Molecular docking and molecular dynamics simulation analyses of urea with ammoniated and ammoxidized lignin

Accepted Manuscript Title: Molecular docking and molecular dynamics simulation analyses of urea with ammoniated and ammoxidized lignin Author: Wenzhuo Li Song Zhang Yingying Zhao Shuaiyu Huang Jiangshan Zhao PII: DOI: Reference:

S1093-3263(16)30378-3 http://dx.doi.org/doi:10.1016/j.jmgm.2016.11.005 JMG 6786

To appear in:

Journal of Molecular Graphics and Modelling

Received date: Revised date: Accepted date:

27-5-2016 3-11-2016 6-11-2016

Please cite this article as: Wenzhuo Li, Song Zhang, Yingying Zhao, Shuaiyu Huang, Jiangshan Zhao, Molecular docking and molecular dynamics simulation analyses of urea with ammoniated and ammoxidized lignin, Journal of Molecular Graphics and Modelling http://dx.doi.org/10.1016/j.jmgm.2016.11.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Molecular docking and molecular dynamics simulation analyses of urea with ammoniated and ammoxidized lignin

Wenzhuo Li*, Song Zhang, Yingying Zhao, Shuaiyu Huang, Jiangshan Zhao Department of Chemistry and Material Science, Nanjing Forestry University, Nanjing 210037, People’s Republic of China

*Corresponding author at Department of Chemistry and Material Science, Nanjing Forestry University, Nanjing 210037, People’s Republic of China. Fax: +86 25 85427703. E-mail: [email protected] (W.Z. Li).

Graphical abstract

Highlights  Ammoniated lignin from Mannich reaction is proposed as urea slow-release carrier.  Molecular dynamics simulations and molecular docking were used.  Interaction of urea with ammoniated and ammoxidized lignin was researched.  Three types of interaction modes exist between urea and ammoniated lignin.  Tg and δ of ammoniated and ammoxidized lignin were researched. 1

ABSTRACT

Ammoniated lignin, prepared through the Mannich reaction of lignin, has more advantages as a slow-release carrier of urea molecules than ammoxidized lignin and lignin. The advantages of the ammoniated lignin include its amine groups added and its high molecular mass kept as similar as that of lignin. Three organic molecules including guaiacyl, 2-hydroxybenzylamine and 5-carbamoylpentanoic acid are monomers respectively in lignin, ammoniated lignin and ammoxidized lignin. We studied the difference between the interactions of lignin, ammoniated lignin and ammoxidized lignin with respect to urea, based on radial distribution functions (RDFs) results from molecular dynamics (MD) simulations. Glass transition temperature (Tg) and solubility parameter (δ) of ammoniated and ammoxidized lignin have been calculated by MD simulations in the constant-temperature and constant-pressure ensemble (NPT). Molecular docking results showed the interaction sites of the urea onto the ammoniated and ammoxidized lignin and three different interaction modes were identified. Root mean square deviation (RMSD) values could indicate the mobilities of the urea molecule affected by the three different interaction modes. A series of MD simulations in the constant-temperature and constant-volume ensemble (NVT) helped us to calculate the diffusivity of urea which was affected by the content of urea in ammoniated and ammoxidized lignin.

Keywords: Ammoniated lignin; Ammoxidized lignin; Molecular dynamics simulations; Molecular docking; Urea

2

1. Introduction

It is well known that urea is an important nitrogen fertilizer to get high crop yields. But urea dissolves in water so quickly that it is readily washed away by irrigation water or rainwater when directly fertilized into the soil. This causes a serious problem of nitrogen wasting [1]. Slow-release urea fertilizer has been applied to solve the problem of urea wasting [2, 3]. The urea in slow-release fertilizer is not quickly dissolved out but slowly released into the soil in water. So the utilization ratio of urea in slow-release urea fertilizer can be effectively improved [1]. Ammoxidized lignin has been applied to make slow-release urea fertilizer [4]. It can be prepared by reactions between lignin, ammonia and oxygen under a certain temperature and pressure [5]. In the preparation of ammoxidized lignin, the effect of oxy-ammonolysis may break the aromatic ring and phenolic hydroxyl of lignin. The chemical effect makes the molecular chain of ammoxidized lignin shorter, and its molecular mass lower [6]. It is not suitable that the ammoxidized lignin with short molecular chain and low molecular mass acts as the slow-release carrier of urea. To solve the issue, ammoniated lignin different from ammoxidized lignin on their structure enters our sights. The ammoniated lignin can be prepared by Mannich reaction and it is a good slow-release fertilizer carrier in practice [7, 8]. This is because the ammoniated lignin obtained after the Mannich reaction still retains a similar hydrophilicity and high a molecular mass as its precursor-lignin [7]. The Mannich reaction consists of an amino alkylation of an acidic proton placed next to a carbonyl group by formaldehyde and a primary or secondary amine or ammonia [9]. 3

In the Mannich reaction, Mannich reagent and phenolic hydroxyl in lignin can establish H-bond interactions. This leads to the methylation reaction preferentially happening at ortho- position other than at para- position when other groups do not occupy the ortho- positions next to the phenolic hydroxyl. If other groups simultaneously occupy both ortho- and para- positions of phenolic hydroxyl, the Mannich reaction cannot happen in the phenyl ring [10, 11]. Lignin has three types of basic structural units including guaiacyl type, syringyl type and p-hydroxyphenyl type [12, 13], as shown in Fig. 1. Among the three structural units, only the guaiacyl and hydroxyl phenyl units can be ammoniated in the Mannich reaction. On the contrary, the syringyl unit cannot be ammoniated through the Mannich reaction because methoxyl groups have occupied the ortho- and para- positions of the phenolic hydroxyl [14]. As it is well known, the distribution ratios of the three different basic structural units are different in different kinds of plants [15]. For example, broadleaf has more content of syringyl units than that of the other two units [16], while needlebush has more guaiacyl units [17]. Here we choose the lignin from the needlebush as the researched model because the needlebush lignin can carry more amine groups through the Mannich reaction. In practice, lignin as starting material to prepare ammoniated lignin can be achieved in a cheaper way from the black liquor produced in the industrial preparation of paper pulp [7, 18]. Therefore, application of the ammoniated lignin in the slow-release urea fertilizer can reduce the cost of preparing fertilizer, and lighten environmental pollution in some degrees. Slow-release urea fertilizers have been prepared experimentally using lignin (or 4

lignin derivatives) and urea as original materials [8, 19, 20]. However, information about slow-release mechanism at atomic and molecular level from the experiments is indirect and inadequate, which limits the practical application of lignin or lignin derivatives as carriers in slow-releasing fertilizer [21, 22]. Molecular simulation techniques, as a complementary method to experiments can help us predict and even directly “watch” the evolution process and the variation trends of properties of slow-release materials at the atomic and molecular level [23]. Our team has researched the interaction between urea molecules and lignin (or lignosulfonate) using molecular simulation techniques [24, 25]. The ammoniated lignin has many differences on its molecular structures and physical-chemical properties, when compared with its precursor-lignin, the lignosulfonate and the ammoxidized lignin. In this work, we carry out studies on interactions between urea and ammoniated lignin prepared from Mannich reaction. We make a comparison between ammoniated lignin, lignin and ammoxidized lignin on some of their properties like Tg, δ, adsorption and diffusivity of urea on them. From molecular docking results, we can find the interaction sites of the urea molecule on the ammoniated lignin and the interaction modes between them. Analyses of RMSD help us learn the effect of different interaction modes of urea with ammoniated and ammoxidized lignin on the mobilities of urea molecules. Mean-squared displacement (MSD) and the calculated diffusion constants are useful to evaluate the influence of the content of ammoniated lignin and water on the diffusivity of urea molecules through NVT MD simulations.

5

2. Methods

2.1. Models of 2-hydroxybenzylamine and 5-carbamoylpentanoic acid

The models of 2-hydroxybenzylamine and 5-carbamoylpentanoic acid as the monomers of ammoniated lignin and ammoxidized lignin, respectively, were built and geometrically optimized in Material studio software 6.0 (MS6) [26]. The geometrical optimizations were performed using polymer consistent force field (PCFF) and the smart minimization algorithm that switched from steepest-decent to conjugated gradient, and then to a Newton-Raphson method with energy convergence criteria of 2×10-5 kcal/mol. Figs.2(a) and (b) show the geometrical conformations of the two monomers. The PCFF was developed on the basis of CFF91 and was specifically applied to polymers and organic materials [25].

2.2. Urea model

The initial model of urea molecule in this work was taken from its known crystal structure [27]. Then the urea model was geometrically optimized in MS6 using the same minimization methods above, and the optimized urea model was used for the following simulation calculations.

2.3. Model of ammoniated lignin

The procedure to set up the model of ammoniated lignin was according to its experimental preparation process reported in literature [28]. The procedure consisted 6

of three steps: (1) We built the model of needlebush lignin [29] with 20-monomer oligomer, in which the content of guaiacyl monomers occupied 80 wt.%, p-hydroxyphenyl monomers reached to 15 wt.%, and syringyl monomers reached to 5 wt.%. Every two monomers combine to form a β-O-4 linkage dimer as basic structural unit of the lignin. (2) We added one methylamino group on the position next to the phenolic hydroxyl to simulate the effect of the ammonization in the Mannich reaction of lignin as starting material. Then the angles of torsion 1, 2 and 3 stayed manually at the angles of the most stable conformation of 2-hydroxybenzylamine obtained before. The nitrogen content in the built model of ammoniated lignin reached to 2.2 wt.%, which was just in the range of total nitrogen content (2.0~2.5 wt.%) in the real ammoniated lignin reported in the literature [7]. Figs.3 (a) and (b) illustrate the structural models of lignin and ammoniated lignin, and present the IUPAC names of their β-O-4 linkage dimers as structural units. (3) The built ammoniated lignin model was geometrically optimized in MS6 using the same method above and the optimized model was further used for the following simulation calculations.

2.4. Model of ammoxidized lignin

We firstly built the model of needlebush lignin with 15-unit oligomer using the same procedure described before. Then the guaiacyl moieties of lignin model were changed into the structures of 5-carbamoylpentanoic acid, according to the reported references. The conformation of 5-carbamoylpentanoic acid in the polymeric structure 7

stayed manually at the most stable conformation of 5-carbamoylpentanoic acid obtained before. The nitrogen content in the built model of ammoxidized lignin reached to 6.01 wt.%, which was just in the range of total nitrogen content (5%~7%) in the real ammoxidized lignin reported in the literature [30, 31]. The structural model of ammoxidized lignin and the IUPAC name of its β-O-4 linkage dimers as structural units are shown in Fig.3 (c). The molecular mass of the built model of ammoxidized lignin was also 12% lower than that of the lignin, which is consistent with reports in previous references [30, 31]. The built ammoxidized lignin model was geometrically optimized in MS6 using the same minimization method above and the optimized model was further used for the following simulation calculations.

2.5. Models of binary and ternary systems containing ammoniated (or ammoxidized) lignin and urea

Models of binary systems composed of three ammoniated (or ammoxidized) lignin polymer chains and urea molecules were built using Amorphous Cell software (Accelrys Inc.) in three-dimensional (3D) unit cells. The modeling details can be found in previous published literature [25]. There are the two types of binary systems established for different purposes: (1) With the aim to study the differences between the interactions of urea molecule with lignin, ammoniated lignin and ammoxidized lignin, we performed the NVT MD simulations for three binary systems. The three binary systems were composed of (1) urea molecules and lignin (molecular ratio of the former to the latter was 100:1), (2) urea molecules and ammoniated lignin (100:1) 8

and (3) urea molecules and ammoxidized lignin (100:1), respectively. (2) In order to study the effect of the content of ammoniated (or ammoxidized) lignin on the diffusion property of urea molecules, the molar ratios of ammoniated (or ammoxidized) lignin to urea molecules in the final binary systems were respectively set to 3:50, 3:90, and 3:130 according to the reference [25]. The specified densities of the binary systems were set to 1.2 g/cm3 [29]. Models of ternary systems composed of three ammoniated (or ammoxidized) lignin chains, urea molecules and water molecules were built in the same way as binary systems, as shown in Fig. 4. Here, the TIP3P model of water molecules was adopted [32]. The molar ratios between ammoniated (or ammoxidized) lignin, urea and water were set to 3:130:160, 3:130:320 and 3:130:640 according to the references [24, 25, 41], respectively. Such an arrangement of molar ratios was set to study the effect of the water molecules content on the diffusivity of urea molecules. The densities of ternary systems were 1.1 g/cm3. All the built binary and ternary systems went through geometrical optimization in MS6 for the following simulations.

2.6. Calculation methods for Tg and δ

All polymers become a brittle solid or glass state below certain temperatures. As the temperature is raised to a certain level, the properties of the polymer change to those of a rubber [33, 34]. The phase transition temperature is called the glass transition temperature, Tg. One can measure Tg by noting a change in slope of plots of specific volume against temperature [35]. In fact, Tg can be determined by the 9

intersection of two least-square fitted lines representing thermal expansion rates of glassy and rubbery states [35]. On the other hand, cohesion energy (Ecoh) and solubility parameter (δ) are also important thermodynamic properties. Ecoh is used to estimate the energy change on mixing two species [36]. The solubility parameter, δ, can be used to numerically estimate the degree of interaction between polymer materials, and indicate their solubility. This makes δ often applied in industry to predict the compatibility, permeation, and swelling, bulk and solution properties of polymers [37]. The cohesive energy density, εcoh, is defined to be the cohesive energy per molar volume, as described in Eq. (1):  co h  E coh V m

(1)

where Vm is molar volume. The relationship between the δ (J/cm3)1/2 and the εcoh (J/m3) is described as Eq. (2) below:  

 c oh

(2)

2.7. Docking between urea and ammoniated (or ammoxidized) lignin

Docking between the urea molecule and the ammoniated (or ammoxidized) lignin by the AutoDock (4.0) was performed in order to find the available interaction sites of the former on the latter [25, 38]. Ammoniated lignin is a branched macromolecule, the structure of which can be twisted, bended and even folded. This makes lignin like protein to form minor grooves. The urea molecule as small intercalator can produce distinctive thermodynamic binding with the host grooves in 10

lignin through different driving forces. The diverse binding sites and binding forces between the urea molecules and the lignin can cause various diffusion cases of urea in lignin. In docking, one urea molecule as a ligand could move freely, whereas ammoniated (or ammoxidized) lignin as a receptor was kept rigid. Several cubic boxes with the size of 5 Å × 5 Å × 5 Å were used to overlay the whole ammoniated (or ammoxidized) lignin molecule and then to calculate atomic interaction energy grid maps. The Lamarckian genetic algorithm (LGA) [39] was selected for the ligand conformational search and then the energy is calculated through a scoring function using the grid maps. The LGA algorithm uses a five-term force field-based function derived from the AMBER force field, and that comprises a Lennard-Jones dispersion term, a directional hydrogen bonding term, a coulombic electrostatic potential, an entropic term, and an intermolecular pair-wise desolvation term. Each LGA job consisted of 200 runs, and the number of generation in each run was 27,000 with an initial population of 50 individuals. The maximum number of energy evaluations was set to 1,000,000. Operator weights for cross-over, mutation, and elitism were 0.80, 0.02, and 1, respectively. The docking results were clustered using a tolerance of 1 Å root-mean-square deviation (RMSD), and the most favorable binding conformation with the lowest free energies was selected within the top-ranked cluster.

2.8. Diffusivity of urea molecules affected by ammoniated or ammoxidized lignin

Diffusivity (or diffusion constant) of urea molecules is an important macroscopic property to characterize the urea release behavior in slow-release fertilizers [40]. In 11

the slow-release fertilizers consisting of urea and ammoniated lignin, the diffusivity of urea can be greatly affected by the molecular ratios between urea and ammoniated lignin. Diffusion constant can be calculated according to Einstein equation or the slope of the MSD against time plot [25, 41], i.e. D 

1 6N

lim

t 

d dt

2

N

  r  t   r 0  i

(4)

i

i

where D is the diffusion constant, N is the total number of atoms, ri is the position of atom i, and t is time.

2.9. MD Simulation details

2.9.1. NPT molecular dynamics

For calculations of Tg and δ of ammoniated (or ammoxidized) lignin, fifteen cells (the size of every cell was set to 27 Å×27 Å×27 Å), each of which was composed of three lignin molecules, were built with Amorphous Cell Calculation. Three out of fifteen cells with the lowest total potential energies were selected for the following simulations. Each selected cell was subjected to 3000 steps of geometry optimization with the smart minimization algorithm that switched from steepest-decent to conjugated gradient, and then to a Newton-Raphson method as the energy derivative was decreased to 2×10-5 kcal/mol [42, 43]. The energy-minimized ammoniated (or ammoxidized) lignin was further annealed using a NPT ensemble at 240 °C for 30 ns until equilibration. Then, we ran the MD simulations at 30 °C intervals from 240 °C to -30 °C using the final equilibrium structure obtained at the higher temperature as 12

the starting structure for the next temperature [29]. At each target temperature, a 100 ns MD simulation was carried out, and in the last 50 ns, simulation data were collected at every 50 fs for the analyses of static and dynamic properties of the molecular systems. The atom-based summation method with a cut off of 9.5 Å was used to evaluate van der Waals interactions [29]. The Ewald summation method with precision of 1×10-4 kcal/mol was used to evaluate coulombic interactions. Initial velocities were randomly designated from Boltzmann distribution [44]. A modified Hoover algorithm was employed to control temperature and pressure with the specific time constants set between 0.5 and 2 ps [45]. The PCFF forcefield and periodic boundary conditions were employed here. Charge equilibration (Qeq) method was used to calculate atomic charges [46]. The basis of the Qeq method is the equilibration of atomic electrostatic potentials with respect to a local charge distribution [46]. The time step was set to 1fs. As a comparison, we also applied the same calculations of Tg and δ described above to ammoniated lignin and ammoxidized lignin. In order to calculate RMSDs between ammoniated (or ammoxidized) lignin and urea, several models composed of one ammoniated (or ammoxidized) lignin molecule and one urea molecule, which were obtained from docking results between ammoniated (or ammoxidized) lignin and urea above, were set up for the subsequent NPT MD simulations. The NPT MD simulations were carried out for 130 ns at 25 °C for the selected models. In the NPT MD simulations, the first 30 ns were used for equilibration, and the next 100 ns were used to collect data. The PCFF forcefield was still used here, and time step was set to 1fs. Other simulation details were the same as 13

the details described above for the calculations of Tg and δ.

2.9.2. NVT molecular dynamics NVT MD simulations were employed to (1) learn the interaction of urea molecules with ammoniated and ammoxidized lignin, and (2) study the diffusivity of urea molecules adsorbed on the ammoniated and ammoxidized lignin. Several binary and ternary systems, with the specific proportions between ammoniated lignin, urea and water, were built as described in the last section. The minimizations of the binary and ternary systems were performed using the smart minimization algorithm with energy convergence criteria of 2×10-5 kcal/mol. The minimization process was found to be efficient and was typically converged in a single minimization run. Non-bonded interactions were simply calculated to a defined cut-off distance (rcut=12 Å) and interactions beyond this distance were ignored [24]. The dynamic simulation was performed using the Verlet leapfrog integrator under fixed composition with periodic boundary conditions [47]. NPT MD simulations for the binary and ternary systems were firstly carried out for 30 ns at 25 °C until equilibration. Then, 100 ns NVT MD simulations with the last 50 ns for data collection were performed to get the diffusivity of urea molecules in the binary and ternary systems. The equation of motion was solved using the Verlet algorithm with a time step of 1 fs [48]. The PCFF forcefield, the Qeq method, the atom-based summation method for van der Waals interactions and the Ewald summation for coulombic interactions were applied here.

14

3. Results and discussion

3.1. Interactions of lignin, ammoniated lignin and ammoxidized lignin with urea molecules

The main differences between the molecular structures of lignin, ammoniated lignin and ammoxidized lignin occur in their guaiacyl moieties. The rest of the studied molecular systems, except the guaiacyl moiety, are very similar on their structures and compositions. After the Mannich reaction, the guaiacyl of lignin changes into 2-hydroxybenzylamine moiety of ammoniated lignin. After the oxy-ammonolysis reaction, the guaiacyl of lignin changes into 5-carbamoylpentanoic acid moiety, and at the same time, the molecular chain of lignin becomes shorter due to the ring cleavage reaction occurring in the guaiacyl. The interaction of urea molecule with guaiacyl moiety, 2-hydroxybenzylamine moiety and 5-carbamoylpentanoic acid moiety in the polymeric structures can indicate the difference between the interactions of urea molecule with respect to lignin, ammoniated lignin and ammoxidized lignin. Fig. 5(a) shows RDF of the H atoms of urea molecules and the N atoms of amine groups in every 2-hydroxybenzylamine moiety of ammoniated lignin, which is derived from the NVT MD simulation results. From Fig. 5(a), we found that some urea molecules can appear within the 2.5 Å radius around the amine of 2-hydroxybenzylamine. This could be explained as a result of the H-bonds established between the 2-hydroxybenzylamine N atoms and urea H atoms, according to our previous researches [24, 25]. Fig. 5(b) shows RDF between the mass centers of the C=O group 15

of urea molecule and the phenyl group of 2-hydroxybenzylamine amine. It can be observed that some urea molecules can appear within the vertical distance of 3.5 Å far from the phenyl plane of 2-hydroxybenzylamine. This is thought to be a result of pi-stacking

interaction

between

the

C=O

of

urea

and

the

phenyl

of

2-hydroxybenzylamine [25]. The number of urea molecules appearing around the 2-hydroxybenzylamine amine in the range of H-bonds and pi-stacking interactions can reflect the interaction between them. The number of urea molecules can be estimated from the integral of the corresponding RDF line [49, 50], as described in Eq. (3): N



 4 

r max



r

2

g  ( r )r dr

(3)

min

where Nαβ is the coordination number of β atoms around α atoms, ρβ is the number density of β atoms in the system, and the ranges of integration (rmax) are normally chosen to less than 2.5 Å for H-bonds and less than 3.5 Å for pi-stacking interactions [25, 51]. Table 1 lists the numbers of urea molecules appearing around guaiacyl moieties, 2-hydroxybenzylamine moieties and 5-carbamoylpentanoic acid moieties in their polymeric structures within the range of H-bonds and pi-stacking interaction, respectively. From Table 1, we can note that the calculated numbers of urea molecules appearing around the every guaiacyl (5.15) moiety and every 2-hydroxybenzylamine (5.13) moiety of their polymeric structures in the range of H-bonds and pi-stacking interaction are similar. This suggests that the ammoniated lignin after the Mannich reaction still retains the ability to interact similarly with urea molecules as lignin. Considering there are more amine groups (containing N element) in the ammoniated 16

lignin, the ammoniated lignin with N element should be more suitable to be slow-release nitrogen fertilizer than the lignin without N element. In the polymeric structures, the calculated number (2.82) of urea molecules adsorbed on the every 5-carbamoylpentanoic moiety of ammoxidized lignin is about one half less than that (5.13) on the every 2-hydroxybenzylamine moiety of ammoniated lignin. This indicates that the interaction of urea molecule with the ammoxidized lignin formed after the oxy-ammonolysis reaction is weaker than that of the ammoniated lignin formed after the Mannich reaction.

3.2. Comparison of Tg and δ values of lignin, ammoniated lignin and ammoxidized lignin

Tg is an important property to influence usability and processing craft of lignin-related polymers. Tg marks the temperature at which major segments of polymer are no longer held in a rigid state with motion restricted to vibrational movement of individual atoms, but sections of polymer chains begin to move [52]. Figs. 6 (a), (b) and (c) show fitting lines (R2 0.9 for (a), R2 0.89 for (b) and R2 0.91 for (c), generated from NPT MD simulations) of specific volume to temperature of lignin, ammoniated lignin and ammoxidized lignin, respectively. It can be seen from Fig. 6 (a) that the Tg (120.1 °C) of lignin calculated from our molecular simulations is in the range of experimental Tgs (120~150 °C) reported by Back and Salmen [53]. This suggests that the built lignin-related models and simulation methods employed in this 17

work are reliable. From Fig. 6 (b), one can know that Tg of ammoniated lignin is 138.0 °C, which is about 15% higher than that of lignin. The phenomenon that Tg (ammoniated lignin)

is higher than Tg (lignin) is considered to be a result of the introduction of

amine group into the ammoniated lignin. The amine groups of the ammoniated lignin cause that the number of the intramolecular and the intermolecular H-bonds from the ammoniated lignin molecules is more than that from the lignin molecules. As the number of the intramolecular and the intermolecular H-bonds in the polymer system increases, the cohesive energy density of the system also increases. So the cohesive energy density of ammoniated lignin should be larger than that of lignin, which has been proved by data listed in Table 2. Fig. 6 (c) shows that Tg of ammoxidized lignin is 109.0 °C, which is about 9% lower than that of lignin. For this, the phenomenon can be explained by two reasons: (1) the molecular weight of ammoxidized lignin is 12% lower than that of lignin; (2) the “stiff” structure of benzene ring is changed into the “soft” structure of 5-carbamoylpentanoic acid in the ammoxidized lignin, which makes the polymer chain of ammoxidized lignin more flexible than that of lignin. The stiff polymer like lignin has generally higher Tg than the flexible polymer like ammoxidized lignin [54]. Table 2 also lists the calculated δs of lignin, ammoniated lignin and ammoxidized lignin obtained by molecular simulations. The calculated δ of lignin here is 21.87 (J/cm3)1/2, just being in the range of experimental values (20.5~26.5 (J/cm3)1/2) reported in literature [55, 56]. This again indicates the validity of the models and force field employed in our work. From Table 2, the calculated δ of ammoniated lignin (22.12 (J/cm3)1/2) is only about 8% slightly lower than δ of 18

ammoxidized lignin (24.16 (J/cm3)1/2). In contrast, the variation between Tgs of ammoniated lignin and ammoxidized lignin reaches approx. 21%, as discussed above. Therefore, it is thought that in the systems containing ammoniated and ammoxidized lignin, their Tgs are more sensitive to the variation of the intermolecular energy (or the cohesive energy density) than their δs.

3.3. Available interaction sites of urea on ammoniated lignin and ammoxidized lignin

Figs. 7(a) and (b) shows the docking results of urea on the surface of ammoniated lignin and ammoxidized lignin. We could find that there are a total of 17 (or 7) interaction sites of urea on one ammoniated lignin (or ammoxidized lignin). In these interaction sites, there are three different interaction modes between urea and ammoniated lignin. The first interaction mode is pi-stacking, interaction sites with which occupy 5.9%, established between the C=O bond of urea molecule and the phenyl ring of ammoniated lignin, as shown in Fig. 8 (Ia). Details about the pi-stacking between the C=O bond of urea and the C=C bonds of phenyl ring of lignin molecule have been discussed in our previous published papers [25]. The pi-stacking only exists in between the urea and the ammoniated lignin, not in between the urea and ammoxidized lignin without the phenyl ring. The second interaction mode is only one H-bond, interaction sites which have an occupancy of 64.7% (or 57.1%), established between the polar groups of ammoniated lignin (or ammoxidized lignin) and the urea molecule, as shown in Figs. 8 (Ib) and (Ic) (or Figs. 8 (IIa) and (IIb)). The third interaction mode is two H-bonds, interaction sites with which occupy 29.4% 19

(or 42.9%), forming between the polar groups of ammoniated lignin (or ammoxidized lignin) and the urea molecule, as shown in Figs. 8 (Id) and (Ie) (or Figs. 8 (IIc) and (IId)). It is worthy to note that the content (5.9%) of pi-stacking interaction sites between urea and ammoniated lignin is far less than the reported content (44%) between urea and lignin in our previous research [25]. It can be concluded that it is the introduction of amine into the phenyl ring of ammoniated lignin that causes the difference between the contents of pi-stacking interaction sites of urea on the ammoniated lignin and lignin. The urea molecule near a phenyl ring with one amine attached prefers to move close to the amine to form H-bond rather than to the phenyl ring to form pi-stacking interaction. This is because the energy of one H-bond is more than that of one pi-stacking interaction, and the formation of one H-bond can make system more stable than that of one pi-stacking interaction [25]. Molecular docking results suggest that there are higher ratios of H-bond interaction sites of urea on the ammoniate lignin (or ammoxidized lignin) than that of urea on the lignin. This also indicates that there are stronger interactions between urea and ammoniated lignin (or ammoxidized lignin) than between urea and lignin.

3.4. Mobility of urea molecules affected by the different interaction modes of urea with ammoniated lignin or ammoxidized lignin

From molecular docking results as mentioned above, three different interaction modes were identified between urea and ammoniated lignin (or ammoxidized lignin): pi-stacking interaction, only one H-bond, and two H-bonds. In order to elucidate how 20

the three different interaction modes affect the mobility of urea molecules, RMSD method was used. The RMSD is a measure of molecular mobility, which is calculated by rotating and translating the coordinates of the instantaneous structure to superimpose it with the reference structure in order to achieve a maximum overlap [25, 57]. Figs. 9(Ia) and (Ib) show the variation of the RMSD value of urea molecules during simulation time in the two interaction sites of ammoniated lignin: 11 (representing the interaction mode of pi-stacking interaction) and 16 (representing the interaction modes of only one H-bond). The larger the degree of the fluctuation amplitude of RMSD curve of small molecule, the larger the degree of mobility of the small molecule, the weaker the interaction between small guest molecule and large host polymer molecule [24,25,41] The fluctuation amplitude of the curve for the pi-stacking interaction is larger than that of the curve for the one H-bond effect in Fig.9. This indicates that the pi-stacking interaction is weaker than the H-bonds effect, which leads to the larger mobility of urea molecule at the pi-stacking interaction site than that at the H-bond interaction site. The same conclusion was also drawn in other reported research [25]. Fig. 9 (Ic) exhibits the variation of the RMSD value of urea molecules with time in the interaction site 16 of ammoniated lignin (representing the interaction mode of two H-bonds). One can see that the fluctuation amplitude of the curve for only one H-bond effect is larger than that of the curve for the two H-bonds effect [24,25]. This is caused by the fact that the two H-bonds effect is stronger than only one H-bond effect. Fig.9 (II) also proves that the same story above also occurs between urea and the ammoxidized lignin. Figs. 9 (IIa) and (IIb) exhibit the variation 21

of the RMSD value of urea molecules on the ammoxidized lignin with time in the interaction sites 1 (representing the interaction mode of one H-bond) and 4 (representing the interaction mode of two H-bonds). So it is suggested that as amine groups (or other polar groups such as carboxyl and carbonyl), which are not contained into lignin structure, are introduced into the ammoniated lignin (or ammoxidized lignin), the formation of more H-bonds between amine groups (or other polar groups) and urea molecules can produce the stronger interaction of urea molecules with the ammoniated lignin (or ammoxidized lignin) than with the lignin.

3.5. Diffusivity of urea molecules affected by the molecular ratios between urea and ammoniated or ammoxidized lignin Figs. 10 (I) and (II) show the MSDs of urea molecules in three binary systems composed of the ammoniated (or ammoxidized) lignin polymer chains and the urea molecules with the molar ratios being (I) 3:50, (II) 3:90 and (III) (or (VI)) 3:130 (or (IV) 3: 50, (V) 3:90 and (VI) 3:130 for ammoxidized lignin), respectively. From Fig.10, the calculated diffusion constants of urea molecules are 6.56×10-11 m2/s, 8.37×10-11 m2/s and 1.01×10-10 m2/s for binary systems I, II and III of ammoniated lignin, and 7.16×10-11 m2/s, 9.15×10-11 m2/s and 1.16×10-10 m2/s for binary systems IV, V and VI of ammoxidized lignin, respectively. Apparently, as the content of urea in these binary systems increases, the calculated diffusion constant of urea molecules increases. And the diffusion constant values of urea molecules in the ammoniated lignin are lower than in the ammoxidized lignin, possible due to the molecular chain (or molecular mass) of the ammoxidized lignin shorter (or lower) than that of the 22

ammoniated lignin. This suggests that the ammoniated lignin is more proper used as slow release carrier of urea than the ammoxidized lignin. We also built three ammoniated lignin-urea-water ternary systems to research how the content of water molecules affected the diffusivity of urea molecules. Molar ratios between ammoniated lignin, urea and water are (a) 3:50:160, (b) 3:50:320, and (c) 3:50:640 in the ternary systems, respectively. In the ternary systems, the calculated average diffusion constant of water was D=0.83×10-9 m2/s, which is lower than experimental value (D=2.3×10-9 m2/s) in bulk water at 298 K [58]. The lower value of D should be caused by the interaction between the water and the ammoniated lignin. Moreover, we found that the calculated diffusion constant of urea molecules in these ternary systems (D=2.41×10-10 m2/s in system a, 3.01×10-10 m2/s in system b, 3.66×10-10 m2/s in system c) increases with the content of water molecules increasing. This could demonstrate a phenomenon where the water molecules can promote the urea molecules to leave away from the ammoniated lignin. The phenomenon is also consistent with experimental observations [59] where the release rate of urea molecules in the lignin-related polymers increases with the increment of the water content in soil.

4. Conclusions

In this work, we have conducted molecular dynamics simulations and molecular docking to study the interaction between urea and ammoniated lignin prepared through the Mannich reaction. (1) From the interaction of urea molecule respectively 23

with ammoniated lignin and ammoxidized lignin, we conclude that the interaction of urea molecule with the ammoniated lignin is stronger than that with the ammoxidized lignin. (2) From the results of NPT MD simulations for Tg and δ of the ammoniated lignin and the ammoxidized lignin, Tg (138.0 °C) of ammoniated lignin is higher than that (109.0 °C) of ammoxidized lignin. And the δ (22.12 (J/cm3)1/2) of ammoniated lignin is similar as that (24.16 (J/cm3)1/2) of lignin. (3) Molecular docking results show the interaction sites and interaction modes between urea molecules and ammoniated (or ammoxidized) lignin. (4) RMSD analyses demonstrate that the introduction of polar groups (such as amine groups, carboxyl and carbonyl) into the ammoniated and ammoxidized lignin can produce the stronger interaction of urea molecules with the ammoniated and ammoxidized lignin than with the lignin. (5) Results from a series of NVT MD simulations indicate that the diffusion constant values of urea molecules in the ammoniated lignin are lower than in the ammoxidized lignin. Finally, results from molecular dynamics simulations and molecular docking in this work provide a certain support that the ammoniated lignin is more proper as a promising slow-release carrier of urea fertilizer applied in the efficient agriculture than the ammoxidized lignin.

Acknowledgements

This work was financially supported by the Natural Science Foundation of China (Grant No. 30871988) and the Jiangsu Provincial Science and Technology Project 24

(BK2014147110).

25

References: 1. L. Wu, M.Z. Liu, and R. Liang, Preparation and properties of a double-coated slow-release npk compound fertilizer with superabsorbent and water-retention. Bioresour. Technol. 99 (2008) 547-554. 2. E.I. Pereira, C.C.T. Da Cruz, A. Solomon, A. Le, M.A. Cavigelli, and C. Ribeiro, Novel slow-release nanocomposite nitrogen fertilizers: the impact of polymers on nanocomposite properties and function. Ind. Eng. Chem. Res. 54 (2015) 3717-3725. 3. H.T. Zou, Y. Ling, X.L. Dang, N. Yu, Y. Zhang, Y. Zhang, and J. Dong, Solubility characteristics and slow-release mechanism of nitrogen from organic-inorganic compound coated urea. Int. J. Photoenergy 2015 (2015) 1-6. 4. K.M. Klinger, F. Liebner, T. Hosoya, A. Potthast, and T. Rosenau, Ammoxidation of lignocellulosic materials: formation of nonheterocyclic nitrogenous compounds from monosaccharides. J. Agric. Food Chem. 61 (2013) 9015-9026. 5. K.M. Klinger, F. Liebner, I. Fritz, A. Potthast, and T. Rosenau, Formation and ecotoxicity of n-heterocyclic compounds on ammoxidation of mono- and polysaccharides. J. Agric. Food Chem. 61 (2013) 9004-9014. 6. L. Cao, J.Y. Quan, and Z.Z. Li, A study on the structure of ammoxidative lignin. J. Nanjing for. Univ. Nat. Sci. Ed. 22 (1998) 66-70. 7. X.Y. Du, J.B. Li, and M.E. Lindström, Modification of industrial softwood kraft lignin using mannich reaction with and without phenolation pretreatment. Ind. Crop. Prod. 52 (2014) 729-735. 8. S. Ariyanti, Z. Man, and B.M. Azmi, Improvement of hydrophobicity of urea modified tapioca starch film with lignin for slow release fertilizer. Adv. Mater. Res. 626 (2013) 350-354. 9. T. Akiyama, J. Itoh, K. Yokota, and K. Fuchibe, Enantioselective mannich-type reaction catalyzed by a chiral brønsted acid. Angew. Chem. Int. Ed. 43 (2004) 1566-1568. 10. S. Laurichesse, and L. Avérous, Chemical modification of lignins: towards biobased polymers. Prog. Polym. Sci. 39 (2014) 1266-1290. 11. Y.Y. Ge, Q.P. Song, and Z.L. Li, A mannich base biosorbent derived from alkaline lignin for lead removal from aqueous solution. J. Ind. Eng. Chem. 23 (2015) 228-234. 12. C.F. Lima, L.C.A. Barbosa, M.N.N. Silva, J.L. Colodette, and F.O. Silvério, In situ determination of the syringyl/guaiacyl ratio of residual lignin in pre-bleached eucalypt kraft pulps by analytical pyrolysis. J. Anal. Appl. Pyrolysis 112 (2015) 164-172. 13. J.K. Xu, Y.C. Sun, and R.C. Sun, Synergistic effects of ionic liquid plus alkaline pretreatments on eucalyptus: lignin structure and cellulose hydrolysis. Process Biochem. 50 (2015) 955-965. 14. Y. Matsushita, and S. Yasuda, Reactivity of a condensed – type lignin model compound in the mannich reaction and preparation of cationic surfactant from sulfuric acid lignin. J. Wood Sci. 49 (2003) 166-171. 15. T. Kishimoto, W. Chiba, K. Saito, K. Fukushima, Y. Uraki, and M. Ubukata, Influence of syringyl to guaiacyl ratio on the structure of natural and synthetic lignins. J. Agric. Food Chem. 58 (2010) 895-901. 16. B. Chavez-Vergara, A. Merino, G. Vázquez-Marrufo, and F. García-Oliva, Organic matter dynamics and microbial activity during decomposition of forest floor under two native neotropical oak species in a temperate deciduous forest in mexico. Geoderma 235 (2014) 133-145. 17. Y. Fujii, H. Kawamoto, S. Tohno, M. Oda, W. Iriana, and P. Lestari, Characteristics of 26

carbonaceous aerosols emitted from peatland fire in riau, sumatra, indonesia (2): identification of organic compounds. Atmos. Environ. 110 (2015) 1-7. 18. G. Gellerstedt, Softwood kraft lignin: raw material for the future. Ind. Crop. Prod. 77 (2015) 845-854. 19. M.C. García, J.A. Díez, and A. Vallejo, Use of kraft pine lignin in controlled-release fertilizer formulations. Ind. Eng. Chem. Res. 35 (1996) 245-249. 20. H.W. Qiu, and H.Z. Chen, Structure, function and higher value application of lignin. J. Cell. Sci. Technol. 14 (2006) 52-59. 21. N. Zhao, Y. Lü, and G. Li, Characterization and three-dimensional structural modeling of humic acid via molecular mechanics and molecular dynamic simulation. Chem. Res. Chin. Univ. 29 (2013) 1180-1184. 22. L. Petridis, S.V. Pingali, and V. Urban, Self-similar multiscale structure of lignin revealed by neutron scattering and molecular dynamics simulation. Phys. Reve. 83 (2011) 061911. 23. A.K. Sangha, L. Petridis, J.C. Smith, A. Ziebell, and J.M. Parks, Molecular simulation as a tool for studying lignin. Environ. Prog. Sust. Energ. 31 (2012) 47-54. 24. W.Z. Li, J.L. Wang, D.J. Xu, and S. Zhang, Study on adsorption and desorption of urea in lignosulfonate with molecular simulations. Comput. Theor. Chem. 1033 (2014) 60-66. 25. W.Z. Li, J.L. Wang, Interactions between lignin and urea researched by molecular simulation. Mol. Simul. 38 (2012) 1048-1054. 26. Accelrys Software Inc. Materials Studio Release Notes, Release 4.3, Accelrys Software Inc., San Diego, CA, 2008. 27. D. Mullen, Electron-density distribution in urea. A multipolar expansion. Acta. Cryst. B36 (1980) 1610-1615. 28. X.L. Sun, X.H. Zhao, Y.G. Zu, W.G. Li, and Y.L. Ge, Preparing, characterizing, and evaluating ammoniated lignin diesel from papermaking black liquor. Energ. Fuel. 28 (2014) 3957-3963. 29. L. Zhang, and E.J. LeBoeuf, A molecular dynamics study of natural organic matter: 1. Lignin, kerogen and soot. Org. Geochem. 40 (2009) 1132-1142. 30.

Z.H. Zhu, D.H. Wang, Z.W. Liao, and Z.L. You, Fertilizer efficiency of ammonia-oxidized

lignin(AOL) a modified lignin from wastewaterin paper-making used as a slow -released Nitrogen Fertilizer. Agro. Environ. Prot. 20(2001) 98-100. 31.

Z.H. Zhu, D.H. Wang, Z.W. Liao, and Z.L. You, Study on ammoxidized lignin used as

nitrification inhibitor. Soil Environ. Sci. 9(2000) 132-134. 32.

J. Azamat, A. Khataee, and S.W. Joo, Molecular dynamics simulation of trihalomethanes

separation from water by functionalized nanoporous graphene under induced pressure. Chem. Eng. Sci. 127(2015) 285-292. 33. M.P. Stevens. Polymer chemistry: an introduction. Addison-Wesley Publishing Company, Inc., Canada, 1975. 34. J.D. Murillo, M. Moffet, J.J. Biernacki, and S. Northrup, High-temperature molecular dynamics simulation of cellobiose and maltose. Aiche J. 61 (2015) 2562-2570. 35. L. Petridis, R. Schulz, and J.C. Smith, Simulation analysis of the temperature dependence of lignin structure and dynamics. J. Am. Chem. Soc. 133 (2011) 20277-20287. 36. A.Y. Alentiev, and Y.P. Yampolskii, Meares equation and the role of cohesion energy density in diffusion in polymers. J. Membrane Sci. 206 (2002) 291-306.

27

37.

X.P. Chen, C. Yuan, C.K.Y. Wong, G.Q. Zhang, Molecular modeling of temperature

dependence of solubility parameters for amorphous polymers. J. Mol. Model. 18 (2012) 2333-2341. 38. M. Awasthi, N. Jaiswal, S. Singh, V.P. Pandey, and U.N. Dwivedi, Molecular docking and dynamics simulation analyses unraveling the differential enzymatic catalysis by plant and fungal laccases with respect to lignin biosynthesis and degradation. J. Biomol. Struct. Dyn. 33 (2015) 1835-1849. 39.

G.M. Morris, R. Huey, and W. Lindstrom, AutoDock4 and AutoDockTools4: Automated docking

with selective receptor flexibility. J. Comput. Chem. 30(2009) 2785-2791. 40. Y. Shih, J. Lin, S. Wu, and L. Lee, Molecular dynamic simulations of the sorption of toluene in a dry humic acid model: a preliminary study. Colloid. Surface. A 275 (2006) 183-186. 41. T. Vu, A. Chaffee, and I. Yarovsky, Investigation of lignin-water interactions by molecular simulation. Mol. Simul. (2002). 42.

A.H. Korayem, M.R. Barati, and S.J. Chen, Optimizing the degree of carbon nanotube dispersion

in a solvent for producing reinforced epoxy matrices. Powder Technol. 284 (2015) 541-550. 43. P.T.V. Nguyen, H.B. Yu, and P.A. Keller, identification of chikungunya virus nsp2 protease inhibitors using structure-base approaches. J. Mol. Graph. Model. 57 (2015) 1-8. 44. C.P. McNew, and E.J. LeBoeuf, The role of attached phase soil and sediment organic matter physicochemical properties on fullerene (nc60) attachment. Chemosphere 139 (2015) 609-616. 45. A. Loya, J.L. Stair, A.R. Jafri, K. Yang, and G. Ren, A molecular dynamic investigation of viscosity and diffusion coefficient of nanoclusters in hydrocarbon fluids. Comput. Mater. Sci. 99 (2015) 242-246. 46. A.K. Rappe, and W.A. Goddard, Charge equilibration for molecular dynamics simulations. J. Phys. Chem. 95 (1991) 3358-3363. 47. H. Wang, X.F. Zeng, W.C. Wang, and D.P. Cao, Selective capture of trace sulfur gas by porous covalentorganic materials. Chem. Eng. Sci. 135 (2015) 373-380. 48. K.Y. Bell, and E.J. LeBoeuf, Mechanistic investigation of non-ideal sorption behavior in natural organic matter. 1. Vapor phase equilibrium. Environ. Sci. Technol. 46 (2012) 6689-6697. 49. B.L. Nguyen, and B.M. Pettitt, Effects of acids, bases, and heteroatoms on proximal radial distribution functions for proteins. J. Chem. Theory Comput. 11 (2015) 1399-1409. 50. D.H. Brookes, and T. Head-Gordon, Family of oxygen–oxygen radial distribution functions for water. J. Phys. Chem. Lett. 6 (2015) 2938-2943. 51. P. Gemünden, C. Poelking, K. Kremer, K. Daoulas, and D. Andrienko, Effect of mesoscale ordering on the density of states of polymeric semiconductors. Macromol. Rapid Commun. 36 (2015) 1047-1053. 52. G. Strobl. The physics of polymers. Springer-Verlag, Berlin Heidelberg, 2012. 53. E.L. Back, and N.L. Salmen, Glass transition of wood components hold implication for molding and pulping processes. Tappi 65 (1982) 107-110. 54.

K. Kunal, C.G. Robertson, S. Pawlus, Role of chemical structure in fragility of polymers: a

qualitative picture. Macromolecules 41(2009) 7232-7238. 55. J. Zhao, and R.M. Wilkins, Controlled release of the herbicide, fluometuron, from matrix granules based on fractionated organosolv lignins. Journal of Agricultural and Food Chemistry 51 (2003) 4023-4028. 56. A.A. MacKay, and P.M. Gschwend, Sorption of monoaromatic hydrocarbons to wood. Environmental Science & Technology 34 (2000) 839-845. 28

57. M.F. Gerini, D. Roccatano, and E. Baciocchi, Molecular dynamics simulations of lignin peroxidase in solution. Biophys. J. 84 (2003) 3883-3893. 58. W.S. Price, H. Ide, and Y. Arata, Self-diffusion of supercooled water to 238 k using pgse nmr diffusion measurements. J. Phys. Chem. A 103 (1999) 448-450. 59. W.Z. Li, J.L. Wang, and M.X. Gao, Slow releasing properties for urea molecules of composite materials made of lignin and clay. J. Nanjing for. Univ. Nat. Sci. Ed. 37 (2013) 91-95.

29

Figure Captions Fig. 1 Mannich reactions of guaiacyl, syringyl and p-hydroxyphenyl units. Fig. 2 Geometrical conformations of (a)

2-hydroxybenzylamine and (b)

5-carbamoylpentanoic acid, O atom in red, N atom in blue, C atom in grey, H atom in white. Fig. 3 2D structures of polymers and IUPAC names of their β-O-4 linkage dimers as structural units of the polymers: (a) lignin, (b) ammoniated lignin and (c) ammoxidized lignin molecules. Fig. 4 3D model of the ammoniated lignin-urea-water system used in molecular simulation, lignin in red, urea in blue, water in green. Fig. 5 RDFs between (a) the H atoms of urea molecules and the N atoms of amino groups in 2-hydroxybenzylamine of ammoniated lignin, (b) the mass centers of C=O group of urea molecule and phenol group of 2-hydroxybenzylamine of ammoniated lignin from NVT MD simulations for the ammoniated lignin and urea molecules. Straight dash lines and arrows denote the positions of rmaxs of H-bonds or pi-stacking interaction, and shadow areas represent the numbers of urea appearing around 2-hydroxybenzylamine in the range of H-bonds or pi-stacking interaction. Fig. 6 Plots of specific volume against temperature for (a) lignin, (b) ammoniated lignin and (c) ammoxidized lignin. Fig. 7 Interaction sites of urea molecule located on the polymeric structures: (a) ammoniated lignin and (b) ammoxidized lignin, through molecular docking. Fig. 8 Scheme of interaction modes (I) between urea molecule and ammoniated lignin, (a) pi-stacking (see interaction site 11 in Fig. 7 (a)), (b) only one H-bond established between amine of ammoniated lignin and oxygen atom of urea (see interaction site 16 in Fig. 7 (a)), (c) only one H-bond forming between hydroxyl of ammoniated lignin and hydrogen atom of urea (see interaction site 5 in Fig. 7 (a)), (d) and (e) two 30

H-bonds forming between urea and ammoniated lignin (see interaction sites 3 and 14 in Fig. 7(a)); (II) (a) only one H-bond established between hydroxyl of ammoxidized lignin and oxygen atom of urea (see interaction site 1 in Fig. 7 (b)), (b) only one H-bond forming between carbonyl of ammoxidized lignin and hydrogen atom of urea (see interaction site 5 in Fig. 7 (b)), (c) and (d) two H-bonds forming between urea and ammoxidized lignin (see interaction sites 3 and 4 in Fig. 7(b)). Fig. 9 RMSD against time of urea molecule (I) in ammoniated lignin: (a) at the interaction site 11 with pi-stacking interaction, (b) at the interaction site 16 with only one H-bond, and (c) at the interaction site 3 with two H-bonds; (II) in ammoxidized lignin: (a) at the interaction site 1 with only one H-bond, and (b) at the interaction site 3 with two H-bonds. Fig. 10 MSDs of urea molecules in three binary systems with three different molar ratios of polymers (: (I) ammoniated lignin and (II) ammoxidized lignin) to urea molecules: (a) 3:50, (b) 3:90, (c) 3:130.

31

Figure 1

32

Figure 2

33

Figure 3(a)

Figure 3(b)

34

Figure 3(c)

35

Figure 4

36

Figure 5

37

Figure 6

38

Figure 7(a)

Figure 7(b)

39

Figure 8

40

Figure 9

41

Figure 10

42

Table 1 The number of urea molecules, appearing around guaiacyl, 2-hydroxybenzylamine and 5-carbamoylpentanoic acid moieties in lignin, ammoniated lignin and ammoxidized lignin, respectively; are in the range of H-bonds and pi-stacking interaction according to results from NVT MD simulations for the systems containing the polymeric structures and urea molecules. the numbers of urea molecules in the range of H-bonds

pi-stacking interaction

H-bonds and pi-stacking interaction

3.87

1.28

5.15

3.11

2.02

5.13

2.82

0

2.82

structure units

guaiacyl moieties in lignin 2-hydroxybenzylamine moieties in ammoniated lignin 5-carbamoylpentanoic acid moieties in ammoxidized lignin

43

Table 2 Summary of calculated Tg, εcoh and δ values of lignin, ammoniated lignin and ammoxidized lignin obtained by molecular dynamics simulations. samples

Tg (°C)

εcoh (×108 J/m3)

δ (J/cm3)1/2

lignin

120.1(+/- 10)a,b

4.78(+/-0.5) a,b

21.87(+/-0.7) a,b

ammoniated lignin

138.0(+/- 8) a,b

4.89(+/-0.1) a,b

22.12(+/-0.3) a,b

ammoxidized lignin

109.0 (+/- 9)a,b

5.81(+/-0.1) a,b

24.16(+/-0.5) a,b

native lignin

120~150 c

4.20~7.0 c

20.5~26.5 c

a

95% confidence interval. over three cells. c Reported experimental values of isolated “native” lignins by Back and Salmen [53]. b Averaged

44