Article pubs.acs.org/Macromolecules
Absence of Sum Frequency Generation in Support of Orthorhombic Symmetry of α‑Chitin Yu Ogawa,†,‡ Christopher M. Lee,§ Yoshiharu Nishiyama,*,†,‡ and Seong H. Kim*,§ †
Univ. Grenoble Alpes, Cermav, F-38000 Grenoble, France CNRS-Cermav, F-38000 Grenoble, France § Department of Chemical Engineering and Materials Research Institute, Pennsylvania State University, University Park, Pennsylvania 16802, United States ‡
ABSTRACT: The native polymorphic structures of chitin, namely αand β-chitin, were studied with X-ray diffraction (XRD), infrared and Raman spectroscopies, and sum-frequency generation (SFG) vibrational spectroscopy. The currently proposed model of α-chitin crystal based on X-ray and electron diffraction study has an antiparallel chain arrangement imposed by the orthorhombic symmetry in contrast to parallel chain arrangements in β-chitin and native cellulose; however, the natural occurrence of antiparallel chain arrangement for α-chitin is controversial to widely accepted biosynthetic mechanisms for structural polysaccharides such as chitin and cellulose, where chains are successively polymerized and crystallized in a unidirectional way. We compared the SFG signals among samples with similar crystallinity, morphology, and textures but differing in allomorphs. The SFG signal from α-chitin was ∼40-fold weaker compared to β-chitin having a similar lateral dimension. The strong SFG signals arising from β-chitin can be explained by a net polar ordering of parallel packed chains. These results strongly supports the antiparallel chain orientation and high symmetry (orthorhombic) of α-chitin suggested by diffraction analysis.
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INTRODUCTION Chitin, a linear polymer of β-1,4-linked N-acetylglucosamine, is one of the most widespread and abundant biopolymers. Chitin is biosynthesized as nanometer thick crystalline fibers, called microfibrils. Based on X-ray diffraction (XRD) and infrared (IR) vibrational spectroscopy, two crystalline allomorphs have been identified for native chitin: α- and β-form. The industrially predominant form is α-chitin which occurs in the exoskeleton of arthropods and fungal and yeast cell walls, and the more rare form, β-chitin, is found in aquatic organisms such as squid pens, mollusk shells, diatom spines, and the housing tubes of deepsea tubeworms.1 The structures of chitin allomorphs have been extensively studied using X-ray and neutron diffraction,2−6 electron microscopy and diffraction,7−9 solid state nuclear magnetic resonance (NMR) spectroscopy,10 Fourier-transform infrared (FTIR),2,5,11−13 and Raman spectroscopies.13 The crystal structure of β-chitin is well established from diffraction studies with highly crystalline specimens.3,14 This allomorph is known to form several hydrate crystals at high humidity conditions1 and to be dehydrated upon drying to form an anhydrous form.15 The β-chitin anhydrous and dihydrate crystal structures have been determined based on X-ray and neutron diffraction study. Both of the structures have a monoclinic one chain unit cell with P21 symmetry with all chains arranged in “parallel” where all reducing ends point in the same direction along the caxis (green arrows in Figure 1c,d). This is similar to the parallel chain polarity found in the native cellulose polymorphs.16−18 © XXXX American Chemical Society
There is high similarity in molecular arrangement with cellulose IIII,19 and the structure contains only one anhydrous sugar residue in asymmetric unit without any disorder. On the other hand, the structure of α-chitin is less established involving inherent disorder in the model.2,20 Based on systematic absence of odd order reflections of h00, 0k0, and 00l, an orthorhombic unit cell with P212121 symmetry is proposed,2 which requires an antiparallel chain arrangement where the reducing ends of two adjacent chains are pointing in the opposite direction along the c-axis (green and red arrows in Figure 1a,b). Naturally occurring polysaccharide crystals have predominantly parallel structure (Table 1), corroborating the fact of high density of enzymatic activities and the systematic addition of monomer units to the reducing ends of existing polymer, which results in the crystal structures containing parallel packing of polysaccharide chains. For cellulose, a transmembrane protein complex, namely cellulose synthase complex (CSC), was identified as the synthetic machinery.21 The crystal structure of the transmembrane synthetic enzyme from Rhodobacter composing CSC was determined, and a polymerization mechanism was recently reported.22 The cellulose chains are extruded out of the cell wall with leading nonreducing end which are then crystallized into cellulose Received: July 22, 2016 Revised: August 10, 2016
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DOI: 10.1021/acs.macromol.6b01583 Macromolecules XXXX, XXX, XXX−XXX
Article
Macromolecules
Figure 1. Molecular models of the crystal structure of α-chitin and β-chitin showing the chain directionality. Chains are aligned along the c-axis in the unit cell. (a) α-Chitin bc projection, (b) α-chitin ab projection, (c) β-chitin bc projection, and (d) β-chitin ab projection. The arrows are in the direction from the nonreducing to reducing ends. In (c) and (d) the “up” arrow in green points out of the page and the “down” arrow in red points into the page. Hydroxyl and amide hydrogen atoms in α-chitin are inserted based on the model by Sikorski et al.2
Table 1. Proposed Crystal Structures from XRD and Corresponding Chain Polarity from XRD and SFG for Cellulose, Amylose, and Chitin
a
crystalline polysaccharide
produced in nature?
crystal structure (XRD)
chain or helix polarity (XRD and SFG)
ref (XRD)
ref (SFG)
cellulose Iα cellulose Iβ cellulose II amylose A-type amylose B-type amylose V-type α-chitin β-chitin (anhydrous)
yes yes very rare34 yes yes very rare35 yes yes
triclinic monoclinic monoclinic monoclinic hexagonal hexagonal orthorhombic monoclinic
parallel parallel antiparallel parallel parallel antiparallel antiparallel parallel
17 16 24 35 36 37a 2 3
33 26 26 28 28 28 this work this work
Based on electron diffraction analysis.
resolved X-ray diffraction diagram with additional reflections which are not expected from the currently proposed model,6 indicative of a more complex crystal structure. Thus, it is still fairly challenging to determine the crystal structure of α-chitin with the diffraction technique alone. The lack of complementary techniques that can characterize chain polarity in molecular crystals unambiguously has been a challenge in crystal structure determination. For polysaccharide crystals, electron microscopy using silver or gold labeling of reducing ends of glucan chains is one approach for discrimination of chain polarity.9,23,25 However, experimental difficulty that lies in labeling efficiency and accuracy restricts the application of this method to highly purified cellulose or chitin crystallites. Recently, sum-frequency-generation (SFG) vibrational spectroscopy was shown to strongly correlate with the polarity of cellulose chains,26 collagen fibril bundles,27 and starch helices.28 SFG vibrational spectroscopy is a second-order nonlinear optical technique which is selective to noncentrosymmetric
microfibrils with all reducing ends pointing in the same direction or with “parallel” chain polarity.23 Unlike cellulose, the details of chitin polymerization are not as well understood. For β-chitin, unidirectional spinning of the microfibril was reported using combination of electron diffraction and gold labeling of reducing end, so that the mechanism for its biosynthesis has been proposed to be similar to cellulose.9 In contrast, the natural occurrence of antiparallel chain arrangement in α-chitin is somewhat counterintuitive, considering the fibrous morphology of the crystal which is similar to that of native cellulose and β-chitin. The antiparallel chain arrangement would require a very different synthetic machinery or mechanism.8 For these reasons, it is critical to have an independent technique that is sensitive to the chain polarity of cellulose and chitin. The fiber diffraction analysis has been the primary technique to determine the polysaccharide crystals.16,17,24 Highly crystalline α-chitin specimen is found as an extracellular spine of a phytoplankton, Phaeocystis. This crystal provides well B
DOI: 10.1021/acs.macromol.6b01583 Macromolecules XXXX, XXX, XXX−XXX
Article
Macromolecules
of purified diatom spine β-chitin was treated in 35% NaOH aqueous solution at room temperature for 1 h to convert to α-chitin.5 The treated sample was then washed with water. The droplets of chitin water suspensions were deposited onto glass plates and air-dried to make film shaped specimens for FT-IR, Raman, and SFG measurements. For X-ray diffraction measurements the suspensions were freeze-dried, and about 100 mg of each dried chitin was then compressed to disk-shaped pellets. Sum-Frequency-Generation (SFG) Vibrational Spectroscopy. In SFG, two high intensity laser pulses are irradiated simultaneously in the sample which combine forming a new photon at the sum of the two input frequencies (ωSFG = ω1 + ω2). In SFG vibrational spectroscopy, ω1 is fixed (typically 532 nm) and ω2 is tunable in the infrared (2.3−10 μm). SFG spectra were obtained from α- and βchitin pellets using a SFG spectrometer (EKSPLA) as described previously.32,38 The 532 nm visible laser pulses were generated by frequency doubling the fundamental 1064 nm beam from a Nd:YAG laser (repetition rate = 10 Hz, pulse duration = 27 ps). Tunable IR pulses (2.3−10 μm,
