine Complex: Dynamic Hydrogen Bonds and Improvement of Polymeric Crystallinity

∗Daisuke Sawada† ,+, ∗Yu Ogawa¶ ,+, Yoshiharu Nishiyama¶, Eiji Togawa§, Satoshi Kimura‡, Paul Langan†

†

Biology and Soft Matter Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, 37831, USA; ¶Univ. Grenoble Alpes, Cermav, F-38000 Grenoble, France; §Forestry and Forest Products Research Institute (FFPRI), Tsukuba, Ibaraki 305-8687, Japan; ‡Kyoto University, Laboratory of Biomass Morphogenesis and Information Research Institute for Sustainable Humanosphere, Uji, Kyoto 611-0011, Japan. +Equally contributing authors Abstract: The high-resolution structure of α-chitin/hydrazine complex has been determined to reveal the molecular interactions between hydrazine molecules and chitin chains. The complexation with guest hydrazine molecules improves the crystallinity of α-chitin from original form, so that the primary hydroxyl group is clearly located in the gt position on the chitin chains arranged in anti-parallel manner in contrast to original α-chitin structure having the disorder in gt and gg positions. During the complexation process, α-chitin chains translate by c/4 to incorporate hydrazine molecules. Molecular dynamics calculations based on the refined structure show that hydrazine nitrogen atoms are disordered with competing hydrogen bonds. These observations suggest a mechanism for the structural conversion of polysaccharides to their crystal solvate forms during penetration by amine molecules. CORRESPONDING AUTHORS ∗Daisuke Sawada Biology and Soft Matter Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, 37831, USA E-mail: [email protected] Phone: +1-865-560-6263 * Yu Ogawa CNRS-CERMAV, Univ. Grenoble Alpes, Cermav, F-38000 Grenoble, France E-mail: [email protected] Phone: +33476037608

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Molecular Interactions in an α-chitin/Hydrazine Complex: Dynamic Hydrogen Bonds and Improvement of Polymeric Crystallinity Daisuke Sawada† ,+, Yu Ogawa¶ ,+, Yoshiharu Nishiyama¶, Eiji Togawa§, Satoshi Kimura‡, Paul Langan† †

Biology and Soft Matter Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, 37831, USA; ¶Univ. Grenoble Alpes, Cermav, F-38000 Grenoble, France; §Forestry and Forest Products

Research Institute (FFPRI), P.O. Box 16, Tsukuba Norin Kenkyu, Ibaraki 305-8687, Japan; ‡Kyoto University; Laboratory of Biomass Morphogenesis and Information Research Institute for Sustainable Humanosphere Kyoto University, Uji, Kyoto 611-0011, Japan. +Equally contributing authors AUTHOR EMAIL ADDRESS ∗Daisuke sawada: [email protected] *Yu Ogawa: [email protected] Yoshiharu Nishiyama: [email protected] Satoshi Kimura: [email protected] Eiji Togawa: [email protected] Paul Langan: [email protected]


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Abstract

The high-resolution structure of α-chitin/hydrazine complex has been determined to reveal the molecular interactions between hydrazine molecules and chitin chains. The complexation with guest hydrazine molecules improves the crystallinity of α-chitin from original form, so that the primary hydroxyl group is clearly located in the gt position on the chitin chains arranged in anti-parallel manner in contrast to original α-chitin structure having the disorder in gt and gg positions. During the complexation process, α-chitin chains translate by c/4 to incorporate hydrazine molecules. Molecular dynamics calculations based on the refined structure show that hydrazine nitrogen atoms are disordered with competing hydrogen bonds. These observations suggest a mechanism for the structural conversion of polysaccharides to their crystal solvate forms during penetration by amine molecules.


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Introduction Crystalline polysaccharides such as cellulose and chitin are often found in nature as a fibrous component of complex tissues. The major role of these polysaccharides is to confer mechanical strength on the tissues. The crystal structures have been analyzed to understand the structure - properties relationship in such systems. Fiber diffraction methods have been developed in the past 100 years to obtain a molecular structure from uni-axially oriented polycrystalline or amorphous samples. In the last fifteen years, many of the native structures of crystalline polysaccharides have been resolved at atomic resolution1–4 thanks to recent developments in the field of fiber diffraction: High-brightness synchrotron X-ray equipped with a modern detector, background estimation and peak fitting method of diffraction intensity3, and direct determination of hydrogen atoms using neutron fiber diffraction1,2,4,5. In the previous studies polysaccharide crystals with large lateral crystallite sizes were used to obtain highly resolved diffraction patterns allowing the structural determination with atomic resolution1–3. However, crystalline polysaccharides in nature often have small crystallites, reducing the quality of diffraction intensity data. Thus the structural determination of such small polysaccharide crystals is still challenging to date. Under certain conditions, small solvent molecules can penetrate crystals of polymers, such as syndiotactic polystyrene6,7, poly (L-lactide)8, cellulose9,10, cyclodextrin11–13, and starch14–17, to form crystalline complexes which we refer to here as crystal solvates. Crystal solvate can be considered as an intermediate phase between neat solid crystal and solution state, and thus can be used to investigate molecular interactions between solutes and solvent molecules, which are not accessible in the solution system. Such information can be utilized to understand dissolution mechanisms and design novel solvent system. This approach is especially useful for crystalline polysaccharides, since they are mostly insoluble in ordinary solvents, and only soluble in specific multicomponent solvents. We are particularly interested in crystal solvates formed by amines with polysaccharides. When combined with copper or thiocyanate salts18–20, amines are effective cosolvents of polysaccharides. ACS Paragon Plus Environment

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Amines can be also used as reagents to enhance the chemical21,22 and enzymatic23–25 reactivity of polysaccharides. Studies of several different amine-polysaccharide crystal solvates have provided detailed information on the conditions of complex formation, stoichiometry, stability26–29 as well as their crystal structures9,10,30–34. We have contributed to these studies by determining the high resolution X-ray crystal structure of the complex formed by ethylenediamine (EDA) and β-chitin35, the neutron and X-ray crystal structures of the complex formed by EDA with cellulose10,36 and the neutron and X-ray crystal structures of the complex formed by ammonia and cellulose9,37. In all cases, the polysaccharide chains pack in hydrophobic stacks without chain stagger, with the primary hydroxyl groups in the gt conformation, with disordered hydrogen bonding. The amines mostly accept protons rather than donate them to hydrogen bonds from the primary hydroxyl groups. The similar features of the different complexes suggest that there may be common organizing principles in the way the complexes are formed and stabilized at a molecular level. To investigate this possibility further we determined the structure of another amine-polysaccharide crystal solvate, namely that formed by hydrazine with α-chitin. The current model of α-chitin has anti-parallel chain packing with roughly P212121 symmetry with one acetylglucosamine residue as the asymmetric unit, but with disordered primary hydroxyl group conformation38. Interestingly the crystallinity improves as the native fibers are complexed with hydrazine while still forming an orthorhombic unit cell with a P212121 symmetry. We report the structure of the α-chitin crystalline complex with hydrazine determined using high-resolution synchrotron X-ray diffraction and solid-state 13C nuclear magnetic resonance (NMR) spectroscopy. Based on the refined crystal structure we further investigated molecular interactions between chitin and hydrazine molecules by using density functional theory calculation and force field molecular dynamics simulations.


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Experimental Section Sample Snow crab tendon α-chitin was purified as previously described38. The purified samples were immersed in 98 % anhydrous hydrazine, resulting in rapid complex formation. The samples were kept in anhydrous hydrazine until use.

CP/MAS 13C NMR spectroscopy Solid-state 13C cross-polarization/magic angle spinning (CP/MAS) NMR spectra were recorded using a CMX 300 spectrometer (Chemagnetics, USA) operating at 75.6 MHz. The sample was placed in a 4.0 mm zirconia rotor and sealed using a Teflon cap to avoid drying. The rotor was spun at a frequency of 5 kHz in a solid-state probe at the magnetic angle. All the spectra were obtained using a 1H NMR 90° pulse length of 2.5 µs, with a cross-polarization time of 1.0 ms and a 60kHz CW proton decoupling. The recycle time was 3 s. the spectra were calibrated using adamantane as a standard.

Figure 1. Solid-state

13

C NMR spectra from α-chitin and α-chitin hydrazine complex. Carbon

numbers are shown in Figure 3.


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Figure 1 shows the NMR spectra of α-chitin and the α-chitin/hydrazine complex. The peak assignment is based on a previous two-dimensional study of α-chitin39 assuming that the order of the chemical shift does not change by the complex formation. The hydrazine complex produces narrow peaks compared to original α-chitin, with single peaks for each carbon atoms except for the C4 atom whose signal splits in two with 0.8 ppm difference.

X-ray Studies Synchrotron X-ray fiber diffraction data were collected at the beam line BL40B2 at SPring-8 (Hyogo, Japan). The sample was mounted perpendicular to the beam (λ = 0.7 Å) on a goniometer. A fiber diffraction pattern was recorded using a flat imaging plate (IP) (R-AXIS IV++, Rigaku) (Figure 1a). The sample-to-IP distance, about 196 mm, was calibrated using Si powder (d = 0.31355 nm)38. The positions of 66 reflections were measured using R-AXIS software (RIGAKU), indexed with an orthorhombic unit cell, and then used for least squares refinement of the unit cell parameters; a = 4.723 Å b = 22.527 Å c = 10.337 Å; P212121. A polarization correction, background subtraction, and peak fitting were carried out as previously described.3 Briefly, the polarization factor P was used for the azimuthal angle ρ and the scattering angle θ with linear polarization coefficient A of 0.83 as follows40: =

( )

−

The diffraction pattern was converted into polar coordinate with applying the polarization correction. The background was evaluated for each radial trace of polar coordinate using bicubic spline function with grid points every 50 pixels. A Bayesian approach was used to evaluate the background and the first guess was given by Sonnevelt algorithm. Linear least-squares fit cycles with weighting scheme to scale down the intensity from diffraction peaks were applied to maximize the likelihood function. The orientation distribution of the fiber axis was approximated by a single Gaussian function whose width was estimated from the azimuthal intensity distribution of equatorial reflection. The radial width of each peak was given based on the peak position as follows: ACS Paragon Plus Environment

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= + ( ∗ , )/ 2" " = # + $% &' + ∗ where w0 corresponds to the broadening of the peak from the size of beam, wm is the width of the peak along the meridian and weq is the width along the equator, and c is the paracrystallinity parameter. Given the peak profile of each diffraction, the diffraction data should be a linear combination of the peak profile with positive peak intensities, which was determined in a block-wise manner (360 (W) x 150 (H)) by least-squares fitting. A LAPACK driver function DGELSD was applied to compute the minimum norm solution for under-determined linear least-squares problem. The fitting was iterated on the residuals with negative intensities set to zero. The fitted intensity of each Miller index was kept in a “crude intensity list” for Fourier syntheses. For structure refinement we grouped neighboring reflections on the same layer line that were close enough to overlap into composite intensities in a “regrouped intensity list”. X-ray structure refinement was carried out using previously described strategies3,41 and restraints42 for applying SHELX-9743 to fiber diffraction data. The backbone atoms of native α-chitin were taken as the starting model38. An initial refinement was carried out with the primary hydroxyl group O6 removed from the model and using the crude intensity list up to 1.09 Å resolution (atomic numbers are described in Figure 3) with the meridional intensities omitted. Both Fo-Fc and 2mFo-Fc (σA) omit maps were calculated and then visualized by using Coot44 software.


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Figure 2. (a) The X-ray diffraction pattern from crab tendon α-chitin complex with hydrazine. Section through the X-ray σA map (indicating electron density around the molecules) and the X-ray FoFc omit map (showing positive and negative density in green and red, respectively). (b) Calculated using only the backbone (omitting O6). Density indicated by the circle can be associated with the primary hydroxyl O6 in gt position. (c) Calculated after introduction of O6. Density indicated by the circles can be associated with hydrazine N11 and N12. (d) Calculated with the complete molecular model (except hydrogen atoms).

Except for the primary hydroxyl groups, the only degree of freedom that offers significant movement is the translation along the c-axis. Other movement is inhibited because of the rigidity of the pyranose ring and the steric hindrance with the neighboring chains. The chain was therefore translated along caxis (chain) direction to reduce the R1 value from 0.51 to 0.46 at a translation of 2.5 Å. At this translated position, a clear density peak appeared in the Fourier omit map corresponding to the primary hydroxyl oxygen O6 in gt position (Fig. 1b). When the O6 was included in the refinement, the value of R1 decreased to 0.42, Figure 1c, and two density peaks appeared that could be associated with the


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hydrazine nitrogen atoms. Incorporation of these N atoms resulted in R1= 0.39 (Fig. 1d). During the refinement, the hydrazine N-N bond length was restrained to 1.44945. Further refinement was carried out using the regrouped intensity list, resulting in a reduction in the number of data (Fo > 4σ(Fo)) from 206 to 118, and R1 = 0.1723. Incorporation of hydrogen atoms attached to carbon atoms at standard positions using HFIX options gave R1 = 0.1578. Finally, the intensity data above 1.14 Å resolution, or those with Fc / Fcmax < 0.083 were omitted41. This resulted in small changes in atomic coordinates of hydrazine molecule and R1 = 0.1332 for 99 data. The coordinates of the X-ray structure are available in pdb format in the supplementary information. Table 1 shows the list of potential hydrogen bonds predicted from the X-ray structure. The criterion for the donor O (N) atom to the acceptor O (N) atom was set to 4 Å.

Table 1. List of potential hydrogen bonds in α-chitin hydrazine complex. Donor

Acceptor

N2

O7

Do…A distance (Å) 2.74

O3

O5 O6 N11 N11

O6

N11

N12

-

acceptor residue x-1,y,z

2.81 3.29 3.23 3.08

94 141 87 142

3/2-x, -y, -1/2+z 3/2-x, -y, -1/2+z x-1, y, z x, y, z

N12

2.96

168

x, y, z

O3 N12 N12

3.29 3.23 2.49

111 100 141

3/2-x, -y, 1/2+z 5/2-x, -y, 1/2+z 3/2-x, -y, 1/2+z

N11

3.79

121

5/2-x, -y, 1/2+z

O3 O3

3.08 3.23

72 105

x, y, z x+1, y, z

O7

3.37

140

x, y, z

O3 O6

2.96 2.49

81 151

x+1,y,z 3/2-x, -y, -1/2+z

O6

3.18

96

5/2-x, -y, -1/2+z

R-Do...A (°)


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Periodic density functional theory calculation Hydrogen atoms scatter X-rays weakly, and their positions could not be located in the crystallographic electron density maps. Therefore we used DFT calculation and MD simulations to study the hydrogen bonding system. As already described in the literature an amine group is a weak Hydrogen bond (HB) donor and a strong HB acceptor. Indeed, this HB property of amines has been demonstrated based on neutron crystallography and MD simulations for two other crystalline complexes of polysaccharides and amines, namely cellulose-ammonia complex37 and cellulose-EDA complex36. Thus, we assume that the hydrazine molecule is most likely to act as HB acceptor. Possible HB donors in the X-ray structure are H-O3, H-O6 and H-N2 where H-N2 can form HBs only with the neighboring acetamide groups O7 to support the hydrophobic stacking. Although H-O6 has several possible HB acceptors, the HB to the hydrazine N12 is the most geometrically favorable. On the other hand, the H-O3 has two favorable HB acceptors, O5 and N11 with similar geometric conditions. Therefore, in the DFT calculation we considered two different HB systems, namely pattern A and B (Figure 3). H-O3 donates a HB to N11 atom of hydrazine in pattern A, and to O5 in pattern B. In both patterns the H-O6 donates a hydrogen atom to the N12 atom.

Figure 3. Superimpositions of the structures of α-chitin hydrazine complex before (black) and after (colored) DFT optimizations with (a) pattern A and (b) pattern B hydrogen bonding systems. Arrows denote hydrogen bonds. ACS Paragon Plus Environment

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These two possible HB patterns were analyzed with periodic DFT calculations with ultrasoft pseudopotentials and plane wave basis set using Quantum Espresso, ver 5.046 as previously described47. The optimizations were performed with one unit cell structure, resulting in the structures shown in Figure 3. The cell dimensions with relative potential energy of the optimized structures are given in Table 2. In both structures the HB patterns were similar to the initial patterns, but the cell dimensions were different after the optimization. The parameter b was not sensitive to the HB pattern since there are no HB interactions between chains in the b direction. On the other hand, parameters a and c were sensitive to the HB pattern. The sensitivity of the parameter c is related to the glycosidic angle, τ (C1-O1-C4). The angle τ of the structure with HB pattern A, 117.3° is larger than that with HB pattern B, 116.3°, so that the molecular chains are more extended with the HB pattern A. Note that the parameter c of both of the optimized structures is different from the experimental value, and this discrepancy is discussed below in this paper. The optimizations did not change any major structural features; the gt position of the primary hydroxyl groups, intermolecular HBs of the H-O6 to the hydrazine nitrogen N12, and initial HBs of the H-O3 to the ring oxygen O5 or the hydrazine nitrogen N11. The hydrazine molecules are slightly displaced from crystallographic positions when not involved as a HB acceptor. The conformation of hydrazine molecules in both of the optimized structures is gauche which is its most stable conformation48. The structure with the pattern A HB is energetically favorable, and the energy difference, 12.7 kJ/mol is about 5 times larger than kBT, 2.4 kJ/mol at 300 K.

Table 2. Cell dimensions and relative potential energy of the optimized structures b (Å)

c (Å)

4.723

22.527

10.337

Pattern A

4.636

22.048

10.504

0

Pattern B

4.728

21.929

10.280

12.2

300 K

4.828

22.782

10.240

Exp DFT-D2

MD

ΔE (kJ/mol)

a (Å)


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Crystal Growth & Design 250 K

4.816

22.666

10.255

Molecular dynamics simulation Molecular dynamics simulations were performed using GROMACS, ver 5.049. The pattern A HB system was taken as a starting structure of the MD simulations because of its energetic favorability. We constructed an infinite P1 supercell model by duplicating the unit cell structure. The supercell is roughly cubic (4×4×4 nm3), and contains 256 N-acetyl-D-glucosamine (GlcNAc) residues (8×4×8) and 256 hydrazine molecules. To simulate infinite size crystal three-dimensional periodic boundary conditions were applied and each individual chitin chain composed of eight GlcNAc monomers is covalently bonded to its own periodic images. The energy of the system was first minimized by the conjugate-gradient method. A maximum force of 1.0 kJ/mol/nm was used as the convergence criterion that signals the end of minimization. In the MD simulations, the equations of motion were solved by using the standard leap-frog algorithm with time steps of 1 fs. The simulation box was firstly heated up from 0 K to 300 K with a heating rate of 0.5 K/ps, and then kept at 250 K and 300 K for 10 ns. MD frames were saved every 10 picoseconds. The cutoff distance for nonbonding interactions was 0.9 nm, the long-range dispersion forces were corrected for energy and pressure, and a particle mesh Ewald summation was used to calculate the long-range electrostatic interactions. The covalent-bond lengths were constrained by using linear-constraint-solver (LINCS) algorithms50. The pressure was regulated by using the Berendsen algorithm51 with a 2 ps pressure relaxation time and the temperature was controlled by using the Berendsen-type velocity rescaling algorithm52 with a 0.1 ps coupling-time constant. The xx, yy, zz, xz, yz, and xy components of the box compressibility were 6.2×10−6, 7.8×10−6, 2.4×10−6, 6.4×10−5, 5.5×10−6, and 5.0×10−5 bar−1. Lattice parameters and HB occupancy were calculated from the last 2.5 ns of the 10-ns simulation. We used all-atom CHARMM C35 force field53. In the CHARMM force field the Lenard-Jones parameter σ of hydrogen of hydrazine is parameterized as those of hydrogen in hydroxyl group (σ = 0.040 nm), so that hydrogen of hydrazine can donate a strong HB as hydroxyl hydrogen can do. ACS Paragon Plus Environment

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However, in reality the lone pair of the nitrogen atom makes the amine group less accessible and consequently a weaker HB donor in comparison to hydroxyl hydrogen. To simulate this effect in point charge approximation the CHARMM force field adopts a larger σ, 0.155 nm for hydrogen of ammonia to give a stronger repulsion. Since this correction has not been applied to the hydrazine molecule we introduced σ of ammonia hydrogen to hydrazine hydrogen in this study. The averaged values of cell dimensions calculated by MD simulation are listed in Table 2 and a snapshot of the MD simulated structure is shown in Figure 4a. The unit cell parameters were reproduced in good agreement with the experimental values with the largest departure of 2.2 % for the a-axis. The backbone structure of chitin molecules is maintained as the X-ray structure. The hydrazine molecules are more mobile and displaced from the crystallographic positions, but remains on the same site for the duration of the 10 ns simulation.

Figure 4. Snapshot of MD-simulated crystal structure of α-chitin hydrazine complex (a). Thin solid lines denote hydrogen bonds. For clarity hydrogen bond criteria were set as 3.0 Å for Donner-toAcceptor distance and 150° for Do-H…A angle. Spatial distributions of atomic trajectories of HO3 (gray), N11 (blue), and N12 (red) atoms with the average structure of chitin molecule in the last 2.5 ns of the MD trajectory (b). Aliphatic hydrogen atoms were removed in this representation.


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Table 3 lists the HB occupancy values in the last 2.5 ns of the 10-ns MD simulations and Figure 4b shows spatial distributions of atomic trajectories of H-O3 and nitrogen atoms of hydrazine molecule in the same span of the MD trajectory at 300 K. In the MD structure H-O3 donates to two hydrazine nitrogen atoms and the O5 ring atom. The occupancy of the HB between H-O3 and hydrazine is twice as large as that between H-O3 and O5. Thus the HB systems A and B can co-exist in the crystal in the MD simulations. These HBs are not permanent but dynamically exchanged as the H atom of H-O3 fluctuates between the two positions in pattern A and pattern B as shown in Figure 4b. All the other HBs have occupancies greater than 0.8, and we assumed that they represent stable HBs. Note that occupancies of HBs with both nitrogen atoms of hydrazine as acceptors are the same due to the fact that the two nitrogen atoms exchange between two crystallographic positions as described below. Table 3. Hydrogen bond occupancy in the MD simulations at different temperature.

donor HN HO3

HO6

acceptor O7 O5 O6 N11 N12 (Hydrazine) N11 N12

MD Occupancy 300 K 250 K 0.98 0.36 0.05 0.33 0.33 0.66 0.82 0.82

0.98 0.34 0.03 0.35 0.35 0.71 0.85 0.85


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Results and Discussion

Figure 5. Chain translation after hydrazine penetration. (a) Original α-chitin. (b) α-chitin hydrazine complex.

High-resolution structure of an α-chitin hydrazine complex from the tendon of snow crab was reported in this study. The tendon of snow crab contains highly oriented α-chitin fibers, so that diffraction intensities of neighboring layers are well separated. The crystallite size of the tendon, about 8 nm54 was confirmed for the sample used in this study applying Scherrer equation. The crystallite size is smaller than those of the samples used for the previous high-resolution fiber diffraction structure determination of cellulose and β-chitin1–4. An α-chitin hydrazine complex was prepared by simple immersion of naturally occurring α-chitin fibers into anhydrous liquid hydrazine, resulting an increase of the crystallite size estimated using Scherrer equation to 14 nm. This significant increase associated with the complexation may be derived from improvement of crystal perfection and apparent cocrystallization of individual crystallites with the presence of hydrazine molecules between them. The ACS Paragon Plus Environment

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complex was stable at ambient condition, but the measurement was carried out under wet condition without wiping out anhydrous hydrazine on surface to prevent any drying. Strong diffraction intensities from synchrotron X-ray were observed with relatively simple background scattering from air and liquid anhydrous hydrazine. By applying recently developed method for the data processing of fiber diffraction3, reliable diffraction intensities were obtained. The unit cell parameters of α-chitin hydrazine complex (a = 4.723 Å b = 22.527 Å c = 10.337 Å; volume =1099.8 Å3) differ from those of original α-chitin (a = 4.749 Å, b = 18.89 Å, c = 10.33 Å; volume = 920.8 Å3)38 mainly by an expansion in the b-axis direction. The refined structure retained hydrophobically stacked chitin chains along a-axis. It suggests that hydrazine molecules penetrate the space between 0 2 0 planes to expand α-chitin fibrous crystallites while relaxing the intermolecular hydrogen bonds (HBs). However, the chitin stacks along a-axis are translated approximately 2.5 Å with respect to each other along the c-axis direction. As a result of this translation, neighboring GlcNAc residues have acetamide and primary hydroxyl groups on the same side although their chain polarity is reversed (Figure 5). This translation creates a pocket within which the hydrazine molecule can sit, thus allowing for denser packing. Indeed the volume increase is 16 % smaller than expected from the volume of hydrazine molecule, 51.7 Å3, calculated from the liquid density of 1.02 g/cm3 and its formula weight (Table 4).

Table 4. Volumes of amine molecules in bulk states and in crystalline complexes with chitin and cellulose. In α-chitin 3 complex (Å )

3

Bulk (Å )

Hydrazine EDA Ammonia

In β-chitin 3 complex (Å )

In Cellulose I 3 complex (Å )

*1

43.3 (83.6%)

ND

43.1 (83.3%)

*1

89.3 (80.2%)

113.4 (102%)

101.8 (91.4%)

*2

ND

ND

38.8 (93.4%)

51.7

111.3 41.5

*1: at 25°C, *2: at -33.3°C (boiling point)


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Page 18 of 27

In other polysaccharide crystal solvates different types of chain packing rearrangements are observed. In β-chitin there are no intermolecular HBs in the space between hydrophobically stacked 0 1 0 planes and amine molecule penetration does not cause any translation along the c-axis direction of the chitin stacks26,35. In the cellulose EDA complex55 and the cellulose ammonia complex34 penetration of amines translates the stacked cellulose sheets so that they no longer have the stagger found in native cellulose I. In spite of the different chain packing rearrangements observed during amine-polysaccharide complex formation, there are also common structural features. In all cases, incorporation of amine molecules results in the O6 primary hydroxyl group adopting the gt conformation regardless of its conformation in the original structure (original conformation of cellulose I and β-chitin is tg1,2 and gg3, respectively). It should be noted that the gt is the major conformation of the majority of glucose-based small crystals which may be due to the formation of a bifurcated hydrogen bond from H-O3 to O5 and O656. In case of amine-polysaccharide complexes, the occupancy of hydrogen bond from H-O3 to O6 is low. In all amine-polysaccharide complexes, the gt conformation of primary hydroxyl groups allows nitrogen atoms of amine molecules to be located in the space among two secondary hydroxyl groups, O3 and two primary hydroxyl groups, O6. MD simulations36,37 indicate that those HBs are highly dynamic in the amine-polysaccharide complexes with a constant that the H-O6 is always pointing towards the lone pair electrons of amine. In the structure of an α-chitin hydrazine complex, the hydrogen positions of four hydroxyl groups seem to be dominated by local conformational preference. The secondary alcohol hydrogen prefers to sit in the cis position with respect to the neighbor aliphatic hydrogen, while the primary alcohol hydrogen prefers to sit in a trans position with respect to the γ carbon47. The orientation of H-O3 is similar in the H-O3 donating to a water molecule in β-chitin dihydrate. Also, the orientation of H-O6 is similar to that in intermolecular hydrogen bonding in cellulose II and IIII as well as cellulose amine complexes. Thus the conformational freedom of the hydroxyl groups is relatively limited. ACS Paragon Plus Environment

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In the α-chitin hydrazine complex, the ratio of host GlcNAc and guest hydrazine was approximated to be 1 : 1 as indicated in the Fourier-omit map. Further occupancy analysis was performed by using a free parameter for the occupancy of a hydrazine molecule, resulting in the occupancy of 0.86. The smaller occupancy of X-ray diffraction analysis is probably due to the dynamic behavior of hydrazine molecules as indicated by molecular dynamics simulations. That is, widely distributed electron density of a hydrazine molecule reduces the local occupancy of the hydrazine molecule at its coordinate. The ratio of approximately 1 : 1 is the same for most previously reported amine-polysaccharide complexes, namely cellulose I amine complexes9,10,29, cellulose II hydrazine complex28, and β-chitin type II complexes26. Interestingly, the host-guest ratio of the α-chitin EDA complex was reported to be 1 : 0.527 with almost the same unit cell parameters (a = 4.73 Å b = 22.7 Å c = 10.29 Å; volume =1105 Å3) as the α-chitin hydrazine complex. An EDA molecule may occupy the space which is used by two hydrazine molecules in the α-chitin hydrazine complex, so that both nitrogen atoms of an EDA molecule can be involved in sufficient HBs. This would explain the high thermal stability of the α-chitin EDA complex up to approximately 260 °C27. Table 5 shows the isotropic thermal displacement factors calculated from the crystallographic analysis and MD simulation. The experimental values are globally larger than the simulated values. In the MD simulations low-energy phonon modes such as lattice vibration are not reproduced due to the smaller box size of MD crystal than the real crystal. Since such phonon modes contribute more than higher-energy phonon mode at room temperature the underestimation of the displacement factors in the MD simulation is reasonable. Although the absolute values of the experimentally and MD derived thermal displacement factors are different, they have a similar trends; oxygen atoms in side groups have larger displacement factors than the backbone carbon atoms, and the largest value is found in the nitrogen atom in the guest hydrazine. One significant discrepancy is the difference between the two nitrogen atoms of the hydrazine molecule where N11 has a larger value than N12 in the crystallographic analysis while two values are almost equal in the MD simulation.


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Page 20 of 27

Table 5. Isotropic atomic thermal displacement factors (Å2) calculated from crystallographic analysis (Exp) and MD simulation at 300 K (MD). Exp

MD

C1

4.60

0.34

C2 C3

4.61 3.63

0.25 0.41

C4 C5

4.81 2.05

0.41 0.48

C6

2.44

1.21

C7 C8

6.83 5.82

0.89 2.09

O1 O3 O5 O6 N O N11

4.64 6.90 4.71 4.99 4.99 9.76 19.57

0.47 1.22 0.62 1.46 0.49 3.25 14.85

N12

14.69

14.53

This discrepancy can be explained by the dynamic behavior of the hydrazine molecule. Figure 4b shows spatial distribution of two nitrogen atoms, N11 (red dot) and N12 (blue dot) in the MD trajectory at 300 K. Two large and discrete populations denoted as red and blue circles are observed roughly at the crystallographic positions of N11 and N12, respectively. Both of these populations contain N11 and N12 atoms, indicating a fast exchange or rotation of the two nitrogen atoms between the two crystallographic positions. As a consequence of this position exchange the thermal displacement factors of two nitrogen atoms are estimated as almost equal. However, the difference of the thermal displacement factors between two crystallographic positions can be observed in the distribution map as the atomic trajectories are more widely distributed in the red circle region corresponding to N11 position than in the blue circle region corresponding to N12 position. Since the HBs between H-O6 and nitrogen at N12 position are stable (their occupancies are greater than 0.8), the thermal fluctuation of nitrogen atoms is rather restricted by the HBs. On the other hand, instability of HBs between H-O3 and ACS Paragon Plus Environment

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nitrogen atom at the N11 position allows the nitrogen atom to displace and thus results in the larger thermal displacement factor. The dynamic behavior of HBs with H-O3 influences the length of c-axis. DFT optimization with the HB pattern A gave a larger value of parameter c, 10.51 Å than that with the HB pattern B, 10.28 Å. The experimental value, 10.34 Å is in between the values of two DFT optimized structures. Although one can assume the pattern A is dominant in the crystal structure due to its energetic favorability, the smaller experimental values of parameter c than the theoretical value with the pattern A HB suggests an existence of substantial amount of the pattern B in the crystal structure as observed in the MD simulation. The enthalpy difference of patterns A and B in the force-field approximation could not be obtained since energy minimization starting from pattern B results in pattern A. Qualitatively, the higher energy of pattern B HB can be compensated by higher entropy due to the fact that the nitrogen atom at position N11 can explore more space. In the MD simulations the c-axis was about 1% shorter than the experimental value and is also shorter than the DFT optimization of both A and B pattern. The relatively large departure is essentially due to the bias of force-field parameters. On the other hand, the population of H-O3 donating an intramolecular HB to O5 atom reduces from 36% to 34% by the reduction of temperature from 300 K to 250 K and this was accompanied by slight elongation by 0.15% of the c axis, corroborating the above hypothesis that the experimental length of c-axis comes from the mixture of patterns A and B co-existing inside the crystal.

Table 6. 13C chemical shift of chitin and cellulose and their amine complexes. C1 α-chitin

39

C2

C3

C4

C5

C6

C7

C8

EDA

104

54.8

73.4

82.9

75.6

60.6

173

22.6

-

hydrazine complex

104.9

56

75.4/75.0

87.0/86.2

76.8

61.6

173.1

23.5

-

EDA complex

104.9

55.8

75.7

88.0/87.5

76.9

62.2

172.7

25.4/24.4

46.6/45.2


21

Crystal Growth & Design β-chitin

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

57

105.3

55.2/56.0

73

84.4

75.4

59.8

175.5

22.6

104.1

54.9

74.1

86.9

75.8

61.2

171.7

23.4

105.1

55.4/56.3

74.4

83

75.3

59.1

174.9/175.8

23.9

107.6/105.9

73

76.8/76.0

90.6/90.0

74.2/73.0

67.5/66.9

-

-

-

106.6

75.3

77.7

89.6

74.6

64

-

-

-

107

73.4

79.4

86.4

78.3

63.8

-

-

46.1

EDA complex Dihydrate

57

cellulose Iβ

58

cellulose IIII

Page 22 of 27

59

EDA 60 complex

45.0

Finally, there are two open questions for the solid state NMR spectrum of an α-chitin hydrazine complex. One question is the reason for the C4 splitting. Although DFT optimized structure with pattern A and B have different glycosidic angles, this cannot explain the splitting of C4 due to the dynamic nature of the exchange between the two patterns at a time scale much shorter than the radio frequency used in NMR. Another question is about the downfield shift trends of ring carbons accompanied by the complexation with amines. C4 peaks exhibit the most important downfield shift caused by complexation with hydrazine molecules. The complexation of α- and β-chitin with EDA also moves the C4 shift by 5 and 2.5 ppm from original form. Complexation of cellulose with amine molecules move the C4 signal in the opposite direction as the crystalline cellulose exhibits an unusually low magnetic shielding for its chemical environment. Consequently, all amine complexed cellulose or chitin shows the C4 in the rather narrow 86-88 ppm range. Neighboring C3 and C5 of chitin also show downfield shift by 1-2 ppm when complexed with amines. The downfield shift is even more pronounced in cellulose (2-4 ppm). By forming HBs, the lone pair of amine would pull the proton of hydroxyl groups, rendering the donor oxygen more electronegative. That in turn, would shield the neighboring carbon. However, the downfield shift of C3 and C5 suggests that the electrons are pulled from those carbon atoms, which is counter intuitive. The chemical shifts of those ring carbons seem to be weakly correlated with conformational parameters unlike that of primary hydroxyl group which is strongly affected61 by its


22

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conformation (Table S1). Further study is necessary to understand the fine detail of the effect of amine complexation which seems to be relatively universal. Conclusion Hydrazine molecules penetrate and expand α-chitin crystals to form a stable crystal solvate complex. During this process hydrophobically stacked chitin chains are translated with respect to each other along the c-axis direction to create a pocket formed by acetamide and primary alcohols within which the hydrazine molecule can sit. A geometry optimization using dispersion corrected DFT alone cannot predict the dynamic crystal structure, but molecular dynamics simulation taking into account the dynamic nature of the guest molecule combined with DFT optimization could qualitatively explain the average structure obtained by X-ray diffraction. Several common features could be picked up among different structures of cellulose/chitin –amine complex. In particular, all have hydrophobically stacked molecular chains, primary hydroxyl groups in the gt conformation donating hydrogen bond to the amine, locally mobile amine molecules, and dynamic hydrogen bonding. Supporting Information Available. Molecular coordinates in pdb format and conformational parameters in Table s1. Acknowledgment Authors thank the Japan Synchrotron Research Institute (JASRI) for the provision of beam time at BL40B2 in SPring-8. Authors thank Dr. Axel Ettori for support with 13C NMR data collection. Y.O. is financially supported by Japan society for the promotion of science (JSPS). P.L. and D.S. were partly funded by the Genomic Science Program of the Office of Biological and Environmental Research, Office of Science, US Department of Energy, under FWP ERKP752 and by the US Department of Energy, managed by UT-Battelle, LLC under contract No. DE- AC05-00OR22725.


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For Table of Contents Use Only Authors: Daisuke Sawada, Yu Ogawa, Yoshiharu Nishiyama, Eiji Togawa, Satoshi Kimura, Paul Langan

Title: Molecular Interactions in an α-chitin/Hydrazine Complex: Dynamic Hydrogen Bonds and Improvement of Polymeric Crystallinity Synopsis: Solvation with hydrazine molecules improved a crystallinity of α-chitin polymer. The highresolution structure of an α-chitin/hydrazine complex was determined using X-ray fiber diffraction, density functional theory calculation and force field simulations to reveal the molecular interactions in the structure. Molecular dynamics calculations show that hydrazine nitrogen atoms are disordered with competing hydrogen bonds.


27

Hydrazine Complex: Dynamic

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