Preparation of Triglycerol from Glycerol and Epichlorohydrin at Room

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Preparation of triglycerol from glycerol and epichlorohydrin at room temperature. Synthesis optimization and toxicity studies. Andrzej Kamil Milewski, Piotr Dydo, Agata Jakóbik-Kolon, Dymitr Czechowicz, Dorota Babilas, Ma#gorzata Burek, Sylwia Wa#kiewicz, Anna Byczek-Wyrostek, Tomasz Krawczyk, and Anna Kasprzycka ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.8b02817 • Publication Date (Web): 05 Sep 2018 Downloaded from http://pubs.acs.org on September 5, 2018

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ACS Sustainable Chemistry & Engineering

Preparation

of

triglycerol

from glycerol

and

epichlorohydrin at room temperature. Synthesis optimization and toxicity studies. Andrzej Milewski1*, Piotr Dydo1, Agata Jakóbik-Kolon1, Dymitr Czechowicz2, Dorota Babilas1, Małgorzata Burek3, Sylwia Waśkiewicz4, Anna Byczek-Wyrostek3, Tomasz Krawczyk2 and Anna Kasprzycka3 1.

Department of Inorganic, Analytical Chemistry and Electrochemistry, Faculty of

Chemistry, Silesian University of Technology, 6 Krzywoustego St., 44-100 Gliwice, Poland 2.

Department of Chemical Organic Technology and Petrochemistry, Faculty of Chemistry,

Silesian University of Technology, 4 Krzywoustego St., 44-100 Gliwice, Poland 3.

Department of Organic Chemistry, Bioorganic Chemistry and Biotechnology, Faculty of

Chemistry, Silesian University of Technology, 4 Krzywoustego St., 44-100 Gliwice, Poland 4.

Department of Physical Chemistry and Technology of Polymers, Faculty of Chemistry,

Silesian University of Technology, 9 Strzody St., 44-100 Gliwice, Poland

*The email address of the corresponding author: [email protected]

KEYWORDS: triglycerols, oligoglycerols, triethanolamine, epichlorohydrin, toxicity

ACS Paragon Plus Environment

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ABSTRACT: An alternate method of synthesizing oligoglycerols from glycerol epichlorohydrin - KOH mixtures at room temperature is proposed. This method involves using triethanolamine as a catalyst and produced a mixture of low molecular weight oligoglycerols, mainly di- and triglycerols. Secondly, the method was optimized for triglycerol yield and the product obtained was found to be similar to the commercially available Triglycerol. Superior results were achieved using a combination of electrodialysis and ion-exchange as a purification step, which allowed for polyols synthesis with a total chlorine content below 120 ppm. To validate the applicability of oligoglycerol synthetized herein as a food, cosmetic and pharmaceutical additive, preliminary toxicity studies including the cytotoxicity, comet and micronucleus assays are discussed.

INTRODUCTION Poly- and oligoglycerols are attractive and valuable derivatives of glycerol (GL), which are widely used as raw materials for the preparation of polyurethanes, surfactants, cosmetics, detergents, emulsifiers as well as pharmaceuticals1–9. These polyols are highly biocompatible, thus they are widely studied for many biomedical applications1,2,10–13. Their major advantages in the pharmaceutical industry are their ability to endow hydrophobic molecules with sufficient hydrophilicity, increase water solubility and thermal stability of drugs13,14. Moreover, their derivatives–fatty acid esters–comprise an important class of additives in food, cosmetics and pharmaceuticals1,2,8,15–18. Thus, the unique applications of poly- and oligoglycerols make them quite important chemical products. Currently, low molecular weight oligoglycerols are commercially produced by condensation of glycerol19,20. Most of the commercially available oligoglycerols are prepared primarily by Solvay Group as Diglycerol, Polyglycerol-3 or -4 in which main components are


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diglycerols, triglycerols or tetraglycerols, respectively19. Smaller quantities of oligoglycerols are produced by the combination of glycerol with epichlorohydrin (ECH) or 3-chloro-1,2propanediol (glycerol monochlorohydrin, MCH)18,20–22. Industrially applied methods of synthesis are summarized in Figure 1A19,22–28.

Figure 1. A. Scheme of typical industrial synthetic methods of oligoglycerols (n = 1-4); B. Reaction of oligoglycerols preparation with triethanolamine (TEOA) as a catalytic agent proposed in this study (n = 1-2) Production of epichlorohydrin from glycerol (Epicerol®, Solvay process) gives access to biosourced ECH and MCH29, which can be used in the Williamson-type reaction. One of the drawbacks of this strategy is the formation of chloride salts as a byproduct which reduces the conversion efficiency (atom economy) to lower than 80%21. Therefore, oligoglycerols are generally obtained from glycerol at high temperatures (above 200°C) and using acidic (zeolite, H2SO4, cationic resin, etc.)16,17,19,21,30,31 or basic (NaOH, K2CO3, Cs-MCM41, metal oxides, etc.)4,5,16,21,30,32 catalysts (Figure 1). High-temperature, acid-catalyzed glycerol conversion has certain disadvantages such as occurrence of side-reactions like dehydration and oxidation as well


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as formation of by-products which affects final product quality (undesirable color and odor commonly occur)16,21. In order to produce oligoglycerols at lower temperatures the process needs to be conducted at lower pressures21,33. This allows glycerol condensation to take place in the presence of sulfuric acid at temperatures between 110 and 180°C19. The crude product usually contains: glycerol, cyclic and linear diglycerols and triglycerols as well as higher oligoglycerols and are distilled into separate fractions19,21. The high-temperature glycerol conversion catalyzed with bases also yields by-products by competitive dehydration or dehydrogenation reactions of (oligo)glycerols to acrolein, cyclic di- and triglycerols as well as their derivatives. Therefore, glycerol conversion to oligoglycerols is hard to control mainly because of the difficulty in controlling the reaction selectivity. In this process, the by-products always occur in minor amounts and fractional distillation of raw product is necessary. Additionally, other wastes such as the catalyst and the neutralization agent are always produced in both the basic and acidic synthetic routes16,21. Using haloorganic compounds, oligoglycerols were obtained from the reaction between glycerol and ECH or MCH (Figure 1A). The reaction temperature was much below 100°C, which limited the formation of the toxic dehydration products (acrolein)21,24,26,27. Moreover, the reaction with MCH formed diglycerols in high yield and purity. Similarly, triglycerol was obtained as the main product from the reaction between GL and ECH. Unfortunately, these synthetic routes also suffer from the formation of by-products (covalently bonded chlorine and chloride salts). The purification of the crude-mixture is complicated because chloride salts in high amounts severely contaminate the reaction mixture. Therefore, methods based on haloorganic compounds are not often used on the industrial scale, despite their milder reaction conditions, i.e. lower temperature21,23,26. Currently, the use of ECH is controversial because it is a


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highly toxic and reactive epoxide. However, the reactivity of ECH may be controlled by conducting the synthesis at lower temperatures or with an alternative catalyst. Moreover, ECH is an important epoxide, commonly used in many essential industrial processes such as the production of plastics, epoxy glues and resins29. Therefore in 2011, Solvay Group started the glycerol-based production of ECH at the so-called Epicerol process, which is an alternative to traditional routes based on the chlorination of propane at elevated temperatures21,29. The Epicerol process involves the reaction between glycerol and hydrogen chloride in the presence of Lewisacid catalysts; the dichloride intermediates are transformed into epoxide by inorganic alkaline hydrolysis21,29. Over 100 000 tons of ECH were obtained using this process in 2012 alone21. Thus, ECH seems to be a promising substrate for the synthesis of oligoglycerols at low temperatures. Simultaneously, the development of a cheap, easily available, stable and highly efficient catalyst, as well as a new purification strategy of the oligoglycerols mixture based on a Williamson-type reaction, are needed. Chen et. al34 reported the synthesis of triglycidyl glycerol ethers from ECH and GL, performed in 19.2% (w/w) sodium hydroxide at 50-60˚C for 8 h. In this simple reaction, high molecular weight oligo- and polyglycerols were not observed. It was also reported that amine and ammonium salts react with ECH; Ward et. al35 examined the reaction of ECH with triethanolamine (TEOA) in glacial acetic acid at 25°C. Triethanolamine hydrochloride and other epichlorohydrin derivatives were isolated after two months as white, extremely hygroscopic crystals. It was reported that the triethanolamine derivatives had autocatalytic properties in epoxy ring opening, but their concentration in the reaction mixture remained low in the presence of strong bases34–36. The complete oligoglycerols synthesis route from GL and ECH is so far


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revealed only in patents24,26,27,37. In those patents, the molar ratio between GL and ECH varied from 1:2 to 1:1 at temperatures between 20 to 120°C, preferably 50˚C. The objective of this work was to investigate a novel synthesis and purification route for the preparation of triglycerols from GL and ECH with a low chlorine content and at temperatures below 25°C. Oligoglycerols were obtained in the reaction between epichlorohydrin and glycerol dissolved in a 50% solution of potassium hydroxide using TEOA as a catalytic agent. Separation of the salt from post-reaction mixture was found to be the most challenging. To overcome that challenge, a new product purification method employing electrodialysis and ion-exchange has been applied. Development of this new purification strategy should especially contribute to the use of (epi)chlorohydrin for the synthesis of oligoglycerols.

RESULTS AND DISCUSSION In the following subsections the results of our experiments on catalyst selection, optimization of ECH:GL molar ratio, identification of the reaction mechanism, product characterization and reaction times are discussed. Also, the preliminary toxicity results are discussed. Catalyst Selection The reaction between epichlorohydrin (ECH) and glycerol (GL) is an exothermic process, and must be thermally-controlled. In this case, a variety of conditions such as temperature, ECH and GL molar ratio as well as the concentration of strong base all significantly impact the content of cyclic oligoglycerols and high molecular weight compounds. The cyclic oligoglycerols content is an important factor in oligoglycerol synthesis because these compounds are less biodegradable and less reactive; thus, they are undesirable in the industrial product. Moreover, a high content of cyclic fractions in oligoglycerol mixtures used in the production of emulsifiers


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causes deterioration in emulsion stability. High molecular weight oligoglycerols, such as tetraglycerols and higher fractions show similar effects; they limit product miscibility with water. Generally, these undesirable by-products are not limited during industrial process, because the crude product is separated in di-, tri- and tetraglycerols by fractional distillation. Preliminary experiments utilized hexadecyltrimethylammonium bromide (CTAB), methyltricaprylammonium bromide and methyltributylammonium bromide as an phase transfer catalysts. Desirable oligoglycerols were obtained in those experiments, but removing the catalyst from the post-reaction mixture was problematic and likely due to the large size and molecular weights of the quaternary ammonium salts. This suggested the need to use low molecular weight amines. The best oligoglycerols synthesis result was obtained when triethanolamine (TEOA) was used as a catalyst. TEOA activity in epoxy ring opening reactions is commonly known. Several studies have shown that TEOA reacts with epoxide compounds, e.g. ECH and bis-quaternary, with the formation of ammonium salts36,38–42. The presence of such products and their precursors were confirmed in post-reaction mixtures (shown in Figure 2) by means of GC-MS (comparison with library of fragmentation ions of corresponding silyl-derivatives), 1H NMR (signals between 2.5 and 2.7 ppm) and ESI-MS analysis (m/z = 280.1757; 206.1388; 178.1250 (M2+); 224.1492). Products of further reaction of TEOA were also detectable. It should be mentioned that these compounds may act as a good phase-transfer catalyst; moreover, they may also act as surfactants with strong micelle-forming properties42. Moreover, the electrodialytic desalination is an effective method for the removal of derivatives of TEOA from post-reaction mixtures. GC-MS, ESI-MS and NMR analysis confirmed the absence of TEOA and its derivatives (shown in Figure 2) in the purified product.


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Figure 2. Cations of triethanolamine derivatives detected in crude oligoglycerols mixture by ESI-MS. Calculated values of m/z given.

Taking into account the above it was concluded that triethanolamine can be used as an efficient and cheap catalyst for the synthesis of oligoglycerols. Hypothetically, TEOH based bisquaternary ammonium salts (Figure 2) may be responsible for micelle formation and act as a phase

transfer

catalyst,

in

a

manner

analogous

to

previously

studied

CTAB,

methyltricaprylammonium bromide or methyltributylammonium bromide. The mechanism of TEOA catalytic activity was not investigated further in this study, but it was observed that in the absence of TEOA, the synthesis of oligoglycerols did not occur. The total chlorine content in the oligoglycerols was determined by ICP MS43. The total chlorine content for all samples was below 120 ppm, when the post-reaction mixture was desalinated by electrodialysis followed by removal of residual salts from the mixture with a mixed-bed Amberlite MB 20 resin. It should be noted that electrodialytic desalination alone reduced the total chlorine content to approx. 1.1% (w/w) and also the TEOA derivatives were not observed by NMR or GC.


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Selection of ECH:GL molar ratio The effect of molar ratio between ECH and GL on product composition was investigated and reaction data are summarized in Table 1.

Table 1. Summary of the reaction data Entry

molar ratio ECH : GL

ECH

GL

TEOA

KOH

[mol]

[mol]

[mol.%]

[mol]

Selectivity of triglycerols

Glycerol conversion

[%]

[%]

1.

0.5

0.42

0.83

0.5

41.5

95.2

2.

1

0.60

0.60

0.72

28.5

94.9

3.

1.5

0.74

0.49

0.89

23.5

91.3

0.1 4.

2

0.83

0.42

1

20.1

85.4

5.

2.5

0.90

0.36

1.08

15.2

83.8

6.

3

0.95

0.32

1.14

12.9

82.9

These experiments were carried out at room temperature, but with two modifications. In the first case, reagents were stirred for 24 h and each product purified as described in the General procedure of oligoglycerols preparation (Supporting information). In the second modification, the reaction times were expanded over 24 h, which resulted in cross-linked, solid, hydrogel-like materials. The composition of oligoglycerol products obtained after 24 h for different ECH:GL molar ratios are presented in Figure 3.


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Figure 3. Compositions of oligoglycerol products obtained after 24 h at different ECH:GL molar ratios: A. Fraction content (%, w/w) with cyclic oligoglycerols; B. Fraction content (%, w/w) of cyclic di- and triglycerols As shown in Figure 3A, the composition of the oligoglycerol samples strongly depended on the reaction mixture composition. In samples obtained using a high glycerol content (1 and 2; Table 1, Figure 3.) low-molecular weight oligomers (di- and triglycerols) were prevalent with triglycerols the primary components. It should be mentioned that the selectivity of triglycerol formation decrease with ECH:GL molar ratio and an increase in ECH content leads to formation of heavier oligoglycerols. This is the result of ECH hydrolysis which may lead to the formation of hydrogel-like materials and explains a decrease in oligoglycerol yield shown in Figure 1A.


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This suggests that the main reaction mechanism for triglycerol synthesis involves a reaction between two GL molecules and one ECH. Diglycerols and triglycerols can be formed here by direct reaction between GL molecules; reaction between ECH and GL or a direct reaction between ECH molecules. Due to the low temperature, a direct reaction between GL molecules seems unlikely, as does the direct reaction between ECH molecules which would lead to the formation of cyclic oligoglycerols (discussed later). Therefore, we assumed that the mechanism involves a catalytic reaction between ECH and GL as shown in Figure 4, which leads to aqueously soluble halohydrin compound (1). In the presence of potassium hydroxide, the halohydrin would then undergo either substitution reaction (d) to yield a linear diglycerol (4) or dehydrohalogenation reaction (b) that yields an epoxy compound (2) and reacts (c) with another GL molecule to yield triglycerols (3). Alternatively, this epoxy derivative might undergo hydrolysis (e) to yield diglycerols (4). This, along with the halohydrin substitution mechanism, justifies the large fractions of diglycerol in both 1 and 2 (Table 1, Figure 3). Similarly, epoxy compound formation and its reaction with GL explains the predominant formation of triglycerols for both 1 and 2 (Table 1, Figure 3). It also explains large amounts of tetraglycerols in reaction mixtures 1 and 2 (Table 1, Figure 3) through a reaction between the epoxy derivative and already formed diglycerol. As the ECH:GL molar ratio increased, the content of high molecular weight oligoglycerols also increased (Figure 3); this increase comes from the condensation reaction of the epoxy intermediate with GL or di- and triglycerols already formed. Alternatively, condensation of ECH and the epoxy intermediate yields cyclic products as shown in Figure 5. The data in Figure 3B supports this conclusion, which shows an increase in the cyclic diglycerol and triglycerol fractions ECH content. It should be noted that the cyclic oligomers always constituted a tiny fraction of the total oligomers.


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Also noteworthy, high yields of diglycerol cannot be achieved because ECH combines with at least two molecules making the formation of triglycerols more likely (Figure 3A). Figure 4 summarizes the primary triglycerol formation mechanisms (a-c) and side reactions (d-f).

Figure 4. Scheme of three reactions for triglycerol synthesis; a) reaction between epichlorohydrin and glycerol, b) dehalogenation reaction of (1) to epoxy derivatives (2) c) reaction of (2) and glycerol to triglycerol (3), and proposed side-reactions (d-f), n = 1-4. For the clarity, the synthetic route for αα-αα-triglycerol was only presented.

Figure 5. Scheme of the likely reaction mechanism between epoxide molecules (R = -Cl or -OH) Therefore, we concluded that GL – ECH based reactions following the (a) – (c) pathway is an effective method for the synthesis of triglycerols (Figure 4.). An excess of GL should be


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applied to limit side reactions; this will effectively reduce the amount of epoxy- and halohydrin intermediates in the reaction mixture and thus reduce the formation of diglycerols, heavier oligoglycerols and cyclic compounds. The similar results are observed for the selectivity of triglycerols-forming (்ܵீ , Table 1.). In this case, the decrease in selectivity with increasing ECH content is correlated with the content of the heavier oligoglycerols. To validate this assumption, experiments following the General procedure for oligoglycerols preparation (Supporting information) at ECH:GL molar ratios of 0.4, 0.3 and 0.2, corresponding to GL excess ranging from 25 to 75%, were performed. Despite the change in GL excess, the products obtained were characterized by similar compositions; the triglycerol content always exceeded 75% and no penta- and heavier oligoglycerols were observed. Since excess GL would need to be distilled from the reaction mixture, the optimal ECH:GL molar ratio used for triglycerol synthesis was 0.4. If the reaction time for the synthetic method described below increased beyond 72 h and high ECH:GL molar ratios (>2), solid hydrogel-like materials were also obtained; these products were completely cross-linked and insoluble. Composition of the triglycerol samples obtained A Triglycerol (PG3) sample was obtained according to Triglycerol preparation (Supporting information) procedure after 24 hours at room temperature. In this case, linear triglycerol isomers were the major products. GC FID analysis of the raw product and triglycerol samples purified by distillation between 180-205°C at 4 mbar are shown in Figure 6A and B. The composition of the purified PG3 sample (Figure 6B) was compared with commercially available Triglycerol (TGL, Figure 6C).


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Figure 6. GC FID analysis of oligoglycerols; A. raw triglycerol (PG3); G1: glycerol, G2: cyclic diglycerols, G3: diglycerols, G4: cyclic triglycerols, G5: triglycerols, G6: tetraglycerols and G7 pentaglycerols and higher fractions, * - TEOA derivatives; B. Triglycerol (PG3) after purification; C. Commercially available Triglycerol (TGL) D. Chemical structure and


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abbreviation of various triglycerol isomers, with magnified the G4 and G5 regions of triglycerols indicated by GC: (top) PG3; (bottom) TGL According to previous work of De Meulenaer, et. al44, the typical GC chromatogram of the triglycerols region (G4 and G5 in Figure 6A-C) includes five characteristic peaks and represent seven linear isomers of triglycerol (G5) marked as αα-αα, αα-βα, αα-αβ, αβ-αβ, αβ-ββ, βα-αβ and αα-ββ (Figure 6D). Based on previous results39 and our own analysis, PG3 peaks are probably represented by the αα-αα, αα-αβ and βα-αβ (Figure 6D.) The difference between PG3 and TGL chromatograms indicates that TGL was probably obtained during a high-temperature process in which direct reaction between GL molecules which resulted in branched isomers and cyclic fractions. Thus, the PG3 synthetized here had a higher linear triglycerol content than commercially available TGL. The GC-FID PG3 sample analysis showed that the G5 fraction contents of αα-αα, αα-αβ and βα-αβ isomers were 81.7%, 17.5% and 0.8%, respectively, thus the sample obtained contained 78% linear triglycerols. The PG3 fractional composition is shown in Figure 7.

Figure 7. Triglycerols isomer mixture (PG3) fractions This method resulted in formation of triglycerols with lower amounts of high molecular weight fractions and cyclic oligoglycerols as compared to the commercially available product. However,


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our product also contained minute amounts of GL (0.2%), which was difficult to distill due to the wide distillation endpoint. The linear triglycerols obtained from the PG3 sample explains further the mechanism of our synthesis21. Linear oligomers form only when glycerol combines with ECH through a hydroxyl group at the terminal (C1 position) carbon atom, which in the presence of KOH forms an α-alkoxide as shown in Figure 8 Then, this α-alkoxide reacts with ECH by nucleophilic substitution or a ring opening of the epoxide, both of which yield a linear epoxy-intermediate (structure 2 in Figure 4), which reacts further with GL.

Figure 8. Scheme of two competitive reactions of glycerol with epichlorohydrin21 The effect of time on triglycerol composition and yield The effect of time (from 6 to 52 h) on the fractional composition of triglycerol (PG3) samples was evaluated. The samples were obtained as described previously and analyzed by GCFID; the hydroxyl value was analyzed as well and describes the amounts of reactive hydroxyl groups. In addition, GL conversion was calculated by comparing the mass of glycerol in the products with the theoretical mass of GL that should have reacted with ECH in a 2:1 molar ratio.


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The results are shown in Figure 9. A strong increase in GL conversion up to 93% for PG3 samples obtained during the initial 24 h was observed with a simultaneous decrease in hydroxyl value to 820 mg/g. After 24 h, GL conversion and hydroxyl values changed rather slowly to reach values of 95% and 760 mg/g, respectively, after 52 h. In terms of fractional composition, initially (up to 12 h of reaction) diglycerols were the primary product and their formation was accompanied by a relatively small decrease in the hydroxyl value. They were formed at low GL conversions and yields. This initial formation of diglycerols was clearly the result of a direct reaction between GL and ECH, which should initially yield halohydrine. Unfortunately, the presence of halohydrine in the reaction mixture was not confirmed as it underwent hydrolysis during the post reaction treatment, making diglycerol and glycerol-halohydrin indistinguishable. After 12 h, KCl precipitates from the reaction mixture, the triglycerols content increased dramatically and the hydroxyl number sharply decreased with GL conversion exceeding 90% after 24 h. No significant amounts of penta- and heavier oligoglycerols were observed.


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Figure 9. A. The triglycerol (PG3) hydroxyl value and glycerol conversion versus reaction time (h); B. PG3 fraction content (%, w/w) versus reaction time (h) Preliminary toxicity studies of the triglycerols In order to perform preliminary toxicity studies, in vitro cytotoxicity and genotoxicity in relation to immortalized human keratinocytes HaCaT were evaluated. Cytotoxicity was determined by using the MTT assay, which involves an assessment of the activity of mitochondrial dehydrogenase enzymes produced by living cells. Potential genotoxicity was evaluated in two assays: comet assay and the micronucleus assay. The first measures DNA damage, while the latter determines the number of micronucleus formed as a result of anomalous mitotic cell division (Figure 10).


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Figure 10. HaCaT cells without and with micronucleus (cells in red circle) Three aqueous solutions were tested. The first solution contained triglycerol (PG3), the second contained commercially available triglycerol (TGL) and the last solution was prepared by adding 1% (w/w) 3-chloro-1,2-propanediol into TGL (TGL+MCH). MCH was chosen as a possible product of ECH hydrolysis. As determined by MTT assay within the range of applied concentrations (0.25 to 2.5 mg/cm3), the proliferation did not decrease below 82% compared to the non-treated control test for both PG3 and TGL (Figure 11A).


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Figure 11. A. HaCaT cells viability measured with the MTT test after 72-hour treatment with prepared triglycerol (PG3), commercially available triglycerol (TGL) and commercially available triglycerol with 1% (w/w) 3-chloro-1,2-propanediol (TGL + MCH); the red line marks the IC50 value. B. Tail moment (N=100) after treatment of HaCaT cells with etoposide (40 µg/cm3), PG3 and TGL + MCH In turn, when the cells were treated with TGL + MCH, the increased concentrations caused the proliferation to decrease and the IC50 value of 1.4 ± 0.1 mg/cm3 was determined (concentration of a compound that decreased HaCaT cell viability by 50%). Likewise, the comet assay also showed that only the TGL + MCH caused an increase in DNA damage, similar to the positive control – etoposide (Figure 11B, Figure 12).


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Figure 12. Tail moment (N=100) after treatment of HaCaT cells with etoposide (40 µg/cm3), commercially available triglycerol (TGL), prepared triglycerol (PG3) and commercially available triglycerol with 1% (w/w) 3-chloro-1,2-propanediol (TGL + MCH). Tail moment performed using CometScore™ Freeware v1.5 software. DNA of HaCaT cells after the comet assay: control (A), etoposide 40 µg/cm3 (B) as well as TGL + MCH: 0.25 mg/cm3 (C), 1 mg/cm3 (D) 2.5 mg/cm3 (E). The micronucleus test saw no genotoxic damage in interphase cells caused by any of the tested solutions, as the increase in a number of created micronuclei was observed only in the positive control (etoposide) (Table 2).


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Table 2. Average frequency of micronucleus (MN) in HaCaT cells (N=500) Concentration,

MN,

mg/cm3

%

Control

-

2.3 ± 0.7

Etoposide

0.04

13.5 ± 0.3

0.25

3.4 ± 0.9

1

3.0 ± 0.6

2.5

2.6 ± 0.5

0.25

2.3 ± 0.8

1

2.6 ± 0.1

2.5

3.2 ± 0.8

0.25

3.1 ± 0.4

1

2.2 ± 0.1

2.5

3.0 ± 0.1

Tested compounds

TGL

PG3

TGL + MCH

CONCLUSIONS

The oligoglycerols synthesis methods based on haloorganic compounds are not often used on an industrial scale, despite the milder reaction conditions. In this case, major difficulties of Williamson-type ether reaction lie in the formation of by-products with covalently bonded chlorine as well as chloride salts. The purification of the crude-mixture is complicated because high concentrations of chloride salts severely contaminate the reaction mixture. Therefore we propose the electrodialysis followed by removal of residual salts from the mixture with a mixed-bed Amberlite MB 20 resin, as the effective purification method of post-reaction mixture and complement of this synthesis route. This method allows to reuse of waste salt, and isolate product of low chlorine content (less


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than 120 ppm) by distilling off water and glycerol excess, only. Fractional distillation was not used after desalination, because the crude-product included a low amount of cyclic (less than 2%) and heavier fractions (3%, above tetraglycerols). With this purification method, the oligoglycerols based on Williamson-type ether reaction were successfully synthesized from glycerol and epichlorohydrin at room temperature. The reaction between ECH and GL in presence of a phase transfer catalyst was observed and the best result was achieved using triethanolamine. In the reaction mixture, triethanolamine and its derivatives were responsible for interfacial reaction between the unstirred-phases. In this case, bis-ammonium salts formed by the reaction between ECH and TEOA, that was confirmed by GC. The molar ratio of reagents, as well as the amount of the glycerol excess, were successfully matched. The synthetic route focused on triglycerol because this compound is commonly used as an emulsifier agent in the care-products or the food industry. As the result an oligoglycerols mixture with 78% triglycerol isomers was obtained. Moreover, this triglycerols mixture preferably contained linear fractions such as: αα-αα, αα-αβ and βα-αβ and their concentrations in the product were 81.7%, 17.5% and 0.8%, respectively. These results confirm the steps for the ECH and GL reaction are ring opening addition and SN2, both of which preferentially form linear regioisomers. Preliminary toxicity studies proved our product and commercially available triglycerol to be non-toxic in terms of DNA damage. For comparison cyto- and genotoxicity of oligoglycerols with MCH in relation to skin cells (immortalized human keratinocytes HaCaT) were confirmed.


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In summary, oligoglycerols were successfully synthesized under mild conditions from glycerol and ECH. The proposed route is an alternative to established processes where harsh conditions may lead to numerous impurities and overall high cost of production. Our strategy, in combination with the Epicerol process, may constitute a completely innovative route for the preferential production of high-end oligoglycerols in general and triglycerols in particular. The advantages of our method are: 1. Higher reaction selectivity and content of linear oligoglycerol fractions Our product contained mainly triglycerol in consequence of matching the molar ratio of reagents. Moreover, low content of cyclic fraction and preferably linear fractions in triglycerols mixture such as: αα-αα, αα-αβ and βα-αβ were achieved. In the case of traditional oligomerization methods, the oligoglycerols mixture is obtained (usually below 40% of all oligoglycerols and ca. 10% cyclic oligoglycerol), thus proper fraction (e.g. triglycerol) must be distilled off by fractional distillation. 2. The total epichlorohydrin conversion In our method, epichlorohydrin is totally converted to oligoglycerols or non-toxic glycerol and inorganic salts, thus the proposed purification method (electrodialysis with the ion exchange) allows to obtain product of total chlorine content below 120 ppm. 3. Mild process conditions During oligomerization processes under acidic and basic condition, the high temperature is used (>150°C and >200°C, respectively) which result in formation of toxic by-products (acrolein and unsaturated derivatives) caused by decomposition of product. The reaction between glycerol and chlorohydrines (e.g. epichlorohydrin or 3-chloro-1,2-propanediol)


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required lower, but still elevated temperature from 50 to 170°C, preferably 120°C to 150°C. In our method, application of triethanolamine provides to obtain oligoglycerols at a room temperature at which the above-mentioned toxic by-products are not formed.

ASSOCIATED CONTENT Supporting Information. The experimental section (materials, preparation procedure, analytical and experimental methods, purifications of the post-reaction mixture by electrodialysis method and procedure of preliminary toxicity experiments set) are included in supporting information text (PDF).

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] Notes The authors declare no competing financial interest.

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For Table of Contents Use Only

Synopsis This alternative route of oligoglycerols synthesis and purification under mild conditions has a good prospect for environmental sustainable development.


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Preparation of Triglycerol from Glycerol and Epichlorohydrin at Room

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