Preparation of New Glycerol-Based Dendrimers and Studies on Their Behavior toward Essential Oil Encapsulation

Two new families of glycerol-based dendrimers (glyceroladendrimers (GADs) and glyceroclickdendrimers (GCDs)) have been synthesized. Three generations have been isolated for each family with good yields and were fully analyzed. The encapsulation of essential oils (citronella and cinnamon) in GADs, GCDs, and also in previously described glycerodendrimers GD-PAMAMs and GD-PPIs has been studied by dynamic-headspace gas chromatography coupled to mass spectrometry. The retention rates obtained were from −35.8 to 26.65% for citronella essential oil and from 2.14 to 38.84% for the cinnamon essential oil. In addition, the best results were obtained with GD-PAMAMs and GD-PPIs of higher generation. The interaction study between essential oils or more precisely their major components have been performed through NMR spectroscopy (1H NMR and DOSY NMR). No direct interactions between dendrimers and essential oils have been observed, but a surprising behavior of compression of the dendrimer in stable emulsions was observed. Indeed, the hydrodynamic radius of GD-PPI-3 has been reduced in the presence of cinnamon essential oil.


INTRODUCTION
Dendrimers are macromolecules synthesized from monomers in a three-dimensional structure following divergent or convergent approaches. 1−3 Due to their multivalent and monodisperse character, dendrimers have stimulated wide interest in the field of chemistry, physics, and biology. 4 Some recent applications have been reported in the literature especially in the fields of drug delivery, 5−7 photodynamic therapy, 8 electronics, 9 catalysis, 10−12 and water purification. 13 In the past few years, some glycerol-based dendrimers derived from polypropylenimine (PPI) and polyamidoamine (PAMAM) dendrimers were developed and used in catalysis and encapsulation. 14−17 Dendritic ionic entities were also performed to encapsulate metallic species. 18 Glycerol is a very know by-product of the biodiesel industry. 19 As this compound is expensive to purify for food applications, its valorization toward value-added products was largely developed for around 20 years through essentially catalytic processes. 20−25 Next, concerning the use of essential oils (EOs), in a global context of the willingness of a return to green and clean agriculture to feed the population growing very quickly, the research about the creation of an herbicide containing essential oil is in full swing. 26 Cinnamon and citronella EOs are part of these natural products. Concerning the citronella oil, therapeutic 27 and pesticide applications have been reported in the literature. 28 Similar properties have been also revealed for cinnamon oil. 29 However, EOs are very volatile products that is why they need to be retained and protected to allow an effective use of their biological properties. Many matrices can be used to encapsulate these EOs in order to preserve their applicative potential. 30,31 Some are solid, others are liquid, and their release can be very quick or very slow. For the creation of a pesticide, a liquid product with a slow release seems to be a good choice.
In this work, three or four generations of four families of glycerol-based dendrimers have been screened as potential encapsulation matrices of cinnamon and citronella EOs. Among these families, two are new and their synthesis will be described. The encapsulation capacities have been demonstrated through the technique of headspace chromatography coupled to mass spectroscopy. 32 Moreover, NMR (1D, 2D, and DOSY 33 ) investigations were realized to understand potential interactions between major compounds of cinnamon oils and dendrimer moieties.

RESULTS AND DISCUSSION
2.1. Dendrimers Synthesis. Four families of glycerol-based dendrimers have been employed for our study: first of all, the glycerodendrimers (GD)-PPIs and GD-PAMAMs, then the glyceroclickdendrimers (GCDs), and next the glyceroladendrimers (GADs). The two first family syntheses have already been described in the literature. 14,16 The synthesis of GCDs and GADs are presented here following the procedures described respectively in two patents. 34,35 2.1.1. Glycerol-Based PPIs and PAMAMs. The four generations of functionalized PPIs (polypropyleneimines) and PAMAMs (polyamidoamines) (Figures 1 and 2) were synthesized following previously described works. 14,16 Commercial dendrimers (PPIs or PAMAMs) react with glycerol carbonate in methanol with Et 3 N as a catalyst. Products were characterized by NMR and elemental analysis and then used for EO encapsulations.
2.1.2. Glyceroclickdendrimers (GCDs). The starting material for GCD family is a glycerol derivate, the solketal. This compound is classically obtained from glycerol through various methods largely described in the literature. 36 Compound I.3 (Scheme 1) is prepared in two steps starting from solketal: the mesylation of the hydroxyl group is followed by substitution by sodium azide. 37,38 The expected azide was obtained with good yields, without purification steps by simple phase separation and especially without the use of organic solvents such as DMSO or DMF. Next, compound I.4 synthesis was carried out from glycerol in the presence of propargyl bromide and a base, in DMF as a solvent, for 24 h at 50°C. 39 KOH was chosen as base instead of NaH to avoid its disadvantages (explosive in contact with H 2 O, flammable in humid air). During this synthesis, several by-products were observed and identified by NMR as mono-and di-substitution products. Different reaction conditions were tested in order to reduce the formation of these compounds (increase in the number of base and propargyl bromide equivalents, addition of these reagents during the reaction, increase of temperature, and reaction time), but they did not help to promote the tripropynylation. Nevertheless, these various by-products are eliminated easily by flash chromatography on silica gel and the yields in trialcynyl compounds are good.  On the 1 H NMR spectrum of I.4, the 4.15 ppm peak integrates twice as much as the 4.30 ppm peak, confirming the symmetry of only two branches of the molecule. Moreover, the signal integration at 2.42 ppm is low, which is explained by a different relaxation of the protons of the terminal alkyne group. A ″click″ reaction was then performed between compound I.3 and compound I.4 to obtain the first generation of this new family of dendrimers from glycerol, compound I.5. The conditions used for the click reaction are classical (catalytic amounts of CuSO 4 ·5H 2 O (0.1 equiv), sodium ascorbate (0.2 equiv), tBu 4 NBr (0.1 equiv) in a CH 2 Cl 2 /NaHCO 3 (aq) mixture (1:1) at 50°C for 24 h). 40,41 After washing with ammonia and precipitation with petroleum ether, the expected compound I.5 is obtained with an isolated yield of 72%. The 1 H NMR spectrum of I.5 showed two signals at 4.65 and 4.79 ppm corresponding to protons H3 and H12. In the molecule, two branches are thus equivalent two by two. Nevertheless, it should be noted that contrary to compound I.4, this partial symmetry is only observed for the protons of the pyrazole rings. Beyond the cycle, the protons are all equivalent on the three branches. This can be explained by the size of the molecule, which becomes more important. The deprotection of I.5 is then realized in an acid medium and led to the formation of I.6 (GCD-1).
Once compound I.5 is obtained, a last step is necessary to obtain the first generation of this family (I.6): the deprotection of acetal groups. I.5 is dissolved in a minimum of water and one equivalent per protected function of HCl is added at room temperature. The advantage of this method is to obtain very good yields, even quantitative ones. 42 The reaction is monitored by 1 H NMR by observing the disappearance of the signals corresponding to the CH 3 groups.
The reacting sequence was repeated one time to furnish the second generation of GCD (compound I.9, GCD-2, Scheme 2) and another time too for the third one (compound I.12, GCD-3, Scheme 3).
Three generations of new glyceroclickdendrimers from glycerol have been synthesized and purified with good yields. These glyceroclickdendrimers present respectively 6, 12, and 24 OH terminal functions and are soluble in water.

GlycerolADdendrimers (GADs).
GADs are a new family of glycerodendrimers presenting only the pattern glycerol as the core with OH functions at the periphery (6, 12, or 24 for generations 1, 2, and 3, respectively).
At first, the synthesis of glycerol triallyl was carried out from glycerol in the presence of allyl bromide and potassium hydroxyde in DMF (Scheme 4).
Compound II.1 is obtained with an isolated yield of 46%. This yield can be explained by the formation, during the synthesis, of by-products from mono-and disubstitution, which can be easily removed by flash chromatography on silica gel. To reduce the formation of these compounds, we varied the reaction conditions. An increase of the number of equivalents of potassium hydroxide or allyl bromide, the reaction temperature, or even the reaction time unfortunately did not improve the yields. Likewise, a successive addition of the different reagents during the reaction did not increase the reaction yield either.
The 1 H NMR of II.1 at room temperature curiously showed the presence of signals with a complex multiplicity (Figure 3a). For a better understanding of the structure, a homo-nuclear Jresolved NMR (Figure 3b) was realized. Thanks to NMR and more particularly to J-resolved NMR, we were thus able to attribute each signal of the molecule and to observe that compound II.1 presented, just like compound I.4, a central symmetry along the C1-C6 axis. The various results obtained by J-resolved NMR complement those already described in 2006 by Kuzńik et al. concerning the isomerization of allyl alkyls and allyl silyl ethers catalyzed by ruthenium complexes. 43 The J-resolved NMR analysis completed this first analysis by determining the multiplicity of all protons (Table 1).
Next, in order to obtain a first generation of ″poly glycerol″ dendrimers comprising six alcohol functions at the periphery, we realized a dihydroxylation of the three double bonds of compound II.1. We used the classical conditions of Sharpless. 44 The dihydroxylation is carried out in the presence of the ADmix-β (1.4 g for 1 mmol olefin) in a tBuOH/H 2 O mixture (1:1) at 0°C. After 24 h, Na 2 SO 3 was added to reduce K 2 OsO 2 (OH) 4 to OsO 2 .
The same reaction scheme was applied to prepare the second and then the third generation of this glycerol-based dendrimer family (Scheme 4).
Compound II.3 was obtained with a yield of 89%. Up to this generation, the purification was simplified by simple precipitation of the impurities, which were insoluble in diethyl ether, and no silica gel chromatography was necessary. Compounds II.4 and II.6 with 12 and 24 OH functions, respectively, at the periphery were similarly obtained with yields of 65 and 75%.
The first three generations of ″poly glycerol″ dendrimers were synthesized with a glycerol core. The synthesis of each generation is carried out easily in two steps: allylation of the OH functions and then dihydroxylation of the double terminal links. The presence of alcohol functions on the surface of the dendrimer allows increasing its water solubility and then considering the use of these glycerodendrimers for aqueous catalysis or stabilization of nanoparticles. In addition, the presence of double bonds at the periphery of the ″half generations″, easily isolatable, also allows for the possibility of considering for these molecules the transformations by metathesis or ″click″.
2.2. Essential Oils Encapsulations. For retention experiments, the same amount of each EO was used to prepare solutions with or without dendrimers, which are then analyzed by DHS-GC−MS. EO retention by GDs was quantified by comparing the sum of the chromatographic peaks areas of EO components in the presence of GDs to that of the blank experiment. The percentage of retention (r) of EOs by GDs was calculated by the equation and listed in Table 2. 31,45 where ∑A D is the sum of peak areas of EO component in the presence of dendrimers and ∑A 0 is the sum of the peak of the EO component in the free EO solution (control).
Depending on the family and generation of dendrimers, the peak areas of all EO components decreased in the presence of GDs. This decay was attributed to the retention of EOs upon encapsulation formation. 45 Citronella EO in GCDs, GD-PPI-1, and GAD-2 did not show a remarkable retention capacity. The main volatile compounds present in this EO are limonene, geraniol, citronellal, and citronellol. These terpenes are very apolar such as GCD, which could induce repulsion instead of attraction as a salting-out effect of volatile EO components from the GD solution to the vapor phase. Negative retentions were also reported in the literature. 46,47 For the other cases, retention values showed that EOs were retained by GDs. This reflected the tendency of their components to form encapsulation with GDs. The comparison of the retention capacities of GDs toward the two EOs showed that GD-PPI and GD-PAMAM were more efficient than GCD and GAD; the GD retention capacities can be ranked for each family following the generation order G3 > G4 > G2 > G1. These results suggested that GDs, particularly GD-PPI-3, GD-PAMAM-2, GCD-2, and GAD-1, could efficiently retain EOs and consequently protect them from evaporation. Thus, GDs might be considered as efficient materials to encapsulate and improve the release efficiency of volatile compounds enlarging the applications of EOs for pesticides. The rate of retention obtained is comparable to the Lippia sidoides essential oil nanocapsule produced for agricultural purposes. 48 2.3. Interaction Analyses. 1D NMR of dendrimers GD-PPI-3, GD-PAMAM-2, GCD-2, and GAD-1 have been recorded alone and in the presence of citronella and cinnamon EOs. No real shift or interaction could be remarked with GD-PAMAM-2, GCD-2, and GAD-1. On the contrary, considering the solution of GD-PPI-3 and cinnamon EO, a shoulder of the peak relative to the CH 2 -N around 2.5 ppm is observed suggesting an interaction between cinnamon EO or more particularly its major compound, the trans-cinnamaldehyde and this part of the dendrimer (Figure 4).
A similar behavior has been already reported in previous works. 16 So, we decided to realize DOSY experiments to finalize this interaction.
As the evaluation of the ability of glycerodendrimers to encapsulate essential oils is the main goal of this work, the hydrodynamic radii of these macromolecules were determined by DOSY NMR because it was already found that the encapsulation efficiency depends on the dendrimer size. In addition, a variation of this size when dendrimers are in the presence of EOs should demonstrate that encapsulation modifies dendrimer organization.
The determination of the hydrodynamic radius was based on the Stokes−Einstein equation and relied on the measurement of the diffusion coefficient of the dendrimer. 15 For each solution, the diffusion coefficients of the solute and of the solvent were extracted from the 2D DOSY spectra by using the TopSpin software (Bruker) and the solvent viscosity was not changed by the presence of the dendrimer because of their low   concentration. DOSY NMR ( Figure 5) was realized for GD-PPI-3, GD-PAMAM-2, GCD-2, and GAD-1, but more attention has been devoted for the emulsion GD-PPI-3 with cinnamon EO where an interaction was noticed in 1 H NMR. The diffusion coefficients for this study are detailed in Table 3. The diffusion coefficient of the dendrimer is higher in the presence of the cinnamon EO, which suggests that its radius is lower in this solution than alone. This observation led us to think that the dendrimer is compressed with the essential oil and that the latter is even more stabilized. To our knowledge, this behavior has not been reported in the literature, the size of the dendrimers being always bigger when various species (metallic complexes or organic compounds) are encapsulated inside. These studies should be continued by microscopic studies to confirm this phenomenon and preliminary results seemed to confirm this phenomenon.

CONCLUSIONS
Three generations of two new family of dendrimers based on glycerol have been successfully synthetized. Their EO encapsulation abilities have been studied at the same time for two others, more known, and already described glycerol-based dendrimers, GD-PPI and GD-PAMAM. GD-PPI-3, GD-PAMAM-2, GCD-2, and GAD-1 showed good retention properties, so the interaction existing between EOs and GDs had been thoroughly analyzed. The NMR experiments (1D and DOSY) showed an interaction between the GD-PPI-3 and the cinnamon EO, and furthermore, the dendrimer is compressed in a stable emulsion containing this dendrimer and the essential oil. Investigations have to be pursued with GD-PPI-3 using microscopic analyses to confirm this surprising phenomenon.  1 H and 13 C NMR spectra were recorded at room temperature with a Bruker AC 500 spectrometer (500 or 600 MHz for 1 H, 62.5 or 150 MHz for 13 C). High resolution mass spectra were recorded on a Q TOF micro (Micromass) with an electrospray source (injection by infusion: 5 L/min, solvent used: MeOH +0.2% (by volume) of formic acid, source temperature: 80°C and drying gas: nitrogen at 100°C). The processing of the spectra is done with the Masslynx software. I.4. Purification. After addition of water, the aqueous phase is extracted three times with CH 2 Cl 2 . Organic phases are collected and dried on MgSO 4 ; the solvent is then removed under pressure, and the crude mixture is purified on silica gel chromatography with the mixture petroleum ether/AcOEt (7:3) as an eluting mixture. I.4 is obtained as a yellow oil with 80% yield.  . and I.10 Purification. After the addition of water, the aqueous phase is extracted three times with CH 2 Cl 2 . Organic phases are collected and dried on MgSO 4 ; the solvent is then removed under pressure. After verification by NMR 1 H, a simple purification by precipitation in CH 2 Cl 2 with an excess of petroleum ether can be performed. I.7 and I.10 are obtained as yellow oils with respectively 70% and 52% yields. . The mixture is stirred at 4°C under argon for 48 h. After an extraction with CH 2 Cl 2 , the organic phase is washed with NH 4 OH solution (0.8 M) until the aqueous phase gets uncolored and then two times with water. Organic phases are collected and dried on MgSO 4 , and the solvent is removed under pressure. The resulting mixture is dissolved in a minimum of CH 2 Cl 2 , and the addition of an excess of petroleum ether allows the precipitation of the required products (I.5, I.8, and I.11).

EXPERIMENTAL
The desired compounds are obtained as a brown resin with 70, 70, and 43% yields. 4.2.2.7. General Procedure for the Removing of Protection (Synthesis of I.6, I.9, and I.12). I.5, I.8, or I.11 are dissolved with minimum water, and then a HCl solution (1 M, 1.2 equiv per branch) is added. The mixture is stirred at room temperature until the total deprotection (followed by 1 H NMR). Then, a NaOH solution (1 M, 1 equiv per equiv HCl) is added. The mixture is filtered on celite, and water is removed under reduced pressure. The products are obtained as brown wax with quantitative yields. 4.4. Dynamic-Headspace Chromatography−Mass Spectrometry (DHS-GC−MS) Analysis. The percentage of retention (r) of EOs by GDs was determined by dynamic headspace sampling (DHS, Gerstel, Germany) coupled to a thermal desorption unit (TDU, Gerstel, Germany), a gas chromatograph (Agilent Technologies 7890A), and a mass spectrometer (MS, Agilent Technologies 5975C). During treatment in the DHS unit, the vials were conditioned at 25°C for 30 min with agitation (500 rpm). The head-space sampling was performed on Gerstel TDU desorption tubes (OD 6.00 mm, filled with 60 mg of Tenax TA, Gerstel, Germany), for 200 mL at 20 mL/min, followed by 200 mL at 60 mL/min of the drying phase. Desorption then occurred for 10 min at 300°C and coupled to a cooled injection system (CIS, Gerstel, Ger-many) set at −80°C

Glyceroladendrimer
. EOs were then transferred to the GC column (VF-WAXms, Agilent technologies USA; 30 m length, 0.250 mm I.D, 0.25 μm film thickness) for separation with temperature programs as follows: citronella, from 70°C (5 min) to 100°C at a rate of 8°C /min, then 2°C/min to 160°C, and then 20°C/min to 260°C (10 min); cinnamon, from 40°C (4 min) to 80°C at a rate of 3.5°C/min, then 5°C/min to 160°C, and then 20°C/min to 220°C (10 min) with helium as carrier gas at a flow rate of 1.5 mL/min. The MS were recorded in electron ionization mode at 70 eV (scanned mass range: 35 to 300 m/z); source and quadrupole temperature at 230 and 150°C, respectively. The component identification was performed by comparison of the recorded spectra with two data libraries ( The 2D 1 H DOSY NMR experiments were carried out using the Bruker sequence ledbpgp2s, at 298 K. The gradient value G = 0.535 T·m −1 was generated by a 10A amplifier. The strength of the pulsed-field gradient was linearly increased from 2 to 95% in 24 steps. The diffusion time (Δ) and the gradient duration (δ/2) were set at 180 ms and 800 us. The longitudinal eddy current delay and the spoil gradient delay were fixed at 5 and 0.2 ms, respectively. Spectral data were processed via dynamics center version 2.6.2 software (Bruker) or from topspin software for DOSY experiments.