DFT study on host-guest interaction in chitosan–amino acid complexes

Accepted Manuscript DFT study on host-guest interaction in chitosan–amino acid complexes Bhabesh Chandra Deka, Pradip Kr. Bhattacharyya PII: DOI: Reference:

S2210-271X(17)30159-7 http://dx.doi.org/10.1016/j.comptc.2017.03.036 COMPTC 2463

To appear in:

Computational & Theoretical Chemistry

Received Date: Revised Date: Accepted Date:

10 February 2017 28 March 2017 28 March 2017

Please cite this article as: B.C. Deka, P.K. Bhattacharyya, DFT study on host-guest interaction in chitosan–amino acid complexes, Computational & Theoretical Chemistry (2017), doi: http://dx.doi.org/10.1016/j.comptc. 2017.03.036

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DFT study on host-guest interaction in chitosan–amino acid complexes Bhabesh Chandra Deka and Pradip Kr. Bhattacharyya* Department of Chemistry, Arya Vidyapeeth College, Guwahati, Assam, 781016, India

Email: [email protected] (BCD) [email protected] (PKB) * Corresponding author

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Abstract: The present density functional theory (DFT) study delves into the interaction of chitosan (CS) and a few chitosan derivatives (CSDs) with amino acids (AAs) to form CS/CSD-AA complexes. Interaction energy, reactivity descriptors and various thermo-chemical parameters are utilized to illustrate the stability of the complexes. Results suggest comprehensive stability of CS/CSD-AA complexes excluding those formed with lysine in gas phase and of all the complexes in aqueous phase. Increase in polarity of the solvent lowers the reactivity of the complexes (evaluated by using reactivity descriptors). Thermochemical analysis suggests the presence of strong thermodynamic driving force in CS/CSD-AA complexation with a few exceptions .

Keywords: Density functional theory, hydrogen bonding, chitosan, amino acid, interaction energy, solvent effect

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1. Introduction Intra-cellular delivery of functional proteins bear therapeutic implications in terms of disease therapies, vaccination, and imaging [1]. Therefore, development of protein drug delivery systems has been one of the major issue in pharmaceutical and biomedical research because of diverse challenges such as fragility of protein molecules, protein denaturation, complex structure and high molecular weight inter alia [2]. Natural polymeric carrier such as polysaccharides have found an edge among the novel delivery systems owing to their bio-compatibility and biodegradability [3]. Moreover, when the carrier is functionalized by a targeting agent, the encapsulated drug may be selectively released inside or near a specific tissue or organ [4]. However, protein stability in the course of delivery still remains a serious impediment in designing efficient protein delivery system [5]. Chitosan (CS) is a bio-degradable polysaccharide derived by partial de-acetylation of chitin. It has been one of the most recognized candidate for drug delivery because of its several incredible characteristics such as- synthetic versatility, ability to indulge in interactions of different nature with variety of drug molecules and most essentially its amenability toward functional modification [6]. The amine group of the CS of each subunit gets protonated in acidic to neutral solutions making the CS based materials highly water soluble and bioadhesive which readily binds to negatively charged surfaces such as mucosal membranes. This endows CS based carriers with the applicability towards enhancing the transport of polar drugs such as proteins drugs across epithelial surfaces. The major advantage of CS based delivery systems is the hydrophilicity incorporated by the polar groups (OH and –NH2 groups) which are able to form secondary interactions such as hydrogen bond (hb) with drug molecules) [7]. A high positive charge density on CS nano-particles enable them to interact with cell membranes and therefore functioning of CS is restricted at protonated conditions where it is positively charged [8]. Van 3

der Lubben et al. have demonstrated that large amounts of bovine serum albumin (BSA) or vaccine tetanus toxoid (TT) were easily encapsulated in CS nanoparticles [9]. Recent literature reports insightful developments taking place with respect to a number of tailor-made chitosan derivatives (CSDs) envisaged to circumvent some inherent limitation of CS such as poor solubility in physiological pH, low transfection efficiency, and low cell specificity [10]. A number of such CSDs have been investigated for their applicability as protein carrier. Amidi et al. investigated the potentiality of N-trimethyl chitosan (TMC) nanoparticles as a carrier system for the nasal delivery of proteins and concluded that TMC nanoparticles are a potential delivery system for protein transport through the nasal mucosa [11] Huo et al. demonstrated the safe applicability of N-octyl-O-glycol chitosan as the carrier of pacilitaxel for intravenous administration [12]. Yin et al. also outlined the high insulin encapsulation efficiency of thiolated CS like TMC–Cysteine [13]. Currently, studies using density functional theory (DFT) have rekindled investigations of the nature and site of interaction between nanomaterials and biomolecules. Amidst a myriad of such studies a number of computational studies are devoted to illustrating CS and its derivatives as gene carrier [14-16]. Equally good number of studies has shed light on different carrier molecules for protein drugs [17,18]. As amino acids (AAs) are the building blocks of proteins; elucidation of their nature of interactions with CS and its derivatives will mimic to some extent a typical CS/CSD-protein interaction and is the focus of the current study. A thorough understanding of the strength of interaction between CS/CSDs and AA as well as reactivity of CS/CSD-AA adducts is deemed essential in terms of evaluating the efficacy of CS or a particular CSD as protein carrier. Despite the ubiquitous presence of such interactions in biological systems, DFT studies on them in the literature have been sparse. The present study is an attempt

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to provide an insight into the stability (in terms of interaction energy and thermodynamic parameter) and reactivity (quantified by reactivity descriptors) of CS/CSD-AA adducts. Herein, along with CS, three CSDs viz. trimethyl chitosan (TMC), CS-Cysteine (CSC), and O-glycol CS (OGC) have been considered. In addition, AAs are chosen so as to include their four variantsneutral (polar/non-polar), cationic and anionic; viz. leucine (leu), tyrosine (tyr), aspartatic acid (asp) and lysine (lys). In view of the complex physiological medium (from a non-polar cellmembrane to polar cytoplasm) encountered by CS/CSD-AA complex we have also examined the impact of polarity of solvent on interaction energy and reactivity parameters taking four solvents, viz. cyclohexane, THF, acetone and water into considerations. 2. Computational details and theoretical formulae of reactivity descriptors 2.1 Computational details In the present study we have employed the density functional theory (DFT) and density functional reactivity theory [19-22]. Since potentiality of CS and its derivatives as DNA or protein carrier has been restricted only at acidic pH (where chitosan bears a positive charge)[23, 24], it should be borne in mind that the model systems (adducts) with leu and tyr bear unit positive charge, with lys they bear two positive charges, while those with asp are electrically neutral. As regard to the structure of chosen AAs, in modeling the association geometries in gas phase, the neutral form is preferred to their zwitter-ionic form. This is mainly due to the fact that in vacuo, the neutral form is more stable than the zwitterions due to larger intrinsic proton affinity of an oxygen atom of the carboxylate group relatively to a nitrogen atom of the amino group [25]. Dispersion corrected DFT methods are endowed with very good level of accuracy and have been evolved as an alternative to methods that include extensive electron correlation in the 5

calculation juxtaposed with a sufficiently large basis set [26-28]. Various dispersion corrected functionals are proposed and are being applied to assess strength of weak interactions such as hydrogen bonding. Among these DFT functionals, several recent studies have outlined the suitability of CAM-B3LYP for estimating strength of hydrogen bonding interaction[29,30]. CAM-B3LYP combines the hybrid qualities of Becke three parameter exchange and Lee, Yang and Parr correlation functional (B3LYP) and the dispersion correction essential for calculating interaction energy of hydrogen bonded systems [31,32]. In addition to CAM-B3LYP, M06-2X functional has also found extensive application for successful description of hydrogen-bonded systems [33]. Zhao and Truhlar employed M06-2X to characterize the host–guest interactions in supra-molecular complexes [34]. Moreover, in a study to evaluate the performance of conventional and dispersion-corrected DFT methods for estimating hydrogen bonding interaction energies, DiLabio et. al demonstrated the suitability of M06-2X functional for accurate calculation of binding energy in hb23 dimers of S66 benchmark [30]. Herein, optimized geometries of CS, chosen CSDs, AAs and adducts and different moities are obtained using 631++G(d,p) basis set with CAM-B3LYP and M06-2X functional. The molecular systems considered here have large number of geometry degrees of freedom and may exhibit multiminima PES. However the Gaussian algorithm is capable of taking into account of all these local minima and providing global minima thereof. Herein, frequency calculations at the same level of theory of geometry optimization have been carried out to confirm the nature of the stationary points. The respective Hessian (matrix of analytically determined second derivative of energy) for all the geometries is found real which suggests at their global minima on the potential energy surface.

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Molecular electrostatic potential surfaces of CS and its chosen derivatives as well as the AAs depict a markedly electron deficient region around the tertiary amine group of CS/CSD and lys (shown with blue color in Fig. 1d-h) as well as electron rich regions (shown with red color in Fig. 1a-c) around the carboxylate group of leu, tyr and asp.

(a) asp

(b) leu

(c) tyr

(d) lys

(e) CS

(f) CSC (g) OGC (h) TMC H C N O S Fig.1. Iso-surface diagram of considered CS/CSDs and AAs. Blue and red color shows respectively the electron deficient and electron rich regions (iso-surface diagrams are generated with 0.001 a.u. density and in the color scale values are in a.u.). This suggests the plausibility of electrostatic interaction between amine group of CS/CSD and the carboxylate group of AAs. Accordingly, association geometries as depicted in Fig.2 are modeled and optimized at CAM-B3LYP/6-31++G(d,p) level of theory followed by hessian calculation in order to ascertain the geometrical minima of the optimized geometries. The association geometries calculated at CAM-B3LYP/6-31++G(d,p) level of theory are further reoptimized at M06-2X/6-31++G(d,p) level of theory and subsequent determination of geometrical minima is also carried out. 7

Using the super molecule approach and the above mentioned geometries (Fig.2), the interaction energies of the adducts are calculated according to the following equations∆Eint = ECS/CSD-AA – ECS/CSD – EAA +Ebsse where, ECS/CSD-AA,

(1)

ECS/CSD, and EAA are energy of adduct, CS/CSD and

amino acid

respectively. Ebsse is the basis set superposition error (bsse) correction included to eliminate the effect of basis set incompleteness which are achieved by counterpoise correction method [35]. Counterpoise correction was calculated for the global minima ab-initio geometries (at both CAM-B3LYP/6-31++G (d,p) and M06-2X /6-31++G(d,p) level of theory). The effect of solvent on the strength of interactions has been elucidated using polarizability continuum model (PCM) with four different solvents viz. cyclohexane (ε=2.02), THF (ε=7.43), acetone (ε=20.49) and water (ε=78.35) covering a range of di-electric constant. Polarizability continuum model (PCM) implements the self-consistent reaction field (SCRF) approach which describes the solvent polarization in terms of the electrostatic potential [36]. After developed by Tomasi’s group, this model has thus far found successful applications over various molecular systems [37]. In polar solvent especially in water, the amino acids acquire the zwiterionic form and therefore while in case of other solvent interaction energy is calculated by single point calculation on gas phase geometries of adducts, in aqueous phase calculations re-optimization (at M06-2X/6-31G++(d,p) level of theory) is done by re-modeling of the geometries of AAs as well as of adducts taking the zwitterionic form of the AAs into considerations. Binding enthalpy (∆Hint) and free energy of complexation (∆Gint) are calculated using the super molecular model and particularly the following equations∆Hint = HCS/CSD-AA – HCS/CSD – HAA

(2)

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∆Gint = GCS/CSD-AA – GCS/CSD – GAA

(3)

Free energy of solvation (∆Gsol) of complexes is computed using the Truhlar and coworkers’ SMD solvation model [38] and the following equation∆Gsol = ESMD - Egas

(4)

where, ∆Gsol is the solvation energy of the system, ESMD and Egas are the total energies of the system in the solvent and gas phase respectively. All calculations are performed by using Gaussian 09 [39]. 2.2 Theoretical formulae of reactivity descriptors From among several reactivity descriptors defined by DFRT, energy of highest occupied molecular orbital, global hardness, chemical potential and electrophilicity considered for analysis of reactivity of the title systems in the present study. Global hardness (η) is the second derivatives of energy with respect to the number of electrons [40, 41]. The working formula of η, as derived from finite difference approximation and Koopmans’ theorem [42] is- η = ( E LUMO − E HOMO ) / 2 , where, E HOMO is the energy of the highest occupied molecular orbital (HOMO) and E LUMO is the energy of the lowest unoccupied molecular orbital (LUMO). Electrophilicity (ω) [43] is

(

)

expressed as ω = μ2 2η , where μ is called chemical potential and μ = ( E LUMO + E HOMO ) / 2 . 3. Results and Discussion: 3.1 Adduct formation 3.1.1 Gas phase optimized structure of adducts Earlier works by Mao et al. suggest that at acidic pH, the protonated amine group render CS with the ability to bind with negatively charged DNA/RNA via an electrostatic interaction [44]. Besides, a number of earlier studies have outlined the crucial role of hydrogen bonding in adduct formation by protonated CS and CSDs with electrically neutral nucleobases [14,16]. 9

CS-asp

CS-leu

CS-lys

CS-tyr

CSC-asp

CSC-leu

CSC-lys

CSC-tyr

OGC-asp

OGC-leu

OGC-lys

OGC-tyr

TMC-asp TMC-leu TMC-lys Fig. 2 Optimized structure of CS/CSD-AA adducts obtained at M06-2X/6-31++G(d,p) level of theory 10

The calculated group charge distribution (NBO charges) for the three hydrogen atom of amine group is 1.54 au (in CS and OGC) and 1.01 au in case of CSC (two amine H). These values indicate that these hydrogens possess the propensity to form hydrogen bonds (hb) with electronegative centers. Similarly a group charge of 1.54 au for the three hydrogen atoms of positively charged amine group in lys has enabled us to perceive the plausibility of an otherwise atypical CS/CSD-lys interaction through hb between NH 3+ group of lys with -OH group of CS and CSDs. The optimized structure of the considered adducts are presented in Fig. 2. The distance (in Å) between H atom of amine or -OH group of CS/CSD and O atom of –COO or OH of chosen AAs are listed in Table 1. Table 1: Hydrogen bond distance (in Å) in CS/CSD-AA complexes (N-O bond distances in certain TMC-AA adducts) Adduct CS-asp CS-leu CS-lys CS-tyr CSC-asp CSC-leu CSC-lys CSC-tyr OGC-asp OGC-leu OGC-lys OGC-tyr TMC-asp TMC-leu TMC-lys TMC-tyr

Hydrogen bond distance (in Å) CAM-B3LYP/6-31++G(d,p) M06-2X/6-31++G(d,p) 1.03 1.01 1.69 1.64 1.97 1.87 1.69 1.64 0.99 0.97 1.64 1.57 1.88, 2.01 1.83, 2.00 1.86 2.04 1.01 1.02 1.73 1.63 2.25, 2.26 2.22, 2.23 1.80, 2.22, 2.25 1.73, 2.02, 2.03 3.46 (N-O) 3.40 (N-O) 3.58 (N-O) 3.55 (N-O) 1.86, 1.81 1.88, 1.82 (hb) 3.52 (N-O) 3.52 (N-O)

Referring to Table 1, distance between H atom of amino or OH group of CS/CSD and O atom of –COO– or -OH of chosen AAs is found in the range of 1.57 Å-2.22 Å which is characteristics of hydrogen bonds [14, 45]. This suggests that CS/CSDs form hb with the chosen AAs during 11

adduct formation. It is important to note an abnormally shorter HCS/CSD -Oasp distance (0.991.03Å) in all the CS/CSD-asp adducts. This infers the migration of amine hydrogen of CS/CSD to COO– group of asp and thereby neutralizing the systems. When the adducts are formed via hydrogen bonds there is the possibility of H-transfer between the interacting monomers. The minimization algorithms are not always capable of passing the energy barrier and getting the most stable stationary point. Therefore, different protonation states of the adducts as initial points for geometry optimizations was tested. Results confirmed about the proton transfer in adduct with lysine only thus denying the same in case of other adducts. Although cationic or anionic AAs are well known for their ability to form salt bridges with oppositely charged species [25, 46], in the gas phase thus no such salt bridge formation has been observed in the considered adducts.

Furthermore, due to lack of amine hydrogen, TMC does not form any hb with AAs

other than lys. Instead, there is electrostatic interaction between the amino nitrogen of TMC and carboxylate oxygen of AAs. Its positive charge density around the tertiary amine group (Fig. 1(h)) together with a very strong dipole moment associated with it (calculated dipole moment = 6.5 Debye) render it with the ability to indulge in cation-dipole interaction with leu and tyr and a strong coulombic attraction with asp. With lys, alignment of positive charge density of both the molecule along the interacting faces restrict them from engaging in charge-dipole interaction and facilitates hb formation by amine group of lys and hydroxyl group in the opposite face of amine group of TMC. Hydrogen bond distances calculated at M06-2X/6-31g++(d’p) level of theory in most of the adducts are shorter than those corresponding to CAM-B3LYP/6-31++G(d,p) level of theory. Since interaction energy is inversely dependent on the bond distances, it can be presumed that interaction energy calculated at M06-2X /6-31++G(d,p) level of theory will be higher than that obtained at CAM-B3LYP/6-31++G(d,p) level of theory.

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3.1.2 Aqueous phase geometry of adducts An earlier study also reports that ammonium group of low molecular chitosan binds with carboxylate anion of amino acid containing drugs in dilute solution [50]. In aqueous phase, where the amino acids exist in their zwitterionic form, salt bridge is noticed in all the adducts of CS, CSC and OGC with leu and tyr. Optimized geometries of CS-leu, CSC-leu and OGC-tyr (calculated at M06-2X /6-31G++(d,p) level of theory) as representative example are shown in Fig. 3.

CS-leu CSC-leu Fig.3. Salt bridge formation in CS/CSD-AA adducts in aqueous phase.

OGC-tyr

The distance (in Å) between H of positively charged amine group of CS/CSC/OGC and O of carboxylate ion of leu and tyr are-1.49 (in CS-leu), 1.47 (in CS-tyr), 1.48 (in OGC-tyr), 1.45 (in OGC-leu), 1.40 (in CSC-leu and CSC-tyr). Previous theoretical study of adsorption of AAs on graphene and BN sheet in gas and aqueous phase has demonstrated that geometries of both the molecules remain unaffected by inclusion of solvent [57]. Similarly, marginal effect of solvation on hydrogen bond length (variation of around 0.11 Å) is also reported [16, 58] In the present study, presumably due to the zwitterionic form of the AAs, the hb distances of salt bridged complexes in aqueous phase are shorter than the corresponding bond length in gas phase. Similar to their phase geometries, in CS/CSD-asp complexes, migration of H from CS/CSD to asp is observed in aqueous phase also.

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3.2 Interaction Energy 3.2.1 Gas Phase Interaction Energy Several publications have earlier demonstrated that CS and its derivatives form stable complexes with proteins and peptides [47-49]. The magnitude of interaction energy (∆Eint) of CS/CSD- AA adduct is crucial in terms of the protection of complexed protein from degradation in course of passage of the protein payload into the cell as well as the transfer of protein near or within the nucleus of a target cell. A high value of ∆Eint favors strong binding between the two moieties in adduct while decrease in ∆Eint facilitates dissociation of adduct. Moreover, ∆Eint values will help assessing the suitability of a carrier vis-à-vis a particular amino acid. Bsse corrected ∆Eint values in vacuum, calculated at both CAM-B3LYP/6-31++G(d,p) and M06-2X /6-31g++(d,p) level of theory are presented in Table 2. The basis set superposition error (bsse) was evaluated to be less than 4% of the correspondent interaction energy value. As for example bsse correction for the adduct CSC-leu calculated at CAM-B3LYP/6-31++G(d,p) and M062X/6-31++G(d,p) level of theory are 1.1 and 1.4 kcal/mol against ∆Eint values of –26.2 and – 28.2 kcal/mol (corrected value). Similarly for CSC-lys, these are 1.5 and 1.9 kcal/mol against ∆Eint values of 28.0 and 26.2 kcal/mol respectively. As can be seen in Table 2, except in case of adducts with lys, the calculated ∆Eint values are negative in all the cases which favors the formation CS/CSD- AA adducts with asp, leu, and tyr (throughout the manuscript higher value of interaction energy means more negative value of ∆Eint and vice versa); the values ranges from –12.27 to –121.01 kca/mol-1. An earlier work reports the binding energy of the most stable conformation of CS-insulin adduct to be –38.0 kcal/mol [50]. Another related study fixes the energy of doxrubicine release by mono-ethylene glycol chitosan at 122.41 kcal/mol at B3LYP/631G//PM3 level of theory [51].

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Table 2: bsse corrected ∆Eint values in vacuum (calculated at both CAM-B3LYP/6-31++G (d,p) and M06-2X/6-31++G(d,p)level of theory) and in solvents (at M06-2X/6-31++G(d,p) level of theory. Adduct

CS-Asp CS-Leu CS-Lys CS-Tyr CSC-Asp CSC-Leu

∆Eint (gas) CAM-B3LYP/ 6-31++G(d,p)

–110.81 –29.02

∆Eint (solvent) at M06-2X/6-31++G(d,p) level of theory M06-2X/ 6-31++G(d,p)

–115.54 –30.15

Cyclohexane

THF

–70.97 –20.38

–33.34 –16.19 –3.98 –18.85 –81.55 –17.98 –6.06 –8.28 –58.24 –19.88 –2.04 –21.02 –38.55 –10.21 –5.71 –13.91

30.45

19.44

5.14

–18.38 –127.60 –26.22

–31.54 –138.41 –28.26

–22.77 –93.34 –21.31

CSC-Lys

27.98

26.29

6.82

CSC-Tyr

–7.76

OGC-Leu

–115.81 –21.04

–12.27 –121.01 –27.62

–12.05 –72.80 –19.70

OGC-Lys

33.811

31.50

11.56

OGC-Tyr

30.76

TMC-Leu

–14.94 –80.66 –9.17

–84.17 –18.18

–24.71 –44.82 –12.13

TMC-Lys

11.04

19.37

3.47

TMC-Tyr

-18.70

–17.22

–14.39

OGC-A

TMC-A

Acetone

–24.00 –16.69 –5.74 –16.36 –51.51 –14.64 –8.93 –7.23 –27.48 –16.17 –5.16 –17.65 –10.13 –5.38 –7.56 –6.52

Water

–22.08 –16.14 –11.02 –16.06 –25.27 –13.74 –9.46 –2.79 –18.96 –13.15 –6.58 –16.92 –6.73 –5.55 –14.69 –6.23

Adduct formation by protonated CS/CSD with positively charged lys, perceptibly is endergonic, signifying a repulsive interaction between them. The ∆Eint value ranges from 19.37 kcal/mol (in TMC-lys) to 31.5 kcal/mol (in OGC-lys). As regards to other AAs, abnormally high value of ∆Eint with respect to adducts with asp (–84.17 to –138.41kcal/mol) can be attributed to strong coulombic force of attraction that leads to the salt bridge formation. Strength of interaction between CS/CSDs with respect to a particular AA follows different trend. With asp, the order is CSC> OGC > CS> TMC; with leu, the order is CSC> OGC > CS> TMC, with tyr, the order is CSC> OGC > CS> TMC and with lys the order is CSC> OGC > CS> TMC. In vacuo, among the chosen AA carrier, interaction of AAs with CSC is the strongest, while that with TMC is the 15

weakest one. Steric congestion around the amine group of TMC is believed to lower the ∆Eint values of its adduct substantially. Apart from electrostatics, hydrogen bond is associated with charge transfer concomitant upon changes of electronic structure which can be considered as partial covalent bond [52,53] and play a prominent role in governing ∆Eint values of adducts. Therefore, to grasp further insights into the trend shown by the ∆Eint values, we have calculated the charge transfer with respect to the hydrogen atom (∆q, which is calculated as difference in NBO charge of the H atom involved in hb formation in adduct to that obtained in isolated CS/CSD). Higher charge transfer would indicate a relatively stronger interaction. In case of adducts with asp ∆q values (in au) follow the order CSC-asp (0.047)> OGC-asp (0.046) > CS-asp (0.008) which shows clear consistency with the observed trend of ∆Eint. With a view to gauge the effect of functional on ∆Eint , we have carried out geometry optimization using both CAM-B3LYP and M06-2X functionals with the basis set 6-31++G(d,p) and calculated ∆Eint accordingly. Refering to Table 2, except for CS/CSD-lys, in case of adducts with other three AAs, mo6-2x over estimates ∆Eint than CAM-B3LYP at a range of 1-16 kcal/mol depending on the identity of adduct. Tao et al. have demonstrated the greater efficacy of the M06-2X functional over a range of methods like MP2, SCS-MP2, MP3, TPSS-d, PBE-d,

M06-2X, BH&H, tpss and B3LYP [54] against a benchmark energetic database (based on CCSD(T)) developed by Valdes H. [55]. A key reason as outlined by Steinmann et al. for the better performance of M06-2X functional is its improved treatment of medium-range correlation energy, that includes dispersion-like attractive interactions at geometries where orbital overlap

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(and hence exchange repulsion) of the interacting subsystems may not be neglected [56]. In view of this, calculations involved in the all the sections to be followed are performed by using the M06-2X functional. 3.2.2. Solvent Phase Interaction Energy: Role of solvent on interaction energy is critical and while dealing with aspects that provide avenues for experimental validation as well as biomedical applications. Therefore, the effect of solvent phase on interaction of AAs with CS/CSDs has been examined in four different solvents with varying polarity to mimic the trans cellular media in human living system. However, as can be construed from Table 2, the impact of solvent phase has been greatly imposed over the interaction energies of the chosen adducts. There is progressive destabilization of CS/CSD-AA adducts with increasing di-electric constants of the solvents with an exception of adducts with lysine. It is further worth noting that adducts with asp (in which the two moieties are opposite charged) suffer a massive drop in ∆Eint as compared to other adducts. For instance, ∆Eint values (calculated at M06-2X/6-31++G(d,p) level of theory) for the CSC-Asp adduct in the chosen solvents are- –138.41 (in gas), –93.34 (in cyclohexane), –81.55(in acetone), –51.51(in THF) and –25.27 (in water) indicating a drop of about 113 kcal/mol from gas phase to highly polar aqueous medium. In case of CSC-leu, this drop is about 17 kcal/mol and the observed trend is –28.26 (in gas), –21.31 (in cyclohexane), –17.98 (in acetone), –14.64 (in THF) and –13.74 (in water). The results show that monomers (where opposite charges are indeed more separated) are more stabilized than adducts in solvents with higher dielectric constants, which leads to a decrease in ∆Eint. This can also be attributed to the fact that in polar environments, the interaction with the environment (solvation) is probably more important than the electrostatic interaction between the two moieties, leading to their preferential stabilization. The desolvation penalty is

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very high for them to bind with each other. Furthermore, it is interesting to notice an invariably negative solvent phase ∆Eint value for the adducts with lys which is in sharp contrast to what is observed in gas phase. On account of solvation of the positively charged moieties, repulsive interaction between them diminishes which might render them compatible for hb formation. The obtained results of gas and solvent phase ∆Eint is quite significant from the perspective of amino acid/protein delivery. Results advocate a strong interaction between the CS/CSDs and AAs in non-polar medium and gradual weakening of the interaction as the CS/CSD-AA complexes traverses through the cell membrane (protein-lipid bilayer) which is non-polar in nature. In cytoplasm (which is polar in nature) the interaction is thus expected to be the weakest that might facilitate dissociation of adduct into the the corresponding moieties. It is important to note that ability of CSDs in releasing AA in cytoplasmic environment is comparable to that of CS. A substantially high value of ∆Eint in gas phase together with a very low aqueous phase ∆Eint for TMC-AA indicates its greater suitability over the other chosen CSDs. Greater efficacy of TMC as a carrier of nucleobases/gene also is outlined by a couple of earlier studies. [16, 59, 60] 3.3 Reactivity of adducts Delivery of bare therapeutic peptides and proteins suffers from poor bioavailability when administrated orally, mainly due to lack of stability in the gastrointestinal environment. They are vulnerable to electrophilic attack by various ions present therein that leads to degradation of the compound prior to delivery. Therefore understanding of chemical reactivity of the chosen CS/CSD-AA adducts in different environment is important from bio-chemistry perspective. Reactivity descriptors defined by density functional reactivity theory have become a back-of-an-

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envelop tool for computational chemists for interpreting chemical reactivity of a species in changing environment. 3.3.1 Gas phase reactivity descriptors Among the reactivity descriptors, HOMO energy ( EHOMO ), global hardness (η) and chemical potential (µ) are important from the viewpoint of chemical stability of a system. Variations of these parameters among the chosen sustems in gas phase are presented in Table 3. Measurement of E HOMO of a species is an important factor because it measures the electron donating ability i.e. the reactivity of the species A sharp drop of HOMO energy in AAs upon adduct formation with the chosen CS/CSDs is observed which advocates a more stable HOMO in adducts than in bare AAs. This predicts that CS/CSD-AA adducts are less prone to attack by any electrophile than bare AAs. Similarly, more negative value of µ signifies greater stability of the system. As it is evident from Table 3 in line with EHOMO , chemical potential data also portrays greater chemical stability of CS/CSD-AA adducts than bare AAs and show difference in variation pattern with respect to different AAs. Global hardness (global hardness is the half of HOMO-LUMO gap) quantifies the chemical stability of a molecular system in a changing environment [61]. According to the principle of maximum hardness (MHP) harder a species more is the stability[62, 63]. The principle of minimum electrophilicity (MEP), chemical stability is inversely related to electrophilicity[64].

19

Table 3: Gas phase reactivity parameters (in kcal/mol) of CS, CSD, CS/CSD-AA adducts at M06-2X /6-31++G(d,p) level of theory .

EHOMO

η

µ

ω

asp

–88.32

79.78

–8.53

0.46

leu

–207.02

103.44

–103.58

51.87

lys

–291.94

106.18

–185.76

162.49

tyr

–175.87

87.64

–88.23

44.41

CS-asp

–193.50

93.00

–100.50

54.30

CS-leu

–270.93

99.03

–171.90

149.20

CS-lys

–311.99

88.47

–223.52

282.35

CS-tyr

–248.42

88.32

–160.11

145.12

CSC-asp

–204.13

95.08

–109.05

62.54

CSC-leu

–264.48

86.21

–178.27

184.31

CSC-lys

–319.79

77.24

–242.56

380.85

CSC-tyr

–226.16

60.06

–166.10

229.71

OGC-asp

–205.88

97.71

–108.17

59.87

OGC-leu

–257.14

91.87

–165.26

148.63

OGC-lys

–290.14

72.50

–217.64

326.66

OGC-tyr

–242.09

86.57

–155.52

139.69

TMC-asp

–171.95

77.13

–94.82

58.28

TMC-leu

–270.3

104.52

–165.78

131.47

TMC-lys

–323.83

101.15

–222.68

245.10

TMC-tyr

–232.14

85.92

–146.22

124.44

Systems

Refering to Table 3, η values of adducts with asp decreases in the order- OGC-asp> CSC-asp> CS-asp> TMC-asp> asp; indicating greater stability of adducts than bare AA but at the same time shows a difference in the order as shown by ∆Eint. Moreover in all instances, maximum hardness is not associated with minimum electrophilicity. It is further worth noting that stability predicted by variation in gas phase interaction energy coincides with the trend shown by EHOMO 20

and µ. Gas phase stability order of adducts with asp as infered by EHOMO and µ is CSCasp>OGC-asp>CS-asp>TMC-asp which is also the trend shown by ∆Eint. Similar consistency is observed in case of adducts with other AAs also.

However, this variation pattern do not

corroborate with what is outlined by η. As in case of adducts with leu and tyr, highest interaction energies with CSC claim the reverse to what is inferred by their η values. Actually, relatively higher interaction energy of a system suggests that the system is energetically stable while reactivity parameters predict its chemical stability. The discrepencies associated with η values can be explained by the fact that LUMO of the AAs (which acts as H donor/electron acceptor) are interacted to a different extent in different adducts. In essence, although variation pattern of η does not provide for any generic conclusion regarding the chemical stability of the chosen adducts, EHOMO and µ values do assert greater stability of CS/CSD-AA adducts as compared to bare AAs and also the stability orders predicted by these two parameters are in full conformity with those predicted by ∆Eint values. 3.3.2 Effect of solvent on reactivity descriptors EHOMO,

and µ values of the considered AAs and their respective adducts in cyclohexane,

THF, and water are presented in Table 4. Results suggest that in line with gas phase values of reactivity descriptors, magnitude of CS/CSD-adducts is more negative than bare AA, thus predicting greater stability of adducts in comparison to bare AA in solvent phase also. For instance, EHOMO and µ of asp in aqueous medium are calculated to be –187.63 and –92.40 kcal/mol while these values for CSC-asp are –197.13 and –101.35 kcal/mol respectively. Except for adducts of asp, EHOMO and µ values of the other adducts become progressively less negative with increasing di-elecric constant of the solvent. As for example, EHOMO values (in kcal/mol) of OGC-leu are –232.7 in cyclohexane, –216.96 in THF and –210.96 in aqueous media; µ values

21

(in kcal/mol) changes as: –135.58 in cyclohexane, –115.21 in THF and –108.22 in aqueous media. Interestingly, there is a marginal increase in η of these adducts with increasing polarity of the solvent. In case of adducts with asp, all the three reactivity descriptors remains almost insensitive to varying polarity of the solvent. Table 4: Solvent phase reactivity parameters (in kcal/mol) of CS, CSD, CS/CSD-AA adducts at M06-2X/6-31++G(d,p) level of theory.

System

EHOMO

Cyclohexane μ

THF

water μ

EHOMO

μ

EHOMO

asp

–138.83

56.05

–82.78

–175.31

92.58

–82.73

–190.45

94.28

–92.40

leu

–210.24

102.35

–107.89

–210.10

104.11

–105.99

–200.31

99.21

–105.20

lys

–251.64

103.49

–148.15

–224.25

105.56

–118.69

–214.46

105.51

–109.31

tyr

–174.64

84.26

–90.38

–175.92

86.80

–89.12

–176.38

86.99

–89.05

CS-asp

–198.58

97.07

–101.51

–199.78

98.37

–101.39

–204.26

100.85

–101.50

CS-leu

–238.60

100.27

–138.33

–217.41

101.98

–115.44

–208.53

101.40

–108.18

CS-Lys

–262.25

97.00

–165.24

–226.48

102.64

–123.84

–214.71

103.83

–110.41

CS-tyr

–215.98

89.16

–126.81

–192.34

89.33

–103.01

–178.65

86.33

–95.08

CSC-asp

–201.26

95.44

–105.82

–198.42

95.73

–102.69

–193.78

90.54

–101.35

CSC-leu

–232.97

86.70

–146.27

–210.76

87.46

–123.30

–204.23

87.61

–115.55

CSC-lys

–263.67

79.59

–184.08

–221.96

82.30

–139.66

–206.86

86.47

–124.39

CSC-tyr

–201.22

65.88

–135.33

–185.11

71.53

–113.58

–177.59

73.18

–106.40

OGC-asp

–206.36

100.01

–106.34

–204.57

100.57

–104.00

–199.25

97.69

–103.50

OGC-leu

–232.7

97.12

–135.58

–216.96

101.74

–115.21

–210.65

102.13

–108.22

OGC-lys

–249.13

86.67

–162.46

–221.79

98.38

–123.41

–212.39

103.06

–110.18

OGC-tyr

–210.74

87.72

–123.02

–188.92

88.58

–100.34

–178.63

86.36

–93.11

TMC-asp

–180.35

85.47

–94.88

–188.09

92.26

–95.83

–192.19

94.58

–96.88

TMC-leu

–239.86

103.97

–135.88

–218.75

103.70

–115.04

–204.17

99.72

–108.19

TMC-lys

–266.45

102.23

–164.22

–226.6

103.48

–123.12

–214.60

104.68

–109.78

TMC-tyr

–203.87

86.05

–117.82

–184.53

86.47

–98.06

–178.04

86.17

–91.67

22

This is due to the fact that these adducts are formed by strong coulombic force of attraction upon which solvation fails to impinge. In essence, chemical stability of CS/CSD-AA adducts measured in terms of reactivity descriptors is inversely dependent on polarity of solvents. 3.4 Thermochemical analysis 3.4.1 Binding enthalpy and Gibb’s free energy of in adduct formation Earlier experimental studies ascertain that binding enthalpy (∆Hint) is much more precise and informative than ∆Eint in describing the stability of complexes with similar type of interaction [65]. In absence of experimental or any benchmark data for validation, close agreement between interaction energy and binding enthalpy of complexes is usually looked for providing credence to a theoretical calculation. Also ∆Hint and free energy (∆Gint) are key factors in examining the thermodynamic driving forces involved in complexation. Obtained data is summarized in Table 5. Table 5: Gas phase ∆Hint and ∆Gint values for the chosen adducts calculated at M06-2X/631++G(d,p) level of theory. Adduct CS-asp CS-leu CS-lys CS-tyr CSC-asp CSC-leu CSC-lys CSC-tyr OGC-asp OGC-leu OGC-lys OGC-tyr TMC-asp TMC-leu TMC-lys TMC-tyr

∆Gint (kcal/mol) –104.72 –9.63 349.42 –37.68 –108.68 –17.87 6.66 –20.92 –109.43 –15.16 12.07 –39.04 –71.47 –7.29 1.32 –24.40

23

∆Hint (kcal/mol) –113.81 –21.12 52.38 –52.21 –124.11 –27.65 56.11 –32.91 –120.05 –27.24 62.50 –51.61 –82.31 –16.71 50.05 –39.74

It is observed that ∆Hint and ∆Eint values (at M06-2X/6-31++G(d,p) level of theory) both are in close agreement with each other and follow similar trends. For example ∆Hint/∆Eint values in case of adducts with asp are- CSC-asp (–124.11/–138.41> OGC(–120.04/–121.01 > CS (– 113.8/–115.54> TMC (–82.30/–84.17). The negative value of ∆Hint adducts except those with lys implies that the adduct formation is exergonic in nature and also is enthalpy driven. Showing clear consistency with ∆Eint values, ∆Hint values are positive for adducts with lys, thus confirming the endergonic nature of adduct formation of protonated CCS/CSDs with lys. In line with ∆Hint, the calculated ∆Gint values are also found to be negative except in case CS/CSD-lys adducts. This suggests that formation of the CS/CSD-AA complexes via electrostatic interaction and more precisely hydrogen bonding is feasible and complexes do exhibit thermodynamic stability. Comparatively high ΔEint associated with CS/CSD-AA provide substantial contribution toward negative entropy of the systems which ultimately leads ∆G values to become negative. ∆Hint and ∆Gint values obtained for the complexes confirm that CSC-AA and OGC–AA complexes possess similar thermodynamic stability which is higher than CS-AA followed by TMC-AA complexes. However, positive ∆Gint obtained for CS/CSD-lys complexes have raised question about the thermodynamic feasibility of their formation in vacuo. 3.4.2 Free energy of solvation (∆Gsol) of CSD-AA adduct: Thermodynamic stability of adduct in solvent media can be gauged by free energy of solvation which is also indicative of its extent of solubility. Calculated ∆Gsol values for different adducts are presented in Table 6. As is evident from Table 6, adducts of CS and its three chosen derivative poseess strikingly similar values of ∆Gsol with respect to a particular AA in a specific solvent. Calculated values suggest greater thermodynamic stability of adducts in polar media and also agree with results of earlier relevant studies [61, 66]. Notably, the extent of increment in 24

∆Gsol

values with increasing di-electric constant of the solvent is relatively higher in case of

charged adducts than the neutral ones. For instance difference in ∆Gsol values in cyclohexane and water with respect to CS-lys, CS-leu and CS-asp are 92.45, 40.83 and 26.24 kcal/mol. This result suggests that electrically charged nature of adduct which governs its extent of solvation; enormously contribute to its thermodynamic stability. It is important to note that all the CS/CSDlys adducts are associated with highly negative values of ∆Gsol in each of the considered solvent. Table 6: ∆Gsol values of CS/CSD-AA adducts (in kcal/mol) Adduct CS-asp CS-leu CS-lys CS-tyr CSC-asp CSC-leu CSC-lys CSC-tyr OGC-asp OGC-leu OGC-lys OGC-tyr TMC-asp TMC-leu TMC-lys TMC-tyr

∆Gsol (cyclohexane) –15.12 –29.57 –81.98 –30.13 –14.42 –31.06 –84.67 –38.29 –13.37 –32.52 –88.77 –33.92 –17.40 –29.53 –80.14 –32.43

∆Gsol (THF) –24.22 –52.22 –142.15 –54.02 –26.02 –52.92 –145.59 –67.22 –24.71 –56.53 –153.57 –58.23 –31.59 –49.74 –135.04 –54.55

∆Gsol (acetone) –27.59 –59.37 –158.24 –61.70 –30.28 –60.07 –162.33 –76.86 –28.90 –64.16 –171.28 –66.15 –36.84 –56.21 –149.46 –61.97

∆Gsol (water) –41.36 –70.40 –174.43 –74.13 –23.45 –74.46 –172.98 –91.74 –44.16 –73.78 –185.21 –78.72 –52.50 –60.10 –162.12 –63.75

Abnormally high ∆Gsol value of aducts with lys is apparently due to its higher charge (+2) which attracts a large scale of solvation and this data further substantiates the affirmative role of solvents in adduct formation by protnated CS/CSDs with positively charged lys as outlined by solvent phase ∆Eint value associated with CS/CSD-lys adduct in Table 2.

25

4. Conclusion This study is conceived with an objective to have an insight into the interaction of CS and its three chosen derivatives with AAs. Our study confirms the presence of hydrogen bonding between positively charged amine group of CS/CSDs and carboxylate/-OH group of AAs, namely asp, leu, lys and tyr. CS/CSD-adduct possesses greater chemical stability than bare AAs. Reactivity (measured in terms of reactivity descriptors) and stability (measured in terms of interaction energy and thermochemical parameters) of CS/CSD-AA adducts are sensitive to the nature of functional modification as well as to the prevailing media. That adducts with lys are unstable in gas phase but attains substantial stability in solvent media is worth noting. Three CSDs covering three different mode of functionalization were chosen for the study. CS and all the chosen derivatives exhibit stronger interaction in non-polar medium and there is a gradual weakening with increasing polarity of the medium although there is no linear co-relation between interaction energy and di-electric constant of media. Chemical stability (measured in terms of reactivity descriptors) of CS/CSD-AA adducts also is affected by changing polarity of the media while thermodynamically the considered adducts are more stable in polar solvent. Most of the considered CSD-AA adducts exhibit comparable stability with CS-AA adduct. In addition, stronger interaction between CSDs and AAs in non-polar medium and a gradual weakening with increasing polarity of medium predicts efficacy of these derivatives as protein carrier. Among the chosen derivatives, TMC shows greater suitability as a protein carrier. A thorough understanding of various aspects pertinent to the binding and transfering of AA by CS/CSDs is envisaged to play an important role as predictive tool in formulating chitosan aided delivery system for therapeutic protein. This study is believed to boost future research in designing of protein carrier based on chitosan derivatives with new insights.

26

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Graaphical absttractt

Higghlligh ht 1. Innterracttionn ennerggy oof aamiino aciid-cchittosaan aaddductt aree sttudiied usiing DF FT Effect oof ppolaarityy off soolveent on staabiliity of addduct is analyysedd 2. E Reacctivvity of thee addducts aree annalyyzedd inn gaas aas w welll ass aqqueoous phhasee ussingg DF FR T 3. R Therrmoochhem micaal annalyysiss off addducct fform matiionn is carrriedd out 4. T

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