Understanding the effect of deacetylation on chitin by measuring chemical shift anisotropy tensor and spin lattice relaxation time

Journal Pre-proofs Research paper Understanding the Effect of Deacetylation on Chitin by Measuring Chemical Shift Anisotropy Tensor and Spin Lattice Relaxation Time Krishna Kishor Dey, Manasi Ghosh PII: DOI: Reference:

S0009-2614(19)30763-8 https://doi.org/10.1016/j.cplett.2019.136782 CPLETT 136782

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Chemical Physics Letters

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8 August 2019 17 September 2019 20 September 2019

Please cite this article as: K. Kishor Dey, M. Ghosh, Understanding the Effect of Deacetylation on Chitin by Measuring Chemical Shift Anisotropy Tensor and Spin Lattice Relaxation Time, Chemical Physics Letters (2019), doi: https://doi.org/10.1016/j.cplett.2019.136782

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Understanding the Effect of Deacetylation on Chitin by Measuring Chemical Shift Anisotropy Tensor and Spin Lattice Relaxation Time Krishna Kishor Dey, Manasi Ghosh*, Department of Physics, Dr. Hari Singh Gour Central University, Sagar-470003, MadhyaPradesh, India *Corresponding author: [email protected]

Abstract: The structure and dynamics of the functional biopolymer chitosan were investigated by measuring spin-lattice relaxation time, chemical shift anisotropy parameters, and compare those results with that of the chitin. The relaxation time and the correlation time of the carbon nuclei reside at the polysaccharidic backbone of chitosan are decreased due to depolymerization of polysaccharide chain. The effect of the substitution of different functional group at the C2 position of the monomeric sugar unit is portrayed by this type of comparative and comprehensive, which will enlighten the way of finding biodegradable and biocompatible polymers with enormous application in biomedicine. 1. Introduction: The structural homopolysaccharide chitin is fabricated by a repeating N-acetylglucosamine (GlcNAc) units linked by β-(1,4) glycosidic bonds. Shells of crabs and lobsters, exoskeleton of insects, crustaceans, and invertebrates are source of chitin. It is very difficult to utilize this hugely abundant biopolymer because of its insolubility in water and most ionic solvents. On the other hand, chitosan, a product of the alkali deacetylation of chitin, is soluble in dilute hydrochloric, formic and acetic acids solution, and it has potential to form specific complexes with transition and post-transition metal ions [1]. It is formed by (1→4)-2-amino-2-deoxy-β-Dglucopyranose (GlcN) and (1→4)-2-acetamido-2-deoxy- β-D-glucopyranose (GlcNAc) units.

These two monomers differ with respect to the substitution of either amino or acetamide group at the C2 position in the sugar ring. The solubility of chitosan is dictated by the level of protonated amino groups in the glucosamine monomers. The heterogeneous deacetylation reaction leads to a random distribution of GlcN and GlcNAc residues in the chitosan polymer when the degree of acetylation (DA) is greater than 40%. The degree of acetylation (DA) is measured by the presence of GlcNAc units within the polysaccharide chain. Hence, DA is 100% for chitin [2]. In general, chitin with a degree of acetylation below 50% is regarded as chitosan. The degree of acetylation governs the solubility and conformation of the chitosan [3-7]. There exist two crystallographic arrangements in a fully deacetylated chitosan (DA = 0 %) – hydrous and anhydrous [8]. The degree of elongation and tensile strength of a completely deacetylated chitosan ( DA = 0%) is higher than chitin [5]. The electrostatic behavior can be divided into three categories according to the percentage of DA present in chitosan chain. When DA < 20%, chitosan behaves like polyelectrolytes, and it is soluble in aqueous media. The polysaccharide chain constitutes a transition domain, when DA is at the range of 20 to 50 %. When DA > 50%, the hydrophobic character of chitosan is increased due to the absence of huge number of cations [7]. The stiffness of the chain is also substantially influenced by the distribution of N-acetyl groups or on the percentage of DA. If DA < 25 %, the chain is flexible. The stiffness gradually increased with 25% ≤ DA ≤ 50 %. The persistence length of the chain becomes nearly constant when DA > 50% [5, 6]. Chitosan exhibits high hydrophilicity, reactivity, and structural flexibility due to the presence of the reactive groups like hydroxyl, acetamide and primary amino groups. Additionally, the absorptive properties of chitosan make it a potential polymer to be used as heavy-metal chelators in water, removal of pesticide and dyes from water, for absorption of

proteins, as flocculant agent, and most significantly as a catalyst support for biodiesel production [9-12]. Because of the biocompatibility, biodegradability, non-toxicity, antimicrobial and antifungal activity, this hugely abundant renewable resource has wide range applications in pharmaceutical industries, biofabrication [13], delivery systems for macromolecules [14], wound dressing [15], tissue engineering(bone, cartilage, nerve, skin), cosmetics, agricultural materials [16], food industries [17], water purification, chemical industries, drug delivery, platform for neural stem cell growth, immunoprophylaxis, gene therapy, and treatment of infections [18 – 20, 47-50]. The structure and dynamics of polymers have been explored by solid-state NMR experiments emphasizing dipole-dipole coupling, scalar-coupling, principal components of chemical shift anisotropy (CSA) tensor, spin-spin and spin-lattice relaxation time measurements [21-26]. The primary purpose of the present work is to monitor the effect of deacetylation by comparing the CSA parameters measured by

13C

two-dimensional phase adjusted spinning

sidebands (2DPASS) cross-polarization(CP) magic angle spinning (MAS) SSNMR, spin-lattice relaxation time measured by Torchia CP method, and correlation time calculated by using CSA parameters and spin-lattice relaxation time of chitin and chitosan. During the fast magic angle spinning (MAS) experiment all the anisotropic interactions are averaged out, so the information regarding three-dimensional molecular structure and dynamics are lost. The isotropic and anisotropic dimensions can be correlated by employing the two-dimensional phase adjusted spinning sideband (2DPASS) MAS SSNMR experiment followed by shearing transformation and two-dimensional Fourier Transformation [27-29]. In 1989 Tycko et al. [30] had demonstrated a technique of measuring CSA parameters of polycrystalline or amorphous solids by combining magic angle spinning (MAS) with a radiofrequency pulse sequence synchronized with the sample rotation in one time period. In this

2D MAS/CSA NMR experiment, the CSA line shapes appear along one axis and MAS spectrum appears along the other axis. By applying separation of undistorted powder patterns by effortless recoupling (SUPER) MAS NMR [31] experiment CSA parameters can be measured at MAS speeds 2.5 to 5 kHz. In 2003 Chan and Tycko had employed a rotation-synchronized radiofrequency pulse ROCSA (recoupling of chemical shift anisotropy) sequence[32] to recouple CSA interaction by retaining a static CSA powder pattern line shape and attenuating homonuclear dipole-dipole interacting simultaneously at high value of MAS frequencies (from 11 to 20 kHz). For systems with weak homonuclear dipole-dipole interactions, Hou et al. [33] had applied 𝛾-encoded RN𝜈𝑛-symmetry based CSA (RNCSA) recoupling schemes for CSA recoupling under a wide range of MAS frequencies. The CSA parameters of biopolymer with multiple sites can be extracted by using two-dimensional magic angle flipping (2DMAF) experiment [34],two-dimensional magic angle turning (2DMAT) experiment [35], and 2DPASS [27, 28] experiment when MAS speed is very low. But, it is not possible to perform 2DMAF experiment in the commercially available probe. There required complicated probe design. On the other hand, 2DMAT is not a constant time experiment, so the spin-spin relaxation (𝑇2) mechanism makes the spectrum complicated for extracting quantitative information regarding relative abundance of different sites. In this context, 2DPASS MAS NMR experiment can provide a complete separation of spinning sideband with negligible loss of signal intensity. These side bands amplitudes are utilized to determine CSA parameters [24-26, 30, 34]. 2D PASS MAS NMR experiments had been employed to extract the information regarding structure and dynamics of chitin [24], keratin [25], cellulose[26], to characterize oxospirochlorin derivatives [36], to study host-guest interactions in benzodiazacoronands [37].

2. Experiment Section 2.1 NMR measurements Chitin was purchased from Alfa Aesar. Chitin was deacetyled in hot alkali medium to produce chitosan [16].

13C

CP-MAS and

15N

CP-MAS experiments on chitin and chitosan were carried

out on a JEOL EXC 500 NMR spectrometer. The spectrometer is equipped with 3.2 mm JEOL double resonance MAS probe. Contact time for Cross-Polarization (CP) was 2ms, with repetition interval 30s. External referencing for

13C

and

15N

was done by using tetramethylsilane (TMS)

and NH4Cl respectively. 2.2 CSA measurements The pulse sequence of 2DPASS CP MAS NMR experiment is associated with five 𝜋 pulses [28]. The time gaps among these five 𝜋 pluses are calculated according to the solution of PASS equations. The time duration of all the PASS sequences is same irrespective of the sideband phase shift. The experiments were performed on a JEOL ECX 500 NMR spectrometer, equipped with a 3.2 mm JEOL double resonance MAS probe. 2D PASS CP MAS NMR experiments were carried out at two different MAS frequencies 600 Hz and 2000 Hz. The CP condition was optimized on standard sample with 2 ms contact time. Sixteen steps in indirect dimension were used for 2DPASS experiment with 13 steps cogwheel phase cycling [28]. The 90 degree pulse length for 13C was 3 𝜇s [24]. 2DPASS MAS NMR experiment provides a complete separation of spinning sideband with negligible loss of signal intensity. These side band amplitudes are utilized to determine the CSA eigenvalues [27, 28, 30]. Spin-lattice relaxation time measurement was done by Torchia CP method [38] at MAS speed 10 kHz. referencing was done by using Tetramethylsilane (TMS). 3 Results and Discussion

13C

isotropic chemical shift

3.1 NMR spectral analysis Figure 1 shows (a) the structure of chitosan associated with 1→4 linked 2-acetamide-2-deoxy-𝛽D-glycopyranose

(N-acetylglucosamine)

and

2-amino-2

deoxy- 𝛽-D-glycopyranose

(glucosamine) units. These two units differ with respect to the C2 substituent in the sugar ring – an amide or acetamide group. The rotations of the two glycosidic linkages O5-C1-O1-C4 and C1-O1-C4-C5 are defined by torsion angles Φ and ψ respectively. These rotations are coupled with the glycosidic bridge angle, which is varying in the course of conformational refinement of the chitosan chain. The orientation of the primary hydroxymethyl groups O5-C5-C6-O6 is defined by torsion angle 𝒳. Three staggered positions are preferred by conformation of the polysaccharide chain – gauche-trans (gt), gauche-gauche (gg) or trans-trans (tg) [8]. On the contrary, the homopolysaccharide structure of chitin is associated with 1→4 linked 2-acetamide2-deoxy-𝛽-D-glycopyranose (N-acetylglucosamine) i.e. acetamide group at C2 position of sugar ring. Figure 1 (b) and (c) show 13C CP MAS NMR spectrum of chitosan and chitin respectively. For chitin eight well-resolved resonance line corresponds to the eight chemically different carbon sites of GlcNAc unit. In chitosan, the intensity of C7 and C8 lines are decreased (DA < 20 %) and C3 and C5 resonance lines are overlapped with each other. Singlet patterns in the C1 and C4 resonance lines suggests that the configuration of chitosan maintained the two-fold helix like structure of the sugar chains with repeating periods of between two and eight sugar units [6,8]. The stability in the 2-fold helical configuration is maintained by intramolecular O3-O5 hydrogen bonding across glycosidic linkages and by interchain hydrogen bonding between the oxygen atom of the hydroxymethyl group (O6) of one chain and the nitrogen atom of amine group of the D-glucosamine unit of another chain [6]. The degree of acetylation affected the pattern of polymer packing. Two-fold helix is the representative configuration of the polysaccharide chain

at high pH. The values of the torsion angles are Φ = -900, and ψ = + 900 for 2-fold helix chitin. These values are slightly shifted (Φ = -980 and ψ = + 920) for two fold helix chitosan [6,8]. As the torsion angles of the glycosidic linkage are not changed significantly due to deacetylation of chitin, so the CSA parameters of C1 and C4 resonance lines of chitin and chitosan are not varied significantly (see table 1). Figure 1 (d) and (e) represent

15N

CP MAS NMR spectrum of chitin and chitosan

respectively. For chitin 15N NMR resonance line near 110 ppm is due to amide functional group; whereas for chitosan two resonance lines are observed near 10 ppm and 110 ppm due to the presence of amino and amide group respectively. The degree of acetylation (DA) was measured from

15N

CP MAS NMR spectrum by the relative integrals of amide group compared to total

integrals of both amide and amino group. DA can also be evaluated from

13C

CP MAS NMR

spectrum of chitosan as the relative integrals of methyl or carbonyl groups compared to the total integrals of the backbone polysaccharide [39]. This important characteristic parameter influences the conformation of the chitosan molecule, biodegradability and immunological activity. From both 13C and 15N CP MAS NMR spectral analysis, it is determined that DA is less than 20% for the chitosan produced from the deacetylation of chitin in our laboratory. Hence, in this case, chitosan behaves like polyelectrolytes. The electrostatic field generated by the charges in the polyelectrolyte has significantly affected the structure and dynamics of the molecule. We will discuss in the preceding section how the structure and dynamics are getting altered due to the deacetylation of chitin.

Figure 1: (a) Structure of chitosa with glucosamine(GlcN) and N-acetylglucosamine (GlcNAc) units. These two monomers differ with respect to the C2 substituent in the sugar ring, either an amino or acetamide group. The extended helical structure of chitosan is stabilized by an intramolecular O3-O5 hydrogen bonding. (b) and (c) represent 13C CP MAS NMR spectrum of chitosan and chitin respectively; (d) and (e) show

15N

CP MAS NMR spectrum of chitin and

chitosan respectively 3.1 Chemical Shift Anisotropy The shielding of the external magnetic field by the electron cloud density depends on the orientation of the molecule with respect to the direction of the external magnetic field – this is the source of chemical shift anisotropy (CSA) interaction. It is necessary to know the CSA parameters of chemically different carbon nuclei to extract information about the segmental orientations and reorientations, to probe the local structure, to determine molecular conformation, and to distinguish different chemical groups with overlapping isotropic-chemical shift. CSA interaction cannot be represented by a scalar quantity; a second-rank tensor is required to stipulate CSA interaction. The magnitudes of the principal elements of CSA tensor 𝛿11, 𝛿22 and 𝛿33 represent least shielded component with highest frequency, moderately shielded component with moderate frequency, and most shielded component with lowest frequency respectively. As the CSA parameters are related with molecular orientation and electronic distribution surrounding the nucleus by the relations [40]

2

𝛿11 =

〈|

2

〈|

2

2

𝑛

| 〉 ( )∑

2

𝑒 𝑥 + 𝑧 𝑒ℏ 0 0 ― 2𝑚 2𝑚 𝑟3

2

𝛿33 =

| 〉 ( )∑

2

𝑒 𝑦 + 𝑧 𝑒ℏ 0 0 ― 3 2𝑚 2𝑚 𝑟

2

𝛿22 =

〈|

2

2

𝑛

{

{

| 〉 ( )∑

2

𝑒 𝑥 + 𝑦 𝑒ℏ 0 0 ― 2𝑚 2𝑚 𝑟3

2

𝑛

〈| |〉 〈| |〉

〈0|𝐿𝑥|𝑛〉 𝑛

0

𝑟3

0

+

(𝐸𝑛 ― 𝐸0)

2𝐿𝑥 𝑟3

𝑛 〈𝑛|𝐿𝑥|0〉

2𝐿𝑦 𝑟3

0

0

+

(𝐸𝑛 ― 𝐸0)

2𝐿𝑦 𝑟3

𝑛 〈𝑛|𝐿𝑦|0〉

(𝐸𝑛 ― 𝐸0)

〈| |〉 〈| |〉

〈0|𝐿𝑧|𝑛〉 𝑛

2𝐿𝑧 𝑟3

(𝐸𝑛 ― 𝐸0)

0

}

(1)

}

(2)

(𝐸𝑛 ― 𝐸0)

〈| |〉 〈| |〉

〈0|𝐿𝑦|𝑛〉 𝑛

{

2𝐿𝑥

0

+

2𝐿𝑧 𝑟3

𝑛 〈𝑛|𝐿𝑧|0〉

(𝐸𝑛 ― 𝐸0)

}

(3)

Where L𝑥, L𝑦 and L𝑧 are 𝑥, 𝑦 and 𝑧 components of angular momentum (L = i L𝑥 + j L𝑦 + 𝑘 L𝑧) respectively. The first part of the expressions (1), (2), (3) is diamagnetic term, and the second one is paramagnetic term. The diamagnetic contribution comes from spherically symmetric distribution of electron cloud in ground state i.e. when electrons reside in s-orbital. The paramagnetic term arises due to this distortion in spherically symmetric potential when the electrons reside in p and d orbital. Both diamagnetic and paramagnetic term provides different values for the same nucleus resides in the different chemical environment.

{

Isotropic chemical shift 𝛿𝑖𝑠𝑜 =

𝛿11 + 𝛿22 + 𝛿33 3

} imparts the average value of the principal

components of CSA tensor in principal axis system (PAS) and it also corresponds to the centre of gravity

(𝜂 =

of

𝛿22 ― 𝛿33

𝛿11 ― 𝛿𝑖𝑠𝑜,

the

spinning

CSA

)

side-band

pattern.

The

value

of

asymmetry

when𝛿11 > 𝛿22 > 𝛿33 parameter varies from zero to one. η value close to 1

indicates a highly asymmetric CSA side-band pattern and close to 0 indicates axially symmetric CSA side-band pattern [41]. If its value is less than 0.3, then the spinning CSA side-band pattern is closely axially symmetric. On the other hand, if it is greater than 0.7, then the spinning CSA side-band pattern is closely asymmetric [41]. Hence, it basically measures how much the spinning CSA side-band pattern deviates from an axially symmetric pattern. Except C4 and C8 nuclei, the spinning CSA side-band patterns corresponding to other carbon nuclei like C1, C2, C3, C5, and C6 are highly asymmetric (see Table 1). Span (𝛺 = 𝛿11 ― 𝛿33) and skew 𝑘 = 3(𝛿22 ― 𝛿𝑖𝑠𝑜) 𝛺

represents respectively the maximum width of the side-band CSA pattern, and the

orientation of the asymmetry of the spinning CSA side-band pattern. The sign of skew depends on the position of 𝛿22 with respect to the centre of gravity of the CSA side-band pattern. The

[

anisotropy ∆𝛿 = 𝛿11 ―

(𝛿22 + 𝛿33) 2

] parameter describes the largest separation of the side-band

pattern from the centre of gravity(𝛿𝑖𝑠𝑜). The sign of the anisotropy parameter indicates on which side of the centre of gravity the separation is largest. Table 1: Chemical shift anisotropy parameters of chitosan and chitin at chemically different carbon sites.

𝛿11 Carbon from (ppm) differe nt chemic al environ ment C1 119.6

𝛿22 (ppm)

CSA parameters of chitosan 𝛿33 Span Skew (ppm) (ppm)

106

92.4

27.2

0

106

20.4

1

C4

72.8

62

56.8

-0.6

84.5

51.4

0.3

118.8

𝛿𝑖𝑠𝑜 (ppm)

Anisotr opy (ppm)

Asym metry( ppm)

C5 C3 C6 C2 C8

98.4 97.9 85.3 67.7 49.8

76.9 74.2 61.3 58.8 15.6

𝛿11 Carbon from (ppm) differe nt chemic al environ ment C1 124.4

𝛿22 (ppm)

58.5 39.9 -0.1 56.6 41.3 -0.1 43.1 42.2 -0.1 47.9 19.8 0.1 7.2 42.7 -0.6 CSA parameters of chitin 𝛿33 Span Skew (ppm) (ppm)

105.7

87.9

36.6

0

106

27.6

1

C4 C5 C3 C6 C2 C8 C7

73.3 74.8 72.9 61.4 55.5 16.4 161.6 153.9 166.5

60.6 58.4 56 42.8 35.4 5.4 105.7 104.9 106.8

59 41.9 44.0 43.1 42.8 44.4 152.1 156.6 149.8

-0.6 -0.2 -0.2 -0.1 -0.1 -0.5 -0.3 -0.4 -0.2

84.5 77.9 76.3 63.4 56.4 23.9 175.0 173.5 176.6

52.7 33.7 35.6 33.8 32.7 38.9 124.1 132.1 119.9

0.4 0.7 0.7 0.8 0.9 0.4 0.7 0.6 0.8

119.6 100.3 100 85.9 78.2 49.8 257.8 261.5 256.5

77.9 76.2 63.3 58.1 24.2

30.7 32.5 33.1 15.4 38.5

0.9 0.8 0.8 0.9 0.3

𝛿𝑖𝑠𝑜 (ppm)

Anisotr opy (ppm)

Asym metry( ppm)

The anisotropy parameter of C4 carbon site is higher than any other carbon nuclei site due to the relative orientation of the glycosidic bonds around the torsion angles 𝛷, ψ (as shown in the Figure 1(a) )[2]. The CSA parameters of chitosan (as shown in table 1) and chitin [24] are nearly same for C1, C4, C5, C6, C8 carbon nuclei, but it is widely different for C2 nuclei. This suggests that the nature of the functional group at C2 position on the monomeric unit of sugar influences the electronic environment and molecular dynamics of the long-chain polysaccharide. The substitution of the amino group on C2 position disturbs the effective formations of

intramolecular hydrogen bonding between the polysaccharide chain[8]. Hence, the CSAparameters are reduced at C2 position compared to chitin. 3.2 Spin-Lattice Relaxation The spin-dynamics of this long-chain polysaccharide compound (constructed by random distribution of N-acetyl glucosamine and glucosamine units) can be probed by

13C

spin-lattice

relaxation time measurement at each and every crystallographically and chemically different carbon site. The effect of deacetylation on the dynamic of the polysaccharide compound can be estimated by comparing the spin-lattice relaxation time of chitin [24] and chitosan. Table 2:

13C

spin-lattice relaxation time at chemically different carbon sites of chitin and

chitosan. Chitin data is taken from our previously published article [24]. 13C

spin-lattice relaxation time of chitosan at chemically different carbon site

Carbon nuclei

13C

spin-lattice relaxation time of chitin

at chemically different carbon site

Relaxation time at

Relaxation

Relaxation time at

Relaxation time

crystalline region

time at

crystalline region

at amorphous

(Sec)

amorphous

(Sec)

region (Sec)

125 ± 10

16 ± 5

region (Sec) C7 C1

41 ± 3

260 ± 10

36 ± 5

C4

35 ± 3

170 ± 10

16 ± 5

C5

31 ± 2

170 ± 10

18 ± 5

C3

30 ± 2

260 ± 10

36 ± 5

C6

28 ± 2

1 ± 0.5

25 ± 5

1 ± 0.5

C2

34 ± 2

7 ±1

218 ± 10

28 ± 5

C8

13 ± 2

25 ± 5

7 ±1

From Table 2, it is clear that the spin-lattice relaxation time (T1) is remarkably decreased in chitosan compared to chitin at C1, C2, C3, C4 and C5 carbon nuclei sites, reside at the backbone of the polymer. On the contrary, the relaxation time is almost same for the side chain carbon nuclei C6 and C8. This indicates that the partial substitution of amide group in place of acetamide group mostly affects the dynamics of backbone polymer- the dynamics of side-chain (C6 and C8) remain unaffected. Spin-lattice relaxation decay curves of chitin at each and every well-resolved resonance line were well fitted by two exponential equations 𝑀(𝜏) = 𝑀0𝐼𝑒𝑥𝑝( ― 𝜏 𝑇1𝐼) + 𝑀0𝐼𝐼𝑒𝑥𝑝

( ― 𝜏 𝑇1𝐼𝐼), one with slower relaxation and another with faster relaxation rate [24]. The slower relaxation corresponds to the crystalline region, and the faster relaxation corresponds to the amorphous region, which is consistent with the X-ray diffraction studies [42,43]. Although both chitin and chitosan are semicrystalline polymer consists of microfibrils. Each microfibril is associated with crystalline and amorphous domains statistically alternated along the fibril [43]. But, for chitosan the relaxation time of the amorphous region is decreased in such a level which cannot be measured by NMR-relaxometry except at C2 and C6 position. That’s why for C1, C3, C4, C5 and C8 nuclei sites, the relaxation decay curves are well fitted by single exponential. Free primary amino groups are distributed within the polysaccharide chain of chitosan. Hence, the chemical and biochemical reactivity of chitosan is higher than chitin. The

sorption ability of chitin containing hydrophobic acetyl groups is lower than chitosan containing hydrophilic hydroxyl and amine group. The sorption ability is inversely proportional to the degree of crystallinity. Decrease of the degree of crystallinity and increase of amorphous domains [43] are the reasons of the faster rate of spin-lattice relaxation of the backbone polymer of chitosan. The accumulation of charges in flexible polymer chain would eventually lead to a considerable expansion due to electrostatic repulsion [6]. It also was predicted by theoretical calculation that the nature of the substituent at C2 (by amino or acetamide group) on the nonreducing residue of the disaccharide units affects the accessible conformational space of the glycosidic bond [44]. From Monte Carlo conformational sampling of polymeric chain it was predicted that chitosan shows greater flexibility and tortuosity than chitin [44]. The expansion of the polysaccharide chain, increase of flexibility, and the change in accessible conformational space of glycosidic linkage – all these effects are strongly correlated with the increase of spinlattice relaxation rate of the polymer backbone. 3.3 Correlation Time The local magnetic field surrounding the nucleus can fluctuate due to homonuclar dipole-dipole coupling, heteronuclear dipole-dipole coupling, indirect dipole-dipole coupling (J-coupling), spin-rotation coupling, CSA-interactions, quadrupole-electric field gradient interactions. Spinlattice relaxation mechanism is governed by these interactions. The natural abundance of

13C

nucleus is very low (~ 1.1 %), so the influence of homo-nuclear dipole-dipole coupling is very low in the relaxation mechanism. The quadrupole-electric field gradient interaction is also absent because 13C is spin-1/2 nucleus. Hence, the major contributions in the relaxation mechanism of 13C

nucleus come from CSA-interaction and heteronuclear-dipole-dipole coupling.

The relaxation mechanism of

13C

nuclei is mainly and almost entirely dominated by chemical

shift anisotropy (CSA), especially for non-protonated carbon nuclei [46] when the value of external magnetic field is very high. The general expression for relaxation which arises due to chemical shift anisotropy is [45,46] 1

= 𝐶𝑆𝐴

𝑇1

(

)

𝜏2 2 2 2 2 𝛾𝐵𝑆 15 1 + 𝜔2𝜏22

(4)

Figure 2:

13C

2DPASS MAS NMR spectrum of chitosan. The horizontal axis of the two-

dimensional spectrum shows pure isotropic spectrum with zero side band and the vertical axis shows anisotropic spectrum. The spinning CSA side-band patterns corresponding to chemically different carbon sites are also shown in (a) to (g). Where correlation time is defined as the average time taken by the molecule to rotate through one radian is 𝜏𝑐 = 3 𝜏2 and 𝐵 is the applied external field. Where 𝑆2 = (∆𝜎)2(1 + ɳ2 3) and (∆𝜎) = 𝛿33 ―

(𝛿11 + 𝛿22) 2

;ɳ=

(

𝛿22 ― 𝛿11

).

𝛿33 ― 𝛿𝑖𝑠𝑜

The expression of spin lattice relaxation rate arises due to heteronuclear dipole dipole coupling is [45] 2 1 𝛾𝐶𝛾𝑋 ħ 1 6 3 = + + 𝜏 (5) 2 1 + 𝜔𝐶2𝜏22 1 + (𝜔𝐻 ― 𝜔𝐶)2𝜏22 1 + (𝜔𝐻 ― 𝜔𝐶)2𝜏22 𝑇1𝐷𝐷 10 𝑟3𝐶𝑋

1

( ) [

]

If only the first term of this expression is taken into consideration then 2 3 1 𝛾𝐶𝛾𝑋 ħ 𝜏 = 2 𝑇1𝐷𝐷 10 𝑟3𝐶𝑋 1 + 𝜔𝐶2𝜏22

( ) [

1

]

(6)

Hence, the observed relaxation for 13C is 1 1 1 = 𝐶𝑆𝐴 + 𝐷𝐷 𝑇1 𝑇1 𝑇1

(7)

2 𝜏2 2 2 2 2 1 𝛾𝐶𝛾𝑋 ħ 3 1 = 𝛾𝐵𝑆 + 𝜏 2 10 𝑟3𝐶𝑋 𝑇1 15 1 + 𝜔2𝜏22 1 + 𝜔𝐶2𝜏22

(

) ( ) [

]

(8)

Figure 3: Bar-diagram of spin-lattice relaxation time of (a) chitin and (b) chitosan at chemically different carbon sites.

Table 3: Correlation time of chitosan and chitin at chemically different carbon nuclei sites Correlation time of chitosan at chemically different

Correlation time of chitin at chemically

carbon site

different carbon site

Carbon

Correlation time at

Correlation

Correlation time at

Correlation time

nuclei

crystalline region

time at

crystalline region

at amorphous

(Sec)

amorphous

(Sec)

region (Sec)

region (Sec) C1

2.73 × 10 ―6

1.54 × 10 ―5

2.14 × 10 ―6

C4

1.63 × 10 ―5

1.75 × 10 ―5

1.65 × 10 ―6

C5

5.92 × 10 ―6

1.16 × 10 ―5

1.23 × 10 ―6

C3

6.39 × 10 ―6

1.93 × 10 ―4

2.66 × 10 ―5

C6

6.02 × 10 ―6

4.29 × 10 ―7

1.91 × 10 ―6

7.63 × 10 ―8

C2

1.43 × 10 ―6

2.95 × 10 ―7

1.73 × 10 ―5

2.22 × 10 ―6

1.08 × 10 ―4

1.39 × 10 ―5

1.54 × 10 ―6

4.31 × 10 ―7

C7 C8

3.18 × 10 ―6

Figure 4:

13C

spin-lattice relaxation decay curves of chitin and chitosan at chemically different

carbon site. The solid line represents the fitting of the individual relaxation decay curve. Where X in the second term of Equation (8) represents1H or 14N nucleus. 𝑟𝐶𝑋 is calculated from the crystal structure of chitosan [16,46]. Where 𝜔 = 2𝜋𝑓 = 2 × 3.14 × 125.758 MHz = 789.76024 MHz; 𝐵 = 11.74 𝑇, 𝛾𝐶 = 10.7084 MHz/𝑇, 𝛾𝐻 = 42.577 MHz/𝑇, 𝛾𝑁 = 3.077 MHz/𝑇. ℏ = 1.054 × 10 ―34 𝐽𝑠. Since the relaxation mechanism governed by heteronuclear dipole-dipole interaction vanishes with the sixth power of the distance between 13C and 1H or 14N nuclei, so the contribution from the second term is very small for high-value (11.74 Tesla) of magnetic field. Table 3 shows the correlation time at crystallographically and chemically different carbon nuclei of chitosan and chitin [24] calculated by using Equation (8). It would not be possible to measure spin-lattice relaxation time, and CSA-parameters of C7 nuclei correspond to carbonyl group carbon because it values decreased below the limit of NMR measurement as the degree of acetylation (DA) is less than 20 %. For C1, C5, C2, C3 carbon nuclei sites, the correlation time decreased one order of magnitude compared to that of chitin [24] due to the decrease of the degree of polymerization. The correlation time of C4 nuclei of both chitin and chitosan are almost same although their relaxation time is different. The torsion angles are not changed significantly (Φ from -900 to -980 and ψ from + 900 to + 920) for two fold helix chitosan [6] – this may be one of the reason for the same order of correlation time of chitin and chitosan for C4 nuclei. By calculating partition function, torsion angles and coupling constants of chitosan, it was predicted that C1 is the most flexible and C4 the most rigid [44] atom of the polysaccharide. Hence, the increase of flexibility of the polysaccharide chain cannot influence the correlation time of C4 nuclei. As C6 and C8 atom reside at side chain of the polysaccharide, so the

correlation time and relaxation time would not get affected due to the decrease of the degree of crystallinity. The motional degree of freedom of the carbon nuclei which resides at the backbone of the polysaccharide is increased due to deacetylation of chitin. The replacement of acetamide group by amine group at C2 position of the polymer affects the flexibility of the adjacent glycosidic bond toward the reducing end [44], so the correlation of the backbone polysaccharide of chitosan is decreased in one order of magnitude. Determination of spin-lattice relaxation time and calculation of the correlation time at chemically different carbon nuclei site will provide more vivid scenario how the dynamics of the polysaccharide chain is getting altered due to the deacetylation of chitin. Conclusion The effect of deacetylation on chitin was investigated by comparing spin-lattice relaxation time and CSA parameters at chemically different carbon site of chitin and chitosan. The sorption ability of chitin containing hydrophobic acetyl group is lower than chitosan associated with hydrophilic hydroxyl and amine groups. The sorption ability is inversely proportional to the degree of crystallinity. The decrease of crystallinity and increase of amorphous domains in chitosan compared to chitin is vividly portrayed in relaxation behavior of the backbone polymer. The spin-lattice relaxation time of those carbon nuclei reside at the backbone of the polysaccharide chain like C1, C2, C3, C4, C5 are remarkably decreased compared to chitin. Those carbon nuclei (C6, C8) which reside at side chain of the polymer are not affected due to deacetylation. The correlation time of chitosan at C1, C2, C3, C5 sites is decreased one order of magnitude compared to chitin. The chitosan and chitin monomers are differed with respect to the substitution of either an amino or acetamide group at C2 position. The CSA parameters, which basically reflect the molecular orientation and electronic environment surrounding the nucleus,

are nearly same for both chitin and chitosan at C1, C4, C4, C5, C6, C8 carbon nuclei site, but widely varied for C2 nuclei. This type of comprehensive study will provide the detail picture in the molecular level, how the structure and the dynamics of a polysaccharide are getting altered due to deacetylation.

Acknowledgements The author Manasi Ghosh is indebted to Science and Engineering Research Board (SERB), Department

of

Science

and

Technology

(DST),

government

of

India

(File

no.

EMR/2016/000249), and UGC-BSR (File no. 30-12/2014(BSR)) for financial support. We are also grateful to Sophisticated Instrumentation Centre (SIC) of Dr. Hari Singh Gour Central University for providing high resolution solid state NMR facility.

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   

The effect of deacetylation on chitin Structure and dynamics of chitosan by 2DPASS MAS SSNMR measurement Spin-lattice relaxation time measurements Calculation of molecular correlation time

C8

C1

N

C7

13C spin-lattice relaxation decay curves of chitin and chitosan at chemically different carbon sites

O1

O7

O5

C2 C3 C5 O3

C4 resonance line

C1 resonance line

C6

Chitosan

Chitosan

N-acetylglucosamine unit

Chitin

Chitin

O6

ψ

C4

O1 Hydrogen bond

φ

C1

O5

O6

χ O7

C5 resonance line Chitosan

Chitin

Chitin

O3

C4

ψ

O1

φ

Hydrogen bond

C1

C2 resonance line

C8 resonance line

C7 N

O3

C2

O5

C3

C5

Chitosan

χ

C6

Chitin

O6

Chitosan Chitin

C4 O1

C1

13C 2DPASS MAS NMR Spectrum of Chitosan C2

C1

Anisotropic Chemical Shift (ppm)

C8

Chitosan

Glucosamine unit

C3

C5

C6

C3 resonance line

N

C2

C3

C5

C4 -30 -20 -10

C6

0 10 20 30

C8 200

160

120

80

40

Isotropic Chemical Shift (ppm)

0

-40

13C Frequency (ppm)

Declaration of interests

☐The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

We are agree with above declarations

Digitally Signed by Manasi Ghosh, Krishna Kishor Dey