Raman and infrared spectroscopy of carbohydrates: A review

Accepted Manuscript Raman and infrared spectroscopy of carbohydrates: A review

Ewelina Wiercigroch, Ewelina Szafraniec, Krzysztof Czamara, Marta Z. Pacia, Katarzyna Majzner, Kamila Kochan, Agnieszka Kaczor, Malgorzata Baranska, Kamilla Malek PII: DOI: Reference:

S1386-1425(17)30421-3 doi: 10.1016/j.saa.2017.05.045 SAA 15187

To appear in:

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy

Received date: Revised date: Accepted date:

17 March 2017 15 May 2017 22 May 2017

Please cite this article as: Ewelina Wiercigroch, Ewelina Szafraniec, Krzysztof Czamara, Marta Z. Pacia, Katarzyna Majzner, Kamila Kochan, Agnieszka Kaczor, Malgorzata Baranska, Kamilla Malek , Raman and infrared spectroscopy of carbohydrates: A review, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2017), doi: 10.1016/j.saa.2017.05.045

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ACCEPTED MANUSCRIPT Raman and infrared spectroscopy of carbohydrates: a review

Ewelina Wiercigroch, Ewelina Szafraniec, Krzysztof Czamara, Marta Z. Pacia, Katarzyna Majzner, Kamila Kochan, Agnieszka Kaczor, Malgorzata Baranska*, Kamilla Malek*

Faculty of Chemistry, Jagiellonian University, Ingardena 3, 30-060 Krakow, Poland *

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address the correspondence to: Kamilla Malek ([email protected]) and Malgorzata

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Baranska ([email protected])

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Keywords: carbohydrates, sugars, saccharides, FT-Raman, ATR FT-IR, vibrational analysis

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Abstract

Carbohydrates are widespread and naturally occurring compounds, and essential constituents for living organisms. They are quite often reported when biological systems are studied and

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their role is discussed. However surprisingly, up till now there is no database collecting vibrational spectra of carbohydrates and their assignment, as has been done already for other

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biomolecules. So, this paper serves as a comprehensive review, where for selected 14 carbohydrates in the solid state both FT-Raman and ATR FT-IR spectra were collected and

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assigned. Carbohydrates can be divided into four chemical groups and in the same way is organized this review. First, the smallest molecules are discussed, i.e. monosaccharides (D(−)-ribose, 2-deoxy-D-ribose, L-(−)-arabinose,

D-(+)-xylose, D-(+)-glucose, D-(+)-galactose

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and D-(−)-fructose) and disaccharides (D-(+)-sucrose, D-(+)-maltose and D-(+)-lactose), and then more complex ones, i.e. trisaccharides (D-(+)-raffinose) and polysaccharides

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(amylopectin, amylose, glycogen). Both Raman and IR spectra were collected in the whole spectral range and discussed looking at the specific regions, i.e. region V (3600-3050 cm-1), IV (3050-2800 cm-1) and II (1200-800 cm-1) assigned to the stretching vibrations of the OH, CH/CH2 and C-O/C-C groups, respectively, and region III (1500-1200 cm-1) and I (800-100 cm-1) dominated by deformational modes of the CH/CH2 and CCO groups, respectively. In spite of the fact that vibrational spectra of saccharides are significantly less specific than spectra of other biomolecules (e.g. lipids or proteins), marker bands of the studied molecules can be identified and correlated with their structure.

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ACCEPTED MANUSCRIPT Introduction Mid-infrared and Raman spectroscopy are versatile tools in biochemistry and biophysics, with a wide field of applications ranging from the characterization of structural modifications due to biological processes to the identification of biomolecules in animal and plant cells and tissues. It is well-known that infrared and Raman spectroscopic techniques are complementary for the structural analysis of any molecule. Since they differ in selection rules, bands due to vibrations changing molecular polarizability are easily detectable in Raman

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spectra whereas motions, which change of dipole moment, can be better recognized by IR spectroscopy. Both the techniques are rapid, non-destructive and generally do not need special

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protocols for sample preparation. Vibrational spectroscopy is a powerful probe of molecular

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structure and its advantages for biomedical research with a special emphasis on proteins and nucleic acids are widely recognized in the literature [1,2]. Surprisingly, an overall comparison

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of Raman and IR features of fundamental carbohydrates has not been done yet despite their enormous role in several processes of glycobiology such as photosynthesis and oxidation in the energy pathway as well as being protective elements in the cell walls and molecular agents

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in cellular recognition. Structural and analytical investigations on carbohydrates can be difficult hence the physical and chemical properties of the basic units in these polymeric

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chains are very similar. In terms of vibrational spectroscopy methods, both of them provide different set of characteristic bands indicating e.g. the nature of H-bonding in IR spectra or the

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ring configuration in Raman spectra.

In this review, we described complementary FT-Raman and ATR FT-IR spectra of main carbohydrate building blocks that play an important role in glycobiology, biochemistry

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of plant and animal cells, agriculture, food chemistry and many other disciplines. The comparative analysis is divided into four parts describing in detail spectral characteristics of

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monosaccharides possessing structure of pentoses and hexoses, oligosaccharides and polysaccharides. In total, 14 carbohydrates in the solid state are investigated. On the contrary to most reports published so far, our comparison is based on a full spectral region accessible in Raman and IR spectra. The idea was is to provide a comprehensive evaluation of Raman and IR features specific for each sugar and then to present an overall characteristics for the given group of these biomolecules. Spectral changes in oligo- and polysaccharides were are also interpreted in connection with the number and the type of monomers as well as the type of the glycosidic linkage between them. Since spectra of saccharides, especially polysaccharides are generally complex and difficult to interpret directly, we collected detailed 2

ACCEPTED MANUSCRIPT band assignments reported so far in the literature and supplemented with an assignments based on the known modes specific for the a given functional group of carbohydrates. Our discussion on Raman and IR spectra is based on five spectral regions showing specific molecular vibrations of the sugar functional groups to provide a useful guide for identification of carbohydrates and their mixtures as well as for evaluation of their structural modifications (a similar idea of discussion was used before in our reviews on proteins [2] and lipids [3]. Therefore, we specify ied the regions of the stretching modes of the OH (region V, 3600-3050

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cm-1), CH/CH2 (region IV, 3050-2800 cm-1) and C-O/C-C groups (region II, 1200-800 cm-1) as well as we focused provide the spectral characterization of on the deformational motions of

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the CH/CH2 and CCO moieties in the regions III (1500-1200 cm-1) and I (800-100 cm-1),

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respectively. In addition, we referred to their molecular structures discovered by crystallographic studies and we introduced briefly a reader to the fundamental functions of

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studied carbohydrates in biology of living organisms. as well Finally as we also summarized various applications of vibrational spectroscopy techniques in detection and quantification of

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carbohydrates.

Experimental

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All investigated chemicals were supplied by Sigma-Aldrich and used without further purification. The following 14 standards of carbohydrates were measured: D-(−)-ribose, 2-

D-(+)-sucrose,

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deoxy-D-ribose, L-(+)-arabinose, D-(+)-xylose, D-(+)-glucose, D-(+)-galactose, D-(−)-fructose, D-(+)-maltose

monohydrate,

D-(+)-lactose

monohydrate,

D-(+)-raffinose

pentahydrate, amylopectin, amylose and glycogen.

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Raman measurements

Raman spectra of carbohydrates were recorded using a MultiRAM FT-Raman spectrometer

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equipped with a 1064 nm laser line and a germanium detector. 128 to 512 scans were accumulated to obtain the spectra in the a 50-4000 cm-1 range with a resolution of 4 cm-1 and a laser power of 100 mW. Solids of the samples were measured directly from metal discs. ATR FT-IR measurements FT-IR spectra of sample powders were collected using a Bruker Alpha FT-IR spectrometer employing ATR (Attenuated Total Reflection) sampling device. The FT-IR spectrometer was equipped with a globar source, a KBr beam splitter, and a deuterated triglycine sulfate (DTGS) detector. The ATR sampling device utilized a diamond internal reflection element (IRE) in a single-reflection configuration. Spectra were recorded over the spectral range of 3

ACCEPTED MANUSCRIPT 400-4000 cm-1 at a 4 cm-1 resolution, co-adding 128 scans. ATR extended correction implemented in a Opus 7.0 software was applied to spectra to make relative intensity adjustments.

Results and discussion Chemical compounds consisting of carbon, hydrogen and oxygen combined in the ratio expressed by the empirical formula of Cn(H2O)n are generally called carbohydrates or

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rarely saccharides. This term was originally applied only to monosaccharides to reflect their simple structure, but now it is used in a wider sense. Carbohydrates include molecules

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gathered are usually divided into three main subgroups: monosaccharides, oligosaccharides

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and polysaccharides as well as they include compounds derived from its chemical modifications by i.e. the reduction of the carbonyl group or the replacement of one or more

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hydroxyl groups by a hydrogen atom or heteroatomic groups [4]. Additionally, the term 'sugar' is frequently applied to monosaccharides and rarely to oligosaccharides.

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1. Monosaccharides

Monosaccharides, according to the IUPAC, constitute the simplest group of

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carbohydrates composed of a single unit without any glycosidic connection to other of such units. Chemically they represent polyhydroxy aldehydes or ketones with three or more carbon

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atoms [4]. The simplest monosaccharides are D-glyceraldehyde and dihydroxyacetone as well as three compounds composing of four carbon atoms, i.e.

D-erythrose, D-threose

and

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erythrulose. All of them occur as intermediates in the various metabolic pathways such as

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glycolysis or pentose phosphate transformations [5]. In other words, monosaccharides belong to can be divided into subgroups defined according to the number of carbon atoms (e.g.

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tetroses, pentoses, etc.), placement the type of the carbonyl group (aldoses, ketoses) or chiral activities [6]. In general, they are colorless, crystalline solids, insoluble in nonpolar solvents and most of them have a sweet taste. Monosaccharides represent an important class of compounds playing crucial roles in many biological processes, e.g. 2-deoxyribose is a part of nucleotides while glucose is the a ubiquitous fuel in biology and basic unit for oligo- and polysaccharides. and ubiquitous fuel in biology [5]. In this section, seven the most common in nature monosaccharides such as

D-(−)-

ribose, 2-deoxy-D-ribose, L-(+)-arabinose, D-(+)-xylose, D-(+)-glucose, D-(+)-galactose and D(−)-fructose are described in detail. 4

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1.1. Pentoses Aldopentoses, as the name suggests, possess five carbon atoms and the aldehyde group in their structures. Due to the fact that they have three chiral centers it is possible to distinguish eight different stereoisomers divided into two groups of L- and D-aldopentoses. The latter, except D-luxose, are generally the most common monocarbohydrates in nature [5].

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Aldopentoses as well as other monosaccharides with more than five carbon atoms in the backbone, occur predominantly as cyclic (ring) structures, especially in the aqueous solution.

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Five- or six-carbon rings resembling the structure of furan and pyran, respectively, can be

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formed by a covalent linkage between the carbon atom of the carbonyl group and the oxygen atom from one of the hydroxyl groups in the chain [7]. Cyclic structures of D-aldopentoses in

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D

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solid state and discussed presented here in solid state are shown in Figure 1A.

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Figure 1. Molecular structures of monosaccharides (A - pentoses and B - hexoses).

D-(−)ribose

occurs widely in nature and is very essential for life. It is found in all

living cells, because most of fundamental biological molecules, e.g. nucleotides, adenosine triphosphate (ATP) and all forms of ribonucleic acid (RNA), contain D-ribose. Moreover, its derivatives are present in vitamin B12, coenzymes such as nicotinamide adenine dinucleotide (NAD) and coenzyme A, as well as in lipopolysaccharides in the cell wall of some bacteria [8]. Its synthetic mirror image, L-ribose, is not found in nature [9]. The crystal structure of

D-ribose

has remained unknown for years. Šišak et al.,

determined in 2010 that D-ribose occurs in the solid state in two crystal forms that contain β5

ACCEPTED MANUSCRIPT and α-pyranose cyclic structures in various ratios [10]. This is also characteristic for other pentoses studied here and from for this reason their molecular structures are presented in the form of pyranoses (c.f. Figure 1). In solution D-ribose exists as a mixture of various structures with the predominating β-D-ribopyranose form [7]. However, in all naturally occurring biomolecules containing D-ribose, it is present in the form of β-D-ribofuranose. Raman and IR spectra of D-ribose exhibit numerous bands displayed in Figure 2. Most of them are listed and assigned to vibrations of the functional groups in Table 1. In the high-

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wavenumber spectral region (region V, 3050 - 3500 cm-1) of the Raman spectrum, two lowintensity bands attributed to the O-H stretching modes are present at 3366 and 3198 cm-1

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while their IR counterparts appear as high intensity maxima at 3407, 3343 and 3196 cm-1.

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Bands at ca. 3200 cm-1 are assigned to the vibrations of hydrogen bondinged net of the crystal while a the band at 3366 cm-1 (3343 cm-1 in the IR spectrum) arises from vibrations of the

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secondary alcohol groups involved in intermolecular H-bonding. A The band at 3407 cm-1 (absent in the Raman spectrum) originates from the primary alcohol group [11]. The opposite effect on in relative intensity ratio in of Raman/IR bands occurs is found in the spectral region D-ribose

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IV (2800 – 3050 cm-1). A Raman profile of

exhibits the presence of five well-

separated sharp bands assigned to the C-H stretches ing modes. All of them are also present in the IR spectrum, c.f. Table 1 and Figure 2. According to [11], Raman and IR signals at ca.

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2875 cm-1 are is exclusively assigned to the symmetric stretching mode of the C-5 methylene 1

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group. Moreover, based on Raman (IR) spectra of other pentoses [9,12,13], bands at 3001 cm(3000 cm-1) can be assigned to the asymmetric stretching mode of this moiety. The

remaining set of bands in the region IV is attributed to the ν(C-H) vibrations of the

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methanetriyline groups of ribose.

As mentioned above, the fingerprint region of Raman and IR spectra is divided into

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three regions according to the type of vibrations. For D-ribose, we observe the most intense Raman features at 542 cm-1 (region I, the ring deformation motion), 885/1059/1119 cm-1 [region II, combination of (CC) and (CCH)/(CO), (CC), and (COH)/(CO) and (CC), respectively], and at 1273 cm-1 [region III, (CH2)] [8,9]. The bands in the regions I and II can serve as markers for the D-ribose identification, see Table 1 and Figure 2. In turn, the ribose IR spectrum in the region I exhibits the presence of numerous overlapped maxima with the highest intensity signal at 484 cm-1 originating from the COC ring deformations. Furthermore, the most distinctive bands for D-ribose observed in its the IR spectrum occur in the range of 900-1100 cm-1 (region II). Here, bands of high intensity assigned to the coupled ν(CO), 6

ACCEPTED MANUSCRIPT /ν(CC) and β(COH) vibrations are present at 1027 and 1014 cm-1, respectively. Additionally, this spectral region shows a sharp intense band at 949 cm-1 [the combination of ν(CO)ring, ν(CC) and β(CCH)]. Other IR bands, especially in the region III, exhibit medium and weak intensity bands, and they do not markedly contribute markedly to the identification of this carbohydrate, see Figure 2. The bands observed here at 1027, 1014, and 949 cm-1 are also found in IR spectra of biological species as shown in IR imaging studies of cells and tissues [14]. All band positions are in good accordance with band positions of ribose reported in the D-ribose

was studied in solution using other Raman

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literature so far [11,15]. In addition,

techniques such as surface enhanced resonance Raman optical activity (SERROA) [16,17].

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2-deoxy-D-ribose is another essential aldopentose. Structurally 2-deoxy-D-ribose is an

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analogue of D-ribose in that the hydroxyl group attached to the C-2 atom is substituted by the hydrogen atom. The absence of the hydroxyl group reduces the possibility of branching, and

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thereby it causes fewer mutations in the molecular structure as well as it affects mechanical flexibility of large biomolecules containing 2-deoxy-D-ribose. For instance, 2-deoxy-D-ribose as a building block of nucleotides, contributes to the formation of spiral conformation of

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DNA.

Despite the removal of the OH group,

D-ribose

and 2-deoxy-D-ribose form very

similar crystal structures. Both aldopentoses in the solid state as well as in solution

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predominantly exist as the six-membered rings of D-pyranose remaining in equilibrium of α

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and β anomeric forms [7,11,18]. The dominant β-D-deoxyribopyranose form of 2-deoxy-Dribose is shown in Figure 1A. Nonetheless, its β-D-deoxyribofuranose cyclic form is present in DNA [19]. Surprisingly, Raman and IR spectra of 2-deoxy-D-ribose are not widespread in

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the literature and reports published so far were mainly focused on studies of conformational and chiroptical properties supported by quantum-chemical calculations [11,18,20].

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Interestingly, the structural similarity of to D-ribose and 2-deoxy-D-ribose is not reflected in their Raman and IR spectra, see Figure 2. The absence of one hydroxyl groups in 2-deoxy-Dribose causes pronounced changes in its H-bonding network as it is exhibited by a narrow IR band with two maxima at 3368 and 3327 cm-1 (the region V in the IR spectrum) indicating reduced H-bonding compared to D-ribose. Also the shape of this band enables distinguishing 2-deoxy-D-ribose from other aldopentoses, c.f. Figures 1 and 2. The high wavenumber range (the region IV) in the Raman spectrum of 2-deoxy-D-ribose is dominated by an intense bands at 2955 and 2940 cm-1 originating from the in-phase C-H stretches mode of the methine CH groups linked to the hydroxyl groups. Other bands (at 2940 and 2916 cm-1) are assigned to the 7

ACCEPTED MANUSCRIPT methine stretching vibrations of remaining carbon atoms linked to the hydroxyl groups. The presence of two endocyclic CH2 moieties at C-5 is manifested by Raman features at 2986 and 2868 cm-1 and IR band at 2985, 2980, 2891 cm-1 assigned to derived from the asymmetric and symmetric stretching vibrations., respectively. Moreover, due to slightly different surroundings of these groups, two bands at 2985 and 2980 cm-1 appear in IR spectra, see Table 1. Both Raman and IR profiles of 2-deoxy-D-ribose show numerous bands of medium

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and low intensity in the fingerprint region (the regions I-III), see Figure 2. And both spectra are distinctly different from their counterparts for in D-ribose spectra. The scissoring mode

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The presence of two endocyclic CH2 groups manifests by appears as an intense Raman band

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at 1471 cm-1 while the most intense Raman band is located at 808 cm-1 and is assigned to the stretching vibrations of the backbone exocyclic C-C groups [11]. The spectral regions I and II

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in the IR spectrum of this carbohydrate exhibit many well-separated bands, see Figure 2. One can find that several bands are more intense in the spectrum of 2-deoxy-D-ribose than in Dribose, i.e. at 1111, 1086, 892, 629, 521, and 416 cm-1, and some of them i.e. bands with

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maxima at 1011, 979, 812, and 756 cm-1 appear exclusively in the spectrum of this D-ribose derivative. Most of them are associated with various ring deformations, see Table 1 for the

L-(+)-arabinose

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detailed description.

is naturally occurring aldopentose in plants and it is mainly a

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component of hemicelluloses and pectins [12]. L-arabinose, similarly to above mentioned aldopentoses, crystalizes in the β-L-arabinopyranose form [13]. Its structure is presented in Figure 1. Raman and IR spectra of L-arabinose have been described in detail in the literature

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[21–23]. In the high wavenumber region, typical bands for carbohydrates including signals of due to the H-bonded OH stretches ing motions (3524 and 3324 cm-1 in the IR spectrum,

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region V) and the CH stretches of the CH2/CH methylene/methine groups occur (3001, 2970, 2959, 2939 and 2891 cm-1 in the Raman spectrum, region IV). Similarly to the Raman spectrum of D-(−)-ribose, the 3001 cm-1 band is exclusively assigned to the as(CH2) mode of the C-5 atom. Furthermore, Interestingly the network of H-bonding well correlates with the sugars sweetness, and consequently with the IR characteristics of the carbohydrate, and a IR band at 3524 cm-1 is a marker of sweet taste of L-arabinose [22]. The most characteristic features of L-arabinose in the Raman spectrum shown in Figure 2 are observed at 1476, 1260 (region III) and 843 cm-1 (region II). These bands are assigned

to

the

δ(CH2),

τ(CH2)

modes 8

and

complex

combination

of

ACCEPTED MANUSCRIPT νs(CO)ring+ν(CC)ring+ν(CO) vibrations, respectively. This is a specific spectral marker for all aldoses containing the equatorial hydrogen at C-1 [22,23]. In contrast to the Raman spectrum, the IR spectrum exhibits several high-intensity bands in these regions. The spectral region II is dominated by a band at 991 cm-1 attributed to the sum of νas(COC), ν(CC), ν(CO) and β(CCH) vibrations [23]. This band is specific for L-arabinose only, see Table 1. Other intense IR bands in this region are found at 1129, 1089 and 1048 cm-1 and they exhibit the dominant contribution of the stretching modes of the endo- and exocyclic CO groups. Additionally, L-

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arabinose is also characterized by a unique set of IR bands at 782, 671 and 602 cm-1 in the region I, which are attributed to the combination of ν(CO)ring and various ring deformation is also called a ‘wood sugar’. Its name is derived from the Greek word

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D-(+)-xylose

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modes.

xylon meaning ‘wood’ to highlight the material from which it was isolated for the first time. is a principal monomeric unit present in most of hemicelluloses. For instance, xylan

coating contains 85 - 93% of

D-xylose

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D-xylose

[24]. Together with L-arabinose it is found in the

structure of arabinoxylans - predominant non-cellulosic polysaccharides of cell walls in all

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major cereal grains [25]. D-xylose is an essential compound in the carbohydrate industry as a reagent substratum in the production of xylitol – a sweetener for diabetics [26]. In natural polysaccharides D-xylose occurs as a D-xylopyranosyl unit [24]. The same ring structure is

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found both in the solid state and solution [7]. The corresponding β-D-xylopyranose formula in

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Haworth perspective is presented in Figure 1. Raman and IR spectra of D-xylose in the spectral region above 2800 cm-1 (regions IV and V) are similar in general to spectra of other aldopentoses studied here. However, Raman

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bands at 2982 and 2899 cm-1 in the region IV originating from the C-5 methylene stretching mode are significantly more intense than the corresponding bands of other pentoses, see

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Figure 2. In turn, the fingerprint region of the Raman spectrum is dominated by a band at 903 cm-1 (region II) attributed to coupling of the symmetric stretches of the CO and CC moieties [23]. The other characteristic Raman bands of D-xylose are found at 1150, 1107 and 1086 cm1

. The same region in the IR spectrum is also valuable for the identification of that

carbohydrate. Here, we observe a doublet of bands at 929 and 901 cm-1 as well as an intense band at 1034 cm-1 with a shoulder at 1016 cm-1. The region I in the Raman spectrum of Dxylose shows the presence of a unique 527 cm-1 band assigned to the deformation vibrations of the pyranose ring onthe contrary to the IR profile in that numerous broad bands are found. All Raman and IR bands of D-xylose with modes assignments are listed in Table 1. 9

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Figure 2. FT-Raman and ATR FT-IR spectra of pentoses.

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ACCEPTED MANUSCRIPT 1.2. Hexoses D-(+)-glucose

with its molecular formula of C6H12O6 represents one of the most

common hexoses with the aldehyde group in its structure. Its Haworth perspective formula is displayed in Figure 1B. Glucose is mainly synthesized by green plants from water and carbon dioxide using sunlight energy in the photosynthesis process [27]. In the mammalian cells, glucose takes part in two crucial biochemical pathways; in gluconeogenesis which generates glucose from certain non-carbohydrate carbon substrates e.g. amino acids or lipids and in

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glycolysis which converts one molecule of glucose into two molecules of ATP. Moreover, glucose is the only fuel for brain and red blood cells in mammals [28]. As the common fuel

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and basic structural unit in polysaccharides, glucose serves as energy stores (i.e. glycogen in D

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animals and starch in plants) and plays a structural role (e.g. chitin or cellulose). Its

enantiomer, appointed on the basis of right orientation of the hydroxyl moiety attached to the

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atom C-5, occurs widely in nature on the contrary to L-glucose [4]. Raman and IR spectra of glucose are rich but only few signals can be used as marker bands for the detection and quantification of glucose in blood [29] and dried plasma samples [30], i.e. 1123 and 1033 cmbands in Raman and IR, respectively. As long as a clear correlation between the glucose

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signal and concentration in blood has been established, Raman and IR spectroscopy is capable

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to determine the concentration of glucose in blood samples based on chemometric analysis with clinical accuracy [31,32]. Moreover, some reports have shown that Raman spectroscopy

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is useful for direct in vivo measurements of the glucose level in blood [33,34]. Raman and IR spectroscopy have been also successfully applied to identify and quantify glucose in plethora of food products like carrots [35,36], glucose syrups [37], honey [38,39], juices [40,41],

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jellies [42] and milk.

Due to the fact that glucose is optically active, the (+)-glucose enantiomer rotates a

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plane of polarized light clockwise [4]. In the crystalline form, the glucopyranose ring exists in both α and β-anomer forms, whereas the glucofuranose form rarely appears [43]. The structural variety of α and β anomers with the OH moiety directed below or above the ring plane, respectively, is reflected in Raman [44] and IR [45] spectra of glucose. Some Raman bands located at 1333 (the CH2 wagging mode), 843 (the CC stretching mode), and 542 cm-1 (the CCC, CCO and OCO deformations) indicate the domination of the anomer α over the β form as found in the spectrum shown in Figure 3. The counterparts In turn, a characteristic bands of the β anomer should appear at 1360, 893 and 518 cm-1 [44]. An analysis of the IR spectrum also allows for the identification of both anomers. The corresponding IR marker 11

ACCEPTED MANUSCRIPT bands for the α and β anomer are found observed, respectively, at 1338/1320, 1224/1278, and 1109/1162 cm-1 for α/β anomer, respectively [45]. The v Vibrational spectra of glucose in the crystalline form are extremely rich in bands, see Figure 3. Their origin has been previously determined in detail with the support of molecular modelling [43,45,46] and deuteriumsubstitution [47]. We summarized the bands assignment in Table 2. D-(+)-galactose

is an isomer of glucose with different orientation of the C-4 hydroxyl

group, see Figure 1B. To our best knowledge, the crystal structure of free galactose has not

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been yet determined yet. Galactose when combined with glucose by a β-1-4 glycosidic linkage forms a disaccharide – lactose, described below. The key metabolic galactose process

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is its conversion into the more metabolically useful glucose-1-phosphate upon the Leloir

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pathway [48].

The one reversed hydroxyl group orientation in the C-4 atom in the structure of D-(+)-

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galactose in comparison to D-(+)-glucose results in remarkable differences in positions and intensities of several bands in Raman and IR spectra displayed in (Figure 3). The positions of o Only few Raman bands in their Raman spectra are consistent of galactose and glucose

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appear at similar wavenumber, i.e. at 1333 cm-1 (the wagging mode of the CH2 group), ca. 1154 cm-1 (coupling of the CO and CC stretches), ca. 1069/1055 cm-1 (coupling of the CO

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and CC stretches with the bend of the COH group), and ca. 999 cm-1 (coupling of the bending vibrations of the CCH, CCO moieties) c.f. Table 2. Similarities in the IR spectra of D-(+)-

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galactose and D-(+)-glucose are mainly observed in the regions II-III, see (Figure 3 and Table 2). We find found that IR bands of the CH2 scissoring motions exhibit very similar profile at around 1453 cm-1 in the region III while the region II is specific for both carbohydrates with

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bands originating from various coupling of stretching and bending vibrations of the CC and CO bonds (e.g. 1152, 1102, and 836 cm-1), see Table 2 for details. The massive changes in

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Raman and IR spectra for the two compounds with almost identical structures demonstrate that vibrational spectroscopy is a powerful perfect tool for the identification of these saccharides [49].

FT-Raman and ATR FT-IR have been used to identify saccharides and to determine their composition in edible seaweeds [50]. The presence of β-galactose,

D-galactose-4-

sulphate and 3,6 anhydro-D-galactose can be was confirmed based on bands located at 890 (FT-Raman), 845 (FT-Raman) and 930 cm-1 (FT-IR ATR), respectively. This report has shown that the presence of

D-galactose

and anhydro-D-galactose indicates highly sulfated

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ACCEPTED MANUSCRIPT polysaccharides whereas the mixture of D- and L-galactose and as well as anhydro-L-galactose appear in for less sulfated sugars [50]. The chemical formula of D-(−)-fructose, C6H12O6, is identical as for D-(+)-glucose and D-(+)-galactose,

whereas their structures are considerably different. In contrast to the aldoses

described above, the fructose molecule contains the ketone moiety and can create five(furanose) and six-membered (pyranose) cyclic forms [6]. The naturally preferred crystalline form of fructose is β-D-fructopyranose [21,44], what was determined by X-ray [51] and

D-(−)-fructose,

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neutron diffraction [52] techniques.

called also a ‘fruit sugar’, occurs in fruits and plants in free state as

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well as and as a building block of in sucrose. Vibrational spectroscopy is sensitive enough to

SC

distinguish monosaccharides, even they co-exist in a mixture. For example, Raman and IR spectroscopy have been employed for a quantitative analysis of fructose and glucose in honey

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samples (using a PLS regression model in the 1700-700 cm-1 region of Raman spectra) [38], including the differentiation of honey according to its geographic origin (PLS and PCR regression models in the 1500-800 cm-1 region of IR spectra) [39,53]. During the polyol

MA

metabolic pathway, fructose is synthesized from glucose in seminal vesicles and is secreteds into semen as a major energy source. The human semen is a mixture of several components

D

(fructose, albumin, lysozyme, lactate, and urea) [54] whose can be examined by vibrational spectroscopy [29,55]. In particular, a the characteristic Raman region between 800 and 850

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cm-1 with bands assigned to the CC stretching vibrations [43,56] has been used in the fructose detection identification in semen samples [29,55]. The Raman spectrum of solid

D-(−)-fructose

is shown in Figure 3. Raman bands

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positioned at 980 (the deformation mode of the CCH and CCO groups [56]), 820 (the CC stretches [43]) and 527 cm-1 (the CCO, CCC and OCO deformations [43,56]) represent the

AC

fructopyranose form. The IR feature of this type of the ring is found at 923 cm -1 and originates from the CCH and CCO deformations [45].

13

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ACCEPTED MANUSCRIPT

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Figure 3. FT-Raman and ATR FT-IR spectra of hexoses.

2. Disaccharides

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Disaccharides are built of two monosaccharide units joined by glycosidic bonding. They are formed in condensation reaction and can be broken down through hydrolysis catalyzed by enzymes from the disacharidase group. In living organisms they serve as a source of energy through the provision of monosaccharides. In general, they can be divided into non-reducing and reducing saccharides, depending on the presence of free a hemiacetal unit. The most widespread disaccharides are sucrose, maltose and lactose [57]. D-(+)-sucrose

is a disaccharide commonly occurring in plants, existing at room

temperature as colorless crystals. Due to its taste it is widely used as a sweetener. Sucrose is composed of two monosaccharides, glucose and fructose, linked via 1→2 O-glycosidic 14

ACCEPTED MANUSCRIPT linkage between the C-1 atom first carbon of glucose (C-1) and the C-2 atom second carbon (C-2) of fructose, see Figure 4. As described previously, both glucose and fructose can occur in two anomeric forms (α and β), according to stereochemistry of the C-1 atom. However, only the α-glucose is linked to β-fructose through the glucosyl unit. In contrast to other disaccharides, the O-glycosidic linkage in sucrose is formed between two reducing ends of both monosaccharides, inhibiting further linkage with other units. It involves both anomeric carbon atoms thereby making sucrose a non-reducing disaccharide. Sucrose is easily digested

PT

by mammals through breakdown into monosaccharides by sucrase or isomaltase glycoside hydrolases [58,59]. An The enhancing effect of sucrose on the development of metabolic

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syndrome, inducing including the an elevated blood level of triglycerides, hyperglycemia and

SC

insulin resistance, has been reported several times [60–62]. In addition, sucrose is a cryoprotectant, which is particularly important to for living organisms, as it minimalizes the

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damage caused by solution injury and intracellular ice injury [63]. Cryoprotectants are capable to decrease the intracellular water content without dehydrating the cell, thus they playing a key role in anhydrobiosis [63],[64]. As anhydrobiosis is similar to the phenomenon

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occurring due to freeze drying, cryoprotectants are used as stabilizers during dehydration [63]. They interact stronger with water than with themselves and thus affecting the tetrahedral

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coordination network of water and its structural properties.

Figure 4. Molecular structures of disaccharides.

The Raman spectrum of sucrose shows multitude sharp bands, see as displayed in Figure 5. In general, the presence of numerous sharp bands in this vibrational spectrum 15

ACCEPTED MANUSCRIPT indicates a crystalline form [65,66] since amorphous [65] or a mixture of different conformations [66] result in the presence of broad bands. The region I (up to 800 cm-1) was reported as the most discriminating one between disaccharides studied here [67]. The Raman spectrum of sucrose is dominated by bands originating from torsion and deformation modes (τ(CO), τ(OH), β(CCO), β(OCO)) of the glucosyl and fructosyl units [68,69]. The bands at 527, 641 and 737 cm-1 are assigned to ring deformations. The most intense band in this region, located at 403 cm-1, is assigned to the deformation mode of the endocyclic CCO group

PT

in the fructosyl moiety. In fact, this band position (among others mentioned below) is characteristic for the α–anomer [67]. The counterpart for the β–anomer of sucrose appears at

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425 cm-1. The same type of vibration originating from the part of the glycosidic linkage

SC

directly connected to fructose appears as an intense band at 552 cm-1. The region II of the Raman spectrum has a series of bands of similar intensity assigned to the CC and CO

NU

stretches. The most intense band in this region (at 851 cm-1) is, however, attributed to the torsion motion of the CH2 groups [62] and the CC stretches of α-glucose [43]. The position of this band is also characteristic for the α–anomer (897 cm-1 for the β – anomer) [67]. A Raman

MA

band at 872 cm-1 is specific for the C-O bond of the glycosidic linkage attached to the glucosyl unit and it originates from the deformation mode of the CCO moiety. The region III of the Raman spectrum of sucrose exhibits mostly bands attributed to the deformation modes

D

of the OH and CH, as well as CH2 groups with the most prominent band located at 1462 cm-1

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(the CH2 scissoring mode). In turn, the region IV is characterized by a series of very intense bands originated from the symmetric (2897, 2914, 2943 cm-1) and asymmetric (2995 cm-1) stretching of CH and CH2 methine and methylene groups of both units. A characteristic band

CE

appears at 3015 cm-1 and it is quite unique among the studied sugars studied presented here. According to Refs. [69] and [70], this spectral feature occurs due to coupling of the CH

AC

methine stretches of the glucosyl and fructosyl rings, however a similar band is also specific for fructose suggesting that this signal is due to vibrations of fructose alone rather than fructose and glucose. In the ATR FT-IR spectrum of sucrose, bands present located in the regions II and V are clearly more intense than absorbances in the regions I, III and IV, see Fig. 5 and Table 3. A series of intense bands assigned to the CO and CC stretching modes is found in the region II. Here, we can recognize the most dominant bands mainly assigned mainly to the stretches of the CO groups at 987 cm-1 (from the glucosyl unit) and at 1050 cm-1 (from the fructosyl unit) along with the 1065 cm-1 band specific for the ν(CC) vibrations of glucose. A similar differentiation is found in the high-wavenumber region of the IR spectrum 16

ACCEPTED MANUSCRIPT of sucrose. Here, three maxima of a broad band are present which are exclusively assigned to H-bonding formed by the OH groups of the glucosyl (3382 cm-1) and fructosyl (3319, 3559 cm-1) units [61,62]. The region I of the IR spectrum is very rich in structural information regarding sub-units of sucrose., This includesing several bands identifying the anomeric form of both subunits, i.e. for the α form at 399 and 535 cm-1 together with a the band at 848 cm-1 in region II [67]. In general, in- and out-of-plane deformation modes occur in this region involving a variety of coupling and combinations of the CCO moieties from both rings of the

PT

carbohydrate, c.f. Table 3 and Fig. 5. Regions III and IV contain relatively low intensity bands attributed to the several vibrations of the CH/CH2 groups.

is also disaccharide with a sweet taste, approximately one-third as sweet

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D-(+)-maltose

SC

as sucrose. Structurally, maltose is built of two glucose units, cyclized in the pyranose form and linked via the (1→4) α-O-glycosidic bond. Its molecular structure is shown in Figure 4.

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The presence of the O–glycosidic bond in the α or β forms has a crucial effect on biological and chemical function of the molecule. Maltose can be easily hydrolyzed in human body to monosaccharides whereas its β isomer - cellobiose cannot be digested. Maltose is not very

MA

common in nature, although it can be found in a high content in malt (germinated cereal grains). It occurs also in other food products, including potatoes, beer and pasta. Maltose is an

D

important intermediate in the digestion of starch. It is also a the predominant form of carbon produced by exported from plant chloroplasts during night in from starch breakdown [71].

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Similarly to sucrose, maltose plays a role of a cryoprotectant and from this reason is found in several plant and animal species capable to survive extreme cold or drought in response to desiccation or freezing [64]. As already mentioned, several studies have shown that this

CE

cryoprotective mechanism results from an the ability of the sugar to interact strongly with water destroying water tetrahedral network and slowing down its dynamics [47,72,73].

AC

Raman and ATR FTIR spectra of maltose are displayed in Figure 5 and they are in general similar to vibrational spectra of other disaccharides. Bands assignment of maltose has not been reported in detail so far [47,70,74], but they can be assigned based on by comparison to vibrational spectra of sucrose and lactose (see Table 3). In the region I we find two prominent bands at 463 and 552 cm-1, which are clearly more intense than others. These bands originate from the β(CCO) and β(OCO) modes from the vicinity of the C-1 atom in the glycosidic bond. The region II exhibits the presence of a variety of bands assigned to the CC and CO stretches, with the most intense signals found at 1115 cm-1 [coupling of ν(CO) ν(CC) and ν(COC)] and at 849 cm-1 (coupling of the CH2 torsions of the methylene groups and the 17

ACCEPTED MANUSCRIPT C-C stretches) of the α-glucose unit; the corresponding band for β-glucose is located at 901 cm-1 [43,74]. In turn, the region III includes bands attributed to from the in-plane bending of the OH, CH, and CH2 groups whereas the symmetric and asymmetric stretching modes of the CH2 bonds can be found in the region IV [47,70,74]. The IR spectrum of maltose presents a series of bands in the regions I and II, which are mainly assigned to β(CCO), β(COH) and β(COC) modes and the CO and CC stretching vibrations. The bands located in the region II are clearly more intense than the bands in the region I with the most prominent peak at 1024

PT

cm-1. Intensity of this band significantly increases in saturated solutions of maltose due to formation of intramolecular hydrophobic bonding [75]. Interestingly, Raman spectruma of

RI

maltose solution (not saturated) is are reported to be similar to spectruma of the solid sample

SC

[67]. The region III contains a few bands of low intensity corresponding to the β(OH), β(CH) and β(CH2) modes. The bands due to the stretching modes of CH and OH groups are located D-(+)-lactose

NU

in region IV and V, respectively [47,70,74].

is a disaccharide found mainly in milk. It consists of the galactose and

glucose units linked by (1→4) O-glycosidic bond, c.f. Figure 4. The galactose unit occurs in

MA

the β form, whereas glucose can be present in the α as well as β form [69]. From this reason, lactose occurs as in two isomersic forms as, α-lactose and β-lactose. Due to mutarotation, α-

D

and β-lactose are present in equilibrium in the aqueous solution in equilibrium and their solubility in water strongly depends on temperature [69]. In the digestive system of mammals,

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lactose breakdown is catalyzed by lactase. The deficiency of lactase called hypolactasia or lactose intolerance causes the inability to digest lactose and can be developed to various extend, including a complete lack of lactase (congenital alactasia), entirely preventing the

CE

digestion of lactose [76].

Similarly to other discussed here disaccharides discussed here, the Raman spectrum of

AC

lactose is also rich in sharp bands, see (Figure 5). α-Llactose monohydrate has 129 Raman active vibration modes and 107 of them appear are located in the spectral window region below 1500 cm-1 [66,67]. The region I presents a plethora of bands assigned to ring torsions and deformation modes of the OCC, OCO and CCO groups, with the most intense band at 357 cm-1 attributed to τ(COHO) and τ(HOH). This band (absent in the spectrum of

D-(+)

glucose), together with bands around 1100 cm-1 can be used to detect an determine the anomer form of lactose. This results from different rotational isomerism around the COC bridge between anomer forms, which affects positions of their COC stretching and bending modes [66]. The bands at 477 and 633 cm-1 correspond to deformation of the CCO moiety 18

ACCEPTED MANUSCRIPT within the glycosidic linkage [77]. The region II (associated with the CC and CO stretches) and III (assigned to the rocking and wagging vibrations of the CH, OH and CH2 groups) comprise a plurality of bands with similar intensity. A The band at 1086 cm-1 exhibits the highest intensity among all of them and is attributed to the ν(CC) and ν(CO) modes of the glucosyl unit [77,78]. This band can be used to identify crystalline and amorphous forms of lactose as it is depolarized and polarized, respectively [65]. Bands at 953 and 1142 cm-1 originate from the stretching motion of the CO bond within the glycosidic linkage from the

PT

glucosyl and galactosyl units, respectively. The region IV presents a set of bands associated with the stretches of the CH groups of both units, with the most intense band located at 2887

RI

cm-1. A very weak band (3346 cm-1) from the OH stretching mode can also be also observed

SC

[77,78].The ATR FT-IR spectrum of lactose shows a variety of bands with high intensity in the regions I, II and V and with lower intensity in the regions III and IV. Within the region I,

NU

one can find bands assigned mainly to the deformation modes of the endocyclic ring OCC, OCO and CCO groups with the most intense, sharp band located at 551 cm-1 [β(OCO)]. A very wide band is also observed at 757 cm-1 [coupling of τ(CO), τ(COHO), and τ(HOH)]. In

MA

turn, the region II is rich in intense bands originating from the stretching vibrations of the CO and CC groups, with the most prominent one located at 1031 [ν(CC)]. The rocking, twisting

D

and deformation modes of the CH, OH and CH2 groups give rise to bands in the region III. The CH stretching region IV also contains bands of low intensity with only two maxima at

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2899 and 2932 cm-1 [77]. IR bands assigned to the H-bonded OH stretching modes occur at

AC

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positions similar as in to the IR spectrum of maltose monohydrate.

19

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ACCEPTED MANUSCRIPT

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Figure 5. FT-Raman and ATR FT-IR spectra of disaccharides.

3. Trisaccharides Trisaccharides composed of three monosaccharides linked by various glycosidic bonds, are mainly found in higher plants. Here, we discuss only vibrational features of raffinose as an example of the most known trisaccharide that contains three different units, i.e. galactose, glucose and fructose, see Figure 6.

20

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D-(+)-raffinose

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Figure 6. Molecular structure of trisaccharide.

is crystalline pentahydrate oligosaccharide and is composed of three

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monosaccharide units: α-D-galactose, α-D-glucose and β-D-fructose with 1→6 and 1→2 glycosidic bonds, respectively [79,80]. A full chemical name of raffinose is O-α-Dgalactopyranosyl-[1-6]-α-D-glucopyranosyl-[1-2]-β-D-fructofuranoside.

All

five

water

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molecules in raffinose hydrate are linked in chains forming channels between sugar molecules [72]. Two pyranose rings of raffinose exists in a the chair form whereas the furanose ring

D

adopts puckered conformation and structure of all rings they do not significantly deviate from conformations typical for the monosaccharide units. This carbohydrate is widely distributed in

PT E

plant kingdom and it is the smallest member of raffinose family oligosaccharides (RFOs) [81]. The compound was found and crystallized for the first time by Johnston in 1843 from Eucalyptus manna [82]. After that raffinose was identified and quantified in seeds (e.g. in

CE

cotton seeds), roots, underground stems or leaves [83–85]. RFOs are the major agents responsible for flatulence following ingestion of soybean derived products, so and then

AC

removal of RFOs from soy-based foods would then have a positive impact on the acceptance of the legume-derived products [86]. α-1,6 Llinkages present in raffinose are enzymatically hydrolyzed to

D-galactose

and sucrose by α-galactosidase (α-GAL; α-D-galactoside

galactohydrolase) [86]. Humans lack this enzyme and this sugar passes intact into the large intestine where anaerobic microorganisms ferment raffinose and led to flatulence. Raffinose has been also used as a cryoprotectant in biopharmaceutics and as a predominant component of preservation solution to store biological materials and organs [87]. The Raman spectrum of raffinose displayed in Figure 7 is rich in relatively intense bands mostly in the regions II, III and V on the contrary to the region below 800 cm-1. The 21

ACCEPTED MANUSCRIPT most prominent Raman features in the fingerprint of the Raman spectrum of raffinose are bands at 419 [β(CCO), β(CCC) and β(OCO) of the fructosyl unit], 834 [ν(CC) of α-glycosidic linkage], 1051/1075 [ν(CO), ν(CO), β(OCH)], 1269 [τ(CH2)], 1389 [ω(CH2]) scissoring, and 1466 cm-1 [δ(CH2)] (Table 4). The b Bands of due to the skeletal mode vibrations of the glycosidic linkage are observed in the 900-950 cm-1 range (region II) in both Raman and FTIR spectra [56]. The spectral range of 1150-950 cm-1 (region II) is mainly characterized by the contribution of β(COH) modes [75]. The high wavenumber region presents a set of bands

PT

associated with the CH stretching modes of the glucosyl and galactosyl units, with the most intense band at 2911 cm-1. A very weak band due to the OH stretching (3235 cm-1) is also

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observed.

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In the ATR FTIR spectrum of D-(+)-raffinose pentahydrate (Figure 7, Table 4) the bands observed in at the regions I, II and V are more prominent intensive than bands in

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regions III and IV (Figure 7, Table 4). In the region I that which provides information about deformation modes of OCC, OCO, CCH and CCO groups, the most intense, sharp bands are located at 399 [β(CCO), β(CCC), β(OCO)], 537 (deformation of the glucofuran ring), 619 and

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667 cm-1 [β(CCO), β(CCC), β(OCO)], (OH)]. Regions I and II are specific for The region of 1000-40 cm-1 (region I) provides detailed information about the configuration of the

D

glycosidic linkage and vibrations arising from the pyranose or furanose rings of raffinose [88]. Bands at 860 and 873 cm-1 are assigned to the vibrations of the furanose ring while the

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833 cm-1 band is related to α-configuration of the glycosidic bonds [88]. According to a report of Kačuráková et al., the bands due to of the (CO) mode in the COC linkages appear in two spectral ranges of 1160–1130 and 999–965 cm-1 (region II) [75]. The IR spectrum exhibits the

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presence of bands at 935, 996 and 1152 cm-1, which that are exclusively attributed to the (CO) of the α-(1-6) glycosidic bond [75]. An IR band at 955 cm-l also originates from this

AC

type of vibration. Furthermore, the region between 1300 and 1200 cm-1 is sensitive to intermolecular hydrogen bonding and contributes from the CH and CH2 deformation of and in plane OH modes [75]. The presence of crystal water in raffinose pentahydrate can be observed at is exhibited by a IR band 1649 cm-1 and in the region above 3100 of 3500–3100 cm-1 [89]. The network of hydrogen bonding between water and sugar molecules in raffinose is formed by all hydroxyl, glycosidic and ring oxygen atoms and is well ordered [72]. X-ray data has showed that three water molecules bonded to raffinose are located in a tunnel of raffinose; in addition two water molecules are placed outside the channel [72]. Three water molecules are both proton donors and acceptors whereas the remaining two H2O molecules 22

ACCEPTED MANUSCRIPT acting as donors create longer and weaker hydrogen bonding than the other three water molecules [72]. These spectral features have been used to investigate the re- and dehydration processes of raffinose pentahydrate [89]. In particular, absorbance changes and a band shift of the 1649 cm-1 maximum reveal transition from the pentahydrate form to anhydrate species of

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raffinose.

D

Figure 7. FT-Raman and ATR FT-IR spectra of trisaccharide.

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4. Polysaccharides

Polysaccharides consist of at least 10 (up to and sometimes several thousand) of simple monosaccharide units. They form straight or branched chains mainly via 14 and

CE

16 glycosidic bonding. The 13 β-Gglycosidic linkage is also observed in the backbone of β-glucans. Polysaccharides composed of one type of monosaccharide are called homopolysaccharides (i.e. starch, glycogen) whereas heteropolysaccharides contain different

AC

monosaccharides or their derivatives (i.e. heparin, gamma globulin, hyaluronic acid). Here, we compare vibrational spectra of starch components, amylopectin and amylose, and glycogen. Although, all All of them are composed of glucose units only, but they exhibit different molecular structures. These subtle structural variations are illustrated in a scheme in Figure 8 while vibrational spectra with their characteristics are collected in Figure 9 and Table 5. The Raman spectra of polysaccharides exhibit broader bands than the corresponding signals of mono- and disaccharides, c.f. Figs. 2, 3 and 5. For instance, the region of 1200–1500 cm−1 (region III) is mainly specific for the CH deformation vibrations whereas the CC and CO symmetric stretching modes are found in the range between 950 and 1200 cm−1 (region II). On 23

ACCEPTED MANUSCRIPT the other hand, this region in IR spectrum exhibits the presence of bands attributed to the (CO) motion of the C-O-C glycosidic linkage which serve as markers of the configuration of polysaccharides [90]. Polysaccharides represent structural similarity but their differentiation can be performed from vibrational spectra by using marker bands due to the CC and CO symmetrical stretching vibrations in the range of 1000–1200 cm−1 and skeletal breathing

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modes present in the region below 500 cm−1 [91].

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Figure 8. Molecular structures of polysaccharides.

Amylopectin is a branched polysaccharide in that most of the D-glucose residues are

AC

linked through the linear α-(14) glycosidic linkage with some contribution of the α-(16) C-O-C bonding. In contrast, amylose is a linear polymer made of α-D-glucose units, linked to each other through the α(1→4) glycosidic bonds and coiled into a spiral with six glucose units per turn. The α(1→4) structure facilitates the formation of a helicalx structure, with hydrogen atoms inside and amylose as a hydrophobic species. Some amylose molecules can be slightly branched by (1→6)-α linkages [92]. Both polysaccharides play an energy-storage function in plants in the form of starch granules. Amylopectin is the main component of starch granules (~70-80%) coating starch granules and is responsible for an the effect of swelling of starch and its ability to create sols [92,93]. In contrast to amylose that which is the linear polymer, 24

ACCEPTED MANUSCRIPT amylopectin is branched. Branch points appear every 20-25 glucose units; a chain of α-D(1→4) glucose units is linked to the C-6 hydroxymethyl position of a glucose molecule through α-D-(1→6) glycosidic linkage. what This involves ca. 4-6% of all glucose units in amylopectin in branches points [92,94]. The bBranching and its multiplicity is typical of both amylopectin and glycogen. Branched chains in such polysaccharides are grouped and assigned in terms of the A, B and C chains [90]. Side chains of amylopectin belong to two categories, i.e. A-type (outer chains; non-branched chain) and B-type (inner chains; at least

PT

one additional branched chains). Starch is the major energy reservoir in for plants and is an important carbohydrate that humans consume. Amylases, which digest starch in humans, are

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present in pancreatic juice (pancreatic amylase) and saliva (salivary amylase). Amylases are

SC

also synthesized in fruits during ripening of many plants and germination of cereal grains. Amylases from grains are important for the production of malt [95,96].

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FT-Raman and ATR FT-IR spectra of amylopectin and amylose and their along with modes assignments are presented in Figure 9 and Table 5. IR and Raman spectra of both starch components are virtually identical. Only a detailed examination of these spectra shows

MA

a few bands specific for each polysaccharides, i.e. at ca. 2900 cm-1 in IR and at ca. 850 and 520 cm-1 in Raman [94,97,98]. Raman bands are much broader than those in the Raman spectrum of glucose, compare Figures 2 and 9. For amylopectin and amylose, the spectral

D

region up to 600 cm-1 can be used to distinguish these polymers by the presence of a sharp and

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very intense band in the region of 479–483 cm-1. In spectra of both polysaccharides, a band at 479 cm-1 is specific for vibrations of the C-1OC-4 glucoside bond [95][90,93]. Other Raman marker band of the α-linkages is observed at 942 cm-1. The C–O–C vibration

CE

involving α-D-(1→4) linkages mainly contributes to bands in the region of 960–920 cm-1. Liu et al. has also proposed that this band can be also is associated with the α-D-(1→6) linkages in

AC

amylopectin species [94]. In addition, a Raman feature at 866 cm-1 is assigned to the symmetric COC stretching and ring breathing modes. In general, the region II of the Raman spectra is dominated by bands at 1051 (CO and CC stretches), 1086 (COH and CH bends) and 1131 cm-1 (CC, CO and symmetric COC stretches in glycosidic link and with coupling of the ring breathing vibration), see Table 5 for detailed description [91]. In the region III, we found bands attributed to the CH and COH moieties: 1267 cm-1 [bending modes of CH and CH2OH groups], 1339 cm-1 [β(CH) and β(COH)], 1378 cm-1 [β(CH2)] and 1462 cm-1 [δ(CH2) and β(COH)] [91]. Interestingly, the high-wavenumber region specific for the stretching vibrations of the methine and methylene CH and CH2 groups exhibits only the presence of a 25

ACCEPTED MANUSCRIPT broad band at 2911 cm-1 with a shoulder at 2933 cm-1 in contrast to the other carbohydrates discussed above. The comparison of the spectra shows that both amylose and amylopectin have similar Raman bands with exception of some subtle differences in relative intensities of Raman bands and a pronounced shoulder at 2940 cm-1. The is spectral Raman similarity between the starch components suggests that major Raman bands arise from the D-glucose building blocks [89]. It has been reported in [37, 99] that The structural differences between of amylose and amylopectin can be successfully observed by the application of Raman

PT

spectroscopy using the C–H stretching in the Raman region between 2700 and 3100 cm-1, however we do not find any differences in spectra collected here, cf Fig. 9 [37, 99].

RI

Additionally, the amount of amylose can be determined based on Raman intensities of the

SC

bands in the C-H stretching region But this region has been successfully used for quantification of amylose [89].

NU

It has been shown that FTIR spectroscopy can be also applied to studyies on localized arrangements and or conformations of starch the amylopectin polymer chains [100]. Vasko and Koening have demonstrated that folding of the chains occurs in both components of

MA

natural starches [100]. A IR band at 1295 cm-1 (absent in our spectra) of amylose and amylopectin arises from a unique fold structure and its spectral characteristics suggests that

D

some portion of amylopectin also forms regular, adjacent reentry folds. Forming a polymeric chain, amylopectin as well as other polysaccharides can be identified by a A few IR maxima

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in the region of 1150-800 cm-1 (region II), which are is dominated by the ring vibrations coupled with the stretching vibrations of the COH side groups and the glycosidic bonds, are also associated with formation of polymeric chains [101]. In the case of branched

CE

polysaccharides, IR bands of small intensities and located at around 895 and 845 cm-1 are attributed to the C-1H(β) (β-configuration) and C-1H(α) (α-configuration), respectively

AC

[102]. In addition, In the IR spectra of amylopectin/amylose one can found a very small intensity shoulder at ca. 849 cm-1 of the 860 cm-1 band and according to has been proposed by Cael et al. as a marker , we conclude that this band of is attributed to the skeletal mode involving the α-D-(1→4) glycosidic linkage [103]. As shown in Figure 9, the FT-IR spectra of amylopectin and amylose exhibit an intense absorbance at 991 cm-1 [(CO) in glycosidic linkages], which is accompanied by bands at 928 cm-1 (the ring and COH bending modes), 1076 cm-1 [β(COH)], and at 1149 cm-1 [ν(COC), (CC) in glycosidic linkages] [75,101], c.f. Table 5.,In turn, the high wavenumber region of the IR spectra is dominated by a broad maximum at ca. 3300 cm-1 assigned to the OH stretching vibrations. 26

ACCEPTED MANUSCRIPT Glycogen is a polysaccharide which is a readily available reserve of glucose to supply animal organisms. Its molecular structure is similar to amylopectin since glucose molecules in both are linked together by linearly by α(1→4) glycosidic bonding and links whereas branches are bonded to chains by formed via α(1→6) glycosidic bridges, see Figure 8. In comparison to amylopectin, glycogen is more branched – branching off is observed around every 8 to 12 glucose molecules in the main and linear chain. A glycogen molecule has about 12 tiers of branch points that . Branches in the structure of glycogen are created by branching

PT

enzyme which hydrolyses every 8 to 12 the α-1,4 linkage in the linear glucose chain and then it attaches the chain via the α-1,6 linkage. Glycogen is the storage polysaccharide and its main

RI

pools is are deposited in the liver (~ 1% of wet weigh) and in the muscles (~ 2% of wet

SC

weigh) [104]. In mammals, glycogen is produced formed in the liver from glucose supplied in diet. Glycogen from the liver is a generally available source of glucose for tissues and also to

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maintains blood glucose levels between meals. In turn, The second important source of glycogen are produced by skeletal muscles which are is a reserve of glucose used in for synthesis of ATP during intense muscle contractions and is are not involved in the regulation

MA

of the blood sugar level.

Similarity in molecular structures of amylopectin and glycogen results in virtually

D

similar Raman and IR spectra, see Figure 9. Assignments of Raman and IR bands to vibrations are mostly identical, c.f. Table 5. Differences are mostly observed in relative

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intensities. For instance, the intensity ratio of glycogen Raman bands at 484 cm-1 (479 cm-1 in amylopectin) and 1130 cm-1 in the Raman spectrum is significantly higher (I484/I1130≈1.6) for glycogen (I484/I1130≈1.6) than for amylopectin (I479/I1130≈1). Also some shifts in positions of

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Raman features are found,. An example is e.g. a band assigned to the out-of-plane phase bending of hydrogen bonded OH groups which is present appears at 571 and 579 cm-1 in

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spectra of amylopectin and glycogen, respectively. In turn, a band of amylopectin at 614 cm-1 is not observed in the glycogen spectrum. Raman signals in the 820-880 cm-1 region, assigned to the C(1)-H(α) bending mode, show shoulders are present at 856 and 866 cm-1 in the glycogen and amylopectin spectra, respectively. As well lso bands attributed to the CH inplane bending mode of the CH groups (1360-1420 cm-1) exhibit an intense band at occur at different positions, i.e. 1378 cm-1 for amylopectin and less intense one at 1387 cm-1 for glycogen. In addition, the 614 cm-1 band of amylopectin is absent in the spectrum of glycogen. The IR spectra also exhibit some small differences which enable distinguishing both branched polysaccharides. The most prominent variations are observed in the region of 94527

ACCEPTED MANUSCRIPT 1060 cm-1. It has been reported that For instance, all hydroxyl groups in starch are randomly solvated and this fact is manifested by broadening of IR bands, whereas anisotropically solvated OH groups give rise to distinct IR features ; both are observed in the 945-1060 cm-1 region of the IR spectrum of glycogen [105]. Therefore, glycogen possesses two specific bands at 999 and 1017 cm-1 whereas the spectrum of amylopectin exhibits only one broad band at 991 cm-1 [102]. These bands are assigned to the COH stretching vibrations of highly solvated alcohol groups. These bands are not observed in the corresponding Raman spectra

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since they are related to modes of solvated hydroxyl groups of polysaccharides. Other differences in IR spectra of glycogen and amylopectin are found in shifts of a In addition,

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bands attributed to the C-1-H bending mode in α-configuration of the glycosidic bond appear

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at 850 and 860 cm−1 glycogen and amylopectin, respectively.

Vibrational spectra can be also successfully applied to employed for the identification of glycogen in biomedical samples [106–109]. Marker Raman features of glycogen in tissues

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are found at 484, 856, 941, 1130, 1337 and 1460 cm−1 (assignments are given in Table 5) [102]. Shetty et al. showed that the Raman set of glycogen bands is highly sensitive and

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specific for biochemical changes occurred in the carcinogenesis of Barrett’s esophagus [110]. A higher level of glycogen in the squamous area was found in the normal tissue than in

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cancer. Interestingly, positions of Raman bands assigned to glycogen in tissues are very similar to bands recorded for the solid glycogen, see Table 5. In FTIR studies of biomedical

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samples, a band at 1030 cm-1 is proposed as a marker for the glycogen presence in tissues. Gazi et al. have found that the ratio of peak areas at 1030 and 1080 cm−1, corresponding to the glycogen and phosphate vibrations, respectively, can be used as a potential marker method for

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the differentiation of benign from malignant cells [111]. Also Colagar et al. have reported that the level of carbohydrates, including glycogen, is reduced in cancerous tissue in comparison

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to the control [109]. On the contrary to Raman marker bands of glycogen, the FTIR marker band of this polysaccharide (the COH stretching vibrations of the solvated alcohol groups) is shifted to 1030 cm−1 in tissues from ca. 1020 cm−1 for pure glycogen.

28

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Figure 9. FT-Raman and ATR FT-IR spectra of polysaccharides.

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Conclusions

Vibrational spectra of saccharides are considerably less specific than spectra of lipids or proteins [2,3] as functional groups building saccharides give rise to multiple bands spread in the whole fingerprint and high-wavenumber range. Due to ordered structures of mono-, diand trisaccharides in the lattice, IR and, particularly, Raman signals of these carbohydrates are narrow and sharp. Contrarily, an unordered structure of polysaccharides is reflected in considerable broadening of their vibrational bands compared to smaller saccharides studied here.

29

ACCEPTED MANUSCRIPT Five spectral ranges were chosen to analyze obtained spectra of saccharides. Region V (3600-3050 cm-1), IV (3050-2800 cm-1) and II (1200-800 cm-1) are assigned to the stretching vibrations of the OH, CH/CH2 and C-O/C-C groups, respectively. Region III (1500-1200 cm1

) and I (800-100 cm-1) predominantly have contributions from the deformational modes of

the CH/CH2 and CCO groups, respectively. Regions I and II are particularly suited to discriminate IR and Raman spectra of saccharides from spectra of other important classes of biomolecules such as proteins or lipids, as they exhibit signals of moderate to high intensity.

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Notably, the intensity of Raman bands in the region I is relatively the highest for hexoses and disaccharides composed of hexoses as their building blocks. Interestingly, a trisaccharide

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studied here, D-raffinose, does not obey this rule that might be due to its crystal structure

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(characterized, among others by a different conformation of the glycosidic link from that previously found in crystal structures of sucrose and a high content of hydrated water

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molecules in the raffinose lattice) [79]. Raman signatures in the region I for studied hexoses and disaccharides differ markedly, hence this range can be used to differentiate between these sugars. For pentoses and raffinose, the region II has higher diagnostic potential. Contrarily,

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for every studied mono- di- and trisaccharide, relative intensity of bands in IR spectra is comparable for the regions I and II. Due to significant abundances of bands in the region I and

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considerable differences of IR profiles in this range, it is well suited to discriminate studied sugars.

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No particular range for vibrations involving glycosylic bonds can be indicated as formation of di- or trisaccharides is realized with participation of carbon atoms of various groups (C1, C2, C4 or C6) and by various monosaccharides. As mentioned above, the profile of discernible

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both IR and Raman spectra differ considerably for polysaccharides, so they are easily based

on

vibrational

spectroscopy.

Contrarily,

structures

of

studied

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polysaccharides are alike and therefore difficult to discriminate by comparison of their spectral features.

This paper describes only spectroscopic profile of carbohydrates in the solid state since it is a first step in understanding their spectroscopic properties, however it is desired also to discuss an influence of the environment on the structure and spectroscopic profile of these compounds. So the next paper intends to broaden the spectroscopic characterization of carbohydrates when they are dissolved. Since carbohydrates are chiral, when they are in solution, Raman Optical Activity (ROA) spectroscopy can be applied to study their structure.

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ACCEPTED MANUSCRIPT Potential of ROA in characterization and discrimination of sugars will be also covered in the following review paper.

References [1]

A. Barth, Infrared spectroscopy of proteins, Biochim. Biophys. Acta - Bioenerg. 1767 (2007) 1073–1101. doi:http://dx.doi.org/10.1016/j.bbabio.2007.06.004.

[2]

A. Rygula, K. Majzner, K.M. Marzec, A. Kaczor, M. Pilarczyk, M. Baranska, Raman

PT

spectroscopy of proteins: a review, J. Raman Spectrosc. 44 (2013) 1061–1076. doi:10.1002/jrs.4335.

K. Czamara, K. Majzner, M.Z. Pacia, K. Kochan, A. Kaczor, Raman spectroscopy of

RI

[3]

[4]

SC

lipids : a review, J. Raman Spectrosc. 46 (2015) 4–20. doi:10.1002/jrs.4607. A.D. McNaught, Nomenclature of carbohydrates (Reccomendation 1996), Carbohydr.

[5]

NU

Res. 297 (1997) 1–92. doi:10.1016/S0008-6215(97)83449-0. D.L. Nelson, M.M. Cox, Glycolysis, gluconeogenesis, and the pentose phosphate pathway, in: Lehninger Princ. Biochem., Fourth ed., Worth Publishers, New York,

[6]

MA

2004: pp. 521–559.

J. McMurry, Biomolecules carbohydrates, in: Org. Chem., Ninth ed., Cengage

[7]

D

Learning, USA, 2016: pp. 832–869.

K.N. Drew, J. Zajicek, G. Bondo, B. Bose, A.S. Serianni, C-labeled aldopentoses:

PT E

detection and quantitation of cyclic and acyclic forms by heteronuclear 1D and 2D NMR spectroscopy, Carbohydr. Res. 307 (1998) 199–209. doi:10.1016/S00086215(98)00040-8.

K. Sasajima, M. Yoneda, Production of D-ribose by microorganisms, in:

CE

[8]

197. [9]

AC

Biotechnology of Vitamins, Pigments and Growth Factors, Springer, 1989: pp. 167–

K. Okano, Synthesis and pharmaceutical application of L-ribose, Tetrahedron. 65 (2009) 1937–1949. doi:10.1016/j.tet.2008.11.047.

[10] D. Sisac, L.B. Mccusker, G. Zandomeneghi, B.H. Meier, D. Bläser, R. Boese, W.B. Schweizer, R. Gilmour, J.D. Dunitz, The Crystal Structure of D -Ribose — At Last!, Angew. Chemie Int. Ed. 49 (2010) 4503–4505. doi:10.1002/anie.201001266. [11] M. Mathlouthi, A.M. Seuvre, J.L. Koenig, F.T.-I.R. and laser-Raman spectra of Dribose and 2-deoxy-D-erythro-pentose (“2-deoxy-D-ribose”), Carbohydr. Res. 122 (1983) 31–47. doi:10.1016/0008-6215(83)88404-3. 31

ACCEPTED MANUSCRIPT [12] H. Cheng, H. Wang, J. Lv, M. Jiang, S. Lin, Z. Deng, A novel method to prepare Larabinose from xylose mother liquor by yeast-mediated biopurification., Microb. Cell Fact. 10 (2011) 1–11. doi:10.1186/1475-2859-10-43. [13] S.H. Kim, G.A. Jeffrey, The crystal structure of β-DL-arbinose, Acta Crystallogr. 22 (1967) 537–545. doi:10.1107/S0365110X67001094. [14] K. Malek, B.R. Wood, K.R. Bambery, FTIR Imaging of Tissues: Techniques and Methods of Analysis, in: M. Baranska (Ed.), Optical Spectroscopy and Computational

PT

Methods in Biology and Medicine, Springer, New York London, 2014: pp. 419–473. doi:10.1007/978-94-007-7832-0.

RI

[15] P. Carmona, M. Molina, Raman and infrared spectra of D-ribose and D-ribose 5-

SC

phosphate, J. Raman Spectrosc. 21 (1990) 395–400. doi:10.1002/jrs.1250210703. [16] S. Ostovar Pour, S.E.J. Bell, E.W. Blanch, Use of hydrophilic polymer for reproducible

NU

Surface Enhanced Raman Optical Activity spectroscopy (SEROA), Chem. Commun. 47 (2011) 4757–4756. doi:10.1063/1.3482272.

[17] S. Ostovar pour, L. Rocks, K. Faulds, D. Graham, ParchaňskýVáclav, P. Bouř, E.W.

MA

Blanch, Through-space transfer of chiral information mediated by a plasmonic nanomaterial, Nat Chem. 7 (2015) 591–596. doi:10.1038/nchem.2280.

D

[18] M.M. Quesada-Moreno, L.M. Azofra, J.R. Aviles-Moreno, I. Alkorta, J. Elguero, J.J. Lopez-Gonzalez, Conformational preference and chiroptical response of Carbohydrates

PT E

D-ribose and 2-deoxy-D-ribose in aqueous and solid phases, J. Phys. Chem. B. 117 (2013) 14599–14614. doi:10.1021/jp405121s. [19] J. Watson, F.H.F. Crick, Molecular structure of nucleic acids, Nature. 171 (1953) 737–

CE

8. doi:10.1038/171737a0.

[20] T.Y. Nikoalenko, L.A. Bulavin, D.N. Govorun, Quantum mechanical interpretation of

AC

the IR Spectrum of 2-deoxy-D-ribose in the OH group stretching vibration region, J. Appl. Spectrosc. 78 (2011) 805–808. doi:10.1007/s10812-011-9528-4. [21] L.D. Barron, L. Hecht, A.F. Bell, Vibrational Raman Optical Activity of biomolecules, in: G.D. Fasman (Ed.), Circular Dichroism and the Conformational Analysis of Biomolecules, 1996: pp. 653–696. [22] W.A. Szarek, S. Korppi-Tommola, H.F. Shurvell, V.H. Smith, O.R. Martin, A Raman and infrared study of crystalline D-fructose, L-sorbose, and related carbohydrates. Hydrogen bonding and sweetness, Can. J. Chem. 62 (1984) 1512–1518. doi:10.1139/v84-258. 32

ACCEPTED MANUSCRIPT [23] S.L. Edwards, An investigation of the vibrational spectra of the pentose sugars, 1976. [24] K.B. Bastawde, Xylan structure, microbial xylanases, and their mode of action, World J. Microbiol. Biotechnol. 8 (1992) 353–368. doi:10.1007/BF01198746. [25] M.S. Izydorczyk, Arabinoxylans, in: G.O. Phillips, P.A. Williams (Eds.), Handb. Hydrocoll.,

Woodhead

Publishing

Limited,

2009:

pp.

653–692.

doi:10.1533/9781845695873.653. [26] E. Winkelhausen, S. Kuzmanova, Microbial conversion of D-xylose to xylitol, J.

PT

Ferment. Bioeng. 86 (1998) 1–14. doi:10.1016/S0922-338X(98)80026-3. [27] J.M. Berg, J.L. Tymoczko, L. Stryer, The light reactions of photosynthesis, in:

RI

Biochemistry, Seventh ed, W H Freeman, New York, 2012: pp. 565–587.

SC

[28] J.M. Berg, J.L. Tymoczko, L. Stryer, Glycolysis and glucogenesis, in: Biochemistry, Seventh ed, W H Freeman, New York, 2012: pp. 453–496.

NU

[29] K. Virkler, I.K. Lednev, Raman spectroscopy offers great potential for the nondestructive confirmatory identification of body fluids, Forensic Sci. Int. 181 (2008) e1-5. doi:10.1016/j.forsciint.2008.08.004.

MA

[30] C. Petibois, V. Rigalleau, A.M. Melin, A. Perromat, G. Cazorla, H. Gin, G. Déléris, Determination of glucose in dried serum samples by Fourier-transform infrared

D

spectroscopy, Clin. Chem. 45 (1999) 1530–1535. [31] A.J. Berger, I. Itzkan, M.S. Feld, Feasibility of measuring blood glucose concentration

PT E

by near-infrared Raman spectroscopy, Spectrochim. Acta Part A Mol. Biomol. Spectrosc. 53 (1997) 287–292. doi:10.1016/S1386-1425(96)01779-9. [32] A.M.K. Enejder, T.-W. Koo, J. Oh, M. Hunter, S. Sasic, M.S. Feld, G.L. Horowitz,

CE

Blood analysis by Raman spectroscopy, Opt. Lett. 27 (2002) 2004–2006. doi:10.1364/OL.27.002004.

AC

[33] A.M.K. Enejder, T.G. Scecina, J. Oh, M. Hunter, W.C. Shih, S. Sasic, G.L. Horowitz, M.S. Feld, Raman spectroscopy for noninvasive glucose measurements, J. Biomed. Opt. 10 (2005) 31114. doi:10.1117/1.1920212. [34] D.A. Stuart, J.M. Yuen, N. Shah, O. Lyandres, C.R. Yonzon, M.R. Glucksberg, J.T. Walsh, R.P. Van Duyne, In vivo glucose measurement by surface-enhanced Raman spectroscopy, Anal. Chem. 78 (2006) 7211–7215. doi:10.1021/ac061238u. [35] H. Schulz, M. Baranska, Identification and quantification of valuable plant substances by

IR

and

Raman

spectroscopy,

doi:10.1016/j.vibspec.2006.06.001. 33

Vib.

Spectrosc.

43

(2007)

13–25.

ACCEPTED MANUSCRIPT [36] Y. Ozaki, R. Cho, K. Ikegaya, S. Muraishi, K. Kawauchi, Potential of near-infrared Fourier transform Raman spectroscopy in food analysis, Appl. Spectrosc. 46 (1992) 1503–1507. doi:10.1366/000370292789619368. [37] F. de L. Mirouze, J.C. Boulou, N. Dupuy, M. Meurens, J.P. Huvenne, P. Legrand, Quantitative analysis of glucose syrups by ATR/FT-IR spectroscopy, Appl. Spectrosc. 47 (1993) 1187–1191. doi:10.1366/0003702934067946. [38] A.N. Batsoulis, N.G. Siatis, A.C. Kimbaris, E.K. Alissandrakis, C.S. Pappas, P.A.

PT

Tarantilis, P.C. Harizanis, M.G. Polissiou, FT-Raman spectroscopic simultaneous determination of fructose and glucose in honey, J. Agric. Food Chem. 53 (2005) 207–

RI

210. doi:10.1021/jf048793m.

SC

[39] J. Wang, M.M. Kliks, S. Jun, M. Jackson, Q.X. Li, Rapid Analysis of glucose, fructose, sucrose, and maltose in honeys from different geographic regions using Fourier

NU

Transform Infrared spectroscopy and multivariate analysis, J. Food Sci. 75 (2010) C208–C214. doi:10.1111/j.1750-3841.2009.01504.x. [40] J.F.D. Kelly, G. Downey, Detection of sugar adulterants in apple juice using Fourier

MA

transform infrared spectroscopy and chemometrics, J. Agric. Food Chem. 53 (2005) 3281–3286. doi:10.1021/jf048000w.

D

[41] L. Xie, X. Ye, D. Liu, Y. Ying, Quantification of glucose, fructose and sucrose in bayberry juice by NIR and PLS, Food Chem. 114 (2009) 1135–1140.

PT E

doi:10.1016/j.foodchem.2008.10.076. [42] M. Černá, A.S. Barros, A. Nunes, S.M. Rocha, I. Delgadillo, J. Čopıḱ ová, M.A. Coimbra, Use of FT-IR spectroscopy as a tool for the analysis of polysaccharide food

X.

CE

additives, Carbohydr. Polym. 51 (2003) 383–389. doi:10.1016/S0144-8617(02)00259-

AC

[43] S. Söderholm, Y.H. Roos, N. Meinander, M. Hotokka, Raman spectra of fructose and glucose in the amorphous and crystalline states, J. Raman Spectrosc. 30 (1999) 1009– 1018. doi:10.1002/(SICI)1097-4555(199911)30:11<1009::AID-JRS436>3.0.CO;2-#. [44] M. Mathlouthi, D. Vinh Luu, Laser-raman spectra of d-glucose and sucrose in aqueous solution, Carbohydr. Res. 81 (1980) 203–212. doi:http://dx.doi.org/10.1016/S00086215(00)85652-9. [45] M. Ibrahim, M. Alaam, H. El-Haes, A.F. Jalbout, A. de Leon, Analysis of the structure and vibrational spectra of glucose and fructose, Eclética Química. 31 (2006) 15–21. doi:10.1590/S0100-46702006000300002. 34

ACCEPTED MANUSCRIPT [46] P.D. Vasko, J. Blackwell, J.L. Koenig, Infrared and Raman spectroscopy of carbohydrates.,

Carbohydr.

Res.

23

(1972)

407–416.

doi:10.1016/S0008-

6215(00)82690-7. [47] P.D. Vasko, J. Blackwell, J.L. Koenig, Infrared and Raman spectroscopy of carbohydrates,

Carbohydr.

Res.

19

(1971)

297–310.

doi:10.1016/S0008-

6215(00)86160-1. [48] H.M. Holden, I. Rayment, J.B. Thoden, Structure and function of enzymes of the Leloir

PT

pathway for galactose metabolism, J. Biol. Chem. 278 (2003) 43885–43888. doi:10.1074/jbc.R300025200.

RI

[49] J. Góral, V. Zichy, Fourier Transform Raman studies of materials and compounds of

SC

biological importance, Spectrochim. Acta Part A Mol. Spectrosc. 46 (1990) 253–275. doi:10.1016/0584-8539(90)80094-F.

NU

[50] L. Pereira, S.F. Gheda, P.J. a Ribeiro-claro, Analysis by vibrational spectroscopy of seaweed polysaccharides with potential use in food, pharmaceutical, and cosmetic industries, Int. J. Carbohydr. Chem. 2013 (2013) 1–7. doi:10.1155/2013/537202.

MA

[51] J.A. Kanters, G. Roelofsen, B.P. Alblas, I. Meinders, The crystal and molecular structure of β-D-fructose, with emphasis on anomeric effect and hydrogen-bond interactions, Acta Crystallogr. Sect. B. 33 (1977) 665–672.

Acta

Crystallogr.

PT E

fructopyranose,

D

[52] S. Takagi, G.A. Jeffrey, A neutron diffraction study of the crystal structure of β-DSect.

B.

33

(1977)

3510–3515.

doi:10.1107/S0567740877011315. [53] R. Goodacre, B.S. Radovic, E. Anklam, Progress toward the rapid nondestructive

CE

assessment of the floral origin of European honey using dispersive Raman spectroscopy, Appl. Spectrosc. 56 (2002) 521–527. doi:10.1366/0003702021954980.

AC

[54] D.H. Owen, D.F. Katz, A review of the physical and chemical properties of human semen and the formulation of a semen simulant, J. Androl. 26 (2005) 459–469. doi:10.2164/jandrol.04104. [55] K. Virkler, I.K. Lednev, Analysis of body fluids for forensic purposes: from laboratory testing to non-destructive rapid confirmatory identification at a crime scene, Forensic Sci. Int. 188 (2009) 1–17. doi:10.1016/j.forsciint.2009.02.013. [56] M. Mathlouthi, J.L. Koenig, Advances in carbohydrate chemistry and biochemistry, Adv.

Carbohydr.

Chem.

Biochem.

2318(08)60077-3. 35

44

(1987)

7–89.

doi:10.1016/S0065-

ACCEPTED MANUSCRIPT [57] A.D. McNaught, A. Wilkinson, Compendium of chemical terminology, Blackwell Science, Oxford, 1997. [58] J.J. Kaneko, Carbohydrate metabolism and its diseases, in: M.L.B. . Jerry Kaneko, John W. Harvey (Ed.), in: Clinical Biochemistry of Domestic Animals, Fifth ed., Academic Press, San Diego, 1997: pp. 45–81. doi:10.1016/B978-012396305-5/50004-X. [59] G.M. Gray, Intestinal digestion and maldigestion of dietary carbohydrates, in: Annual review of medicine, 22 (1971) 391–404. doi:10.1146/annurev.me.22.020171.002135.

PT

[60] A.A. Aguilera, G.H. Díaz, M.L. Barcelata, O.A. Guerrero, R.M. Oliart Ros, Effects of fish oil on hypertension, plasma lipids, and tumor necrosis factor-α in rats with

RI

sucrose-induced metabolic syndrome, J. Nutr. Biochem. 15 (2004) 350–357.

SC

doi:10.1016/j.jnutbio.2003.12.008.

[61] S. Fukuchi, K. Hamaguchi, M. Seike, K. Himeno, T. Sakata, H. Yoshimatsu, Role of

NU

fatty acid composition in the development of metabolic disorders in sucrose-induced obese rats, Exp. Biol. Med. 229 (2004) 486–493. doi:10.1177/153537020422900606. [62] Y.B. Lombardo, S. Drago, A. Chicco, P. Fainstein-Day, R. Gutman, J.J. Gagliardino,

MA

C.L. Dumm, Long-term administration of a sucrose-rich diet to normal rats: relationship between metabolic and hormonal profiles and morphological changes in

D

the endocrine pancreas, Metabolism. 45 (2012) 1527–1532. doi:10.1016/S00260495(96)90183-3.

PT E

[63] J.P.R. Motta, F.H. Paraguass??-Braga, L.F. Bouzas, L.C. Porto, Evaluation of intracellular and extracellular trehalose as a cryoprotectant of stem cells obtained from umbilical

cord

blood,

Cryobiology.

68

(2014)

343–348.

CE

doi:10.1016/j.cryobiol.2014.04.007. [64] P. Mazur, Freezing of living cells: mechanisms and implications, Am. J. Physiol.

AC

Physiol. 247 (1984) 125–142. [65] B.M. Murphy, S.W. Prescott, I. Larson, Measurement of lactose crystallinity using Raman

spectroscopy,

J.

Pharm.

Biomed.

Anal.

38

(2005)

186–190.

doi:10.1016/j.jpba.2004.12.013. [66] H. Susi, J.S. Ard, Laser-raman spectra of lactose, Carbohydr. Res. 37 (1974) 351–354. doi:10.1016/S0008-6215(00)82924-9. [67] G. J., Fourier-transform raman spectroscopy of carbohydrates, Curr. Top. Biophys. 16 (1992). [68] A.B. Brizuela, L.C. Bichara, E. Romano, A. Yurquina, S. Locatelli, S.A. Brandan, A 36

ACCEPTED MANUSCRIPT complete characterization of the vibrational spectra of sucrose, Carbohydr. Res. 361 (2012) 212–218. doi:10.1016/j.carres.2012.07.009. [69] A.B. Brizuela, M.V. Castillo, A.B. Raschi, L. Davies, E. Romano, S.A. Brandán, A complete assignment of the vibrational spectra of sucrose in aqueous medium based on the SQM methodology and SCRF calculations, Carbohydr. Res. 388 (2014) 112–24. doi:10.1016/j.carres.2013.12.011. [70] R. Srisutherp, R. Brockman, J.A. Johnson, Infrared and Raman spectra of

PT

maltooligosaccharides, Cereal Chem. 53 (1976) 110–117. doi:10.1039/DT9730000599. [71] Y. Lu, T.D. Sharkey, The importance of maltose in transitory starch breakdown, Plant,

RI

Cell Environ. 29 (2006) 353–366. doi:10.1111/j.1365-3040.2005.01480.x.

SC

[72] C. Branca, A. Faraone, S. Magazu, G. Maisano, P. Migliardo, V. Villari, Structural and dynamical properties of trehalose-water solutions: anomalous behaviour and molecular

NU

models, Rec. Res. Dev. Phys. Chem. 3 (1999) 361–403.

[73] C. Branca, S. Magazu, G. Maisano, P. Migliardo, Anomalous cryoprotective effectiveness of trehalose: Raman scattering evidences, J. Chem. Phys. 111 (1999)

MA

281–287. doi:10.1063/1.479288.

[74] B. Özbalci, I.H. Boyaci, A. Topcu, C. Kadilar, U. Tamer, Rapid analysis of sugars in

D

honey by processing Raman spectrum using chemometric methods and artificial neural networks, Food Chem. 136 (2013) 1444–1452. doi:10.1016/j.foodchem.2012.09.064.

PT E

[75] M. Kacurakova, M. Mathlouthi, FTIR and laser-Raman spectra of oligosaccharides in water: Characterization of the glycosidic bond, Carbohydr. Res. 284 (1996) 145–157. doi:10.1016/0008-6215(95)00412-2.

CE

[76] M. Lember, Hypolactasia: a common enzyme deficiency leading to lactose malabsorption and intolerance, Pol. Arch. Med. Wewn. 122 Suppl (2012) 60–64.

AC

[77] M.J. Marquez, A.B. Brizuela, L. Davies, S.A. Brandan, Spectroscopic and structural studies on lactose species in aqueous solution combining the HATR and Raman spectra with

SCRF

calculations,

Carbohydr.

Res.

407

(2015)

34–41.

doi:10.1016/j.carres.2015.01.019. [78] P.H. Rodrigues Júnior, K. de Sá Oliveira, C.E.R. de Almeida, L.F.C. De Oliveira, R. Stephani, M. da S. Pinto, A.F. de Carvalho, Í.T. Perrone, FT-Raman and chemometric tools for rapid determination of quality parameters in milk powder: Classification of samples for the presence of lactose and fraud detection by addition of maltodextrin, Food Chem. 196 (2016) 584–8. doi:10.1016/j.foodchem.2015.09.055. 37

ACCEPTED MANUSCRIPT [79] H.M. Berman, The crystal structure of a trisaccharide, raffinose pentahydrate, Acta Crystallogr. B. 26 (1970) 290–299. doi:10.1107/S0567740870002273. [80] G.A. Jeffrey, D. bin Huang, The hydrogen bonding in the crystal structure of raffinose pentahydrate, Carbohydr. Res. 206 (1990) 173–182. doi:10.1016/0008-6215(90)80058B. [81] F. Keller, D.M. Pharr, Metabolism of carbohydrates in sinks and sources: galactosylsucrose oligosaccharides, in: E. Zamski, A.A. Schaffer (Eds.), Photoassimilate Distrib.

PT

Plants Crop. Source–Sink Relationships, Marcel Dekker Inc., New York, 1996: pp. 157–183.

RI

[82] J.F.W. Johnston, On the sugar of the Eucalyptus, Philos. Mag. 23 (1843) 14–18.

SC

doi:10.1080/14786444308644684.

[83] T.M. Kuo, J.F. VanMiddlesworth, W.J. Wolf, Content of raffinose oligosaccharides

doi:10.1021/jf00079a008.

NU

and sucrose in various plant seeds, J. Agric. Food Chem. 36 (1988) 32–36.

[84] D. French, The raffinose family of oligosaccharides, in: 1954: pp. 149–184.

MA

doi:10.1016/S0096-5332(08)60375-6.

[85] R. Guimarães, L. Barros, A.M. Carvalho, M.J. Sousa, J.S. Morais, I.C.F.R. Ferreira,

D

Aromatic plants as a source of important phytochemicals: Vitamins, sugars and fatty acids in Cistus ladanifer, Cupressus lusitanica and Eucalyptus gunnii leaves, Ind. Crops

PT E

Prod. 30 (2009) 427–430. doi:10.1016/j.indcrop.2009.08.002. [86] V.M. Guimarães, S.T. de Rezende, M.A. Moreira, E.G. de Barros, C.R. Felix, Characterization of α-galactosidases from germinating soybean seed and their use for

CE

hydrolysis of oligosaccharides, Phytochemistry. 58 (2001) 67–73. doi:10.1016/S00319422(01)00165-0.

AC

[87] S. Fischer, D. Hopkinson, M. Liu, A.A. MacLean, V. Edwards, E. Cutz, S. Keshavjee, Raffinose improves 24-hour lung preservation in low potassium dextran glucose solution: A histologic and ultrastructural analysis, Ann. Thorac. Surg. 71 (2001) 1140– 1145. doi:10.1016/S0003-4975(01)02426-2. [88] V.M. Tul’chinsky, S.E. Zurabyan, K.A. Asankozhoev, G.A. Kogan, A.Y. Khorlin, Study of the infrared spectra of oligosaccharides in the region 1,000-40 cm-1, Carbohydr. Res. 51 (1976) 1–8. doi:10.1016/S0008-6215(00)84031-8. [89] W.T. Cheng, S.Y. Lin, Processes of dehydration and rehydration of raffinose pentahydrate investigated by thermal analysis and FT-IR/DSC microscopic system, 38

ACCEPTED MANUSCRIPT Carbohydr. Polym. 64 (2006) 212–217. doi:10.1016/j.carbpol.2005.11.024. [90] N.A. Nikonenko, D.K. Buslov, N.I. Sushko, R.G. Zhbankov, Investigation of stretching vibrations of glycosidic linkages in disaccharides and polysaccharides with use of IR spectra

deconvolution,

Biopolymers.

57

(2000)

257–262.

doi:10.1002/1097-

0282(2000)57:4<257::AID-BIP7>3.0.CO;2-3. [91] K. De Gussem, P. Vandenabeele, A. Verbeken, L. Moens, Raman spectroscopic study of Lactarius spores (Russulales, Fungi), Spectrochim. Acta - Part A Mol. Biomol.

PT

Spectrosc. 61 (2005) 2896–2908. doi:10.1016/j.saa.2004.10.038.

[92] A. Buléon, P. Colonna, V. Planchot, S. Ball, Starch granules: structure and

RI

biosynthesis, Int. J. Biol. Macromol. 23 (1998) 85–112. doi:10.1016/S0141-

SC

8130(98)00040-3.

[93] M.M. Green, G. Blankenhorn, H. Hart, Which starch fraction is water-soluble, amylose

NU

or amylopectin?, J. Chem. Educ. 52 (1975) 729. doi:10.1021/ed052p729. [94] Y. Liu, D.S. Himmelsbach, F.E. Barton, Two-dimensional fourier transform Raman correlation spectroscopy determination of the glycosidic linkages in amylose and

MA

amylopectin, Appl. Spectrosc. 58 (2004) 745–749. doi:10.1366/000370204873006. [95] A. Sundarram, T.P.K. Murthy, α -Amylase Production and Applications : A Review, J. Appl. Environ. Microbiol. 2 (2014) 166–175. doi:10.12691/jaem-2-4-10.

D

[96] H.K. Sodhi, K. Sharma, J.K. Gupta, S.K. Soni, Production of a thermostable α-amylase

PT E

from Bacillus sp. PS-7 by solid state fermentation and its synergistic use in the hydrolysis of malt starch for alcohol production, Process Biochem. 40 (2005) 525–534. doi:10.1016/j.procbio.2003.10.008.

CE

[97] J. De Gelder, K. De Gussem, P. Vandenabeele, L. Moens, Reference database of Raman spectra of biological molecules, J. Raman Spectrosc. 38 (2007) 1133–1147.

AC

doi:10.1002/jrs.1734.

[98] R.G. Zhbankov, S.P. Firsov, E. V. Korolik, P.T. Petrov, M.P. Lapkovski, V.M. Tsarenkov, M.K. Marchewka, H. Ratajczak, Vibrational spectra and the structure of medical biopolymers, J. Mol. Struct. 555 (2000) 85–96. doi:10.1016/S00222860(00)00590-1. [99] B.J. Bulkin, Y. Kwak, I.C.M. Dea, Retrogradation kinetics of waxy-corn and potato starches; a rapid, Raman-spectroscopic study, Carbohydr. Res. 160 (1987) 95–112. doi:http://dx.doi.org/10.1016/0008-6215(87)80305-1. [100] P.D. Vasko, J.L. Koenig, Infrared studies of chain folding in polymers. IX. 39

ACCEPTED MANUSCRIPT amylopectin and amylose, J. Macromol. Sci. Part B. 6 (1972) 117–127. doi:10.1080/00222347208224793. [101] M. Kacurakova, P. Capek, V. Sasinkova, N. Wellner, A. Ebringerova, FT-IR study of plant cell wall model compounds: pectic polysaccharides and hemicelluloses, Carbohydr. Polym. 43 (2000) 195–203. doi:10.1016/s0144-8617(00)00151-x. [102] A. Gałat, Study of the Raman scattering and infrared absorption spectra of branched polysaccharides, Acta Biochim. Pol. 27 (1980) 135–142.

PT

[103] J.J. Cael, K.H. Gardner, J.L. Koenig, J. Blackwell, Infrared and Raman spectroscopy of carbohydrates. Paper V. Normal coordinate analysis of cellulose I, J. Chem. Phys. 62

RI

(1975) 1145–1153. doi:10.1063/1.430558.

SC

[104] S.B. Heymsfield, Z. Wang, R.N. Baumgartner, R. Ross, Human body composition: advances in models and methods, Annu. Rev. Nutr. 17 (1997) 527–558.

NU

doi:10.1146/annurev.nutr.17.1.527.

[105] M. Mathlouthi, J.L. Koenig, Vibrational spectra of carbohydrates, Adv. Carbohydr. Chem. Biochem. 44 (1987) 7–89. doi:10.1016/S0065-2318(08)60077-3.

MA

[106] M. Moreno, L. Raniero, E.Â.L. Arisawa, A.M. Espírito Santo, E.A.P. Santos, R.A. Bitar, A.A. Martin, Raman spectroscopy study of breast disease, Theor. Chem. Acc.

D

125 (2009) 329–334. doi:10.1007/s00214-009-0698-6. [107] N. Bergner, T. Bocklitz, B.F.M. Romeike, R. Reichart, R. Kalff, C. Krafft, J. Popp,

vector

PT E

Identification of primary tumors of brain metastases by Raman imaging and support machines,

Chemom.

Intell.

Lab.

Syst.

117

(2012)

224–232.

doi:10.1016/j.chemolab.2012.02.008.

CE

[108] G. Srinivasan, Vibrational spectroscopic imaging for biomedical applications, First ed., McGraw-Hill, New York, 2010. Colagar,

AC

[109] A.H.

M.J.

Chaichi,

T.

Khadjvand,

Fourier

transform

infrared

microspectroscopy as a diagnostic tool for distinguishing between normal and malignant human gastric tissue, J. Biosci. 36 (2011) 669–677. doi:10.1007/s12038011-9090-5. [110] G. Shetty, C. Kendall, N. Shepherd, N. Stone, H. Barr, Raman spectroscopy: elucidation of biochemical changes in carcinogenesis of oesophagus, Br. J. Cancer. 94 (2006) 1460–1464. doi:10.1038/sj.bjc.6603102. [111] E. Gazi, J. Dwyer, P. Gardner, A. Ghanbari-Siahkali, A.P. Wade, J. Miyan, N.P. Lockyer, J.C. Vickerman, N.W. Clarke, J.H. Shanks, L.J. Scott, C.A. Hart, M. Brown, 40

ACCEPTED MANUSCRIPT Applications of Fourier transform infrared microspectroscopy in studies of benign prostate and prostate cancer. A pilot study, J. Pathol. 201 (2003) 99–108. doi:10.1002/path.1421. [112] C.S. Tsai, Biomacromolecules: Introduction to Structure, Function and Informatics, John Wiley & Sons, Inc., Hoboken, NJ, USA, 2006. doi:10.1002/0470080124. [113] J. Grdadolnik, D. Hadzi, FT infrared and Raman investigation of saccharidephosphatidylcholine interactions using novel structure probes, Spectrochim. Acta. A.

PT

Mol. Biomol. Spectrosc. 54A (1998) 1989–2000. doi:10.1016/S1386-1425(98)001115.

RI

[114] B. Goodfellow, R. Wilson, A fourier transform IR study of the gelation of amylose and

SC

amylopectin, Biopolymers. 30 (1990) 1183–1189. doi:10.1002/bip.360301304. [115] C.H. Evans, Applications of infrared, Raman and Resonance Raman spectroscopy in

AC

CE

PT E

D

MA

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biochemistry, Trends Biochem. Sci. 9 (1984) 289. doi:10.1016/0968-0004(84)90171-3.

41

ACCEPTED MANUSCRIPT Table 1. Raman (RS) and IR bands assignments for pentoses [11,15,22,23]. Bands positions in bold fonts indicate potential markers of each carbohydrate. Molecular structures and vibrational spectra are shown in Figs. 1A and 2, respectively. Region

D-(−)-ribose

RS

V

IV

2-deoxy-D-ribose

L-(+)-arabinose

IR

RS

IR

3407

3375

3368

3366

3343

3335

3327

3198

3196

3001

3000

2984

2982

2959

2956

2955

2955

2970

2967

2943

2943

2940

2938

2959

2955

2919

2917

2916

2914

2939

2878

2874

2868

2891

1433

III 1364

1363

2986

1312

RS

2985

3001

3398

3324

3320

2997

1443

PT

E C

C A

1466

D E

N A

3213 2979

ν(OH)

νas(CH2) of C-5 νas(CH2) of C-2

2958

ν(CH)

2951

2946

ν(CH)

2918

2917

ν(CH)

2961

νs(CH2) of C-5

2899

2891

2894

2890

2888

1476

1473

1476

1475

1446

1449

1397

1394

1449

T P

I R

C S U

2982

M

2938

Assignment

IR

3524

2980

δ(CH2)

1412 1387

1379 1348

1321

3337

IR

3221

1471 1452

RS

D-(+)-xylose

1343

1375

1371

1373

1371

1356

1354

1341

1339

1312

1314

1316

1303

42

ω(CH2)

ACCEPTED MANUSCRIPT Region

D-(−)-ribose

RS

IR

2-deoxy-D-ribose RS

IR

1298

1300

1273

1279

1279

1277

1246

1244

1257

1256

1235

1234

1196

1196

1161

L-(+)-arabinose

RS

IR

D-(+)-xylose

RS

1260

1244

1256

II

1074

1115

1084

1059 1030

1027 1014

1003

1147

1136

1115

1111

1086

1044

E C

PT

1082

C A 1017

1041

D E

N A

986

M

1115

ν(CO), ν(CC)

1147

1125 1113

1107

1096

1089

1051

1048

1086

1081

ν(CO), ν(CC), β(COH)

1034 1017

1011

993

945

C S U

1150

T P

I R

1235

1229

1129 1119

IR τ(CH2)

1159 1148

Assignment

1016 νas(COC), ν(CC), ν(CO), β(CCH)

991

ν(CO)ring, ν(CC)ring, β(CCH)

979

949 43

ACCEPTED MANUSCRIPT Region

D-(−)-ribose

2-deoxy-D-ribose

RS

IR

RS

IR

922

922

922

925

L-(+)-arabinose

RS

IR

D-(+)-xylose

RS

IR

932

929

903

901

Assignment

909

843 885

804

891

899

892

868

878

878

805

808

812 789

727

I

756

677

670

D E

725

656

654

631

626

598

758

590

698

T P E

627

C S U

N A

M

758

671

A

518

521

ν(CC), β(CCH)

ν(CO)ring, β(COC), β(CCO), β(OCO)

759

623

612

602

579

578

608 β(CCC), ring def. 565

562

527

520

505

503

540

542

νs(CO)ring , ν(CC), ν(CO)

667

629

C C

I R

841

782

T P

511 501 44

ACCEPTED MANUSCRIPT Region

D-(−)-ribose

RS

IR

2-deoxy-D-ribose RS

IR

L-(+)-arabinose

RS

IR

D-(+)-xylose

RS

Assignment

IR β(COC)

484 462

460

441 421

430 416

417

431

428

431

I R

399

416

T P

C S U

Key: ν - stretching, as – asymmetric, s - symmetric, β – in-plane bending, δ - scissoring, ω - wagging, τ - twisting, def – deformation.

N A

D E

M

T P E

C C

A

45

ACCEPTED MANUSCRIPT Table 2. Raman (RS) and IR bands assignments for hexoses [22,43–47]. Bands positions in bold fonts indicate potential markers of each carbohydrate. Molecular structures and vibrational spectra are shown in Figs. 1B and 3, respectively. Region

D-(+)-glucose

RS

IR

D-(+)-galactose

RS

IR

D-(−)-fructose

RS

Assignment

IR ν(OH)

3520 V

3395

3392

3383

3368

3265

3296

3207

3195

3243

3140

3122

IV

3396

C S U

3015

N A

2991 2962 2947

2891

2974

2970

2963

2943

2939

2938

2939

2912

2918

2916

2924

1489

1460

1457

III

C A

1346

1371

νas(CH2) of C-6 ν(CH) of C-4 νs(CH2) of C-6 ν(CH) of C-3

2899

νs(CH2) of C-1

1494

1472

1469

δ(CH2)

1453

1455

1452

2673

1422

1428 1398

1373

2936

νas(CH2) of C-1

2901

E C

2853

M

2959

D E

PT

2891

2880

I R

1397

1379 1357 46

ω(CH2)

T P

ACCEPTED MANUSCRIPT Region

D-(+)-glucose

D-(+)-galactose

RS

IR

RS

1333

1338

1333

1296

1310

IR

D-(−)-fructose

Assignment

RS

IR

1341

1333 τ(CH2)

1298 1273 1248

1248

1265

1265

1251

1251

1179

1176

1142

1147

C S U

1224 1204

1150

1203

1146

1109 1074 1055

1048

1022

1014

1001

993

914

1152

D E

1138

1121

II

1154

915

1107

1102

1069

1065

1082

1076

1055

1048

999

E C

1049

972

973

980

976

959

955 926

923

C A

PT

1042

N A

M

I R

(CO), (CC)

(CC), (CO), β(COH)

β(CCH), β(CCO)

47

T P

ACCEPTED MANUSCRIPT Region

D-(+)-glucose

RS

IR

D-(+)-galactose

RS

IR

889 843

774

837

650 615 560

555

542

537

RS

IR

874

874

820

817

781

782

Assignment (CC)

836

775

725

I

830

D-(−)-fructose

793 766

763

705

707

662

652

T P

I R

β(CCC), β(CCO), β(OCO)

C S U

615

684 627 596

D E

534

531

527

440

494

500

465

423

417

N A

626

M

523

PT

421

E C

407

404

Key: ν - stretching, as – asymmetric, s - symmetric, β – in-plane bending, δ - scissoring, ω - wagging, τ - twisting.

C A

48

ACCEPTED MANUSCRIPT Table 3. Raman (RS) and IR bands assignments for disaccharides [65–70,74,77]. Bands positions in bold fonts indicate potential markers of each carbohydrate. Molecular structures and vibrational spectra are shown in Figs. 4 and 5, respectively. Region

D-(+)-sucrose

RS

D-(+)-maltose

IR

monohydrate

RS

D-(+)-lactose

IR

RS

3521 (glc)

3382(glc)

3360

C S U

3330

3320

3252

IV

2943

2940

2947 2934

2914

2913

E C

1462 1433 (fruct)

2899

C A 1428 (glc)

2845

D E

PT

2912

2897

III

2979

νas(CH2)

2978 (glc)

ν(CH)

2947

νs(CH2)

M

2936

2917

2918

2894

2887 (glc)

2932 (glc, gal)

ν(CH) νs(CH2)

2899

ν(CH)

2844 1470 (glc)

β(OH)

1455

(CH2)

1455 1431

ν(CH)

N A

2995 2976

ν(OH)

3260

3015 (glc)

2972 (glc)

T P

I R

3398

3319 (fruc)

Assignment

IR

3559 (fruc) V

monohydrate

ρ(CH)

1432 1423 (gal) 1416 (glc) 49

ω(CH2)

ACCEPTED MANUSCRIPT Region

D-(+)-sucrose

RS

D-(+)-maltose

IR

monohydrate

RS

D-(+)-lactose

IR

RS

monohydrate

Assignment

IR β(COH)

1382 1368 (glc) 1350

ρ(CH)

1379 1344 1279

1348 1271

1346

1340

C S U

1256

1239 (fruc)

1236 (glc, fruc)

D E

1208 (glc, fruc)

1115 (fruc)

M

ρ(CH2)

β(CH), β(CH2) ρ(CH), ρ(OH)

β(CH), β (CH2) β(OH) β(OH), β(CH)

PT

E C

C A

II

1259 (glc)

N A

1240

1161(fruc)

I R

1272 1263 (glc)

T P

1201 (gal)

β(OH), ρ(CH2)

1167 (glc)

ν(CO) β(COC), β(CC)

1150

1142 (glc,

1140

β(OH), ν(CO)

1115

ν(CO), ν(CC), ν(COC)

gal) 1115

ν(CO)

1127 (fruc) 50

ACCEPTED MANUSCRIPT Region

D-(+)-sucrose

RS

D-(+)-maltose

IR

monohydrate

RS

D-(+)-lactose

IR

RS

monohydrate

Assignment

IR ν(CC), ν(CO)

1121 (gal)

β(COC)

1102 1093 (gal) 1088 (glc)

1086 (glc) 1073

C S U

1071

1071 (glc)

1065 (glc) 1053 (glc,

1050 (fruc)

N A gal)

1038 (fruc)

D E

1015 (fruc)

1024

987 (glc) 943 (glc)

I R

PT

942 (glc)

E C

922 (glc)

922

908

C A

M

T P

ν(CO)

ν(CC), ν(CO) ν(CC) ν(CC) ν(CO)

1031 (glc)

ν(CC)

1020 (glc)

1018 (glc)

ν(CO)

953 (glc)

987 (glc, gal) ν(CC)

916 (gal)

915 (gal) τ(CH2) β(CCO), ν(CC),

901

ν(CO) 899 878

51

875

ν(CC)

ACCEPTED MANUSCRIPT Region

D-(+)-sucrose

D-(+)-maltose

RS

IR

872 (glc)

867 (glc)

851

848

monohydrate

RS

D-(+)-lactose

IR

RS

monohydrate IR

β(CCO) τ(CH2), ν(CC) (glu)

851 849

847

780

776

T P

I R

C S U

777 (gal)

757 (glc)

737

N A

731 680 (glc)

D E

641 (fruc)

585 (fruc)

552

Assignment

PT

E C

581 (fruc)

C A

552

M

633 (gal)

673 (gal)

ν(CC), β(CH) ν(CC), β(CH) Ring torsion

τ(CO), τ(COHO), τ(HOH) ν(CO), Ring (glc) def β(CCO) Ring def

630

β(COC)

602 (gal)

β(OCO) β(CCO)

556 (glc) 540

551 (glc)

527 (glcfr)

535 (glcfr)

444 (glc)

470 (fruc)

519 (glc)

518

477 (glc)

467 (glc)

403 (fruc)

399 (fruc)

463 (glc)

462

398 (gal)

435 (glc)

β(OCO) Ring def

52

β(CCO)

ACCEPTED MANUSCRIPT Region

D-(+)-sucrose

RS

D-(+)-maltose

IR

monohydrate

RS

D-(+)-lactose

IR

RS

monohydrate

Assignment

IR

430 (glc) 385 (glc) 378 (gal)

Ring torsion

I R

367 (fruc)

C S U

357 348 (endc) 334 (gal)

N A

259 (gal) 230 (fruc)

D E

PT

118 (fruc)

E C

M

T P

β(CCO) t(OH)

t(COHO), τ(HOH) β(CCO) β(CCO), t(COHO) β(CCO) t(OH)

189 (glc)

β(CCO)

176 (gal)

Ring torsion τ(CO)

114 (glc)

Ring torsion

Key: ν - stretching, as – asymmetric, s - symmetric, β – in-plane bending, δ - scissoring, ω - wagging, ρ - rocking, τ - twisting, t-torsion, def –

C A

deformation; glc-glucosyl unit, glcfr-glucofuran ring, fruc-fructosyl unit, gal-galactosyl unit, endc –endocyclic.

53

ACCEPTED MANUSCRIPT Table 4. Raman (RS) and IR bands assignments for raffinose – trisaccharide [75,88,89,91,102,112–114]. Its molecular structure and vibrational spectra are shown in Figs. 6 and 7, respectively. Region

D-(+)-raffinose

RS

pentahydrate

Assignment

IR

V

3235

T P

ν(OH)

3492

C S U

3210 ν(CH)

2978

IV

2960

2960

νas(CH2)

2939

2936

ν(CH)

2911

2908

νs(CH2)

2891

2888

N A

D E

1649

PT

1466 1433

C A 1361

1389

M

(OH2)crystal water (CH2) β(CH) β(CH), β(OH) ω(CH2) τ(CH2)

1335 1306

E C

1431 1411

III

I R

3285

1306

τ(CH2) 54

ACCEPTED MANUSCRIPT Region

D-(+)-raffinose

RS

pentahydrate

Assignment

IR τ(CH2)

1269 1265

β(CH) of C-1

1152

(CO) in glycosidic linkage, β(CO), (COH), β(CH)

(CO)

1125(Glc)

N A

(CO), (CC), β(COH)

1078 1043

D E

1030

T P E

1014 997 996 967 936 876 (gal, fru)

834

M

ν(CO), ν(CC), β(COH) of C-l

1051 (glc) II

C S U

(CO), ring def.

1106

1075

I R

(CO)endc

1153

C C

965

A

β(COH)

β(COH), ν(CC)

ν(CO), β(COH), ν(CC)

νas(CO) in glycosidic linkage

νs(CO) in glycosidic linkage, β(COH)

935

ν(CO) in glycosidic linkage

873 (gal, fru)

β(CCO), ring def.

860 (fr)

β(CH) of C-1, β(CH2), ring def.

833

ν(CC) of α-glycosidic linkage 55

T P

ACCEPTED MANUSCRIPT Region

D-(+)-raffinose

RS

pentahydrate

Assignment

IR ν(CC)

771 751

ρ(CH2)

706 (gal)

β(CCC), β(CCO), β(OCO)

I R

654 (glc) 618 (gal)

C S U

β(CCC), β(CCO), β(OCO), γ(OH)

667 (gal) 619

β(CCC), β(CCO), β(OCO)

557 (glc) 538 (glcfr) I

N A

537 (glcfr)

ring def.

461

β(CCO), β(CCC), β(OCO)

D E

399 (fruc)

T P E

378 (gal) 349 (endc)

C C

257 (gal) 232 (fruc) 178 (gal) 114

M

skeletal mode, β(CCC)

454 419 (fruc)

T P

A

β(CCO), β(CCC), β(OCO)

t(OH) ring torsion t(CO) in (fruc), ring torsion

Key: ν - stretching, as – asymmetric, s - symmetric, β – in-plane bending, γ – out-of-plane bending, δ - scissoring, τ - twisting, t-torsion, def – deformation; glc-glucosyl unit, fruc-fructosyl unit, gal-galactosyl unit, glcfr-glucofuran ring, fr-furanose, endc –endocyclic. 56

ACCEPTED MANUSCRIPT Table 5. Raman (RS) and IR bands assignments for polysaccharides [91,98,102,114,115]. Bands positions in bold fonts indicate potential markers of each carbohydrate. Molecular structures and vibrational spectra are shown in Figs. 8 and 9, respectively. Region

amylopectin1/amylose2 RS

IR

RS

2933

2926

2938

2911

2901

1

2908

2896

2

1460

1387 1338

T P E

2

1262

1262

1149

II

11272 10861

C C

A

D E

1337

12671

M

1363

C S U

N A

1411

1378

11311

I R

ν(CH/CH2)

2931

1415

1339

T P

ν(OH)

3313

1462

III

Assignment

IR

3279

V

IV

glycogen

(CH2), β(COH) β(CH) β(CH2) β(COH), β(CH)

β(CH), β(CCH), β(OCH), β(COH)

1149

ν(COC), ν(CC) in glycosidic linkage, asymmetric ring breathing ν(CO), ν(CC), β(COH), νs(COC) in glycosidic

1130

link, ring breathing β(COH), β(CH)

1084

10822

57

ACCEPTED MANUSCRIPT Region

amylopectin1/amylose2 RS

glycogen

IR

RS

Assignment

IR

1076

β(COH)

1078

1051

ν(CO), ν(CC)

1051

T P

ν(COH)solvated

1017 991

I R

ν(COH)solvated, β(CH2), β(COH), ν(CO) in COC

999

C S U

glycosidic linkage

942

νs(COC) of α-D-(1→6) glycosidic linkages, ring

941

928

866

PT

7911 7662 I

714 614

E C

762

C A 708 603

2

577

5711

D E

860 856

7701

N A

931

M 850

def.

β(COH), β(CH) of C-1, νs(COC) in glycosidic linkage, ring modes νs(COC), ring breathing ν(COC), β(CC), ring breathing, β(CH) of C-1 ν(COC)

759

ρ(CH2)

707

γ(OH)Hbonded

596

605

579

574

572

58

ACCEPTED MANUSCRIPT Region

amylopectin1/amylose2 RS

IR

521

523

glycogen RS

Assignment

IR

β(CCC), β(COC) in glycosidic linkage, skeletal

524

modes 479

484

442

446 432

T P

skeletal modes, β(CCC)

430

2

409

I R

C S U

432

406

4131 361

N A

363 326

303

D E

307

M

T P E

Key: 1,2- Bands specific exclusively for amylopectin and amylose, respectively; ν - stretching, s – symmetric, β – in-plane bending, γ – out-ofplane bending, δ - scissoring, ρ – rocking, τ - twisting, t-torsion, def – deformation

C C

A

59

ACCEPTED MANUSCRIPT

Graphical abstract

T P

I R

C S U

N A

D E

M

T P E

C C

A

60

ACCEPTED MANUSCRIPT

Highlights



A description of Raman and IR features of 14 carbohydrates is given.



FT-Raman and ATR FT-IR spectra of solids are collected and assigned in detail.



Applications of vibrational spectroscopy in detection and quantification of carbohydrates are shown.

C S U

I R

N A

D E

M

T P E

C C

A

61

T P