Glycerol acetals with antioxidant properties

Preparation of the acetals

The acetals were prepared by reacting glycerol and aromatic aldehydes in the presence of an Amberlyst-15 acid resin. To improve the yield, toluene and a Dean-Stark apparatus were used to remove the water formed. This procedure was successfully applied for both benzaldehyde and anisaldehyde. However, for furfural the reaction medium darkened after a few minutes and attempts to characterize and isolate the products were laborious. Furfural may undergo oligomerization in the presence of acidic catalysts [19], therefore to reduce this problem a smaller mass of catalyst was used along with cyclohexane, which has a lower azeotrope boiling temperature of 70 °C [20].

The products were characterized by GC/MS and NMR with four isomers of each acetal observed by both techniques. Table 1 shows the yields of the combined isomers products after isolation. The NMR spectra display multiplet bands between 4.7 and 3.4 ppm due to the methine, methylene and hydroxyl protons of the dioxacycle, with multiplets at higher chemical shifts from protons on the furan and benzene rings. Four singlet peaks between 6.0 and 5.4 ppm are characteristic of the H-2 protons of the Z + E isomers of the five-membered ring and six-membered ring compounds (Fig. 1 and Table 1) [21]. In addition, enantiomers of these compounds will be present which could not be separated on the GC column used. We did not attempt to isolate the isomers, assuming that they should present similar antioxidant activities. For simplicity, the mixture of isomers will be referred to as glycerol/aldehyde acetals. The yields of all three acetals were high (Table 1) and similar to the yields found for the C₃-C₆ aldehydes reacted with glycerol previously [16]. Following distillation the products all had a purity of 98–99 %.

Table 1

Isolated yields of the glycerol acetals.

Acetal		Yield (%)
Glycerol/benzaldehyde (GB)		70
Glycerol/anisaldehyde (GA)		75
Glycerol/furfural (GF)		80

Fig. 1

¹H NMR spectra of the acetal isomers from the reaction of furfural and glycerol highlighting the characteristic singlet peaks from the H-2 protons.

Antioxidant activity

The antioxidant activity of the acetals was initially tested following a standard procedure using DPPH· [22]. During the test the spectrometric decay of the diphenyl-picryl-hydrazine radical (DPPH·) is followed in the presence of the potential antioxidant under controlled conditions. Scheme 3 shows the reaction that is monitored. The DPPH· radical shows a strong absorption at 516 nm, giving the solution a deep violet color [22]. Upon abstraction of a hydrogen atom and formation of diphenyl-picryl-hydrazine, the violet color is lost with a residual pale yellow color remaining. The kinetics of the radical decay due to the antioxidant can be monitored with a UV/visible spectrometer and a comparative behavior among several compounds can be established [23].

Scheme 3

DPPH test of antioxidant activity (H-R stands for a potential antioxidant molecule).

The decay of the DPPH· radical over time was studied using various concentrations of the three acetals formed in this study, with the percentage of DPPH· remaining at the steady state observed (Fig. 2). With addition of sufficient concentration of the acetal, a sharp initial drop in the DPPH· concentration is observed which reaches the steady-state over 2 h.

Fig. 2

Antioxidant activity of glycerol/anisaldehyde acetal (GA) with varying ratios of GA/DPPH·.

The antiradical activity was then determined as the amount of antioxidant necessary to decrease the initial DPPH· radical concentration by 50 %, EC₅₀ (Fig. 3). Only the glycerol/anisaldehyde acetal (GA) displayed a significant antioxidant activity with an EC₅₀ = 80. This is similar to coumaric acid or vanillin, but higher than that of BHT [23]. With an acetal/DPPH molar ratio of 32 351 the glycerol/furfural acetal was able to reduce the DPPH· concentration to 50 % at the steady state (Fig. 4). However, even at this high molar ratio the DPPH· radical concentration was only reduced to 70.4 % at the steady state using the glycerol/benzaldehyde acetal. Higher ratios could not be achieved due to the viscosity of the acetal and poor miscibility with the DPPH· solution. This therefore indicates significant differences in the antioxidant activities between the three acetals in the order GA >> GF > GB.

Fig. 3

Determination of EC₅₀ for glycerol/anisaldehyde acetal, the ratio of GA/DPPH· at which the percentage of DPPH· remaining at the steady state is 50 %.

Fig. 4

Change in the percentage of DPPH· remaining at the steady state with acetal/ DPPH· molar ratio using glycerol/furfural and glycerol/benzaldehyde acetals.

The antioxidant activities and the kinetics of DPPH· radical abstraction indicate that the antioxidant capabilities of the glycerol acetals studied rely on the abstraction of their benzylic hydrogen atom to form a delocalized radical (Scheme 4). The presence of a methoxy group in the para position of the glycerol/anisaldehyde acetal will greatly increase the stability of the radical formed, as well as the two oxygen atoms of the dioxane and dioxolan ring (oxygen atoms of the acetal function), thereby contributing to its increased antioxidant activity.

Scheme 4

Free radical formed upon the abstraction of the benzylic hydrogen atom, showing electron delocalization in the aromatic ring.

Acetals

Cyclic acetals of glycerol were synthesised by mixing glycerol (62 g), an activated Amberlyst-15 catalyst (3.8 g) and either a toluene or cyclohexane solvent (70 mL) in a Dean-Stark apparatus. The solution was stirred and once reflux was reached the required aldehyde (0.75 mol) was added. The reaction mixture was heated under reflux for 3–4 h. Once cooled the product was filtered and the solution transferred to a separation funnel. The residual glycerin was separated by washing with 0.1 M NaHCO₃ and water. Finally, the acetals were recovered by distillation under reduced pressure with the products stored in a freezer until use.

Analysis of samples

All of the samples were analyzed by GC-MS and NMR. An Agilent 6850 GC was used fitted with a HP-5MS 5 % Phenyl Methyl Siloxane column with a nominal size of 30 m × 250 μm × 0.25 μm. 0.2 μL of sample was injected onto a column heated to 120 °C, with a split of 25:1 with the temperature heated linearly to 220 °C. ¹H NMR was carried out on a Bruker DPX 200 using CDCl₃ or deuterated acetone as a solvent.

The antioxidant activity of the acetals was analyzed using a UV-vis spectrophotometer Biospectro SP-22. Solutions containing DPPH (3.9 mL, 0.00006 M) and acetals of various concentrations (0.1 mL) were analyzed at a wavelength of 516 nm after 5–120 min reaction. The absorbance of the initial DPPH· solutions were also analyzed. The percentage of DPPH· remaining was calculated.