**3.1.1 Synthetic approaches**

Phenolic acids, such as caffeic acid, have been esterified with good chemoselectivity in the presence of strong protic acids (Fischer esterification), but the severe reaction conditions together with the large excess of alcohol required make this strategy of limited applicability (Burke et al., 1995). Under basic catalysis, phenols can be easily deprotonated, so the esterification of phenolic alcohols and phenolic acids via acyl nucleophilic substitution requires previous protection of the phenolic hydroxyl groups, due to the competition between aliphatic and phenolic hydroxyl groups (Appendino et al., 2002; Gambacorta et al., 2007).

#### **3.1.1.1 Protection of phenolic hydroxyl groups**

378 Olive Oil – Constituents, Quality, Health Properties and Bioconversions

integrase. Thus, these agents provide an advantage over other antiviral therapies in which

HT and its derivatives are also useful, when applied topically, as microbicide for preventing HIV-infection, as well as other sexually transmitted diseases caused by fungi, bacteria or viruses (Gómez-Acebo et al., 2011). Furthermore, it has been reported that hydroxytyrosol inactivated influenza A viruses, suggesting that the mechanism of the antiviral effect of HT

The antinitrosating properties of hydroxytyrosol and other plant polyphenols of dietary relevance have been investigated (De Lucia et al., 2008). It has been shown that HT reacts with sodium nitrite at pH 3 to give 2-nitrohydroxytyrosol, supporting a protective role of

Many different hydroxytyrosol lipophilic analogues occur naturally in olive fruit and in virgin olive oil. The amount of these compounds is related to olive variety and ripeness, climate, location, type of crushing machine and oil extraction procedures. As an example, the concentration of hydroxytyrosyl acetate is similar to that of HT in some olive oil varieties such as Arbequina, twice as high in the Picual variety, and between one third and one

Due to the limited solubility of HT in lipid media, the search for new lipophilic hydroxytyrosol esters with enhanced properties is of great interest, both in food industry and in medicine. Studies on olive polyphenols have shown the importance of the lipophilicity of the antioxidants on the cell uptake and membrane crossing, and on the substrate to be protected (membrane constituents or LDL), (Grasso et al., 2007). These facts explain the efforts made in

Phenolic acids, such as caffeic acid, have been esterified with good chemoselectivity in the presence of strong protic acids (Fischer esterification), but the severe reaction conditions together with the large excess of alcohol required make this strategy of limited applicability (Burke et al., 1995). Under basic catalysis, phenols can be easily deprotonated, so the esterification of phenolic alcohols and phenolic acids via acyl nucleophilic substitution requires previous protection of the phenolic hydroxyl groups, due to the competition between aliphatic and phenolic hydroxyl groups (Appendino et al., 2002; Gambacorta et al.,

both, viral entry and integration, are inhibited (Lee-Huang et al., 2007a, 2007b, 2009).

might require the presence of a viral envelope (Yamada et al., 2009).

**2.7 Hydroxytyrosol as an antinitrosating agent** 

HT as an efficient scavenger of nitrosating species (Fig. 2).

Fig. 2. 2-Nitrohydroxytyrosol formed by nitrosation of HT

fourth in the Manzanilla and Hojiblanca oils (Romero et al., 2007).

the development of new synthetic analogues with increased lipophilicity.

**3. Hydroxytyrosol derivatives 3.1 Lipophilic hydroxytyrosol esters** 

**3.1.1 Synthetic approaches** 

2007).

As an example, benzyl groups have been used to carry out the HT esterification under basic conditions, followed by catalytic hydrogenation to remove the protective groups (Gordon et al., 2001), as depicted in Scheme 2.

Scheme 2. Synthesis of hydroxytyrosyl acetate via benzylation of phenolic hydroxyls

A two-step procedure involving the reaction of methyl orthoformate-protected hydroxytyrosol with acetyl chloride, and hydrolytic deprotection in phosphate buffer under very mild conditions (pH=7.2) to get hydroxytyrosyl acetate (87% overall yield) (Scheme 3) was also described as a successful procedure for the preparation of HT-derived esters (Gambacorta et al., 2007). The key synthetic orthoester intermediate was also used for the synthesis of HT upon reduction with LiAlH4 and acidic deprotection.

Scheme 3. Synthesis of hydroxytyrosyl acetate via methyl orthoformate-protected hydroxytyrosol.

Biological Properties of Hydroxytyrosol and Its Derivatives 381

procedure with 48% and 50% yields, respectively. The esterification is carried out using DIAD (diisopropyl azodicarboxylate) and TPP (triphenylphosphine) in THF (Scheme 6). The removal of byproducts arising during the Mitsunobu reaction, a major problem of this type of reactions,

could be solved by gel-permeation chromatography on Sephadex LH-20.

Scheme 6. Mitsunobu esterification of hydroxytyrosol and vanillyl alcohol

such as hexanoyl, palmitoyl, oleoyl and linoleoyl chlorides.

esters is under protection of two patents (Bernini et al. 2007, 2008c).

**3.1.1.5 Syntheses of hydroxytyrosol esters from tyrosol and homovanillyl alcohol** 

The syntheses previously described in the previous sections had all in common hydroxytyrosol as a precursor of its esters, but some efforts have also been done to get hydroxytyrosyl esters starting from different and cheaper reagents. In this context, the syntheses of hydroxytyrosol esters from tyrosol and homovanillyl alcohol have been proposed (Bernini et al., 2008b). This procedure involves the selective esterification of tyrosol and homovanillyl alcohol with acyl chlorides in dicholoromethane as solvent, to give tyrosyl and homovanillyl acetates in 90% and quantitative yields, respectively, by using only a little excess of acetyl chloride in dichloromethane without any catalysts. The authors suggested acid catalysed acylation due to traces of hydrochloric acid derived from the hydrolysis of the acetyl chloride under the experimental conditions. A similar selectivity was observed by using several saturated or unsaturated acyl chlorides with longer chains

The subsequent oxidation with 2-iodoxybenzoic acid (IBX) or Dess-Martin periodinane reagent (DMP) and *in situ* reduction with sodium dithionite (Na2S2O4) of tyrosyl and homovanillyl esters led to the corresponding hydroxytyrosol derivatives. In general, the oxidation of tyrosol derivatives proceeded with higher yields (92-77%) compared to those of homovanillyl derivatives (88-58%). The use of DMP gave similar results to those obtained with IBX. The procedure of oxidation/reduction with IBX/Na2S2O4 to obtain the different

Scheme 7. Synthesis of hydroxytyrosol esters from tyrosol and homovanillyl alcohol

In order to overcome the problems associated to the protection and deprotection steps of the phenolic hydroxyl groups, different methods for the preparation of hydroxytyrosyl esters by reaction of hydroxytyrosol with various acylating agents have been described, such as esterification with free acids (Appendino et al., 2002), transesterification with methyl or ethyl esters (Alcudia et al., 2004; Trujillo et al., 2006), acyl chlorides (Torregiani et al., 2005) and the use of enzymatic methodologies (Grasso et al., 2007; Mateos et al., 2008; Torres de Pinedo et al., 2005; Buisman, 1998).

#### **3.1.1.2 Acid catalyzed transesterification**

HT transesterification using methyl or ethyl esters and *p*-toluenesulfonic acid as catalyst has been described as a method without the need of protection of the aromatic hydroxyl groups due to its total chemoselectivity (Alcudia et al., 2004; Trujillo et al., 2006). This method involves heating a solution solution of hydroxytyrosol in the corresponding ethyl or methyl ester, containing a catalytic amount of *p*-toluenesulfonic acid (Scheme 4). This protocol has been optimized for HT acetate (86%), and also for longer aliphatic chains like hydroxytyrosyl butyrate, laureate, palmitate, stearate, oleate and linoleate, obtained in acceptable to good yields (62-76%) (Mateos et al., 2008).

Scheme 4. General procedure of acid-catalyzed transesterification

#### **3.1.1.3 Acylation of polyphenolic alcohols with the couple CeCl3–RCOCl**

Cerium (III) chloride has been reported to be an efficient promoter for the chemoselective esterication of unprotected polyphenolic alcohols with acyl halides as acyl donors, thereby making it possible to avoid the protection of phenolic hydroxyl groups and providing polyphenolic esters of interest (Torregiani, 2005). This reaction is one example of the socalled Lewis acid catalysis by lanthanide salts (Ishihara et al., 1995). The reaction presumably involves the formation of an electrophilic Lewis adduct between acyl chlorides and cerium (III) chloride, which is quenched by the more nucleophilic aliphatic hydroxyl group of the substrate, with formation of the ester, and regeneration of the lanthanide promoter. The yields obtained are acceptable for HT using nonanoyl and oleoyl chlorides (53 and 52%), respectively (Scheme 5).

Scheme 5. Acylation of hydroxytyrosol with acyl chlorides and Ce(III)

#### **3.1.1.4 Esterification with free acids: Mitsunobu esterification**

The Mitsunobu reaction has been also applied to the chemoselective esterification of phenolic acids with phenolic alcohols (Appendino et al., 2002) as demonstrated by the condensation of hydroxytyrosol with gallic acid, and of vanillyl alcohol with caffeic acid in a one step

In order to overcome the problems associated to the protection and deprotection steps of the phenolic hydroxyl groups, different methods for the preparation of hydroxytyrosyl esters by reaction of hydroxytyrosol with various acylating agents have been described, such as esterification with free acids (Appendino et al., 2002), transesterification with methyl or ethyl esters (Alcudia et al., 2004; Trujillo et al., 2006), acyl chlorides (Torregiani et al., 2005) and the use of enzymatic methodologies (Grasso et al., 2007; Mateos et al., 2008; Torres de

HT transesterification using methyl or ethyl esters and *p*-toluenesulfonic acid as catalyst has been described as a method without the need of protection of the aromatic hydroxyl groups due to its total chemoselectivity (Alcudia et al., 2004; Trujillo et al., 2006). This method involves heating a solution solution of hydroxytyrosol in the corresponding ethyl or methyl ester, containing a catalytic amount of *p*-toluenesulfonic acid (Scheme 4). This protocol has been optimized for HT acetate (86%), and also for longer aliphatic chains like hydroxytyrosyl butyrate, laureate, palmitate, stearate, oleate and linoleate, obtained in

Cerium (III) chloride has been reported to be an efficient promoter for the chemoselective esterication of unprotected polyphenolic alcohols with acyl halides as acyl donors, thereby making it possible to avoid the protection of phenolic hydroxyl groups and providing polyphenolic esters of interest (Torregiani, 2005). This reaction is one example of the socalled Lewis acid catalysis by lanthanide salts (Ishihara et al., 1995). The reaction presumably involves the formation of an electrophilic Lewis adduct between acyl chlorides and cerium (III) chloride, which is quenched by the more nucleophilic aliphatic hydroxyl group of the substrate, with formation of the ester, and regeneration of the lanthanide promoter. The yields obtained are acceptable for HT using nonanoyl and oleoyl chlorides

The Mitsunobu reaction has been also applied to the chemoselective esterification of phenolic acids with phenolic alcohols (Appendino et al., 2002) as demonstrated by the condensation of hydroxytyrosol with gallic acid, and of vanillyl alcohol with caffeic acid in a one step

Pinedo et al., 2005; Buisman, 1998).

**3.1.1.2 Acid catalyzed transesterification** 

(53 and 52%), respectively (Scheme 5).

acceptable to good yields (62-76%) (Mateos et al., 2008).

Scheme 4. General procedure of acid-catalyzed transesterification

**3.1.1.3 Acylation of polyphenolic alcohols with the couple CeCl3–RCOCl** 

Scheme 5. Acylation of hydroxytyrosol with acyl chlorides and Ce(III)

**3.1.1.4 Esterification with free acids: Mitsunobu esterification** 

procedure with 48% and 50% yields, respectively. The esterification is carried out using DIAD (diisopropyl azodicarboxylate) and TPP (triphenylphosphine) in THF (Scheme 6). The removal of byproducts arising during the Mitsunobu reaction, a major problem of this type of reactions, could be solved by gel-permeation chromatography on Sephadex LH-20.

Scheme 6. Mitsunobu esterification of hydroxytyrosol and vanillyl alcohol

#### **3.1.1.5 Syntheses of hydroxytyrosol esters from tyrosol and homovanillyl alcohol**

The syntheses previously described in the previous sections had all in common hydroxytyrosol as a precursor of its esters, but some efforts have also been done to get hydroxytyrosyl esters starting from different and cheaper reagents. In this context, the syntheses of hydroxytyrosol esters from tyrosol and homovanillyl alcohol have been proposed (Bernini et al., 2008b). This procedure involves the selective esterification of tyrosol and homovanillyl alcohol with acyl chlorides in dicholoromethane as solvent, to give tyrosyl and homovanillyl acetates in 90% and quantitative yields, respectively, by using only a little excess of acetyl chloride in dichloromethane without any catalysts. The authors suggested acid catalysed acylation due to traces of hydrochloric acid derived from the hydrolysis of the acetyl chloride under the experimental conditions. A similar selectivity was observed by using several saturated or unsaturated acyl chlorides with longer chains such as hexanoyl, palmitoyl, oleoyl and linoleoyl chlorides.

The subsequent oxidation with 2-iodoxybenzoic acid (IBX) or Dess-Martin periodinane reagent (DMP) and *in situ* reduction with sodium dithionite (Na2S2O4) of tyrosyl and homovanillyl esters led to the corresponding hydroxytyrosol derivatives. In general, the oxidation of tyrosol derivatives proceeded with higher yields (92-77%) compared to those of homovanillyl derivatives (88-58%). The use of DMP gave similar results to those obtained with IBX. The procedure of oxidation/reduction with IBX/Na2S2O4 to obtain the different esters is under protection of two patents (Bernini et al. 2007, 2008c).

Scheme 7. Synthesis of hydroxytyrosol esters from tyrosol and homovanillyl alcohol

Biological Properties of Hydroxytyrosol and Its Derivatives 383

**Phenol Acylating agent Product Time** 

O

O

O

8

Phenol: acylating agent 1:20, *C. antarctica* lipase*, t*-BuOMe, 40 ºC

assay of the radical scavenging activity (Grasso et al., 2007).

Table 1. Enzymatic esterification of HT and homovanillyl alcohol (Grasso et al., 2007)

The order of the scavenging activities toward the ABTS radical was hydroxytyrosyl esters ≥ -tocopherol > hydroxytyrosol > tyrosyl >tyrosyl esters BHT. In a similar trend, comparison of FRAP values obtained for the free hydroxytyrosol and tyrosol with the corresponding esters revealed that while hydroxytyrosyl esters showed a significantly higher reducing activity than their precursor, all the tyrosyl esters showed a lower antioxidant activity than that of tyrosol. The same conclusion was obtained from DPPH

In connection with the size of the acyl chain, the reported literature seems to conclude that the antioxidant capacity of hydroxytyrosyl esters is better for medium-sized (C4C9) alkyl chains in comparison with HT, whereas further elongation of the acyl chain does not improve the antioxidant activity. This confirms that antioxidant capacity does not depend

O

**(min)** 

**Yield (%)** 

35 95.0

35 96.5

75 93.3

180 92.3

60 96.8

90 90.9

90 97.5

240 98.0

### **3.1.1.6 Lipase-catalyzed transesterification**

The use of enzymes, like lipases, as catalysts in non-aqueous solvents to prepare lipophilic derivatives directly from HT has been widely described in the last few years (Grasso et al., 2007; Torres de Pinedo et al., 2005; Mateos et al., 2008; Buisman et al., 1998). This procedure avoids the use of toxic reagents and allows mild reaction conditions.

The esterication of phenols with carboxylic fatty acids and lipases as biocatalysts was firstly investigated by Buisman et al., (1998), using hydroxytyrosol, octanoic acid in hexane, and immobilized lipases from *Candida antartica* (CAL-B). Furthermore, a strong dependence of the yield on the solvent used was observed; so, in diethyl ether a conversion of 85% was obtained within 15 hours (35 ºC), while conversions of roughly 20% were found in the case of solvents like chloroform, dichloromethane or THF. Yields of 70–80% were observed using *n*-pentane and *n*-hexane, in spite of the poor solubility of HT in such solvents.

Different enzymes have been tested on hydroxytyrosol (Grasso et al., 2007) including lipases from *A. niger, C. cylindracea, M. javanicus, P. cepacia, M. miehei, C. viscosum, P. uorescens, R. arrhizus, R. niveus, C. antarctica*, porcine pancreas and wheat germ, using vinyl acetate as reagent and *tert*-butyl methyl ether as solvent. The best results were obtained with *C. antarctica* in terms of short reaction time, chemioselective conversion and good yield. *C. antarctica* lipase (CAL) was selected for acylation of hydroxytyrosol and homovanillic alcohol with vinyl esters of different acyl chains on a preparative scale, as shown in Table 1. The use of *C. antarctica* with increasing alkyl chain length required longer reaction times. The homovanillyl alcohol and its esters were found to exhibit scarce effectiveness both as radical scavengers and antioxidant agents.

Transesterification of HT with ethyl saturated, mono- and poly-unsaturated fatty acid esters, catalized by Novozym 435 (immobilized *C. antarctica* lipase B), in vacuum under solventless conditions, has been successfully developed (Torres de Pinedo et al. 2005). This procedure gave hydroxytyrosyl esters in 59-98% yield for the saturated fatty acid esters, and 32-97% yield for the mono- and poly-unsaturated fatty acid esters.
