Ab initio computational study of –N-C and –O-C bonding formation : functional group modification reaction based chitosan

Chitosan application in pharmaceutics and cosmeceutics industries is limited by its solubility issue. Modification of -NH2 and -OH fuctional groups of chitosan by adding carboxyl group has been shown to improve its solubility and application. Attempt to synthesize carboxymethyl chitosan (CMC) from monocloroacetic acid (MCAA) has been done prior this report. However no information is available wether –OH (-O-C bonding formation) or -NH2 (-N-C bonding formation) is the preference for - CH2COOH to attach. In the current study, the reaction mechanism between chitosan and MCAA reactants into carboxymethyl chitosan (CMC) was examined by computational approach. Dimer from of chitosan used as a molecular model in calculation All the molecular structure involved in the reaction mechanism was optimized by ab initio computational on the theory and basis set HF/6-31G(d,p). The results showed that the - N-C bonding formation via SN2 than the -O-C bonding formation via SN2 which have activation energy 469.437 kJ/mol and 533.219 kJ/mol respectively. However, the -O-C bonding formation more spontaneous than the -N-C bonding formation because ΔG the formation of O-CMC-2 reaction is more negative than ΔG of formation N-CMC-2 reaction is -4.353 kJ/mol and -1.095 kJ/mol respectively. The synthesis of N,O-CMC first forms -O-CH2COOH, then continues to form -NH-CH2COOH. This information is valuable to further optimize the reaction codition for CMC synthesis.


Introduction
Chitosan is a copolymer of glucosamine and N-acetyl glucosamine monomer linked by 1,4-glucoside bonds [1]. It is commonly obtained from deacetylation of chitin produced by crustacean. Chitosan has high biocompatibility, relatively non toxic and biodegradable, promoting wide use of this polymer in drug delivery, antibacterial, cosmetic, food film and water purifier [2,3]. This polymer has reactive groups -NH2 and -OH that been associated with its insolubility at neutral and alkaline solutions [3]. Some strategies has been performed to increase the solubility of chitosan. One of them by reacting chitosan with monocloro acetic acid (MCAA) to produce carboxymethyl chitosan (CMC) [4]. The hydrophilic group (-CH2COOH) in MCAA group increased chitosan solubility. Modification of chitosan into CMC occurs via nucleophilic substitution reactions with the formation of -N-C and -O-C bonds resulting in N-CMC (-NHCH2COOH) and O-CMC (-O-CH2COOH) [1]. The O-CMC synthesis occurs 2

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The 12th Joint Conference on Chemistry IOP Publishing IOP Conf. Series: Materials Science and Engineering 349 (2018) 012049 doi: 10.1088/1757-899X/349/1/012049 at 30 °C with 40-50% NaOH concentration, while N-CMC synthesis occurs at 90 °C with 25% NaOH and at 60 °C yields N,O-CMC product [5]. Previous studies done in our lab shown that modification of chitosan to CMC improved its solubility and encapsulation efficency [6]. However from those studies it is not known if the substitution present on -NH2 and -OH groups. In real experimental setting it is costly to study intermediate. Thus, in this studies, the formation preference between -N-C with -O-C bond will be examined through ab-initio computational method.
The term ab-initio means "from first principle" which means the total wave function, molecular orbital, and energy are calculated respectively without empirical parameters [7]. The ab initio computational method has been efficiently predicted the formation mechanism of-N-C(= O)-on the cadherin peptide synthesis [8]. Computational methods are also able to determine the mechanism of glycine formation of CO2, H2 and H2C=NH which lead to a conclusion thet glycine formation was via joint reaction pathway where CO2, H2 and H2C=NH react together [9].

Computational Metods
All molecular calculations are performed with linux-based Gaussian 03 applications and all molecules are visualized with chemcraft software with input files created using a notepad++. In this research all calculations using the theory and basis set RHF / 6-31G (d, p). The stable structure at the stationary point in PES (Potential Energy Surface) as well as the minimum energy is determined by the "opt (CalcFC)" command, while the TS structure is determined by "opt (calcfc, ts, noeigentest)" command. Frequency of harmonic vibration is calculated by the "freq" command to determine the correction of zero-point vibration energy (ZPVE) for all molecules. Frequency is also used to determine the TS structure by ensuring that there is only one imaginary frequency, whereas the molecule at the stationary point has no imaginary frequency. In addition, the frequency calculation is also to know the free energy of Gibs of each molecule involved in the reaction mechanism [10,11].

Results and Discussion
Before studying the mechanism, computational study of reactants was done to determine bond characteristic. Reactant in this synthesis was chitosan dimer and MCAA. Chitosan dimer has two types of -NH2 bound to C4 and C9 and two types of -OH bound to C17 and C22 (Figure 1.a). We found that substitution of carboxymethyl groups occurred at -NH2 bound to C4 and -OH bound to C17. Substitution reactions did not occur in -C9-NH2 and -C22-OH, although N and O atoms have a more negative charge than -C4-NH2 and -C17-OH but the steric blockages in C9-NH2 and C22-OH are larger. Large steric hindrances make substitution reactions difficult because the chitosan molecule undergoes a flip that makes the transition state structure difficult to form.

The mechanism of synthesis reaction of O-CMC
The mechanism of synthesis reaction of O-CMC is a CMC synthesis substituted through the -OH group. This synthesis passes through two paths of reaction mechanism is SN1 path and SN2 path. The mechanism of synthesis reaction of O-CMC-1 is the reaction of SN1 pathway. The mechanism of this reaction begins with a weakened C-Cl bond which is indicated by the longer bonding distance of the ܶܵ ைିଵିଵ molecule the bond length of C-Cl is 2.5 Å (Figure 1.c), compared to in MCAA( 1.787 Å). This result is supported by an imaginary vibrational frequency on the center reaction area (ʋ = -394.64 cm -1 ). Weak bonds cause the C-Cl bond to break into carboxymethyl ions (+CH2COOH) and Cl ions, which are the first intermediates ‫ܫ(‬ ைିଵିଵ ).
The second stage is the attack of carbocation by the nucleophile group of chitosan (-OH) through the transition state (ܶܵ ைିଶିଵ ) with the bond length between -O-C is 1.8 Å. The transition state structure is confirmed by the presence of an imaginary vibrational frequency of the freq calculation (ʋ = -288.53 cm -1 ). After the Ea from the second state is passed the molecule will formed ‫ܫ‬ ைିଶିଵ with the formation of the -OH-CH2COOH bonds with the bond length -O-C to 1.437 Å, and the length of the oxygen bonds with hydrogen weakened from 0.947 Å to 0.98 Å (Figure 2.a). This is because the molecules ‫ܫ‬ ைିଶିଵ is charged +1 where the oxygen atom binds 3 atoms [13].  Figure 2. The stable structure of the O-CMC synthesis via SN1 and SN2 pathway The ‫ܫ‬ ைିଶିଵ molecules is highly reactive and unstable, so they must release an atom is hydrogen atom, this is the third reaction step. The third reaction stage begins with the formation of ܶܵ ைିଷିଵ which H atoms begin to move away from O with bond lengths to 1.178 Å (Figure 2.b) (Figure 2.c). The minimum energy of all molecules involved in the reaction mechanism of the O-CMC bond formation of SN1 path is presented in the energy energy potential (Figure 3) [14].
The mechanism of synthesis reaction of O-CMC-2 is the reaction of SN2 pathway. The mechanism of this reaction begins with the Clion starting away and the nucleophile groups of chitosan (-OH) begin to approaching simultaneously, forming ܶܵ ைିଵିଶ . As a result of this process there is a change in the length of bond and charge shown in Figure 2.d. The most significant changes are in the reaction areas (-OH-C-Cl). The Clcharge on the MCAA molecule is -0.086, while in the ܶܵ ைିଵିଶ molecule to -0.765, which makes the Clion more easily released. It is supported by an imaginary vibrational frequency on the center reaction area ʋ = -506,29 cm -1 . ܶܵ ைିଵିଶ through the formation of intermediates ‫ܫ‬ ைିଶ which is the +1-charged molecules at the reaction center that are formed -OH-CH2COOH ions (Figure 2.e ). In this stage the bonds between -O-H is getting weaker with each bond length to 0.987 Å.
The intermediate substance is not a stable molecule so release its hydrogen forming ܶܵ ைିଶିଶ . The second stage transition state (ܶܵ ைିଶିଶ ) was confirmed by an imaginary vibrational frequency is -1408.61 cm -1 . The weak hydrogen bonds with bond length O-H to 1.161 Å make the hydrogen easily loose to form O-CMC-2 (Figure 2.f) with a by-product of HCl. The minimum energy of all molecules involved in the reaction mechanism of the O-CMC bond formation of SN2 path is presented in the energy energy potential (Figure 3) [14]. , the greater the value of the activation energy(Ea) the value of the rate constant (k) will be smaller and the reaction rate will be slow at that stage. The value of the rate constant (k) obtained is presented in Table  1. From Table 1, it can be seen that the mechanism of the reaction of O-CMC formation most preferred is through the path SN2. This is because The Ea2 of SN2 pathway is lower than SN1 pathway, It is also because the value of the k of SN2 pathway is greater than the SN1 pathway, so the reaction is faster. Abou-zied et al., (2013) stated that carboxymethyl groups are formed by the reaction of SN2 [1]. The SN2 reaction path is preferable because the positive C atoms of the MCAA are primary C-type in the reaction path SN2 has the fastest reaction rate [15,16]. From the data of gibbs free energy (ΔG) known that the O-CMC-1 reaction is not spontaneous and the O-CMC-2 reaction is spontaneous [14]. The O-CMC-1 formation reaction is an endothermic reaction, whereas the O-CMC-2 formation reaction is an exothermic reaction.

The mechanism of synthesis reaction of N-CMC
The mechanism of synthesis reaction of N-CMC is a CMC synthesis substituted through the -NH2 group. This synthesis passes through two paths of reaction mechanism is SN1 path and SN2 path. The mechanism of synthesis reaction of N-CMC-1 is the reaction of SN1 pathway. The first stage of this mechanism is the same as the first stage of O-CMC-1 synthesis via the SN1 pathway.
The second stage is the attack of carbocation by the nucleophile group of chitosan (-NH2) through the transition state (ܶܵ ேିଶିଵ ) with the bond length between -N-C is 2.162 Å. The transition state structure is confirmed by the presence of an imaginary vibrational frequency of the freq calculation (ʋ = -338.657 cm -1 ). After the Ea from the second state is passed the molecule will formed ‫ܫ‬ ேିଶିଵ with the formation of the -NH2-CH2COOH bonds with the bond length -N-C to 1.487 Å, and the length of the -N-H bond weakened from 0.947 Å to 0.98 Å (Figure 4.a). This is because the molecules ‫ܫ‬ ேିଶିଵ is charged +1 where the nitrogen atom binds 4 atoms.
The ‫ܫ‬ ேିଶିଵ molecules is highly reactive and unstable, so they must release an atom is hydrogen atom, this is the third reaction step. The third reaction stage begins with the formation of ܶܵ ேିଷିଵ which H atoms begin to move away from N with bond lengths to 1.42 Å (Figure 4.b). The transition state is confirmed by an imaginary vibrational frequency of -1295.77 cm -1 . The length of the H bond with N will be longer and finally H is released forming N-CMC-1and H + ions. H + ions reacts with Clion to form HCl, as a by product. The length of the N-C bond becomes 1.434 Å (Figure 4.c). The minimum energy of all molecules involved in the reaction mechanism of the N-CMC bond formation of SN1 path is presented in the energy energy potential (Figure 5).
The mechanism of synthesis reaction of N-CMC-2 is the reaction of SN2 pathway. The mechanism of this reaction begins with the Clion starting away and the nucleophile groups of chitosan (-NH2) begin to approaching simultaneously, forming ܶܵ ேିଵିଶ . As a result of this process there is a change in the length of bond and charge shown in Figure 4.d. The most significant changes are in the reaction areas (-NH2-C-Cl). The Clcharge on the MCAA molecule is -0.086, while in the ܶܵ ேିଵିଶ molecule to -0.768, which makes the Clion more easily released. It is supported by an imaginary vibrational frequency on the center reaction area ʋ = -563.23 cm -1 . ܶܵ ேିଵିଶ through the formation of intermediates ‫ܫ‬ ேିଶ which is the +1-charged molecules at the reaction center that are formed -NH2-CH2COOH ions (Figure 4.e ). In this stage the bonds between -N-H is getting weaker with each bond length to 1.012 Å.
The intermediate substance is not a stable molecule so release its hydrogen forming ܶܵ ேିଶିଶ . The second stage transition state (ܶܵ ேିଶିଶ ) was confirmed by an imaginary vibrational frequency is -1558.81 cm -1 . The weak hydrogen bonds with bond length N-H to 1.378 Å make the hydrogen easily loose to form N-CMC-2 (Figure 4.f) with a by-product of HCl. The minimum energy of all molecules involved in the reaction mechanism of the O-CMC bond formation of SN2 path is presented in the energy energy potential (Figure 5). Figure 5 shows the reaction rate determinant for N-CMC-1 and N-CMC-2 is the second stage, because it has the greatest activation energy. According to the Arrhenius equation (݇ = ‫݁ܣ‬ ି ா ோ் ൗ ), the greater the value of the activation energy(Ea) the value of the rate constant (k) will be smaller and the reaction rate will be slow at that stage. The value of the rate constant (k) obtained is presented in Table  1. From Table 1, it can be seen that the mechanism of the reaction of N-CMC formation most preferred is through the path SN2. This is because The Ea2 of SN2 pathway is lower than SN1 pathway, It is also because the value of the k of SN2 pathway is greater than the SN1 pathway, so the reaction is faster. The SN2 reaction path is preferable because the positive C atoms of the MCAA are primary C-type in the reaction path SN2 has the fastest reaction rate [15,16]. From the enthalpy energy (ΔH) and gibs free energy, both the N-CMC-1 and N-CMC-2 forming reactions are exothermic and spontaneous.

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The     in the -NH2 group, while N,O-CMC-2 begins substitution in the -NH2 group then substituted in the -OH group. The results obtained are presented in Figure 6. In the N,O-CMC-1 synthesis, the third and fourth phase Ea is smaller than the second stage, so the reaction rate determinant stage is the second stage such as O-CMC-2. As well as the N,O-CMC-2, Ea3 and Ea4 are smaller than Ea1 and Ea2. It's means substitution via the -NH2 group is easier after the chitosan is substituted in the -OH group, because the atomic charge N of -NH2 is initially -0.678 become more negative that is to be -0.685. As well as the N,O-CMC-2 synthesis with the stage of reaction rate at the second stage. It's means substitution of -OH groups is easier to occur after substitution of the -NH2 group.
ΔH and ΔG the synthesis reaction of N,O-CMC-1 is -33.47 kJ/mol and -17.48 kJ/mol, whereas ΔH and ΔG the synthesis reaction of N,O-CMC-2 is -4.95 kJ/mol and 15.47 kJ/mol. From the ΔG data it suggest that the N,O-CMC-2 synthesis is positive so that the reaction is not spontaneous and difficult to occur so that the reaction mechanism of N,O-CMC occurs through substitution -OH first, then new -NH2 substituted. Oleh karena itu, untuk mensintesis N, O-CMC secara eksperimental dengan membuat kondisi reaksi untuk O-CMC terbentuk terlebih dahulu, kemudian lanjutkan untuk mengendalikan kondisi reaksi yang akan diganti pada kelompok -NH2.

Conclusion
A mechanism for the carboxymethyl chitosan synthesis by dimer chitosan and monocloro acetic acid by HF calculation was presented. The results showed that the reaction of CMC synthesis via SN2 reaction pathway, based on the kinitecs data, the N-CMC-2 synthesis is most easily formed with Ea determinant of reaction rate is 469.44 kJ/mol, while from the thermodynamic data, ΔG of the O-CMC-2 synthesis is -4.353 kJ/mol more spontaneous than the N-CMC-2 synthesis. Both N-CMC-2 and O-CMC-2 are exothermic reactions. The synthesis of N,O-CMC occurs through the formation of -O-CH2COOH the first and then proceeds to form -NH-CH2COOH, which is indicated by low and negative ΔH and ΔG values of -33.47 kJ/mol and -17.48 kJ/mol.