Chemoenzymatic Synthesis of the New 3-((2,3-Diacetoxypropanoyl)oxy)propane-1,2-diyl Diacetate Using Immobilized Lipase B from Candida antarctica and Pyridinium Chlorochromate as an Oxidizing Agent

To exploit the hydrolytic activity and high selectivity of immobilized lipase B from Candida antarctica on octyl agarose (CALB-OC) in the hydrolysis of triacetin and also to produce new value-added compounds from glycerol, this work describes a chemoenzymatic methodology for the synthesis of the new dimeric glycerol ester 3-((2,3-diacetoxypropanoyl)oxy)propane-1,2-diyl diacetate. According to this approach, triacetin was regioselectively hydrolyzed to 1,2-diacetin with CALB-OC. The diglyceride product was subsequently oxidized with pyridinium chlorochromate (PCC) and a dimeric ester was isolated as the only product. It was found that the medium acidity during the PCC treatment and a high 1,2-diacetin concentration favored the formation of the ester. The synthesized compounds were characterized using IR, MS, HR-MS, and NMR techniques. The obtained dimeric ester was evaluated at 100 ppm against seven bacterial strains and two Candida species to identify its antimicrobial activity. The compound has no inhibitory activity against the bacterial strains used but decreased C. albicans and C. parapsilosis growth by 49% and 68%, respectively. Hemolytic activity was evaluated, and the results obtained support the use of the dimeric ester to control C. albicans and C. parapsilosis growth in non-intravenous applications because the compound shows hemolytic activity.


Introduction
Glycerol (propane-1,2,3-triol) is the most abundant by-product generated during transesterification reactions of vegetable oils and animal fats with methanol to produce biodiesel [1][2][3]. Due to the global population growth and the demand for new energy sources with a low impact regarding CO 2 emissions [4,5], the biodiesel industry is developing rapidly, resulting in a high excess of glycerol. Therefore, new processes to convert this alcohol into added-value chemicals have been increasingly explored [6][7][8][9]. of glycerol and the esterification with fatty acids in the presence of homogenous or heterogenous catalyst [57][58][59][60].
To exploit the regioselectivity of the immobilized lipases and the efficiency of the chromium (VI) oxidants, in this work we propose a new chemoenzymatic methodology in which we used Candida antarctica lipase B immobilized on octyl-agarose support (CALB) to hydrolyze triacetin to 1,2-diacetin, which was subsequently transformed, using PCC, to obtain the new dimeric glycerol ester 3-((2,3-diacetoxypropanoyl)oxy)propane-1,2-diyl diacetate, a molecule which to date has no reported synthesis. To the best of our knowledge, this is the first time that a diglyceride prepared via catalytic hydrolysis with immobilized lipases has been used as the starting material for new and more complex molecules. Figure 1 shows the immobilization course of CALB on octyl-agarose, which is a quite rapid immobilization process. As we used an excess of the enzyme, immobilization only accounts for around 50% of the offered enzyme, with an expressed activity of around 50% [61]. To exploit the regioselectivity of the immobilized lipases and the efficiency of the chromium (VI) oxidants, in this work we propose a new chemoenzymatic methodology in which we used Candida antarctica lipase B immobilized on octyl-agarose support (CALB) to hydrolyze triacetin to 1,2diacetin, which was subsequently transformed, using PCC, to obtain the new dimeric glycerol ester 3-((2,3-diacetoxypropanoyl)oxy)propane-1,2-diyl diacetate, a molecule which to date has no reported synthesis. To the best of our knowledge, this is the first time that a diglyceride prepared via catalytic hydrolysis with immobilized lipases has been used as the starting material for new and more complex molecules. Figure 1 shows the immobilization course of CALB on octyl-agarose, which is a quite rapid immobilization process. As we used an excess of the enzyme, immobilization only accounts for around 50% of the offered enzyme, with an expressed activity of around 50% [61].

Immobilization of CALB on Octyl-agarose
Compared to other lipases [62,63], in our case there was no hyperactivation of the enzyme upon immobilization, probably since CALB has no large helical lid fully covering the active site and does not exhibit a strong interfacial activation [33,64,65]; therefore, its activity in solution is very similar to the activity of the enzyme immobilized on octyl-agarose [66][67][68], and even decreased due to substrate diffusional problems by the high enzyme loading.  Figure 2 shows the enzymatic hydrolysis of triacetin catalyzed by CALB-OC. In this study, we use CALB-OC, since it has been shown that, like most lipases, CALB-OC is a sn-1,3 regioselective biocatalyst in the hydrolysis of triacetin, so that the hydrolysis of the triglycerides with lipases will produce 1,2-diglycerides and 2-monoglycerides [69][70][71]. It is interesting to note that, based on previous studies, hydrolysis of triacetin by Novozym 435 (a commercial immobilized preparation  of CAL B) exhibited a low yield of diacetin [72], presumably due to high isomerization and production of monoacetins [73,74]. Compared to other lipases [62,63], in our case there was no hyperactivation of the enzyme upon immobilization, probably since CALB has no large helical lid fully covering the active site and does not exhibit a strong interfacial activation [33,64,65]; therefore, its activity in solution is very similar to the activity of the enzyme immobilized on octyl-agarose [66][67][68], and even decreased due to substrate diffusional problems by the high enzyme loading. Figure 2 shows the enzymatic hydrolysis of triacetin catalyzed by CALB-OC. In this study, we use CALB-OC, since it has been shown that, like most lipases, CALB-OC is a sn-1,3 regioselective biocatalyst in the hydrolysis of triacetin, so that the hydrolysis of the triglycerides with lipases will produce 1,2-diglycerides and 2-monoglycerides [69][70][71]. It is interesting to note that, based on previous studies, hydrolysis of triacetin by Novozym 435 (a commercial immobilized preparation of CAL B) exhibited a low yield of diacetin [72], presumably due to high isomerization and production of monoacetins [73,74]. To assess the effect of acetonitrile on the hydrolysis of triacetin by CALB-OC and to improve the solubility of triacetin, we evaluated the addition of 20% acetonitrile to the reaction mixture. Table 1 shows the yield of 1,2-diacetin (2) in the absence and the presence of 20% acetonitrile (entry 1). After 90 min of reaction, 1,2-diacetin (2) and 2-monoacetin were the only detected products and 46.5% of the triacetin remained non-hydrolyzed. On the other hand, the hydrolysis without acetonitrile (entry 2) increased the yield of 1,2-diacetin (2) to 71% with an increase in the accumulation of 2-monoacetin. 1,3-diacetin was not detected in any of the two cases [75]. The reduction in the production of 1,2diacetin (2) in the presence of acetonitrile can be attributed to the fact that organic solvents alter the conformation of the immobilized lipases [76] and could also act as competitive lipase inhibitors [69]. We isolated 1,2-diacetin (2) from 2-monacetin by a liquid-liquid separation in which 2monacetin remained in the aqueous phase and 1,2-diacetin (2) in the organic phase. To ensure complete hydrolysis of triacetin (1), we increased the reaction time to 3 h; finally, a purified 1,2diacetin (2) structure was corroborated by infrared spectroscopy, 1 H NMR and, GCMS (all spectra are found in the Supporting Information, Figures S1-S3).
The formation of ester (5) can be explained if we take into account that the acidity of PCC can promote a rapid addition of 1,2-diacetin (2) to the glyceraldehyde diacetate (3) to form the dimeric hemiacetal (4), from which subsequent oxidation produces the dimeric ester (5) (Figure 3) [77][78][79]. To assess the effect of acetonitrile on the hydrolysis of triacetin by CALB-OC and to improve the solubility of triacetin, we evaluated the addition of 20% acetonitrile to the reaction mixture. Table 1 shows the yield of 1,2-diacetin (2) in the absence and the presence of 20% acetonitrile (entry 1). After 90 min of reaction, 1,2-diacetin (2) and 2-monoacetin were the only detected products and 46.5% of the triacetin remained non-hydrolyzed. On the other hand, the hydrolysis without acetonitrile (entry 2) increased the yield of 1,2-diacetin (2) to 71% with an increase in the accumulation of 2-monoacetin. 1,3-diacetin was not detected in any of the two cases [75]. The reduction in the production of 1,2-diacetin (2) in the presence of acetonitrile can be attributed to the fact that organic solvents alter the conformation of the immobilized lipases [76] and could also act as competitive lipase inhibitors [69]. We isolated 1,2-diacetin (2) from 2-monacetin by a liquid-liquid separation in which 2-monacetin remained in the aqueous phase and 1,2-diacetin (2) in the organic phase. To ensure complete hydrolysis of triacetin (1), we increased the reaction time to 3 h; finally, a purified 1,2-diacetin (2) structure was corroborated by infrared spectroscopy, 1 H NMR and, GCMS (all spectra are found in the Supporting Information, Figures S1-S3).
To increase the yield of the glyceraldehyde diacetate (3) and to avoid the formation of the hemiacetal (4), which could be promoted by the acidity of PCC, we carried out the reaction adding two equivalents of sodium acetate [80] (Table 2, entry 2) and it was found that decreasing the acidity of the medium promotes the accumulation of the aldehyde (3) with a consequent decrease in the reaction rate. Slow addition of 1,2-diacetin (2), and a higher solvent/PCC ratio ( Table 2, entry 3), avoids the formation of ester (5); probably the low concentration of alcohol (2) slows the formation of hemiacetal (4) [78]. Accordingly, the best aldehyde proportions were obtained when the reaction was carried out adding only 1 equivalent of sodium acetate and simultaneously reducing the 1,2-diacetin (2) concentration by slow addition and high dilution (  To increase the yield of the glyceraldehyde diacetate (3) and to avoid the formation of the hemiacetal (4), which could be promoted by the acidity of PCC, we carried out the reaction adding two equivalents of sodium acetate [80] (Table 2, entry 2) and it was found that decreasing the acidity of the medium promotes the accumulation of the aldehyde (3) with a consequent decrease in the reaction rate. Slow addition of 1,2-diacetin (2), and a higher solvent/PCC ratio ( Table 2, entry 3), avoids the formation of ester (5); probably the low concentration of alcohol (2) slows the formation of hemiacetal (4) [78]. Accordingly, the best aldehyde proportions were obtained when the reaction was carried out adding only 1 equivalent of sodium acetate and simultaneously reducing the 1,2diacetin (2) concentration by slow addition and high dilution (Table 2, entry 4) (all chromatograms and mass spectra are shown in the Supporting Information, Figures S10-S16). PCC 2 equiv, AcONa 1 equiv, silica gel 2 g 20 mL CH2Cl2/g PCC 1:5:1 Note: * 3 Å molecular sieves [81] were used in all reactions at room temperature for 12 h. **GC analyses were performed before the reaction workup, as described in Section 3.
It is worth mentioning that glyceraldehyde diacetate (3) could never be isolated for the NMR analysis because the reaction workup promotes the accumulation of the ester (5). Figure 4 shows the reaction's crude (using conditions shown in Table 2 entry 4) composition before and after filtration through a silica pad. During the reaction workup, the unreacted 1,2-diacetin and the produced glyceraldehyde diacetate reacted , to generate compound (5) as the major product in the reaction, no matter which reaction conditions are initially used. We evaluated the effect of silica gel and found that it promotes the accumulation of the dimeric ester (5) ( Table 2 entry 5). If celite [82] is used for filtration, the reduced chromium species are not eliminated from the reaction crude; consequently, a silica gel pad must always be used for filtration [83,84].  PCC 2 equiv, AcONa 1 equiv, silica gel 2 g 20 mL CH 2 Cl 2 /g PCC 1:5:1 * 3 Å molecular sieves [81] were used in all reactions at room temperature for 12 h. ** GC analyses were performed before the reaction workup, as described in Section 3.
It is worth mentioning that glyceraldehyde diacetate (3) could never be isolated for the NMR analysis because the reaction workup promotes the accumulation of the ester (5). Figure 4 shows the reaction's crude (using conditions shown in Table 2 entry 4) composition before and after filtration through a silica pad. During the reaction workup, the unreacted 1,2-diacetin and the produced glyceraldehyde diacetate reacted, to generate compound (5) as the major product in the reaction, no matter which reaction conditions are initially used. We evaluated the effect of silica gel and found that it promotes the accumulation of the dimeric ester (5) ( Table 2 entry 5). If celite [82] is used for filtration, the reduced chromium species are not eliminated from the reaction crude; consequently, a silica gel pad must always be used for filtration [83,84].

Biological Activity of 3-((2,3-Diacetoxypropanoyl)oxy)propane-1,2-diyl diacetate (5)
Antimicrobial activity against MRSA, S. aureus ATCC 29213, E. coli ATCC 25922, E. coli O157:H7, S. typhimurium ATCC 14028, S. enteritidis ATCC 13076, P. aeruginosa ATCC 27853, C. albicans ATCC 10231, and C. parapsilosis ATCC 22019 was evaluated. 3-((2,3-diacetoxypropanoyl)oxy)propane-1,2-diyl diacetate does not show growth inhibitory activity at 100 ppm in any of the bacterial strains. However, an increase in the growth of E. coli O157: H7 and Salmonella spp. was evidenced. On the other hand, evaluation of the fungal activity of the dimeric ester showed growth inhibition of both Candida strains at 100 ppm; the strain C. parapsilosis being the most sensitive with a 68% inhibition within 48 h of culturing. Finally, hemolytic activity was evaluated; the new compound presented hemolysis of 18.5 ± 2.6% at a concentration of 100 ppm, which is considered high, taking into account that a percentage of hemolysis greater than 10% is considered hemolytic or not hemocompatible [85] (biological activity can be found in the Supporting Information, Figures S17-S19).
To date, this is the first report that describes the synthesis of a new glycerol derivative obtained by enzymatic production of 1,2-diacetin and its posterior oxidation with PCC. The dimeric glycerol ester (5), whose synthesis is herein reported, could be of use for the cosmetics, pharmaceutical, and food industries, and could serve as the starting material for the synthesis of new molecules.

Materials and Methods
Soluble CALB was kindly donated by Novozymes. The octyl Sepharose 4BCL beads were obtained from Sigma Aldrich. Pyridinium chlorochromate was prepared using the Corey and Suggs protocol [46]. Silica gel, 230-400 mesh, molecular sieves of 0.4 nm, dichloromethane, n-hexane, ethyl acetate, and diethyl ether were obtained from Merck. The p-nitrophenylbutyrate (p-NPB) was obtained from Sigma Aldrich. Reaction monitoring was performed using silica gel TLC plates (silica Merck 60 F254), and the spots were visualized using Vanillin-HCl staining. Reaction monitoring was also performed using HPLC-DAD (Agilent Santa Clara, CA, United States) with a Zorbax C-18 (5 µm × 250 mm × 4.6 mm) column. 1 H and 13 C NMR spectra were measured at 25 • C on a Bruker Advance III-400 spectrometer, using CDCl 3 as the solvent. Chemical shifts (δ) and coupling constants (J) values are reported in ppm and Hz, respectively. Chemical shifts are relative to the solvent peaks used as reference (CDCl 3 : δ 7.26 for 1H and δ 77.23 for 13 C). 1

Standard Activity Determination
p-NPB hydrolysis was used as a model reaction to determine the standard activity during immobilization. A 20-100 µL lipase suspension or solution was added to 2.5 mL of 25 mM sodium phosphate at pH 7 and 25 • C and the increase in absorbance at 348 nm produced by the release of p-nitrophenol during the hydrolysis of 0.4 mM p-NPB was measured [86]. One international unit of activity (U) was defined as the amount of enzyme that hydrolyzes 1 µmol of p-NPB per minute under the conditions described previously. The protein concentration was determined by Bradford's method using BSA as standard [87].

Immobilization of CALB on Octyl agarose beads
CALB was immobilized on octyl agarose beads at low ionic strength as previously described [34]. A total of 1 g of octyl sepharose support resuspended in 20 mL of an enzymatic solution (containing 0.54 mg/mL of protein and specific activity 10.57 U/mg) was gently stirred in a shaker at 250 rpm in phosphate buffer sodium (5 mM and pH 7). The activities of both the supernatant and suspension were followed using a p-NPB assay. After the indicated time, the immobilized enzyme (protein concentration 4 mg/g of CALB-OC, specific activity 15.5 U/mg) was recovered by being filtered and washed several times with distilled water (3 × 20 mL).

Triacetin (1) Hydrolysis with CALB-OC
A total of 3.8 g of CALB-OC were stirred for 3 h with a solution of 1.9 mL (9.95 mmol) triacetin in 100 mL of 500 mM sodium phosphate at pH 5.5 at room temperature. Upon completion of the reaction monitored by HPLC, the biocatalyst was filtered, washed with 30 mL of distilled water, and stored for reuse. The filtrate was saturated with NaCl and extracted with dichloromethane (3 × 30 mL), the organic layer was washed with brine (1 × 20 mL) and dried over sodium sulfate, the solvent was 3.4. Synthesis of 3-((2,3-Diacetoxypropanoyl)oxy)propane-1,2-diyl diacetate (5) A total of 2.44 g (11.35 mmol) of PCC, 1 g of activated molecular sieves, and 23 mL of dichloromethane were stirred for 10 min in a round bottom flask, and then a solution of 1 g (5.68 mmol) of 1,2-diacetin in 5 mL of dichloromethane was added and the mixture was stirred at 25 • C for 12 h. Upon completion of the reaction monitored by TLC, 10 mL of diethyl ether were added to the reaction mixture, which was later filtered through a pad of silica and washed with diethyl ether (3 x10 mL). The solvents were removed under reduced pressure and the remaining crude was purified by column chromatography in silica gel using a 5:2 hexane:ethyl acetate as eluent to afford 0.623 g (yield 63%, 1.79 mmol) of the product as a colorless liquid. 1  3.5. Biological Activity of 3-((2,3-Diacetoxypropanoyl)oxy)propane-1,2-diyl diacetate (5)

Antibacterial Activity
The antimicrobial activity of 3-((2,3-diacetoxypropanoyl)oxy)propane-1,2-diyl diacetate (5) was evaluated by the micro dilution method described in previous works [88,89]. Briefly, a pre-inoculum in Luria Bertani (E. coli ATCC 25922, E. coli O157:H7, S. typhimurium ATCC 14028, S. enteritidis ATCC 13,076, and P. aeruginosa ATCC 27853) and in Mueller Hinton (MH) for MRSA and S. aureus ATCC 29213, were grown at 37 • C during 12 h at 200 rpm; then, the culture of each strain was set at 0.5 in the McFarland scale (10 5 CFU mL −1 ); 100 µL aliquots of these cell suspensions were mixed with 100 µL of compound (5) at 100 ppm in a 96-well microplate and incubated at 37 • C in an orbital shaker (200 rpm for 8 h). The bacterial growth kinetics of these microbial cultures was performed measuring changes of absorbance at 595 nm over time in an Elisa reader for these cultures (Thermo Fisher Scientific Waltham, MA, United States). MIC 50 was defined as the lowest concentration of dimeric ester (5), inhibiting 50% of the bacterial growth of these bacterial strains. After incubation for 8 h, 100 µL of these bacterial cultures were poured in 900 µL of BHI, incubated at 37 • C for 24 h, and then a 10 µL of these cultures were seeded over BHI-agar petri dishes, incubated for 1 day at 37 • C, after which the appearance of colonies were determined. The MBC was the lowest concentration producing a >99.9% reduction in colony-forming units (CFU).

Antifungal Activity
The antifungal activity of 3-((2,3-diacetoxypropanoyl)oxy)propane-1,2-diyl (5) diacetate was evaluated over C. parapsilosis ATCC 22,019 and C. albicans ATCC 10,231 by the microdilution method described in previous works [90]. From a fresh culture, a suspension was adjusted to an absorbance of 0.09-0.13 at 490 nm in sterile saline solution (10 6 CFU mL −1 ). Once adjusted, a dilution was carried on Roswell Park Memorial Institute medium (RPMI) to obtain a concentration of 10 3 CFU mL −1 , which was inoculated in 96-well plates with 100 ppm of compound (5) and incubated at 37 • C for 48 h. Once the incubation time was over, the absorbance at 490 nm was measured in a microplate reader (Thermo Scientific™, Multiskan Sky) and compared with the selected growth controls.

Hemolytic Activity
The hemolytic activity of compound 3-((2,3-diacetoxypropanoyl)oxy)propane-1,2-diyl diacetate (5) on sheep erythrocytes was evaluated, adapting the method described in previous works [91,92]. From 1 mL of defibrinated blood, three serial washes were carried out with 0.9% saline solution and the cell density was adjusted to 1% v/v by using cell counting in a Neubauer chamber. Then, 100 µL aliquots of the adjusted suspension were transferred to 96-well microplates and incubated with one containing a 100 ppm solution of compound (5), and were incubated for 3 h at 37 • C. The microplates were centrifuged at 500× g in 5 min and the absorbance of the released hemoglobin was measured at 543 nm using a microplate reader (Thermo Scientific™, Multiskan Sky). Saline solution and 0.5% triton X-100 were used as the negative and positive controls, respectively. To determine the percentage of hemolysis, Equation (1) was used.
where Am is the absorbance of the sample, Acp is the absorbance of the positive control, and Acn is the absorbance of the negative control.

Conclusions
In this work, we have shown that immobilized lipases are very useful tools for the synthesis of new glycerol value-added derivatives. We developed a chemoenzymatic methodology for the synthesis of a new glycerol dimeric ester, in which we made use of CALB immobilized on octyl-agarose, which proved to be an efficient biocatalyst for the regioselective hydrolysis of triglycerides, to synthesize under mild conditions 1,2-diacetin, which was subsequently oxidized with PCC. Interestingly, this oxidation did not afford the expected aldehyde but a dimeric ester through a mechanism that involves the condensation of the starting diglyceride and the product aldehyde. Finally, the dimeric glycerol ester obtained has antifungal activity; however, it does not show activity on bacterial growth, which allows us to establish that it is a biologically active compound on eukaryotic cells. The hemolytic activity was evaluated, and the results obtained support the use of the dimeric ester to control C. albicans and C. parapsilosis growth in non-intravenous applications because the compound produces hemolysis at the evaluated concentration.