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Enantiospecific synthesis of sn-1,2-, 2,3-, and 1,3-diacylglycerols as naphthylethylurethane derivatives

The sn-diacylglycerols (DAGs) are important intermediates from a biological point of view. For this reason, the derivatization of DAGs with optically active protective groups represents an important strategy for their characterization. In this work a high-yielding enantiospecific synthesis of stable samples of DAGs as (S)-(1naphthyl)-ethyl urethane derivatives is reported.


Introduction
In the last decades, the interest of consumers and scientific community in developing drugs and nutraceuticals from vegetable sources has grown significantly. Especially in recent years, there has been a return to natural remedies using compounds and plant extracts with relevant health properties 1,2,3,4 that have been used against many diseases and illnesses. Many natural compounds, belonging to different chemical classes, have been studied and fully characterized 5,6 starting from different parts of plants. 7 Diacylglycerols (DAGs) are compounds that may exist in three isomeric forms two of which, sn-1,2-and sn-2,3-DAGs, are optically active enantiomers, while the form of sn-1,3-DAGs is optically inactive. 8,9 Among these, the sn-1,2-DAGs certainly play an important role from a biological point of view, as they can be second messengers in many cellular processes, intermediates in biosynthesis and catabolism of triglycerides and in the biosynthesis of some phospholipids (phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine) 10,11 and they represent important membrane modulators. 12 The sn-DAGs are also the subject of numerous synthetic studies, with particular focus on the development of protective groups that allow the preparation of optically active glycerols. 13,14 The synthesis of some digalactosyldiacylglycerols, which have antihyperlipidemic activity and other important biological activities, has also been reported. 15 No less important is their role in the field of food chemistry; indeed, the determination of the relationship between sn-1,2-, sn-2,3-, and sn-1,3-DAGs 16,17 is an index for the evaluation of state of fresh olive oils (age and conservation methods). 18,19,20 Different methods are available for the preparation of DAG-rich oils. 21,22 From nutritional point of view, consumption of DAG-rich oil enhances loss of body weight and fat in comparison with consumption of a triacylglycerol control oil. 8,23,24 Various techniques that allow the isolation of the fraction containing DAGs and, subsequently, their separation in the form of suitable derivatives such as, for example, (R)-or (S)-1-(1-naphthyl)-ethyl urethanes have been developed. 25,26 The diastereomers character of (R)-or (S)-1-(1-naphthyl)-ethyl urethane derivatives of sn-1,2-and sn-2,3-DAGs makes the properties of these compounds sufficiently different to allow their separation and characterization. Indeed, (R)-or (S)-1-(1-naphthyl)-ethyl urethane moiety is usually used in order to clarify some stereochemical details of natural occurring compounds, for example to determine the absolute stereochemistry of umbraculumins A and C 27 and an unusual DAG (i.e. archidorin), isolated from the mantle of the mollusk Archidoris tuberculate 28 or to separate monoacylglycerol classes from extravirgin olive oil. 29 Nuclear magnetic resonance (NMR) analysis was demonstrated to be a valuable spectroscopic technique in the field of lipid analysis, for example, to establish composition and lipid classification, to provide information on the regiospecific distribution of fatty acids in triacylglycerols (TAGs) and phospholipids and to verify the authenticity and adulteration of food products. 30,31,32 In this field, very few spectroscopic data on diacylglycerol isomers and enantiomers are reported in the literature. 33 In 2007 we performed a study on HPLC separation and NMR structural elucidation of sn-1,2-, 2,3-, and 1,3-diacylglycerols from olive oil derivatized as (S)-1-(1-naphthyl)-ethyl urethanes. 34 To the best of our knowledge, this is the first study that reports complete mono-and two dimensional spectroscopic NMR data of the three individual classes of diacylglycerol derivatives mentioned above.
Then, the final compounds 4a, 4b and 4c were characterized by 1 H-and 13 C-NMR experiments and the results were compared with the data previously reported for the naphthylurethane derivatives of sn-1,2-, sn-2,3-, and sn-1,3-DAGs from olive oil. 34 This comparison allowed us to confirm the structures and the configuration of the latter.

Results and Discussion
To perform the synthesis of (S)-1-(1-naphthyl)-ethyl urethane derivatives of sn-1,2-, sn-2,3-and 1,3-DAGs is of key importance to choose a suitable glycerol protective group which must be compatible with the derivatizing reagent. The latter must be stable to the hydrolytic reaction conditions of protective groups and not give migration phenomena.
Compounds 1a or 1b were initially reacted with (S)-(+)-(1-naphthyl)-ethyl isocyanate in toluene at 50 °C in the presence of catalytic amount of 4-pyrrolidinopyridine. 25   Despite compounds 2a and 2b are diastereomers, minimal differences in the 1 H-and 13 C-NMR spectra carried out in deuterated chloroform (CDCl3) between the two isomers were observed.
The superimposed 1 H-NMR spectra of 2a and 2b ( Figure 1) show slight difference of the NH at 5.21 and 5.15 ppm, respectively. Notably, it is possible to observe differences also in the range of 4.0-4.4 ppm ascribed to the glycerol moiety multiplets, in which tighter signals for compound 2b were observed. To the best of our knowledge, only limited spectroscopical data were reported in the literature for these kind of compounds as described by Gavagnin  In order to clarify that, we applied spectroscopic NMR techniques for a complete characterization of these derivatives. For this purpose, the homonuclear 2D 1 H (COSY) and heteronuclear 2D 1 H/ 13 C HMQC and HMBC experiments were particularly useful for the assignment of the chemical shift of the glycerol moiety hydrogens and carbons.
Heteronuclear multiple quantum correlation spectroscopy (HMQC) 36,37 is an inverse chemical shift correlation experiment that, like heteronuclear (X, H) shift correlation spectroscopy (XHCORR), is used to determine which 1 H of a molecule is bonded to which 13 C nuclei (or other X nuclei). In this case, the HMQC spectra of 2a and 2b ( Figure 2) allowed a better monitoring of the minimal differences existing between the two isomers resolving the problem related to the partial overlapping of hydrogens H-1a and H-3b of the glycerol moiety ( Figure 1). Furthermore, it was possible to establish a more accurate chemical shift of glycerol moiety protons when correlated with the corresponding carbons ( Figure 2). As it is possible to observe in Figure 2, the major differences are related to the chemical shift of glycerol moiety hydrogens, rather than carbons.
Heteronuclear multiple bond correlation spectroscopy (HMBC) 36,38,39 is a modified version of HMQC suitable for determining long-range 1 H-13 C connectivity. This experiment is useful in determining the structure and 1 H and 13 C assignments of molecules. In this case, the assignment of C-1 of 2a was possible because there is a correlation between carbamic carbon at 155.1 ppm and the protons at 4.03 and 4.25 ppm. These protons were previously correlated with the carbon at 65.2 ppm by HMQC experiment (Figure 2). Thus, the results obtained by NMR experiments were particularly useful for the assignment of hydrogens and carbons of the glycerol moiety, as reported in Table 1.  The subsequent removal of the acetonide protective group of 2a and 2b was an important key step. We found that a mild hydrolysis performed with a 0.5N trifluoroacetic acid solution in THF/H2O (4:1, v/v) at room temperature did not affect naphthyl-ethyl urethane moiety, leading to the intermediates 3a or 3b with high yields (78 and 82%, respectively). Under these reaction conditions, migration phenomena of the naphthylethyl urethane moiety were not observed.
Also the diastereoisomers 3a and 3b present minimal NMR spectroscopical differences. Indeed, the 1 H-NMR spectra in DMSO-d6 of 3a and 3b show little differences in the range of 3.7-4.1 ppm attributed to the methylene (C-1) bearing the carbamic ester of the glycerol moiety. These signals appear as two doublets of doublets with a chemical shift at 3.82 and 3.99 ppm for 3a and, 3.87 and 3.95 for 3b (Figure 3). Homonuclear 2D 1 H (COSY) and heteronuclear 2D 1 H/ 13 C (HMQC) experiments allowed us a better monitoring of the minimal differences existing between the two isomers and to confirm the chemical shift assignment of hydrogens and carbons. As it is possible to observe in the HMQC spectra of 3a and 3b (Figure 4), the major differences are related, again, to the chemical shift of glycerol moiety hydrogens, rather than carbons ( Table 2).  Finally, the esterification process to obtain DAG derivatives 4a or 4b was performed by reaction of the intermediates 3a and 3b with oleic acid in the presence of N,N'-dicyclohexylcarbodiimide (DCC) and dimethylaminopyridine (DMAP), in dichloromethane at room temperature under nitrogen atmosphere. After purification by column chromatography on silica gel final compounds 4a or 4b were isolated with very good yields of 91 and 90%, respectively.
DAG derivatives 4a and 4b have been investigated by means of 1 H-NMR and 13 C-NMR, resulting identical to the samples obtained by HPLC separation of natural DAGs derivatized with (S)-1-(1-naphthyl)-ethyl urethanes as we previously mentioned. 34 The synthetic approach to prepare sn-1,2-and sn-2,3-DAG derivatives 4a and 4b was also applied to the synthesis of 1,3-DAG derivative 4c (Scheme 2).
Thus  In Table 3 are summarized the 1 H and 13 C chemical shift values of glycerol moiety of 4a, 4b and 4c. In Figure 5, the superimposition of the 1 H-NMR spectra, in the range of 4.0-4.5 ppm, evidencing the differences at level of glycerol moieties of compounds 4a-c, is reported for both synthetic and semi synthetic (obtained from natural DAGs) Error! Bookmark not defined. derivatives. These differences are particularly important for the characterization of the three DAG-(S)-1-(1-naphthyl)-ethyl urethane derivatives, as previously demonstrated. 33 As shown in Figure 5, the pattern of the glycerol moiety signals of semisynthetic (obtained from sn-1,2-, sn-2,3-, and 1,3-DAGs from olive oil) and synthetic DAGs derivatized as (S)-1-(1-naphthyl)-ethyl urethane are identical.

Conclusions
In this paper a very high-yielding and manageable enantiospecific synthesis of stable sample of DAG derivatives 4a, 4b and 4c has been performed in order to confirm the structure proposed for the DAG (S)-1-(1naphthyl)-ethyl urethane derivatives previously obtained from sn-1,2-, sn-2,3-, and sn-1,3-DAGs from olive oil and separated by normal phase-high performance liquid chromatography. Full characterization of final compounds and intermediates was performed by NMR experiments. The comparison of 1 H and 13 C-NMR spectra of DAG (S)-1-(1-naphthyl)-ethyl urethanes obtained from the enantiospecific synthetic pathway and from natural deriving isolated DAGs, allowed us to confirm the structures and the configuration previously proposed.

Experimental Section
General. All chemicals were purchased from the major chemical suppliers as highest purity grade and used without any further purification. Solvents were dried over standard drying agent and freshly distilled prior to use. Column chromatography was performed with Merck silica gel 60 (70-230 mesh ASTM) and monitored by thin layer chromatography (TLC) on silica gel 60 F254 with detection by charring with 8% phosphomolibdic acid in EtOH. 1 2a (2b or 2c) (1 mmol) was treated with a 0.5N THF/H2O (4:1) solution of trifluoroacetic acid (5 mL) at 0 °C. After 30 minutes, the reaction was warmed at room temperature and stirred for 20 h. The reaction mixture was treated with a 5% solution of NaHCO3 to reach pH 8, then was extracted with EtOAc (3mL×3). The organic layer was dried on Na2SO4 and then concentrated under vacuum. The crude was purified by column chromatography on silica gel eluting with a mixture of CH2Cl2/MeOH (97:3, v/v). -oleate derivatives (4a, 4b and 4c). To a stirred solution of the glyceryl naphthyl urethane derivative 3a (3b or 3c) (1 mmol) and oleic acid (2.1 mmol) in CH2Cl2 (13 mL), at 0 °C and under nitrogen, a solution of DCC (7 mmol) and DMAP (0.2 mmol) in CH2Cl2 (10 mL) was added dropwise. The reaction was monitored by TLC (CH2Cl2/MeOH 9:1). After 3 h at room temperature the reaction mixture was concentrated under vacuum. The crude was treated with CH2Cl2 (10 mL) and filtered on Buchner to remove solid residue. The CH2Cl2 solution was concentrated under vacuum and purified by column chromatography on silica gel eluting with a mixture of CH2Cl2/hexane (1:1, v/v). In order to avoid possible misunderstanding about the numbering of the glycerol moiety, in this paper we applied IUPAC rules numbering. Thus, the C-1 position of glycerol moiety of sn-1,2-and sn-2,3-DAGs always refer to the carbon bearing the naphthyl urethane group.