Structural Analysis of Carbohydrate Polymers by Using Infrared Free Electron Laser

1. Introduction

Carbohydrate is one of key players along with protein, nucleic acid, and lipid among biological polymers in biochemical system [1,2,3]. Especially, polysaccharides (containing more than 20 monomeric sugars) exist at various states in nature, for example, as components of plant tissues [4,5], cellular matrices of animal tissues [6,7], outer shells of insect and crustacean [8,9,10], and cell wall of microorganism [11,12]. There are two types of natural polysaccharides regarding the chemical composition: homo-glycan and hetero-glycan. In the former type, amylose, amylopectin, and glycogen are composed of glucosyl bonds that are linked via α-1, 4 or α-1, 6 bonds [13,14,15]. Cellulose is polymerized by β-1,4 bonds [16,17], and chitin is constructed by β-1,4GcNAc units [18,19]. Both polysaccharides are structured by fiber-like carbohydrate chains, contrasting to globular chains in starch. In the hetero-glycan, heparin is composed of glucosamine and iduronic acid [20,21], hyaluronic acid is constructed by glucuronic acid and N-acetylglucosamine [22,23], and glucomannan is polymerized by glucose and mannose [24,25]. Those carbohydrate polymers perform important roles in the expression of biological functions in addition to potential resources of pharmaceutical agents and food additives [26,27]. To understand the functional roles of those carbohydrates in biological system, it is much necessary to disclose the detailed structure of polysaccharides.

In many cases, it is important for characterization of whole structures of parent carbohydrate polymers to analyze a part of the oligomerized or the monomeric states. For detection of monomeric sugars, colorimetric assay is conventionally used [28,29,30,31,32,33]. For example, anthrone-sulfuric acids can be widely applied for aldose and reducing sugars [28,29,30], Elson-Morgan method using Ehrlich reagent is used for detection of glucosamine [31], and carbazole-sulfuric acid is effective for detection of uronic acids [32,33]. In the clinical chemistry, fluorescence labeling method was developed due to requiring for detection of small amounts of sugar ingredients in biological tissues [34,35]. Quantification of glucose in blood by using fluorescent-labeling boronic acid derivatives [34] and detection of glucose and lactose in serum by using fluorescent iron complex combined with oxidases are developed [35]. For analysis of oligosaccharide structures, various fluorescent labeling reagents are developed as well, and especially, 2-amino pyridine is useful in derivatization of oligosaccharides for detection by high performance liquid chromatography (HPLC) [36,37]. Capillary electrophoresis (CE) can also be a powerful tool for investigating molecular weights of various types of sugars more sensitively than the conventional HPLC [38,39].

As for structural analysis of natural polysaccharides, major analytical techniques are listed in Table 1.

Amylose content is analyzed by various quantitative and qualitative methods including near infrared (NIR) and Raman spectroscopies, CE, and atomic force microscopy (AFM) [40]. Also, size-exclusion chromatography (SEC), multiple-angle laser light scattering (MALLS), and ¹H nuclear magnetic resonance (NMR) are employed for analysis of the soluble fraction in hot water from rice flour [41]. Amylopectin from corn starch is analyzed by SEC, MALLS, and differential scanning calorimetry (DSC) [42]. In addition, starch alteration by enzymatic hydrolysis is thoroughly investigated by scanning-electron microscope (SEM), X-ray diffraction (XRD), Fourier transform infrared (FT-IR) spectroscopy, ¹H NMR, high-performance anion exchange chromatography with pulsed amperometry detection (HPAEC-PAD), DSC, and Rapid Visco Analyzer (RVA) [43]. In particular, HPAEC-PAD is a reliable tool for determining carbohydrate chain length distribution in natural polysaccharides [44]. Glycogen associated with lung disease can be analyzed by matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) combining with N-glycosidase hydrolysis [45]. Photoacoustic spectroscopy (PAS) and ¹H NMR can be utilized for investigating fiber and crystallin conformations of cellulose [46]. In addition, thermogravimetric (TG) analysis was introduced as a new method for determining the relative contents of cellulose, hemicellulose, and lignin in biomass samples [47]. Chitin is analyzed by various methods by using FT-IR, XRD, SEM, UV-Vis-spectroscopy, mass spectrometry (MS), and NMR spectroscopy [48,49]. Heparin can be quantitatively analyzed by enzymatic digestion followed by liquid-chromatography (LC)-MS and strong anion exchange (SAX) chromatography [50]. Particularly, attenuated total reflectance (ATR) FT-IR is employed for analysis of solid-phase samples from a library of various glycosaminoglycan [51]. The molecular distribution of hyaluronic acid can be analyzed by using HPLC-SEC system [52], and solid-state nanopore sensor is developed as a more sensitive detector [53]. Sugar composition of glucomannan can be analyzed by ion exchange chromatography and gel permeation chromatography followed by combining thin-layer chromatography (TLC), FT-IR, and ¹³C NMR [54]. N-glycan is analyzed by CE combined with MALDI-MS and electrospray ionization (ESI)-MS [55].

In general, deproteinization and extraction using water and organic solvents at low- or high- temperatures are frequently employed as pre-treatments for collecting polysaccharides from natural resources prior to spectroscopic studies [56]. In addition, enzymes are applied to cleave the co-valent bonds in the glycol-polymers to separate monolithic functional carbohydrates from the complex polymerized materials [57]. Those procedures are actually necessary for isolating and analyzing polysaccharides distinct from other ingredients such as proteins, but it is undeniable that the stepwise routine process is often time-consuming.

Here, a unique spectroscopic approach by using high energy IR laser for direct analysis of solid polysaccharides is shown. This method demonstrates effects on unveiling chemical components of biological polymers rapidly by combining IR microscopy and ESI-MS [58,59,60].

2. Feature of Method

Many biological and organic molecules possess mid-and near- IR absorption bands from 1000 to 4000 cm⁻¹ (= 2.5 to 10 μm): C=O stretching at 1600−1800 cm⁻¹ (5.5−6.0 μm), N−H bending at 1400−1600cm⁻¹ (6.0−7.0 μm), H−C−O bending at 1200−1300 cm⁻¹ (7.0−7.5 μm), and O−H, N-H, and C-H stretching vibrational modes at 2500−3000 cm⁻¹ (3.0−4.0 μm) [61,62]. If an intense IR laser is irradiated onto an organic molecule, IR multi-photon dissociation (IRMPD) reaction can be induced by the vibrational excitation (VE) energy with the corresponding resonant wavelength, and the chemical bond can be dissociated by the VE energy that exceeds the bond energy [63,64,65]. The IRMPD can be facilitated by IR free electron laser (FEL) [66,67].

Oscillation of FEL is based on a successive interaction of accelerated electron beam (EB) with synchrotron radiation (SR) in a periodic magnetic field called as undulator (Figure 1) [68,69,70,71,72,73,74]. The EB is originated from a radiofrequency (RF) electron gun (2,856 MHz) and is accelerated to 100 MeV by an RF linear accelerator. The SR is stored in an optical cavity, which possesses concave mirrors at both ends. The stored SR is amplified through FEL interactions with EB. As the result, FEL lasing and power saturation can be achieved and highly coherent intense laser light is extracted through a coupling hole in one of the concave mirrors (Figure 1).

Samples are typically put on a slide base or put in a glass tube, and the laser beam can be transported from a beam port onto the sample surface as shown in Figure 2. Since the size of the transported FEL beam is large (>10 mm in diameter), a focusing lens or an off-axis parabola mirror is used for focusing FEL beam onto the sample.

The infrared FEL has unique characteristics compared to thermal light source in laboratory-level IR spectroscopy instrument as follows (Figure 3):

(1) Tunable wavelengths

The oscillation wavelengths of the FEL are tunable generally within the 2.0 - 20 μm (5,000 – 500 cm^-1) range, which covers the absorption frequencies of various vibrational modes of biomolecules (Figure 3a). Many organic molecules have a variety of vibrational modes such as C=O stretching, N-H bending, H-C-O bending, and O-H stretching vibrational modes at 1000-3000 wavenumbers (cm^-1). The FELs can excite those vibrational modes wavelength-selectively, and the IRMPD reaction can modify the chemical structure of many molecules in biological matters, gas-phase chemicals, and organic materials. A wavelength of the oscillation beam can be tuned by adjusting the space interval of the undulator or changing the kinetic energy of EB. The typical FEL spectra at 5.8 μm (1,724 cm^-1) and 9.6 μm (1,041 cm^-1) are shown in Figure 3b [75]. The full-width at half maximum was approximately 100–300 nm in both wavelengths.

(2) Pulse structure

The FEL has complex temporal structure called “pulse train” or “burst pulse,” where several thousands of micro-pulses having 0.1-2 ps duration are bunched in one macro-pulse [70,71]. One macro-pulse has 2-15 μs duration, and the repetition rate is ranged from 2 to 5 Hz. The interval of the micro-pulse is 350 ps that is originated from the frequency of electronic gun (2,856 MHz). In the burst mode experiments, micro-pulse rate is divided by 64 (44.6 MHz), where one macro-pulse consists of several hundred micro-pulses [72,73,74].

(3) High laser energy

The macro-pulse energy is ranged from 5 to 25 mJ, and the beam diameter can be set to approximately 200−400 μm on the sample surface by using a parabolic mirror or a focusing lens made of CaF₂ (f = 100 mm) in general. The irradiation effect of the FEL at various fluences on the sample can be investigated by changing the laser energies.

We applied the FEL to analyze solid-phase carbohydrate polymers with combination of synchrotron-radiation infrared microscopy (SR-IRM) and ESI-MS. It can be suggested that the FEL can unveil the persistent structure of the carbohydrates accompanied by releasing their monomeric units without any pre-treatments under atmospheric conditions. The use of SR-IRM analysis improves the spatial resolution with a high signal-to-noise (S/N) ratio compared to that using a thermal radiation beam in laboratory-level instrument because high-energy light can be focused onto a small area (several micro-meters squares) of a dry sample film [76,77].

Analytical studies of two representative carbohydrates by using FELs are shown as described below.

3. Analysis of Chitin

Chitin is a main component of outer shell of crustaceans including shellfish, shrimp, crab, and beetle [78,79]. We analyzed natural powder of the outer shell of crayfish by using the FEL oscillation system (Figure 4-6) [58]. Several adult crayfishes (Procambarus clarkii) were collected from local ponds and maintained in separate plastic containers under a dark/light cycle of 12 h/12 h, and fresh water was changed during feeding. Five molting shells were collected and one solid arm was rip out from the shell and mashed in a mortar, and the resulting powder (ca. 50 mg) was placed in a 5 mL triangular flask. This powder was directly subjected to the FEL irradiation (Figure 4). For detection of conformational changes of polysaccharide structure, SR-IRM in BL6B of UVSOR was used [80]. The instrument was composed of an IRT-7000 IR microscope combined with an FT/IR-6100 series spectrometer, and the mid-IR spectra were measured by Michelson-type interferometer in reflection mode. The sample powder (ca.10 mg) was suspended in water (1 mL), and the mixture (20 μL) was placed on a stainless-steel base. After drying, the plate was put on a horizontally mutable stage, and observations were performed by 16 × Cassegrain lens with an aperture of 50 μm × 50 μm.

The broadband peak around 1100 cm^-1 corresponds to the glycoside bond (νC-O), and the intensity was largely decreased after the irradiation at 9.8 μm compared to the non-irradiation (Figure 5). In addition, both peaks at 1550 cm^-1 and at 1650 cm^-1 were clearly decreased by the irradiation. This region contains N-H bending vibrational mode and C=O stretching vibrational mode of N-acetylglucosamine residues in chitin, respectively. Therefore, it can be implied that a part of carbohydrate chain in crayfish was cleaved, and the N-acetylglucosamine was released by the irradiation.

LC-ESI-MS analysis is shown (Figure 6). An elution peak at 4.6 min was more increased in the case of irradiation at 9.8 μm than the case of irradiation at 5.0 μm (low-absorption wavelength), and other peaks are hardly changed. The increased eluate indicates N-acetylglucosamine (b). Mass chromatograms showed that the intensity of mass peaks corresponding to 243 Da was more increased at 9.8 μm than at 5.0 μm (c). The mass value of 243 Da corresponds to a sodium ion adduct of N-acetylglucosamine residue (221 Da) because of negative-ion mode measurement.

Next, commercially available chitin powder was irradiated by the FEL, and SR-IRM spectra of the chitin is shown in Figure 7. A peak at 1005 cm^-1 corresponding to νC-O was decreased (upper) compared to the irradiation at 5.0 μm (2,000 cm^-1) (middle) and non-irradiation (bottom) after irradiation at 9.8 μm (1020.4 cm^-1). Amide carbonyl band at 1651 cm⁻¹ (νC=O) in N-acetyl glucosamine residues was more increased than the peak at 1617 cm^-1 after irradiation at 9.8 μm (upper) compared to the irradiation at 5.0 μm (middle). Therefore, the glycoside bonds were cleaved and the main chain of chitin was deformed by the C-O targeting irradiation similarly with the outer shell of crayfish. Taken together, it can be concluded that the FEL irradiation at 9.8 μm can degrade the chitin chain and release N-acetylglucosamine units. This method requires no pretreatment such as boiling in acidic water or solubilization using organic solvents.

4. Analysis of Cellulose

Cellulose is a major element of woods, and the degradation product, mono- and oligo-saccharides, attract attentions as the carbon source of bacteria fermenting bioethanol [81,82]. In addition, cellulose nanofibers are developed as functional biomaterials such as biocompatible cell membranes, antibacterial sheets, and food packaging composites in healthcare and pharmaceutical industry [83,84]. However, the cellulose is structured by many glycoside linkages and is generally difficult to be chemically regulated. As shown in SR-IRM spectrum of cellulose (Figure 8), there are four bands at 9.1, 7.2, 3.5 μm, and 3.0 μm [59]. These bands can be assigned to νC−O, δH−C− O, νC−H, and νO−H around the acetal carbon in cellulose, respectively. The FEL was tuned to those wavelengths and irradiated to the cellulose fiber in a glass bottle at room temperature under atmosphere.

The cleavage of the glucoside bonds was revealed by SR-IRM analysis (Figure 9). The νC−O modes are observed from 1000 to 1100 cm⁻¹ (right panel), and after irradiations at 9.1 μm, and 9.1 μm following 7.2 μm, or 3.5 μm, these bands were clearly decreased compared to the non-irradiation (top) and irradiation at 3.0 μm (bottom). In the NIR region at around 3400 cm⁻¹ (gray dotted line), the half width (as indicated by a double-headed arrow) was about 350 cm⁻¹ in the non-irradiation sample (top) and 400 cm⁻¹ in the sample after irradiation at 3.0 μm (bottom). On the contrary, all three irradiations at 9.1 μm, 9.1 μm following 7.2 μm, and 9.1 μm following 3.5 μm shortened the half width to about 300 cm⁻¹. These changes in the hydroxy group region mean that the FEL irradiations except for 3.0 μm largely altered the structure of cellulose from the non-irradiation sample. Together with the spectral change in the mid-IR region, it is surely that the irradiations targeting the acetal group caused the dissociation of the glucoside bonds in the cellulose.

ESI-MS profiles of the non-irradiated sample (a) and samples after irradiation at 9.1 μm following 7.2 μm (b) and 3.5 μm (c) are shown in Figure 10. There were many peaks detected after irradiations compared to the non-irradiation. This indicates that these irradiations caused fragmentation of the cellulose chain. Mass peaks at 689.2, 527.2, 365.1, and 203.0 Da can be assigned to tetra-saccharide, tri-saccharides, cellobiose, and glucose as each sodium ion adduct, respectively.

Productions of cellobiose and glucose can also be investigated by mass chromatography analysis (Figure 11). The continuous irradiations at 9.1 μm following 3.5 μm (light green) and 7.2 μm (deep blue) afforded more amount of cellobiose than the singe irradiation at 9.1 μm (brown), and the continuous irradiations following 3.5 μm was most effective for production of glucose. It can be considered that the VE at 3.5 μm can disrupt the interchain hydrogen bonds and unveil the fiber structure, which leads to cleavage of the glucoside bonds by the IRMPD reaction at C−O stretching vibrational mode. The irradiation at 3.0 μm (light blue) that corresponds to the O−H stretching mode was not effective for production of those saccharides. This implies that the glucoside bond was not affected by the activation of the hydroxy group.

5. Discussion

In this study, we show a physicochemical approach using an intense IR laser as a “molecular scalpel” to analyze solid-phase carbohydrate polymers using chitin and cellulose as model samples. In the case of chitin, the glycoside bonds were cleaved by laser irradiation targeting the C−O stretching vibrational mode (9.8 μm), and N-acetylglucosamine residue was released from the crude shells of crayfish. In the case of cellulose, the continuous irradiation targeting C-O and C-H stretching modes was effective for the cleavage of the glucoside bonds. It can be estimated that the VE giving to the acetal group is effective in degrading polysaccharides into their monomeric sugars.

The physical method using intense IR laser requires no specific conditions such as acidic or alkaline solutions, organic solvents, high pressures, and high temperature, although high electric power for maintaining the instrument operation is needed. One beneficial point of the use of FEL for analysis of complex polymers is the wavelength-tunability. For example, both cleavage of glycoside bonds and dissociation of the hydrogen bonds in the assembled carbohydrate chains can proceed in one sample tube by changing the irradiation wavelengths. Furthermore, the laser-induced dissociation reaction can be completed within several shots of macro-pulse (several milliseconds) due to the pulse structure of the FEL. These physical characteristics have an advantage over the biochemical system using microbial enzymes with regard to the rapidness and the simplicity of the experiment setup. By the similar approach, we can modify lignin that is one of major components of woods alongside cellulose, and the rigid aromatic polymer can be analyzed by the FEL irradiation [60].

Finally, we suggest other possible applications of the FEL in polymer science. Similarly with natural carbohydrates, textile fiber is also rigid polymers. For example, fiber plastics such as polyester and polyamide, fiber proteins such as wool keratin and silk fibroin are polymerized by co-valent linkages of C-C, C-O, and C-N bonds [85,86,87,88]. Textiles often contain dyes, and those insoluble materials cause environmental pollution [89]. As for analysis of dyes, CE is frequently used, and heating with strong acids or alkaline solutions are employed as the pre-treatments [90]. A direct analysis of textile dyes was pioneeringly performed by using matrix-assisted IR laser desorption electrospray ionization mass spectrometry [91]. In that study, the IR laser was tuned to 2.9 μm corresponding to O-H stretching mode of water, which induced desorption of particles of textile fiber. We can suppose the use of FEL as molecular dissection tool for analysis of textile fibers. The textile organic materials exhibit many IR absorption bands as C-O stretching and N-H bending modes, and the irradiation experiments can be performed on the above organic fibers if the FEL can be tuned to those wavelengths. It can be expected that the fragmentation patterns of fibers are different dependent on the irradiation wavelengths, which can allow us to estimate the molecular compositions of the fiber structures. In addition, the monomer materials can be produced from the rigid polymerized fibers by the laser irradiation, and the system can be applied to regenerate the textiles that are wasted in the soil and ocean environments.

As another persistent polymers, per- and poly-fluorinated alkyl substances (PFAS) can be represented, and those small amounts of contaminations including in drinking water are analyzed by solid-phase extraction followed by liquid chromatography/tandem mass spectrometry (LC/MS/MS) [92]. In addition, PFAS contents along with volatile organic compounds in kitchenware are investigated by using GC-MS [93]. We can suppose that the FEL irradiation can affect the conformation of PFAS dependent on the specific wavelengths. Stretching vibrational mode of C-F is observed at 1000-1500 cm^-1, and the VE energy targeting the C-F bonds can be deposited onto the fluorinated molecules, which can induce the degradation of the backbone conformation. This pre-treatment by using the FEL can be applied to the following analyses by the conventional chromatography and mass spectrometry. These studies are now undergoing in our laboratories.

6. Conclusions

We summarized various analytical techniques for carbohydrate polymers briefly and described an original spectroscopic approach by employing intense IR laser in polymer analysis. The FEL oscillates in IR region from 1000 cm^-1 (10 μm) to 4000 cm^-1 (2.5 μm) and can be applied to irradiate rigid carbohydrates represented as chitin and cellulose. After the wavelength-specific irradiations to their solid-phase materials under atmospheric conditions, each monomeric sugar and oligosaccharides are detected by ESI-MS, and the conformational changes of the parent polymeric structures are observed by SR-IRM. The fragmentation and degradation mechanisms are based on IRMPD reaction that is induced by the VE energy from a linear accelerator. The IR laser described herein can be used not only for unveiling rigid carbohydrate polymers but also as a pre-treatment method for dissolving the intact polymerized structure prior to separation from other ingredients by LC and molecular analysis by MS. In addition, the FEL technique can be adopted to irradiate many types of biological and organic molecules by tuning the irradiation wavelengths to their specific vibrational modes. Nonetheless, the current FEL system is installed at a synchrotron radiation facility, and it is expected to develop a laboratory-level IR laser instruments that possess both high laser energy and wavelength tunability in future.