Raman Optical Activity and Raman spectroscopy of carbohydrates in solution

Accepted Manuscript Raman Optical Activity carbohydrates in solution

and

Raman

spectroscopy

of

Monika Dudek, Grzegorz Zajac, Ewelina Szafraniec, Ewelina Wiercigroch, Szymon Tott, Kamilla Malek, Agnieszka Kaczor, Malgorzata Baranska PII: DOI: Reference:

S1386-1425(18)30788-1 doi:10.1016/j.saa.2018.08.017 SAA 16392

To appear in:

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy

Received date: Revised date: Accepted date:

23 March 2018 2 August 2018 11 August 2018

Please cite this article as: Monika Dudek, Grzegorz Zajac, Ewelina Szafraniec, Ewelina Wiercigroch, Szymon Tott, Kamilla Malek, Agnieszka Kaczor, Malgorzata Baranska , Raman Optical Activity and Raman spectroscopy of carbohydrates in solution. Saa (2018), doi:10.1016/j.saa.2018.08.017

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ACCEPTED MANUSCRIPT Raman Optical Activity and Raman Spectroscopy of carbohydrates in solution

Monika Dudeka, Grzegorz Zajaca, Ewelina Szafranieca, Ewelina Wiercigrocha, Szymon Totta, Kamilla Maleka,b, Agnieszka Kaczora,b, Malgorzata Baranskaa,b,*

Faculty of Chemistry, Jagiellonian University, Gronostajowa 2, 30-387 Krakow, Poland

b

Jagiellonian Centre for Experimental Therapeutics (JCET), Jagiellonian University,

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a

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Bobrzynskiego 14, 30-348 Krakow, Poland

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*correspondence to: M. Baranska ([email protected])

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Key words: ROA, optical activity, Raman spectroscopy, carbohydrates, sugars, saccharides,

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vibrational analysis

ACCEPTED MANUSCRIPT Abstract This comprehensive study on selected 14 carbohydrates in water solution is an extension of previously published one focused only on solid state analysis. Here, Raman spectroscopy was used as a dedicated method for analysis of carbohydrates in solution, both using a normal effect

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(RS) and its chiral analogue: Raman Optical Activity spectroscopy (ROA). The compounds were selected as biologically important and representative of all groups: monosaccharides,

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disaccharides, trisaccharides, cyclodextrines and polysaccharides. RS and ROA spectra are

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presented together with an expanded discussion on various structures and conformations of studied carbohydrates in the solution taking into account particular regions, i.e. (1) low

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wavenumber region (250-600 cm-1), (2) anomeric region (600-950 cm-1), (3) fingerprint region

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(950-1200 cm-1) and (4) CH2 and COH deformations region (1200-1500 cm-1). So, the following information can be obtained about: (1) the absolute configuration of the anomeric centre; (2) the configuration of the anomeric centre and the orientation of the anomeric

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hydroxyl group; (3) the ring structures and the relative orientation of substituents and (4) the conformation of the exocyclic CH2OH (4), respectively. Raman spectroscopy and Raman

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Optical Activity were shown as unique tools to study complex structures of carbohydrates.

ACCEPTED MANUSCRIPT 1. Introduction Carbohydrates, beside proteins and lipids, are common compounds and essential constituents for living organisms. Very recently we have published a comprehensive review on selected 14 carbohydrates in the solid state analysed by Raman and Infrared spectroscopy [1]. Since

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biological relevance of carbohydrates should be studied also taking into account their various structures and conformations, this papers continues the discussion on a spectroscopic analysis

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of carbohydrates, but in the solution. Moreover, multiple chiral centres in their structures make

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carbohydrates ideal compounds for chiroptical studies. Out of various chiroptical methods, Raman Optical Activity (ROA) seems most suited to investigate the chiral dimension of

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carbohydrates, as both Electronic Circular Dichroism (ECD) and its vibrational analogy (VCD)

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possess serious limitations for saccharides studies (e.g. a lack of the ECD signal of sugars above ca. 190 nm and interfering infrared absorptions from water in VCD) [2,3].

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ROA spectra of various mono- di- and polysaccharides have been recorded and analysed by

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Barron and co-workers in the nineties [2–4]. This very important pioneer work on carbohydrates enabled assigning various bands in their ROA spectra and draw numerous

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conclusions regarding their structures. Overall, it has been demonstrated that due to delocalization of normal modes over the chiral arrangement in the carbohydrate structures and

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due to the presence of the C-O-C linkages, the ROA signal is intense. It has been highlighted also that adding of the chiral dimension yielded a plethora of new information compared to the data obtained using classical Raman spectroscopy. Unfortunately, revealing of these information is not trivial, mostly due to the very complicated equilibria in the aqueous solutions of carbohydrates (chain  ring, anomers: α  β, ring tautomers: pyranose  furanose, chair conformers: 4C1  1C4 and 5C2  2C5, Fig. 1) resulting in multiple and overlaid ROA (and Raman) features. Raman spectra of carbohydrates in aqueous solution are, therefore, very different than Raman spectra in the solid state [1], particularly comparing a practically

ACCEPTED MANUSCRIPT negligible impact of the excitation line and the spectral resolution on the shape of the obtained

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spectra, Fig. 1S, Supplementary Materials).

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Fig. 1. Diversity of D-(-)-fructose equilibrium occurring in aqueous solution.

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Wavenumbers of Raman and ROA bands of carbohydrates are alike (vide infra, Figs. 2-5), but the richness of information obtained from ROA spectra is multiplied due to the additional

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dimension of the spectra, i.e. the sign. Also, the nomenclature of the Raman and ROA spectral

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ranges is quite different; ROA spectra have been conveniently divided into four regions [3,4]: 1. low wavenumber region (250-600 cm-1), 2. anomeric region (600-950 cm-1), 3. fingerprint

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region (950-1200 cm-1) and 4. CH2 and COH deformations region (1200-1500 cm-1). The key information that can be extracted from ROA signatures is: the absolute configuration of the

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anomeric centre, available by analysing of the 250-600 cm-1 region, the anomeric preference, the configuration of the anomeric centre, and the orientation of the anomeric hydroxyl group that are encrypted in the 600-950 cm-1 region as well as the conformation of the exocyclic CH2OH group at C5 that is encoded in the 1200-1500 cm-1 range. The so-called fingerprint region, although rich in multiple bands, is much less understood. Additionally, less examined features of ROA spectra of carbohydrates, such as circular intensity difference (CID), the shape

ACCEPTED MANUSCRIPT of bands and their relative intensity, can possibly provide the additional information about their structure. The twenty year progress in ROA instrumentation in conjunction with application of the SCP (scattered circular polarization) mode gives rise to a significantly better signal-to-noise and

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enables recording the low-wavelength range of ROA spectra. The low-wavenumber range, important from the point of view of the absolute configuration of the anomeric centre was

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analysed only for ketose monosaccharides [4] and a few other saccharides [5], while for many

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aldose monosaccharides, some di- and oligosaccharides, this range was not studied due to

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technical problems with recording it in the nineties [2,3].

In this paper, fourteen carbohydrates selected from different groups (mono- aldoses and

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ketoses, di-, tri- and oligosaccharides) were measured in aqueous solution using Raman spectroscopy and collecting high signal-to-noise SCP spectra. Among them, for the first time

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ROA spectra were recorded for 2-deoxy-D-(-)-ribose and glycogen, two carbohydrates of key

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functions from the point of view of living organisms, and 2-deoxy-D-(+)-glucose, and D-(+)raffinose important in view of the structural analysis of carbohydrates. Collected spectra

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enabled a detailed structural analysis of carbohydrates, including an assignment of some weak spectral features as well as the ones occurring in the ROA low-wavenumber range.

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2. Experimental 2.1 Materials

All carbohydrates were purchased from Sigma-Aldrich. Accordingly, we measured 14 standards of the following saccharides: D-(-)-ribose, 2-deoxy-D-(-)ribose, D-(+)-xylose, L-(+)arabinose,

D-(-)-fructose,

D-(+)-glucose,

2-deoxy-D-(+)-glucose,

D-(+)-galactose,

D-(+)-

maltose, D-(+)-sucrose, D-(+)-lactose, D-(+)-raffinose, glycogen, and γ-cyclodextrin. Solutions were prepared by dissolving saccharides in 2x distilled water. Solutions were gently heated in

ACCEPTED MANUSCRIPT water bath (up to 60°C) to increase solubility, and cooled down to room temperature before measurements. All solutions were filtered in use of MilliporeTM Millex® syringe PTFE filters (pore size 0.45 μm).

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2.2 ROA measurements Measurements of Raman Optical Activity of aqueous solutions of carbohydrates were

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performed by using a ChiralRAMAN-2XTM spectrometer from BioTools Inc., with 7 cm-1

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resolution, in optical cells with anti-reflective coating and the 532 nm wavelength excitation. Simultaneously, on the same spectrometer and from the same sample, Raman spectra were

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collected. If it was necessary, activated charcoal and fotobleaching were used for decreasing

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background fluorescence. Power of laser, integration time, concentration and total registration

Table 1. The ROA measurement parameters.

Concentration

Integration time [s]

Power of laser [mW]

Registration time [h]

1000

2.0580

200

20

1000

0.5145

500

24

1000

2.0580

90

24

1000

2.0580

150

24

D-(-)-fructose

1000

2.0580

120

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D-(+)-glucose

1000

2.0580

105

24

2-deoxy- D-(+)-glucose

330

2.0580

120

24

D-(+)-galactose

200

2,0580

600

24

D-(+)-maltose

1000

0.5145

600

24

D-(+)-sucrose

1000

0.5145

480

10

D-(+)-lactose

330

0.5145

800

12

D-(+)-raffinose

500

0.5145

1000

24

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Carbohydrate

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time were chosen for each sample individually, cf. Table 1.

[mg/mL]

2-deoxy- D-(-)ribose D-(+)-xylose

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L-(+)-arabinose

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D-(-)-ribose

ACCEPTED MANUSCRIPT glycogen

500

0.5145

200

24

γ-cyclodextrin

100

2.0580

300

20

3. Results and Discussion

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3.1. Monosacharrides Monosaccharides, the simplest and a very soluble form of sugars, exist in the aqueous solution

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in many equilibrium conditions between α ↔ β anomers, furanose ↔ pyranose ring tautomers

galactose, D-(-)-fructose,

D-(-)-ribose,

D-(+)-glucose,

and 2-deoxy-D-(-)-ribose) and four hexoses (D-(+)-

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(+)-xylose, L-(+)-arabinose,

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and different chair conformers (1C4 ↔ 4C1 and 2C5 ↔ 5C2) [6]. Here, we study four pentoses (D-

and 2-deoxy-D-(+)-glucose), which differ from each

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other, particularly in the mutual orientation of the hydroxyl group and the anomeric configuration. For the first time ROA spectra of 2-deoxy-D-(-)-ribose and 2-deoxy-D-(+)-

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glucose in aqueous solution are presented. The backscattering Raman and ROA spectra of these

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monosaccharides are discussed in reference to their molecular structures (Figs. 2 and 3), and are related to four spectral regions [3,7]. Accordingly, the low wavenumber region (~350-600 cm-1) contains sign patterns of backbone conformation assigned to skeletal bending and

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twisting vibrational modes. The anomeric region (~600-950 cm-1) enables specification of α- or

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β-anomer and gives information about the nature of ring’s chiral centres. The fingerprint region (~ 950-1200 cm-1) shows ROA bands characteristic for the ring structures and the relative orientation of substituents. And finally, the CH2 and COH deformations region range of 12001500 cm-1, which reflects also the exocyclic conformation of the CH2OH group. The origin of vibrational modes of registered Raman/ROA spectra is shown in Tables 2 and 3. All discussed here monosaccharides exist in the aqueous solution mostly as pyranoses (Fig. 1) [6]. Moreover, strongly in favour is the less sterically hindered equatorial orientation of the anomeric hydroxyl groups. However, a key to the ROA data interpretation is the classification of monosaccharides

ACCEPTED MANUSCRIPT according to their configuration (based on the position of groups around the sugar ring), i.e. the xylo-, the arabino- and the ribotype [4]. It is also worth noting that the computation of carbohydrate ROA spectrum is difficult due to their solvent sensitivity. Recently, Zielinski F. et. all have shed a new light on a deeper applicability of ROA to investigate the dynamic structure of carbohydrate in aqueous solution [8]. They have resolved vibrational bands of β-D-

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(+)-xylose using a simulation strategy based on the combination of density functional theory

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(DFT) and molecular dynamics (MD) taking into account the crucial hydration effect.

3.1.1 Pentoses

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Xylose (the xylo-type sugar, Fig. 2), called a ‘wood sugar’ is the main building block for the

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hemicellulose xylan, that is found predominantly in hardwood trees. In industry, xylose is produced from hemicellulose and hydrogenated in a form of xylitol – a sugar alcohol widely

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used as a sweetener [9]. D-(+)-xylose in solution exists predominantly in the β-D-xylopyranose

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form in an equilibrium with α-D-xylopyranose (Fig. 2). In fact, Raman spectrum of the β anomer exhibits a very weak band at ~763 cm-1, which is in agreement with the presence of the α form in solution. The most prominent bands in the Raman spectrum displayed in Fig. 2 are

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observed at 1470 cm-1 (CH2 scissoring mode), 1123 cm-1 (coupled CO and CC stretching

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modes), 904 cm-1 (coupled CO and CC stretching modes of pyranose ring) and 539 cm -1 (CCC bending and ring deformation modes) [10]. The Raman spectrum of solid state D-(+)-xylose exhibits two prominent bands at similar positions, i.e. 903 and 527 cm-1, however with the opposite ratio of intensity comparing to the spectrum of solution. Additionally, two intense bands at 1107 and 1086 cm-1 are present only in the spectrum of solid state, whereas a band at 1123 cm-1 characteristic for aqueous solution is not observed in solid [1].

ACCEPTED MANUSCRIPT As mentioned, a dominant form of D-(+)-xylose in aqueous solution is the β-anomer (64%) in a 4

C1 chair conformation with all OH groups in the equatorial orientation [2,3]. It has been

reported, that ROA bands, especially in the range of 750-950 cm-1, are sensitive to the absolute configuration of the anomeric carbon [3,4]. Therefore, an intensive and positive ROA band observed here at 901 cm-1 signifies the equatorial position of the anomeric OH group, as well as

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confirms the majority of the β form of xylose in aqueous solution [4]. The most intensive ROA

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bands of D-(+)-xylose are in the fingerprint region, cf. Fig. 2. The characteristic sign pattern is composed of negative at 983 cm-1, positive at 1018 cm-1, positive at 1061 cm-1, negative at

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1093 cm-1, and positive at 1129 cm-1, mainly assigned to the coupled CO and CC stretches with

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contribution of the weak OH wagging band [8,11]. Comparing to ROA spectra of other monosaccharides, the pattern in the fingerprint range of D-(+)-xylose is the most similar to D-

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(+)-glucose, since both carbohydrates are a homomorphic pair, only differences are positions of respective bands in D-(+)-glucose that are found at higher wavenumbers than for D-(+)-xylose,

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see Figs. 2 and 3. Clearly, D-(+)-xylose does not possess the exocyclic CH2OH group, thus the

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ROA ‘fingerprint’ bands are slightly shifted and probably not related with the motions of this group. Furthermore, the sign pattern in the 1200-1500 cm-1 region is also very similar to D-(+)-

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glucose, despite the lack of the CH2OH substituent at C5. It follows that the apparent ROA bands in the deformation region of D-(+)-xylose, are mainly attributed to the C-H and C-O-H

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deformation modes [11]. In contrast to the homomorphic monosaccharides, the epimers of D(+)-xylose and

L-(+)-arabinose

exhibit different ROA patterns, practically over the entire

spectrum, due to a different disposition of the C4 hydroxyl group, cf. Fig. 2. For example, D(+)-xylose exhibits three negative ROA bands in the 1200-1500 cm-1 region whereas L-(+)arabinose only one at 1270 cm-1 [3], However, some similarities can be observed in the low wavenumber region. The presence of a couplet centred at ~430 cm-1, positive in low and negative in high frequency, may be associated with an absolute configuration of the anomeric

ACCEPTED MANUSCRIPT centre [4]. Both D-(+)-xylose and L-(+)-arabinose exhibit the absolute ‘R’ configuration and share the same sign of the mentioned ROA couplet. Overall, more bands in the low wavenumber region of

D-(+)-xylose

are observed than in the spectrum of L-(+)-arabinose.

Endocyclic bending deformations of the CCO, CCC, COC and OCO groups are probably more

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involved in the case of D-(+)-xylose, see Table 2. Arabinose co-occurs with xylose in arabinoxylans - a type of hemicellulose which is present

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along with cellulose in almost all plant cell walls [12]. Interestingly, L-arabinose is one of a

arabinopyranose [14], but in aqueous solution

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few L-carbohydrates that occurs naturally [13]. The crystalline form of L-arabinose is β-LL-arabinose

predominantly exists as α- L-

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arabinopyranose [15]. This difference is reflected in the respective Raman spectra, i.e. the βanomer exhibits bands at ~845 and ~900 cm-1 [10] (and both bands are found in the spectrum of

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solid state L-arabinose [1]), when the band at 873 cm-1 in the spectrum of L-arabinose in aqueous solution results from the vibrational mode of the anomer α, Fig. 2 [10]. This band

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(coupled CO and CC stretching modes) together with the ones at 848 cm-1 (coupled CC and CO

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stretching modes of the pyranose ring) and 578 cm-1 (CO and ring deformations) are the most intensive ones in the Raman spectrum of L-(+)-arabinose, Fig. 2. [10].

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Furthermore, the ROA spectrum of L-(+)-arabinose (Fig. 2), especially the anomeric region

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shows the presence of two characteristic, opposite in sign, sets of bands. First, the positive band at 894 cm-1 indicates the predominant α form (60% in aqueous solution) with the equatorial orientation of the anomeric OH group [3,4]. Next, two negative bands at 920 and 848 cm-1 correspond with the axial position of the β anomeric OH group [4]. Moreover, there is also a low-intensity and negative band at 874 cm-1 specific for monosaccharides with the other than anomeric hydroxyl group located in the axial orientation. Here, this band results from the axial orientation of the C4-OH group in relation to the equatorial position of the anomeric group in α-pyranose with 4C1 chair conformation (Fig. 1). It is noteworthy that the ROA spectra of

ACCEPTED MANUSCRIPT homomorphic pair L-(+)-arabinose and D-(+)-galactose are not much alike as shown above for D-(+)-xylose

and D-(+)-glucose. However, some similar ROA features are recorded, Fig. 2. L-

(+)-Arabinose and D-(+)-galactose have four ROA bands at similar positions, i.e. ca. 1010 (-), ca. 1068 (-), ca. 1107 (-) and ca. 1148 (+) cm-1. A significant difference in the intensity, wavenumber and the opposite sing of the ROA couplet in L-(+)-arabinose (1270 (-) and 1306 D-(+)-galactose

(1271 (+) and 1339 (-) cm-1) probably results from the

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(+) cm-1) and

D-Ribose

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contribution of the CH2OH vibrations of galactose, Table 2 and 3 [3].

is widespread in Nature as a building block of RNA and its phosphorylated

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derivatives, such as ATP and NADH, key molecules in the cell metabolism. Despite such significance, the crystal structure of D-ribose has not been determined until 2010 [16]. In solid

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state, D-(-)-ribose exists in two crystal forms as α- and β-anomers of D-ribopyranose [1] while in aqueous solution as a mixture of α-pyranose, β-pyranose, α-furanose, and β-furanose. The

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predominance of the pyranose over furanose form has been established by NMR spectroscopy

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[15]. However, the configurational preference is confusing. In accordance with the NMR data the major component of D-(-)-ribose in aqueous solution is β-D-ribopyranose in the 4C1 chair

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conformation (41-60 %) [17–20]. Moreover, the α/β ratio is temperature-dependent [18]. For example, 20 % of α-pyranose and 56 % of β-pyranose exist at 35o C and this ratio changes to

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23%:41%, respectively, when temperature drops down to 30o C. In contrast to NMR data, theoretical calculations performed by Quesada-Moreno et al. have suggested that

D-ribose

occurs in aqueous solution mainly as α-pyranose (69%) and β-pyranose (31%), both in the 1C4 chair conformation [21]. In addition, the authors have not found features of furanose and openchain forms in IR and Raman spectra of this carbohydrate. In our study, the most prominent Raman bands, characteristic for the pyranoid ring of D-(-)-ribose, are observed at 1123 (CO stretching mode), 1082 (COH bending mode), 879 (CC stretching mode) and 546 cm-1 (CCC

ACCEPTED MANUSCRIPT bending mode), cf. Fig. 1 and Table 2 [22]. The corresponding bands of the furanoid ring are also exhibited in the Raman spectrum at 1159 (sh), 1050 (sh), 918 and 599 cm-1, respectively. Their lower intensity indicate the minor content of the furanose forms in the

D-(-)-ribose

solution. In turn, the hemiacetal character of D-(-)-ribose is manifested by bands due to the O4C1-O1 deformation modes at 679 and 653 cm-1 for the β- and α-anomer, respectively, with the D-(-)-ribose

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band of the β form showing higher intensity. The ROA spectrum of

reveals two

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negative bands at 913 and 851 cm-1 assigned to the axial orientation of the anomeric OH group and thus associated with the α anomeric orientation, Fig. 1 [4]. Next, we observe also a positive

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ROA shoulder at 891 cm-1, which indicates the equatorial orientation of the OH group, i.e. the β

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configuration at the anomeric C1. The ROA spectrum of D-ribose shows additionally a positive band at 874 cm-1, characteristic for the monosaccharides with one of the hydroxyl groups

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located in the axial orientation, but other than the anomeric one, here it is the C3 OH group. On account of the complexity produced by the ring tautomeric equilibrium, a negative ROA band

) spectra, is attributed here to furanose forms in aqueous solution [3]. Similarly to D-(+)-xylose

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at 651 cm-1, which is also observed in the D-(-)-fructose (634 cm-1) and D-(+)-sucrose (639 cm-

and L-(+)-arabinose, positive/negative couplet at 409 and 445 cm-1 likely indicates the absolute

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‘R’ configuration at the anomeric centre [4]. 2-deoxy-D-(-)-ribose structurally differs from D-ribose only by the presence of the hydrogen

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atom instead of the OH group at C2. Functionally, 2-deoxy-D-(-)-ribose is essential as a component of DNA, where it is present as β-D-deoxyribofuranose. On contrary, 2-deoxy-D-(-)ribose exists in aqueous solution as a mixture of nearly equal amounts of α- and β-pyranose forms present in their low-energy 4C1 and 1C4 chair conformations, respectively (Fig. 1) [23]. The relative proportions of the anomeric forms (α-pyranose : β-pyranose : α-furanose : βfuranose) in 20% D2O solution at 21o C is 39 : 40.5 : 11 : 8.6 [24]. It should be highlighted here

ACCEPTED MANUSCRIPT that complex composition and conformation of 2-deoxy-D-(-)-ribose in solutions has not been fully resolved so far. The Raman spectrum of 2-deoxy-D-(-)-ribose in solution displayed in Fig. 1 shows the presence of three bands attributed to the pyranoid ring, i.e. at 1142 (CO stretching mode), 1078 (COH bending mode) and 560 cm-1 (CCC deformation mode) [22]. The existence of the

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furanose forms in solution are confirmed by the equivalent bands located at 1189, 1050 and 508

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cm-1. The almost equal content of anomeric forms of 2-deoxy-D-(-)-ribopyranose could

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correspond to two Raman bands assigned to the O4-C1-O1 deformation modes at 640 and 619 cm-1 for α and β anomers, respectively. The lack of the OH group at C2 is apparent as

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modification of positions and intensities of Raman bands, especially in the region up to ~ 700 cm-1. In turn, the most dominant Raman band at 816 cm-1 in solution is assigned to the CC

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stretching mode and is also very intense in the corresponding spectrum of solid. [1]. The ROA spectrum of 2-deoxy-D-(-)-ribose is reported here for the first time (Fig. 1). The

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anomeric region reveals the presence of four bands at 859, 881, 899 and 918 cm-1 associated with orientation of the OH groups around the sugar ring. Negative ROA bands at 859, 899 and 918 cm-1 confirms the axial orientation of the anomeric OH group in α- and β-pyranose forms

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with 4C1 and 1C4 conformation, respectively [4], while a positive ROA feature at 881 cm-1

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indicates the axial orientation of the OH group at C3 also in both anomers. Similarly to D-(-)ribose, the 625 cm-1 ROA band probably expresses a furanose content in aqueous solution [3]. In total, α anomer of 2-deoxy-D-(-)-ribose exists as pyranose (40.5 %) and furanose (11 %) forms [24]. Thus one may expect that a small-intensity couplet, negative at 366 and positive at 425 cm-1 with opposite signs to other discussed here pentoses, results from the absolute ‘S’ configuration at the anomeric centre of 2-deoxy-D-(-)-ribose [4].

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ACCEPTED MANUSCRIPT

Fig. 2. Raman and ROA spectra of pentoses in aqueous solution.

ACCEPTED MANUSCRIPT 3.1.2 Hexoses D-(+)-galactose

is aldohexose monosaccharide commonly called “brain sugar” or “cerebrose”

[25]. This sugar rarely occurs free in Nature, where it is found only in small amounts in fermented milk products, fruits and wine. However, it is simultaneously one of the few sugars

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that are distributed commonly in the animal kingdom. D-(+)-galactose is not only a frequent component of several oligosaccharides, particularly raffinose and lactose (described below),

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and polysaccharides of primary cell walls like galactans and pectins [26–28], but also an

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important sugar constituent of cerebrosides, glycoproteins in brain and nerve tissues and glycolipids of mammals. In mammalian oligosaccharides, D-(+)-galactose occurs only in the

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pyranose form, however it can be found as the disfavoured furanose in some microorganisms

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[27,29]. Raman and ROA spectra of hexoses along with bands assignment are collected in Fig. 3 and Table 3.

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Preferred conformation of D-(+)-galactopyranose in aqueous solution is a 4C1 chair tautomer

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with β configuration of the anomeric OH group (64 %) (Fig. 3) [6,30]. This is reflected in Raman spectrum in solution (Fig. 3), in which the 882 cm-1 band of β-D-galactopyranose is more intense than the 829 cm-1 band characteristic for α isomers [31]. In turn, intensities of

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these bands are almost identical in spectrum of solid

D-(+)-galactose

[1]. Other prominent

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Raman bands of D-(+)-galactose in aqueous solution are found at 1466 (CH2 deformations), 1269 (CH2 twisting mode), 1068 (coupled CC and CO stretches with contributions of COH deformations) and 526 cm-1 (CCO deformations) and are also presented in the solid spectrum; only two of them are significantly shifted to 1489 and 1248 cm-1, respectively [1,32]. ROA spectrum of

D-(+)-galactose

exhibits two characteristic bands in the anomeric region

(~950-800 cm-1), which represent both anomeric configurations [4]. A positive band at 900 cm1

is associated with the equatorial orientation of the OH group at C1. A similar band is also

apparent in spectrum of β-D-methylgalactoside and this supports a high content of β anomer.

ACCEPTED MANUSCRIPT Next, a negative band at 829 cm-1 is a marker of the axial orientation of the α anomeric OH group. Moreover, the negative band at 885 cm-1 is typical for the monosaccharides with one of the hydroxyl groups (but other than the anomeric one) adopting the axial orientation (here, the axial orientation of the OH group at C4 and the equatorial for the anomeric one). In contrast to homomorphic α-arabinopyranose, the sugar ring of β-galactopyranose have an

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additional exocyclic CH2OH group, which is manifested in ROA spectrum by the intense

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positive/negative couplet at 1271 and 1339 cm-1 [3]. The strong and positive band at 1271

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cm-1 is in turn attributed to a normal mode involving the C4-O-H and C6-O-H groups [11]. A high intensity of this band results from the close proximity of the C4-OH and C6-OH groups

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in the trans-gauche rotamer. This rotamer along with the gauche-trans form is energetically preferable on contrary to gauche-gauche. The absence of the latter is confirmed by the lack of a

and

D-(+)-glucose

MA

negative band at ~1229 cm-1 as observed for D-(+)-glucose. As epimeric sugars, D-(+)-galactose reveal dramatically different ROA features, especially in the fingerprint

D

region. This observation results from the different disposition of the C4 hydroxyl group, which

D-(-)-fructose

PT E

is axial in β-D-galactopyranose and equatorial in β-D-glucopyranose. is a simple ketonic monosaccharide with the same chemical formula as D-(+)-

CE

glucose and D-(+)-galactose. Fructose is found in tree and vine fruits, flowers, berries, most

AC

root vegetables and honey. Dry fructose is very sweet and is the most water-soluble sugar [33]. It is known that only β-D-fructopyranose occurs in the crystalline form. In solution, D-fructose exists as an equilibrium mixture of β-D-fructopyranose (a dominant form) and α-Dfructofuranose with some contribution of three other forms, including acyclic structures [13]. This equilibrium is reflected in the solution Raman spectrum by the presence of a set of bands specific for β-D-fructopyranose (1149 cm-1, C-O stretches; 984 cm-1, CCH deformations; 825 cm-1, CC stretches; 523 cm-1; CCO deformations; 423 cm-1, CCC deformations) that are more intense than the corresponding bands of β-D-fructofuranose (1183, 923, 873, 708, and 460 cm-

ACCEPTED MANUSCRIPT 1

), see Fig. 3 and Table 3 [34]. The absence of bands originated from the C=O stretching

vibrations confirms the lack or a small content of the chain form. The most prominent bands in the Raman spectrum of solution at 1459, 1269, 1087/1068, and 631 cm-1 are also present in the spectrum of solid state (1472/1455, 1265, 1082, 627 cm-1, respectively) [1]; however they are slightly shifted.

PT

The ROA spectrum of D-(-)-fructose displayed in Fig. 3 exhibits characteristic bands for the

RI

arabino-type sugar. In the anomeric region, positive bands observed at 923 and 825 cm-1 are

SC

typical for axial orientation of the anomeric OH group in β-D-fructopyranose [4]. According to the NMR data, the predominant β form (68%) exists in solution as 2C5 chair conformation

NU

[11,35]. In turn, the occurrence of a negative ROA band at 881 cm-1 indicates the axial position of the OH group at C4 [4]. The fingerprint pattern is reversed relatively to the spectrum of DD-(-)-fructose

and

D-(+)-galactose

MA

(+)-galactose.

represent a nearly mirror-image fingerprint

profiles due to the same orientation of the ring OH groups, but different absolute configurations

D

at the ring carbons, except from the anomeric ones. A negative band at 1188 cm-1, appearing

PT E

exclusively for ketose sugars, is assigned to the CO stretching and H-C-O bending vibrations of the exocyclic CH2OH group attached to the anomeric carbon [4]. In addition, the negative sign

CE

of this band highlights an absolute ‘R’ configuration at the anomeric carbon.

AC

Glucose plays an important role in all animals bodies, as it serves as an immediate source of energy and a stabilizer of the blood osmotic pressure. It is also a precursor for production of glycogen and fat and constitutes a building block of complex carbohydrates such as cellulose, starch, sucrose and lactose [36].

D-glucose,

among the carbohydrates, has been the most

extensively studied by means of spectroscopic methods including IR and Raman spectroscopy [37]. This sugar exists in an equilibrium mixture of α and β anomers with domination of β-Dglucopyranose [13]. The presence of the anomeric mixture is reflected in Raman spectrum of D(+)-glucose in aqueous solution, see Fig. 3 and Table 3. Some Raman bands at 1368

ACCEPTED MANUSCRIPT cm-1 (CH2 wagging mode), 1066 cm-1 (CO stretching mode) and 521 cm-1 (CCO deformations) indicate a higher concentration of β anomer than the α one. For the latter, the most specific Raman signals are found at 850 (CC stretching mode) and 542 cm-1 (CCO deformations) [37]. In comparison to the Raman spectrum of solid D-(+)-glucose, the most intense bands at 1460, 1121, 1074, 914 and 542 cm-1 observed in the solid state are also present in aqueous solution

PT

[1].

RI

It should be highlighted that D-glucopyranose generates very weak ROA pattern between ~700-

SC

950 cm-1, cf. Fig. 3. Additionally, the prevalent β-anomeric form of D-glucose does not give ROA bands in the anomeric region [4]. Visible but weak ROA signals at 850 (-), 903 (-) and

NU

923 (+) cm-1 are attributed to the α-D-glucopyranose form and assigned to the α-anomer ring mode coupled to C-O stretches and C1-H and CH2 vibrations. In the ROA fingerprint region, D-

MA

glucose generates a negative/positive/negative/positive sign pattern at 995, 1051, 1114 and 1156 cm-1, respectively. These features are similar to the spectrum of D-xylose and are assigned

D-glucose-1-d

PT E

spectrum of deuterated

D

mainly to the CC and the endocyclic CO stretching vibrations [11]. An analysis of ROA has indicated that the C1-H bending motions also

contributes to a positive ROA band at 1156 cm-1 [11]. Vibrations of the exocyclic CH2OH

CE

group are manifested in the ROA spectrum of D-glucose by a positive/negative couplet centred around 1240 cm-1 and are markers of rotameric forms [11]. A positive band at 1264 cm-1 is

AC

characteristic for the gauche-trans rotamer in the β anomeric form while a negative band at 1229 cm-1 is typical for the gauche-gauche rotamer in both α and β anomeric forms. The 12001500 cm-1 region shows the presence of two intense ROA bands with opposite signs, i.e. (-) at 1316 cm-1 assigned mainly to the C1-H deformation mode and (+) at 1362 cm-1 originated from the C-O-H deformations.

D-xylose

exhibits a similar ROA pattern in the 1200-1500

deformation region, however bands originate from different vibrations since it does not possess the exocyclic hydroxymethyl groups. In turn, the ROA spectrum of D-galactose differs from D-

ACCEPTED MANUSCRIPT glucose nearly in the whole spectral region since both sugars have the different OH orientation at C4, i.e. equatorial in β-D-galactopuranose and axial in β-D-glucopuranose, cf. Fig. 3 [3]. It is widely known that modelling of ROA spectra of carbohydrates is difficult due to their sensitivity to complex hydration effects. Cheeseman et al. have reported calculations of Raman and ROA spectra of methyl-β-D-glucose [38]. They involved a full MD simulation of aqueous

PT

environment with ROA QM/MM computations using extracted snapshots. A single snapshot

RI

was represented by the explicitly hydrated methyl-β-D-glucose surrounded by 150 water

SC

molecules (a cut off distance of 8 Å). Using this computational approach, a very good agreement between the experiment and simulation was achieved.

NU

2-Deoxy-D-glucose is a glucose molecule which has a hydroxyl group at the C2 atom replaced

MA

by a hydrogen atom. As a structural analogue of 2-deoxyglucose it can be useful in the examination of initial reactions involved in metabolism of glucose [39]. A fluorescent analogue of 2-deoxy-D-glucose has been reported as a successful tumour targeting agent for optical

PT E

D

imaging [40]. Interestingly, the structural similarity to D-glucose is not reflected in the Raman spectrum of 2-deoxy-D-glucose presented in Fig. 3. Instead of two intense bands at 1128 and 1066 cm-1 observed for D-glucose, three less intense bands at 1139, 1100 and 1078 cm-1 are

CE

found 2-deoxy-D-glucose. These bands are assigned to coupled CC and CO stretching modes

AC

(Table 3). Other characteristic bands 2-deoxy-D-glucose are present at 539 and 494 cm-1 (coupled CCC and CCO bending modes). Interestingly, ROA profiles of D-glucose and 2-deoxy-D-glucose are similar only in the 12001500 cm-1 region originating from the presence of exocyclic CH2OH groups, see details in Fig. 3 and Table 3. Main differences correspond to the lack of the D-glucose ROA pattern in the fingerprint region, especially two intense ROA bands at 1156 and 1114 cm-1 assigned mainly to the CO stretching vibrations [11]. The anomeric region also exposes some differences. A positive/negative ROA couplet at 916 and 894 cm-1 in the spectrum of 2-deoxy-D-glucose is

ACCEPTED MANUSCRIPT much more intense and red-shifted in comparison to D-glucose. These bands are attributed to the CC and CO stretches coupled with the CH2 deformation modes and their higher intensity probably results from the presence of the additional CH2 group in 2-deoxy-D-glucose. A similar situation is found for a positive/negative couplet at 1285 and 1227 cm-1 that is the ROA signal of the CH2 deformation mode, see Table 3 [11]. The low-wavenumber region, assigned to the

PT

endo- and exocyclic CCC, CCO, COC and OCO bending deformations, also reveals few

RI

differences, for instance negative at 505 cm-1 and positive at 473 cm-1 bands of 2-deoxy-D-

AC

CE

PT E

D

MA

NU

SC

glucose are absent in ROA of D-glucose.

AC

CE

PT E

D

MA

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

Fig.3. Raman and ROA spectra of hexoses in aqueous solution.

ACCEPTED MANUSCRIPT 3.2 Disaccharides Disaccharides are built by two monosaccharide residues connected via O-glycosidic linkage with a variety of possible regioisomers since it is formed between the anomeric carbon atom of one monosaccharide unit and C1, C2, C3, C4 or C6 of another one. For example, D-maltose and D-isomaltose

are formed by two glucose units via α(1→4) and α(1→6) glycosidic linkages,

PT

respectively. Secondly, the glycosidic bond can by formed by α- or β-anomeric forms of sugar

RI

units, giving different stereoisomers as observed for D-maltose and D-cellobiose having two

SC

glucose units connected via α(1→4) and β(1→4) glycosidic linkages, respectively. Furthermore, disaccharides belong to reducing or non-reducing groups. For the former, the

NU

anomeric carbon of one residue involved in glycosidic linkage is blocked as a non-reducing residue while the anomeric carbon atom of the second residue is not blocked (a reducing

MA

residue, e.g. D-maltose) and this fact yields to equilibrium between α and β anomers due to mutarotation. Reducing disaccharides have only one reducing residue - the reducing end. In the

PT E

D

case of non-reducing disaccharides both residues are linked via anomeric carbon atoms, and consequently both anomeric carbon atoms are trapped in a single anomeric form and are nonreducing (e.g. D-sucrose). In living organisms disaccharides act as an energy source; among the

CE

most abundant in Nature, we study here D-maltose, D-lactose and D-sucrose [41–43].

AC

The backscattering Raman and ROA spectra of the disaccharides, together with their molecular structures and bands assignment, are presented in Fig. 4 and Table 4. As previously, Raman and ROA spectra of disaccharides are subdivided into four spectral regions since each of them provides different information about the disaccharide structure, Table 4. In general, Raman spectra of dissolved disaccharides are less resolved than the corresponding spectra of solids due to the presence of mixture of rotational isomers [1,44].

ACCEPTED MANUSCRIPT Only a few publications related to Raman optical activity of disaccharides can be found [41,42]. Barron and co-workers have studied a variety of aqueous solutions of disaccharides build of D-glucose units only in order to determine an effect of glyosidic link on ROA spectra. They have proposed ROA marker bands characteristic for regioisomerism as well as stereoisomerism of the glycosidic bond [41,42]. One of the most important ROA features of

D-glucose.

This couplet is positive at low- and negative at high-

RI

found in the spectrum of

PT

glucose-based disaccharides is a low wavenumber couplet observed at ca. 375-475 cm-1 and not

SC

wavenumbers for α-glycosidic linkages while a reverse signs are specific for β-linked species [41,42]. Both signals are assigned deformation vibrations around the anomeric carbon atom.

NU

One must be aware in using this couplet as a marker of isomerism for other disaccharides since it is also observed in spectra other monosaccharides and could not be simply transferred to the

MA

spectral analysis of other disaccharides [4]. Another glycosidic couplet at 900-950 cm-1 observed in glucose-based disaccharides is specific for α linkages only. The two signals are

D

positive at low- and negative at high-wavenumbers for α(1→4) links in D-maltose whereas

PT E

reverse order of signs is found for α(1→6) links in D-isomaltose. Moreover, the intensity of the 900-950 cm-1 couplet increases along with a number of glycosidic linkages, e.g. it is doubled

CE

for maltotriose and much more higher for cyclodextrins [41,42,45]. A plethora of mannosebased disaccharides have been also studied by means of ROA spectroscopy, for instance in the

AC

study of the high mannose glycan structure of glycoproteins i.e. intact yeast external invertase and ribonuclease B [46,47]. Miller and co-workers have synthesised and characterised a variety of differentially sulfated heparin-related,

D-glucosamine-L-iduronic

acid disaccharides by

means of Raman and ROA spectroscopy. They have showed that a specific sulfation-site affects conformational freedom at the disaccharide level [48]. Another work reporting Raman and ROA study combined with quantum-chemical calculations has been focused on molecular structures of benzyl and methyl glucoside, galactoside and lactoside [49].

ACCEPTED MANUSCRIPT D-(+)-maltose

is disaccharide with sweet taste (three times less than sucrose) and built of two

D-(+)-glucopyranose

moieties connected via the α(1→4) O-glycosidic linkage. It is a reducing

sugar and exists in equilibrium between α- and β-anomers (ca. 60%) in aqueous solution [50]. The Raman spectrum of D-maltose in the aqueous solution is quite similar to the spectrum of its building blocks, namely D-glucose, see Figs. 3 and 4. Only minor changes in positions and

PT

intensities of some bands are observed, mainly due to the presence of the glycosidic link in D-

RI

maltose [43]. Raman spectrum of maltose in solution is similar to the spectrum of solid state sample, cf. Fig. 3 and in [1]. In general, bands in the spectrum of dissolved D-(+)-maltose are

SC

broadened and slightly shifted with features below 600 cm-1 of lower intensity than in solid

NU

state [1,51].

The most important ROA pattern in the low-wavenumber region for D-maltose is the couplet

MA

around 400-450 cm-1, originating from the endo- and exocyclic CCC, CCO, COC and OCO bending deformations coupled with the endo- and exocyclic CO torsions and COC motions in

D

the glycosidic link, cf. Fig. 4 and Table 4. This profile is characteristic for configuration of the

PT E

glycosidic link and conformation of disaccharide composed of glucose units. In contrast to βlinked disaccharides (e.g. for

D-maltose),

this couplet exhibits positive and negative ROA

CE

bands at the low and high wavenumbers, respectively, in α-linked disaccharides. In the ~700-

AC

880 cm-1 range of the anomeric region (Fig. 4) we observe three ROA bands, (-) at 712 and 850 cm-1 and (+) at 792 cm-1 specific exclusively for α-anomer of D-glucose subunits [41]. Another important feature in the anomeric region is a positive-negative couplet (from low to high wavenumbers) centred at 921 cm-1 in the D-maltose ROA that indicates the presence of α(1→4) links. This couplet originates from the COC glycosidic link stretching vibrations and the anomeric C(1)H deformations. In the fingerprint region of the ROA spectrum associated with vibrations of the backbone structure,

D-maltose

generates similarly to

D-glucose

negative/positive/negative/positive sign patterns at 999, 1051, 1110 and 1151 cm-1,

ACCEPTED MANUSCRIPT respectively. Additionally a negative band of low-intensity at 1080 cm-1, not observed for Dglucose, is generated by motions of the glycosidic link. It is worth to stress here that β-linked glucose-based disaccharides modify significantly the ROA profile in the fingerprint region on contrary to the anomeric region. Furthermore, the ROA fingerprint region of disaccharides is not only a sum of monosaccharide units but also gives information about glycosidic links and

PT

their conformations [41]. The CH2 and COH deformation region (~1200-1500 cm-1) of

D-

RI

maltose shows a similar spectral profile to D-glucose, cf. Figs. 3 and 4. A positive ROA band at

SC

1256 cm-1 is a marker for β-anomers and its intensity correlates well with this anomeric population. From this reason an intensity of that ROA band for D-maltose is lower than for D-

NU

glucose because of a lower total content of β-anomers in the D-maltose solution. As described above, a non-reducing residue is blocked in the α-anomeric form whereas the reducing residue

MA

has the same proportion of β-anomers as in D-glucose (~64%). The ~1300-1500 cm-1 region of Raman and ROA spectra of β-linked disaccharides (D-cellobiose and D-gentiobiose) is almost

D

identical to the D-glucose spectra due to the predominance of β-anomeric forms in solutions of

PT E

these compounds. In the case of α-linked D-maltose, a Raman band at ~1342 cm-1 increases its intensity in comparison to the D-glucose spectrum and its ROA profile slightly changes [41,42].

CE

β-D-(+)-Galactopyranose and

D-(+)-glucose

connected via the β(1→4) O-glycosidic linkage

AC

constitute D-(+)-lactose. It is a reducing sugar and its equilibrium mixture in aqueous solution consists of α and β anomers with a slight predominance of β-lactose (57.3 - 62.3%) [50]. Most of Raman bands detected in solid state are observed in solution, except two strong bands at 398 and 378 cm-1 that are characteristic for α-lactose in the solid state [1,44]. As lactose is composed of two different monosaccharides, its ROA spectrum supposed to be more complex than maltose spectrum, but surprisingly it is not, see Fig. 4. The comparison of ROA spectra of disaccharides studied here indicates that D-lactose ROA profile exhibits low-intensity bands below 600 cm-1. In this region, we found two most prominent negative features at 350 and 378

ACCEPTED MANUSCRIPT cm-1, present also in the galactose spectrum (Fig. 3), which are attributed to the CCOH, COHO torsions and glycosidic CCO deformations; as well negative (434 cm-1) and positive (410 cm-1) bands originating from the CCC, CCO, COC and OCO deformations, Table 4 [52]. The 600800 cm1 range of the anomeric region is not prominent, similarly to the ROA spectrum of galactose. Only weak bands at 629(-) and 704(+) cm-1 are found. In the 800-950 cm-1 range of

PT

the anomeric region a strong negative-positive couplet (from low to high wavenumbers) with

RI

maxima at 884 and 903 cm-1, and weak bands at 923(+) and 950(-) cm-1 are present. The couplet is also observed in ROA spectra of galactose (with lower intensity than for lactose) and

SC

glucose (slightly shifted to higher wavenumbers). The negative band at 884 cm-1 could be

NU

related with the presence of β-D-galactose anomer in D-lactose structure, since a similar feature has been observed in methyl-β-D-galactoside [3]. The corresponding Raman bands in this

MA

region of the lactose spectrum are assigned to the COC glycosidic link stretching, CC stretching and CH2 twisting vibrations, cf. Table 4 [52]. In comparison to the ROA spectrum of

D

galactose, a strong and negative band at 829 cm-1 is absent in the lactose spectrum since this

PT E

band is associated with vibrations of α-D-galactopyranose. Next, the fingerprint region is dominated by a positive/negative/negative/negative/positive sign pattern located at 986, 1024,

CE

1069, 1117 and 1161 cm-1. Three negative and one positive features are also observed in ROA of galactose. The CH2 and COH deformation region (~1200-1500 cm-1) shows vibrations of the

AC

monosaccharide units and its profile is almost superposition of ROA spectra of glucose and galactose. The only noticeable difference is a negative band located at 1412 cm-1, observed also in the D-(+)-cellobiose spectrum (not shown here), that could by associated with the β(1→4) linkage [41]. D-(+)-sucrose

is disaccharide consisted of α-D-(+)-glucopyranose and β-D-(-)-fructofuranose

linked together via α(1→2)β O-glycosidic linkage. It is a non-reducing sugar as both anomeric carbon atoms are blocked by the glycosidic linkage at the single anomeric form. Almost all

ACCEPTED MANUSCRIPT prominent Raman bands of solid sucrose are also present in the spectrum of aqueous solution however their shape, position and relative intensities considerably change due to dissolving, see Fig. 4 and [1]. As sucrose is composed of two different monosaccharides, its ROA spectrum becomes more

PT

complex than for other disaccharides studied in this work. Furthermore, blocked anomeric carbon atoms of both monosaccharide units, as well as the furanose form of the fructose unit

RI

(that in the aqueous solution occurs mainly as a pyranose form: 68%), have a significant

SC

influence on Raman and ROA spectra of sucrose, i.e. obtained spectra are not a superposition of the corresponding monosaccharide units spectra, that was observed, with a few exceptions,

NU

for maltose and lactose. Only small parts of the whole spectrum are similar to the constituent

MA

monosaccharide spectra, or their superposition. The low wavenumber region is definitely more rich in comparison to the ROA spectra of other disaccharides shown here. It is worth to stress that the ROA pattern in this region shows bands with reverse signs comparing to D-fructose, in

D

particular bands at 391, 425, 451, 534, and 590 cm-1, cf. Figs. 3 and 4. Moreover, a positive-

PT E

negative couplet centred at 406 cm-1 is also observed for ketose monosaccharides and glucosebased disaccharides and is sensitive to configuration at the anomeric centre [4]. Free fructose

CE

that occurs in aqueous solution mainly as the β-pyranose form with the R configuration,

AC

exhibits the opposite sign of this couplet comparing to D-sucrose because its fructose unit is as β-furanose with the S configuration. The anomeric region is also very rich in structural information. A negative band at 639 cm-1 is associated with vibrations of the furanose ring since it is present in ROA spectra of other furanoses such as D-fructose, D-ribose and D-talose [3]. A Raman band at 745 cm-1 is assigned to the glucose ring deformations [53]. Since, this band is not observed in the ROA spectra neither for D-fructose nor D-glucose, one can assume that it could be associated also with the vibrations of the glycosidic link. In the contrary to the methyl-β-D-glucoside, this positive feature is observed also for the methyl-α-D-glucoside,

ACCEPTED MANUSCRIPT therefore it could be characteristic for α-D-glucose, that forms

D-sucrose

[3]. The ROA

spectrum of D-sucrose between 850 and 950 cm-1 is very similar to that observed for D-fructose, reflecting that this region does not distinguish furanose and pyranose form of

D-fructose,

however it is sensitive to the anomeric configuration [4]. The fingerprint region is dominated by negative/positive/negative/negative/positive patterns located at the 1020, 1051, 1071, 1124,

PT

and 1144 cm-1, respectively. Only two positive bands (1051 and 1144 cm-1) have the same sign

RI

as the corresponding signals of D-fructose. Two above-mentioned D-sucrose bands have also the same sign for D-glucose, as well as features at 1124 and 996 cm-1. Another interesting

SC

observation is a lack of the strong negative 1188 cm-1 band, observed in the

D-fructose

NU

spectrum assigned to the CO stretching and HCO bending vibrations of the exocyclic CH2OH group bonded to the anomeric carbon. In general, this band is a characteristic ketose marker,

MA

and similarly to the couplet at ~400 cm-1, is sensitive to the absolute configuration of the anomeric centre of ketose monosaccharides [4]. Since this band is not prominent in the

D-

D

sucrose spectrum, we propose assigning it to the pyranose form. One of the features observed

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in the CH2 and COH deformation region is a positive band at 1277 cm-1 that is also observed in the D-glucose and D-fructose ROA. Since the β anomeric form of the D-glucose generates a

CE

positive signal at ~1260 cm-1 that is absent in sucrose this positive 1277 cm-1 band could be assigned to the pyranose form of the D-fructose unit [3,11]. Another positive band at 1321 cm-1

AC

very likely corresponds to the C1-H bending mode of the D-glucose unit as was identified with opposite sign in ROA spectrum of D-glucose [11]. The ~1350-1500 cm-1 region of the ROA spectrum, assigned to the CH2 and COH deformation vibrations, shows the presence positive/negative/positive/negative patterns at 1375, 1406, 1455, 1474 cm-1, respectively, and is almost a perfect sum of spectra D-glucose and D-fructose, see Figs. 3 and 4.

ACCEPTED MANUSCRIPT 3.3 Trisaccharides Trisaccharides are oligosaccharides composed of three monosaccharides that are linked to each other by a variety of O-glycosidic links giving possibility to form various regio- and stereoisomers. As for disaccharides, trisaccharides form reducing as well as non-reducing

PT

carbohydrates which depends on the type of O-glycosidic linkages. Trisaccharides are mainly found in higher plants. Up to now, a few trisaccharides have been examined by means of ROA D-maltotriose

and a variety of mannose-based trisaccharides constituting

RI

spectroscopy, e.g.

D-(+)-

SC

high mannose glycoproteins [2,46]. Here we present Raman and ROA spectra of

D-(+)-raffinose

NU

raffinose in aqueous solution. ROA spectra are presented for the first time. contains α-D-galactose, α-D-glucose and β-D-fructose connected via α(1→6)

MA

and α(1→2)β O-glycosidic linkages, respectively. Because all of the anomeric centres are blocked by the glycosidic linkages, D-raffinose is a non-reducing sugar. All of the prominent

D

Raman bands measured in solid state are present in aqueous solution of D-raffinose (Fig. 4), D-raffinose

is

PT E

however, some differences are found below 700 cm-1[1]. ROA spectrum of

almost a sum of spectra of D-sucrose and D-galactose units. Major differences are observed in

CE

the region between 800 and 1000 cm-1, assigned to vibrations associated with the glycosidic linkages. The low wavenumber region, dominated by bands at 395 (+), 440 (-), 507 (-), 534 (+)

AC

and 592 cm-1 (+), as well as the anomeric region resemble to a large extent the corresponding regions of the D-sucrose spectrum. A broad and intense couplet at 828 (-) and 889 (+) cm-1 is observed in the higher range of the anomeric region in the D-raffinose spectrum. A 828 cm-1 signal likely highlights the presence of α-D-galactose because a similar ROA feature is found for methyl-α-D-galactoside and D-lactose containing the β-D-galactose unit [3]. However the bands of the latter are positive [3]. This couplet also appears in D-isomaltose spectrum (not shown here) that also possesses the α(1→6) glycosidic linkage but between two D-glucose units [41]. The fingerprint region of

D-raffinose

is also very similar to that observed for the

D-

ACCEPTED MANUSCRIPT sucrose and D-galactose spectra, except a positive band at 1107 cm-1 which is opposite in sign to

D-galactose.

The ROA region between 1130 and 1500 cm-1 of

D-raffinose

AC

CE

PT E

D

MA

NU

SC

RI

PT

superposition of the spectra of D-sucrose and D-galactose units, cf. Figs. 3 and 4.

is simply a

AC

CE

PT E

D

MA

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

Fig.4. Raman and ROA spectra of di- and trisaccharides in aqueous solution.

ACCEPTED MANUSCRIPT 3.4 Cyclodextrins Cyclodextrins (CDs) are cyclic, non-reducing oligosaccharides, built by several

D-glucose

units, connected via α(1→4) O-glycosidic links. The most common CDs are α-, β- and γ-CD, containing in their structure six, seven and eight D-glucose units, respectively [54]. The three

PT

dimensional torus of CDs structure, including hydrophobic inside and hydrophilic outside, enables the formation of inclusion complexes with a wide range of organic compounds. From

RI

this reason CDs are designed as drug delivery systems improving bioavailability of

SC

pharmaceuticals [55]. The secondary hydroxyl groups are situated in the wider cavity whereas the primary groups are present in the narrower cavity of the CDs conical cylinder. The C(2)-

NU

OH group of one glucose unit links via H-bonds to the C(3)-OH group of the neighbouring glucose leading to the formation of a complete intramolecular H-bonding system for β-CD

MA

(rigid and less soluble in water) or an incomplete system for α-CD (more soluble). The structure of γ-CD is more flexible and is the most soluble species from the three cyclodextrins

D

mentioned above [54]. These three CDs, methylated derivatives of β-CD, inclusion complexes

PT E

as well as α-CDs bridged/interconnected by disulfide bridges have been already elucidated by means of ROA spectroscopy [2,45,56–58]. Recently, the α-CD structure has been studied by

CE

MIM-ROA (Molecules-in-Molecules) computational methodology [59]. The most significant

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ROA feature of CDs spectra is a positive-negative anomeric couplet, observed around 890-960 cm-1 with the CID value bigger by an order of magnitude than for other ROA bands [2,45]. Intensity of this couplet (absent in ROA of D-glucose) increases with a number of the α(1→4) O-glycosidic links in oligosaccharide structures. For instance, intensity of this couplet for Dmaltotriose is twice as for D-maltose [2,41,45]. In this work, we discuss in detail Raman and ROA features of γ-D-cyclodextrin in aqueous solution to show a general spectral characteristic of this group of carbohydrates. The spectra are shown in Fig. 5 while the assignment of bands to vibrational modes are summarised in Table 5.

ACCEPTED MANUSCRIPT γ-D-cyclodextrin (γ-CD) is an cyclic oligosaccharide build by eight α-D-(+)-glucopyranose units connected via α(1→4) O-glycosidic links. In general, ROA features of γ-CD are less structured and broader in comparison to β-CD due to the flexibility of γ-CDs ring [45]. The most important Raman feature in the low wavenumber region is a sharp and very intense band at 483 cm-1 which is not observed for glucose and associated with the skeletal and α(1→4)

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glycosidic link vibrations. This feature is also specific for other oligo- and polysaccharides

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linked by the 1→4 glycosidic bonds (e.g. maltodextrin, amylose, amylopectin, glycogen) [1,60,61]. On contrary to maltose and other α-linked oligosaccharides and polysaccharides, the

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ROA spectrum of γ-CD does not possess a glycosidic couplet at ca. 400-450 cm-1 but it is

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shifted to lower wavenumbers, and centred at 379 and 358 cm-1 [41]. In the anomeric region one can find negative/positive/negative features at 709, 783 and 853 cm-1, respectively,

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characteristic for α-anomeric configuration, with an additional negative band at 760 cm-1. The most intense ROA band in the spectrum is a negative/positive couplet, centred at 929 cm-1 that

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is associated with modes related to the glycosidic COC stretching and the anomeric C(1)-H

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deformation vibrations [45]. Intensity and position of this couplet depend on temperature, methylation, solvent and complexation, reflecting a conformational flexibility around the

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glycosidic bonds in CDs [56]. The fingerprint region attributed to the CO and CC stretching as well as to the COH and CCH bending vibrations shows similar ROA features like for D-glucose

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and D-maltose, Figs. 3, 4 and Tables 3, 4. The same situation is found in the 1200-1500 cm-1 region, where a negative/positive couplet centred at 1349 cm-1, assigned to the CH2OH deformations is observed [2,41,45]. The couplet in the spectrum of γ-CD is broader and much less structured than for the β-CD and D-maltose, while similar to the one observed in the Dmaltotriose spectrum, reflecting higher conformational flexibility for γ-CD [45]. Furthermore, the lack of a positive band at 1256 cm-1 found previously for glucose and maltose c the absence of β-anomers in structure of γ-CD.

ACCEPTED MANUSCRIPT 3.5 Polysaccharides Polysaccharides are macromolecules that consist of the large number of monosaccharide units connected via glycosidic linkages. Homopolysaccharides are composed of one type of monosaccharide (i.e. glycogen, cellulose) while heteropolysaccharides are built of two or more

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different kinds of monosaccharides or their derivatives (i.e. heparin, chondroitin sulphate) [62]. Broad structure of bands in Raman spectra of solids is not affected by dissolving

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polysaccharides in water [1].

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Reported so far ROA studies of polysaccharides, glycosaminoglycans and glycoproteins have

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showed a great potential of this technique in the complex analysis of their secondary and tertiary structures [46–48,63–67]. As an example we present here Raman and ROA spectra of

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glycogen in aqueous solution, cf. Fig. 5 and Table 5.

Glycogen is a multi-branched polysaccharide built of α-D-(+)-glucopyranose units only.

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Glycogen is composed of linear chains of D-glucose connected via α(1→4) O-glycosidic links

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and branches bonded to the chains by α(1→6) O-glycosidic linkages. Branching off in glycogen is observed in every 8 to 12 glucose units in chains, so it is much more branched than

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other polysaccharides, i.e. amylopectin. Glycogen is the energy storage polysaccharide, found in liver and muscles of mammals. It is synthesized in liver from D-glucose units and provided

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with a diet [68]. In contrary to monosaccharides (e.g. D-glucose), Raman spectra of solid and dissolved glycogen are almost identical, see Fig. 5 and in [1]. Similarly to the CDs spectrum, the most prominent Raman feature in the low wavenumber region is a band at 482 cm-1, associated with the skeletal and α(1→4) glycosidic links vibrations. In the corresponding region of ROA spectra, a positive/negative couplet at 407 and 439 cm-1, respectively, is found and is characteristic for α-linked saccharides containing glucose units only. In the anomeric region, negative/positive/negative features at 712, 796 and 862 cm-1, respectively, indicate α-

ACCEPTED MANUSCRIPT anomeric configuration of glycogen [41]. Next, the most intense ROA feature, a negative/positive couplet centred at 918 cm-1 is associated with modes related to the glycosidic COC stretching and the anomeric C(1)-H deformations of α(1→4) glycosidic links. [41]. Surprisingly, the β anomer ROA marker at 1253 cm-1 (+) appears in the glycogen spectrum. Since only one glycogen molecule over 100 000 glucose monomers connected via α linkages

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contains the reducing end in either α or β configuration, this observation could indicate a high

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sensitivity of ROA for the detection of β anomeric species [41]. In general, the ROA spectrum

D-glucose

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of glycogen reveals the presence of most features found in spectra of other α(1→4) linked and based oligosaccharides (e.g. maltose) [41,63]. Minor differences are probably related

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with α(1→6) O-glycosidic linkages in the structure of glycogen. Nonetheless, Raman spectra of aqueous solution of γ-CD and glycogen are almost identical (Fig. 5), whereas ROA reflects

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many differences, showing much greater potential in the structural studies of carbohydrates.

ACCEPTED MANUSCRIPT Fig.5. Raman and ROA spectra of γ-D-cyclodextrin and glycogen in aqueous solution.

4. Conclusions Raman spectra of carbohydrates differ slightly depending on the phase what is mostly related

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with the fact that usually more anomers and tautomers of carbohydrates appear in the aqueous

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solution than in the solid state. The additional dimension, i.e. sign, significantly increases the

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complexity of spectra, therefore ROA spectra, particularly for carbohydrates that exist in the aqueous solution in complicated anomeric, tautomeric and conformational equilibria, are

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considerably more difficult to analyse compared to the Raman ones. Nevertheless, due both the intense ROA signal of carbohydrates and a plethora of structural information, coded in their

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signatures, first ROA spectra of carbohydrates were recorded quite early, i.e. in the nineties by Barron and co-workers [2–4,11,41,42]. These very important pioneer works enabled to find the

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marker regions that could be correlated with the various structural features of carbohydrates.

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Therefore, the low wavenumber region (250-600 cm-1); anomeric region (600-950 cm-1); fingerprint region (950-1200 cm-1) and CH2 and COH deformations region (1200-1500 cm-1)

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contain the information about the absolute configuration of the anomeric centre; the configuration of the anomeric centre and the orientation of the anomeric hydroxyl group; the

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ring structures and the relative orientation of substituents; and the conformation of the exocyclic CH2OH, respectively. The low wavenumber region is quite interesting due to correlation of the sign of the couplet around 410-420 cm-1 and the configuration of the anomeric centre of the dominating anomer. It was proposed by Bell et al [4] for ketose monosaccharides, but it was not verified for other carbohydrates. In this work, in a careful comparison of spectra of several carbohydrates we demonstrate that this rule is quite universal, but not without exceptions. For all studied here pentoses and some of disaccharides, the sign of

ACCEPTED MANUSCRIPT the couplet is in agreement with the configuration at the anomeric centre according the our observation that when the negative band of the couplet (at ~ 420-430 cm-1) is at the higher wavenumber than the positive, the configuration of the anomeric centre is R and vice versa. This observation disobeys somehow for some hexoses, i.e. is in disagreement for D-fructose and is verifiable for D-glucose and its 2-deoxy derivative, as both the latter possess a broad

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band at ca. 340 cm-1 that influences the shape of the couplet at 420 cm-1. For β-D-maltose and

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β-D-lactose, though, for which β-D-glucopyranose adopts predominantly R anomeric

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configuration and the broad band at ca. 340 cm-1 is not present, the sign of the couplet follows the observation. The sign of the couplet might be considered as an further information on the

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configuration at the anomeric centre, in addition to signals in anomeric region and, in particular, in the 800-950 cm-1 range that provides also indications about the axial and

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equatorial orientations of the hydroxyl groups at the anomeric centre [4]. For all studied monosaccharides, the presence of two forms, i.e. with the axial and equatorial orientations of

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the hydroxyl groups can be demonstrated based on this region. For di- and trisaccharides the

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anomeric region changes considerably compared to monosaccharides and concluding about the orientation around the anomeric centre becomes less obvious. Two other ranges, i.e. fingerprint

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(950-1200 cm-1) and CH2 and COH deformations (1200-1500 cm-1) regions are much more difficult to generalize. Further studies, possibly based on quantum-chemical calculations, may

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shed more light on assignments of signals in these ranges and enhance analytical capabilities of ROA of carbohydrates. Nevertheless, already at the moment, this spectroscopy emerges as a unique tool to study structures of this group of compounds.

Acknowledgements This work was supported by National Science Center (grants: DEC-2013/08/A/ST4/00308 and UMO-2017/25/B/ST4/00854).

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Johannessen, V.P. Shastri, S. Lüdeke, Unravelling a Direct Role for Polysaccharide βStrands in the Higher Order Structure of Physical Hydrogels, Angew. Chemie Int. Ed. 56 (2017) 4603–4607. doi:10.1002/anie.201701019. [66] A.F. Bell, S.J. Ford, L. Hecht, G. Wilson, L.D. Barron, Vibrational Raman optical activity of glycoproteins, Int. J. Biol. Macromol. 16 (1994) 277–278. doi:https://doi.org/10.1016/0141-8130(94)90033-7. [67] F. Zhu, N.W. Isaacs, L. Hecht, L.D. Barron, Polypeptide and Carbohydrate Structure of

ACCEPTED MANUSCRIPT an Intact Glycoprotein from Raman Optical Activity, J. Am. Chem. Soc. 127 (2005) 6142–6143. doi:10.1021/ja051048l. [68] D.J. Manners, Recent developments in our understanding of glycogen structure, Carbohydr. Polym. 16 (1991) 37–82. doi:10.1016/0144-8617(91)90071-J.

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[69] M. Mathlouthi, A.M. Seuvre, J.L. Koenig, F.T.-I.R. and laser-raman spectra of d-ribose and 2-deoxy-d-erythro-pentose (“2-deoxy-d-ribose”), Carbohydr. Res. 122 (1983) 31–

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47. doi:10.1016/0008-6215(83)88404-3.

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[70] J.L.K. P.D.Vasko, J.Blackwell, Infrared and raman spectroscopy of carbohydrates. : Part

doi:10.1016/S0008-6215(00)82690-7.

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II: Normal coordinate analysis of α-D-glucose., Carbohydr. Res. 23 (1972) 407–416.

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[71] M. Mathlouthi, J.L. Koenig, Vibrational Spectra of Carbohydrates, Adv. Carbohydr. Chem. Biochem. 44 (1987) 7–89. doi:http://dx.doi.org/10.1016/S0065-2318(08)60077-3.

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[72] H.A. Wells, R.H. Atalla, An investigation of the vibrational spectra of glucose, galactose

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and mannose, J. Mol. Struct. 224 (1990) 385–424. doi:http://dx.doi.org/10.1016/0022-

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2860(90)87031-R.

ACCEPTED MANUSCRIPT Table 2. Raman (RS) and ROA bands assigned for aqueous solution of selected monosaccharides (pentose): D-(+)-xylose, L-(-)-arabinose,, D-(-)-ribose and 2-deoxy-D(-)-ribose. Molecular structure and vibrational spectra are shown in Fig. 2 [1,4,8,69]. D-(+)-xylose

L-(-)-arabinose

D-(-)-ribose

2-deoxy-D-(-)-ribose

ROA

RS

ROA

RS

ROA

RS

ROA

1470 (m)

+1466

1457 (m)

+1460 (w)

1465 (s)

+1458 (w)

1457 (s)

+1433 (w)

1386 (m)

-1360 (w)

1360 (m)

1376( m)

+1382 (w)

1319 (m)

1324 (s)

+1306 (w) +1294 (sh)

-1246 (m)

1265 (s)

-1270 (w)

1212 (w) +1221 (w)

1144 (s)

+1148 (w)

+1129 (s)

1071 (s)

+1018 (s)

1009 (s)

-983 (m)

948 (w)

ν(CC)

-1156 (w)

ν(CO)

+1120 (m)

ν(CH)

1082 (s)

-1070 (m)

1078 (s)

-1097 (w)

1050 (sh)

+1049 (s)

1050 (m)

+1075 (m)

1011 (m)

-1010 (s)

1009 (w)

+1013 (s)

970 (w)

+967 (s)

984 (w)1

+984 (s)

δ(COH) δ(OCH)

-1010 (s)

+952 (w)

60% of β-pyranose , 4C1

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64% of β-pyranose, 4C1

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1021 (w)

δ(CCH)

943 (w)

+940 (w)

904 (s)

-920 (vw)

-920 (w) β

918 (w)

-913 (w) α

897 (m)1

+894 (m) α

879 (s)

+891 (sh) β

868 (m)

-874 (w)

834 (w)

+874 (m)

816 (s)

-848 (m) β

802 (m)

-851 (m) α

754 (m)

+789 (m)

727 (w)

-805 (w)

720 (m)

+639 (w)

679 (w)

+756 (w)

640 (w)

+605 (w)

653 (w)

-651 (m)

619 (w)

-576 (w)

599 (w)

-918 (sh) α

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788 (w) 699 (w)

+605 (m)

1

δ(CCH)

α- and β-pyranose

848 (s)

-765 (w)

δ(OCH)

40% of each

873 (s)

+901 (m) β

1

~ 60% of β-pyranose, 4C1

921 (w)

640 (m)

wavenumber

-1190 (w)

-1068 (m) +1061 (w)

τ(C-5-H2)

1142 (w)

-1109 (m)

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1092 (s)

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the fingerprint region

-1093 (vs) 1073 (m)

605 (w)

1

1222 (w)

1189 (w)

+1132 (s)

1123 (s) -1107 (m)

763 (vw)

τ(CH2)

+1270 (w) -

def. (CH2)

+1164 (m)

1159 (sh)

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1123 (s)

816 (vw)

δ(CH) -1304 (w)

+1264 (s) +1205 (sh)

984 (w)

1269 (s)1

1269 (s)*

ω(CH2)

1324 (m)

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1251 (w)

+1170 (w)

the anomeric region

-1326 (w)

-1324 (m)

+1297 (w)

ω(C-2-H2) +1338 (w)

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+1357 (m)

+1116 (w)

-899 (s) α

δ(CH)

+881 (w)

ν(C-C)

-859 (m) α

δ(CCO)

+818 (w)

δ(OCO)

-799 (w)

+593 (m)

539 (s)

region

the low

δ(CH2)

-1387 (w) 1372 (m)

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the CH2 and COH deformations region

Assigment RS

513 (w)

-539 (m)

578 (s) 512

560 (w)

563 (vw)

δ(CCC)

508 (w)

509 (vw)

ring def.

+552 (w) -510 (m)

(m)

-508 (w)

546 (s) +509 (m)

ACCEPTED MANUSCRIPT 469 (w)

-471 (w)

-435 (w) 435 (m)

419 (m)

-437 (w)

+338 (w)

+405 (m)

462 (w)

-445 (m)

+400 (w) 398 (w)

+425 (w)

δ(COC)

-366 (w)

OH twist

419 (w) 416 (m)

+409 (vw)

+332 (w)

Key: s – strong, m – medium, w – weak, sh – shoulder, ν - stretching, δ – bending, ω - wagging, τ - twisting, def. – deformation, α anomer,

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β anomer.

ACCEPTED MANUSCRIPT Table 3. Raman (RS) and ROA bands assigned for aqueous solution of selected monosaccharides (hexoses): D-(-)-fructose, D-(+)-glucose, 2deoxy-D-(+)-glucose. Molecular structure and vibrational spectra are presented in Fig. 3 [1,11,34,70,71] D-(+)-galactose

D-(-)-fructose

D-(+)-glucose

2-deoxy-D-(+)-glucose Assigment

RS

ROA

RS

1466 (m)

+ 1463 (w)

1459 (s)

ROA

RS

ROA

RS

1461 (m)

+1464 (w)

1461 (m)

+1386 (w)

-1426 (w)

δ(CH)

1338 (m)

-1339 (m)

+1386 (w)

1368 (m) β

+1362 (s)

1374 (s)

+1360 (m)

+1350 (w)

1333 (sh)

-1316(m)1

1331 (m)

-1321 (w)

τ(CH2)

+1271 (s)

1269 (s)

+1264 (m)1

1206 (w)

-1229 (m)1

+1270 (s)

+1129 (m)

1183 (w)

-1108 (m)

1149 (w)1

1149 (m)

+1143 (w)

1082 (s)

+1107 (m) -1081 (s)

1087 (s)

+1046 (w)

1068 (s)

1068 (s)

+1083 (m)

1021 (m)

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984 (m) 2

973 (w) -954 (m) 1

923 (w)

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954 (w)

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-973 (m)

70% of β-pyranose, 2C5

882 (s)

873 (s)

825 (s) 3

1060 (s) β 1022 (sh)

AC +776 (w)

1

def. (CH2)

-1227 (m)* and (COH) +1156 (w) +1129 (w) δ(COH)

1194 (w)

-1098 (m)

1139 (s)

-1085 (m)

ν(CO) 1

-1114 (s) 1100 (s)

+1071 (w)

1078 (s)

+1056 (w)

ν(CO) pyr ν(CC)

+1051 (m) -1034 (w) +1032 (w) -1015 (w)

72% of β-pyranose, 4C1

β-pyranose, 4C1

-995 (s) δ(COH)

1

+976 (m) +923 (w) α 984 (m) 918 (m) β

+954 (m)

δ(CCH) pyr

2

-903 (w) α

903 (sh)

-850 (w) α

850 (m) α

*β form

-881 (s) +825 (m) β

-829 (m) α

708 (w)

τ(CH2)

+1285 (s)*

+1156 (m)

+923 (m) β

-885 (w) 829 (m)

1128 (s)

+984 (s)

+900 (m) β

def. (C1-H)

+1200 (w)

+1017 (m) -1021 (s)

1

1269 (m)

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-1188 (s)

64% of β-pyranose, 4C1

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1269 (s)

1265 (w)

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1372 (w)

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the fingerprint region

δ(CH2)

ω(CH2) 1372 (m)

+1150 (m)

the anomeric region

+1467 (w)

-1415 (w)

SC

the CH2 and COH deformations region

+1459 (w)

ROA

3

ν(CC) pyr

913 (m)

+916 (s)

850 (m)

-894 (s)

δ(CH)

829 (m)

+857 (m)

ν(CC)

-842 (w)

ν(C1-H)

783 (w)

-784 (w)

δ(OCO)

670 (w)

+680 (w)

δ(CCO)

628 (w)

+639 (w)

don’t give ROA -784 (w)

786 (w)

774 (w) -709 (w)

670 (w)

+702 (w)

708 (m)

713 (w)

-712 (w)

- 643 (w) 613 (w)

-616 (w)

631 (s)1

646 (w)

1

δ(CCO)exo

590 (w) region

wavenumber

the low

+607 (w)

+560 (w)

-582(w) 521(m) β

524 (m)1

526 (s) -525 (w)

+595(w)

-522(w)

δ(CCO)

-595 (w) 539 (s)

-500(w)

-505 (m) 494 (s)

1

δ(CCO) pyr

ACCEPTED MANUSCRIPT +473 (w)

δ(CCC)

444 (m)

-444 (m)

δ(CCC) pyr

394 (w)

-396 (w)

δ(CCO)endo

+335 (w)

δ(COC)

+461 (w) 467 (w)

460 (w) -466 (w)

428 (m)

-452 (w)

423 (m)

-406 (w)

352 (w)

+350 (w)

+429 (w) 423 (m)*

+400 (w) 348 (m)

448 (m)

-396 (w) 332 (w) -330 (w)

Key: s – strong, m – medium, w – weak, sh – shoulder, ν - stretching, δ – bending, ω – wagging, τ – twisting, def. – deformation, pyr – pyranose,

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α anomer, β anomer, endo – endocyclic CH2OH group, exo – endocyclic CH2OH group.

ACCEPTED MANUSCRIPT Table 4. Raman (RS) and ROA bands assigned for aqueous solution of selected disaccharides: D-(+)-maltose, D-(+)-sucrose, D-(+)-lactose and D(+)-raffinose (trisaccharide). Molecular structure and vibrational spectra are shown in Fig. 4 [1,3,32,37,41,42,53,71,72]. D-(+)-maltose

D-(+)-lactose

D-(+)-sucrose

D-(+)-raffinose

Assigment RS

ROA

RS

ROA

RS

ROA

RS

ROA

1461 (s)

+1458 (w)

-1474 (w) δ(CH2)

1461 (m)

+1464 (w)

1465 (m)

1459 (s)

* g.l. β(1-4)

-1412 (w)* -1406 (w)

ω(CH2) 1377 (s)

+1375 (s) 1374 (m)

+1367 (s)

1368 (s)

-1340 (m) 1342 (s) 1

1343 (m) 1

-1331 (m)

1336 (s) 1

1212 (w)

-1222 (w)

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1269 (m)

-1214 (w)

+1151 (m)

1149 (sh) -1139 (w) 1123 (s)1

1081 (s)

1087 (s)

-1024 (s)

-937 (m) 2

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1027 (sh) -999 (m)

1133 (s)

+1144 (s)

δ(COH) 1139 (s)

-1137 (w) 1

v(CO) Glc

2

v(CO) Gal

+1124 (sh)

-1071 (s)

δ(COH)

-1073 (s)

+1051 (m)

1082 (s)

+1051 (m)

-1020 (m)

982 (w) 2

-1020 (s)

δ(COH) Glc

1

1071 (s) ν(CO)exo

+986 (w) -996 (w)

939 (w) +923 (m)

854 (m)

-850 (m) α

884 (m)

+903 (w)

840 (s)

-876 (m)

CE

927 (w)

-884 (s) β

+704 (w) -629 (w)

2

δ(COH)

-923 (sh) 2

ν(COC) g.l.

877 (m)

+886 (m)

836 (s)

-828 (s) α

ν(CC)

-794 (sh)

τ(CH2)

+746 (m) α

749 (m)

+726 (m)

+693 (w)

702 (w)

-655 (w)

-639 (s) 2

640 (w)

-639 (m)

745 (w) 1

711 (w) +633 (w)

δ(COH) Gal

1

-843 (m)

777 (w)

-712 (w) α

δ(CCH) Fru

2

-947 (w)

+923 (w)

+792 (w) α

-978 (s) -952 (sh)

-950 (w)

952 (w) 1

AC

ν(CO)endo +1151 (m)

+1107 (m)2

+910 (m) 2

717 (w)

-1170 (w)

-1124 (m)

923 (m)

765 (w)

τ(CH2) -1224 (w)

-1069 (s)

+1051 (m) 1044 (w) 1

+1277 (s)

1270 (m)

-1170 (w)

-1117 (m)

-1080 (m)

δ(C1-H) Glc

2

-1212 (w)

MA

-1110 (s)

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the fingerprint region

1126 (s)1

+1243 (m)

NU

+1161 (w)

τ(CH2) Glc

1

+1324 (m)

+1277 (s) β

+1256 (w) β 1265(m)

r(CH2)

-1345 (w)

1347 (s) 1 +1321 (s) 2

+1267 (s) 1265 (w)

1373 (sh) -1338 (w)

-1323 (m)

+1384 (m)

PT

+1377 (s)

-1204 (w)

the anomeric region

+1455 (s)

SC

the CH2 and COH deformations region

+1462 (w)

1

ring def. Glc

2

ring def. Fru δ(CCO)

638 (w) 2

653 (w)

wavenumbe

548 (m)

+595 (w)

517 (w)

+525 (w)

r region

the low

δ(COC)

517 (w)

+517 (w)

596 (w)

+590 (m)

592 (w)

+592 (s)

δ(OCO)

548 (m)1

+534 (m)

546 (w)

-556 (w)

δ(CCO)

524 (m)1

-502 (m)

526 (w)

-507(s)

1

δ(CCO) Glc

ACCEPTED MANUSCRIPT * g.l. α(1-4) -468 (w)

-451 (m) 453 (w) 1

462 (w) -442 (w)*

441 (w)

-378 (m)

432 (w)

414 (w) +415 (m)*

354 (m)

-356 (m)

345 (w)

δ(CCC)

469 (w) -425 (w)

2

-440 (w) 423 (w)

+391 (m) 366 (m)

+350 (w)

1

δ(CCC) Glc

+395 (w) 361 (w)

2

δ(O-H-O)

-345 (w) β(COC)

Key: s – strong, m – medium, w – weak, sh – shoulder, ν - stretching, δ – bending, ω – wagging, τ – twisting, def. – deformation, g.l. –glyosidic

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link, Glc – glucose, Gal – galactose, Fru – fructose, α anomer, β anomer, endo – endocyclic CH2OH group, exo – endocyclic CH2OH group.

ACCEPTED MANUSCRIPT Table 5. Raman (RS) and ROA bands assigned for aqueous solution of γ-cyclodextrin and glycogene (polysaccharides). Molecular structure and vibrational spectra are shown in Fig. 5 [1,2,32,41,45,60,61,63,64]. γ-cyclodextrin

Glycogene Assigment

ROA

RS

ROA

+1461 (vw)

1461 (m)

+1461 (m)

1462 (m)

def. (CH2) δ(CH)

1409 (m)

+1365 (w)

1381 (s)

+1381 (s)

δ(COH)

1339 (s)

-1338 (w)

1339 (s)

-1343 (m)

def. (CH2)

1266 (m)

+1253 (w) β

1212 (w)

-1212 (m)

-1212 (w) 1211 (w)

+1164 (vw) 1131 (s)

1129 (s)1

δ(COH)

ν(CO) Glc ν(CC)

-1106 (m)

NU

1113 (s) -1106 (m) -1069 (m)

ν(COC) δ(COH)

-1088 (m)

1051 (s)

1

+1042 (w) -1013 (w)

1088 (s)

MA

1088 (s)

1050 (m)

-1001 (w) ν(COH)

942 (s) *

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+917 (s) *

CE

866 (m) α

-853 (w) α

* g.l α(1-4) ν(C-H) Glc 865 (m) α

AC

-862 (m) α δ(C1-H)

+783 (vw) α 764 (w)

+796 (m) α

717 (w)

-712 (w) α

-

+605 (w)

γ(OH)Hbonded

-576(w)

δ(CCC)

+528(w)

δ(CCO)

482(m) *

-485(w)

* g.l α(1-4)

442(w)

-439(w) *

δ (COC)

408(w)

+407(w) *

δ(CCO)endo

358(w)

-374(w)

δ(CCC)

δ(OCO) Glc

-760 (w)

712 (w)

590(w)

def. (C1-H) +905 (m) *

762 (w)

654 (w)

ν(COC) -942 (w) *

D

948 (s) *

ν(CO)endo Glc ν(CC)

1

-942 (s) *

1

+1047 (m)

+1000 (w)

the anomeric region

1

δ(CCH)

+1143 (m)

+1141 (w) the fingerprint region

RI

δ(CH)

1265 (m)

1

the low wavenumber region

PT

1388 (m)

SC

the CH2 and COH deformations region

RS

-709 (w) α +594(w) -570(w)

597(w)

+532(w)

-482(w) 483(s) * -379(w) * 445(w) +358(w) *

Key: s – strong, m – medium, w – weak, sh – shoulder, ν - stretching, δ – bending, γ – out of plane bending, def – deformation, Glc – glucose, endo – endocyclic CH2OH group, g.l. – glycosidic link, α anomer.

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ACCEPTED MANUSCRIPT

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Graphical abstract

ACCEPTED MANUSCRIPT Highlights 

Raman and Raman Optical Activity (ROA) spectra of 14 carbohydrates in aqueous solution were collected and assigned in detail.



Studied carbohydrates were selected as biologically important and representative from among mono-, di-, tri- and polysaccharides. An expanded discussion on various structures and conformations of studied

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

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Raman and ROA spectroscopy are shown as unique tools to study complex structures of

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carbohydrates.

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

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carbohydrates in the solution is described based on collected Raman and ROA spectra