Chemoenzymatic synthesis and antimicrobial activity evaluation of monogalactosyl diglycerides

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European Journal of Medicinal Chemistry 43 (2008) 210e221 http://www.elsevier.com/locate/ejmech

Laboratory note

Chemoenzymatic synthesis and antimicrobial activity evaluation of monogalactosyl diglycerides Francesca Cateni a,*, Paolo Bonivento a, Giuseppe Procida b, Marina Zacchigna a, Luciana Gabrielli Favretto b, Giuditta Scialino c, Elena Banfi c a

Department of Pharmaceutical Sciences, University of Trieste, P.zle Europa, 1, 34127 Trieste, TS, Italy Department of Materials and Natural Resources, University of Trieste, Via A. Valerio 6, 34127 Trieste, Italy c Department of Biomedical Sciences, Microbiology Section, University of Trieste, Via A. Fleming 22, 34127 Trieste, Italy b

Received 25 October 2006; received in revised form 5 March 2007; accepted 8 March 2007 Available online 2 April 2007

Abstract Monogalactosyl diglycerides with medium to long fatty acid acyl chains, were prepared and examined for antimicrobial activity against Gram positive, Gram negative bacteria and fungi. The study of their in vitro antimicrobial activity confirms the significant activity of some monogalactosyl diacylglycerol analogues and establishes for the galactose series that the 1,2-disubstitution and the octanoyl chain are the proper structural features for the maximum activity. Ó 2007 Elsevier Masson SAS. All rights reserved. Keywords: Synthesis; Monogalactosyl diglycerides; 1,2-O-Diacyl-3-O-b-D-galactopyranosyl-glycerols; Antimicrobial activity; Lipases

1. Introduction Glycoglycerolipids are common constituents of plant cell membranes and bacterial cell walls [1e4]. Various glycolipids have been isolated from plants [5,6], algae [7] and bacteria [4,8]. Recently, we investigated the active principles from various species of Euphorbiaceae and isolated monogalactosyl diacylglycerols (MGDGs), monogalactosyl monoacylglycerols (MGMGs) and digalactosyl diacylglycerols (DGDGs) as the active components, after repeated chromatography [9]. MGDGs, MGMGs and DGDGs, a class of glycolipids, have attracted much attention in recent years because of their biological activities, such as anti-tumor-promoting [5,6,8,10,11], oxygen scavenging [12], anti-viral [13,14], anti-inflammatory [15] and anti-hyperlipemic * Corresponding author. Tel.: þ39 040 5583722; fax: þ39 040 52572. E-mail address: [email protected] (F. Cateni). 0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.ejmech.2007.03.012

activities [16]. The activity of such compounds seems strictly related to the acyl chain length [10,11,16]. Previous pharmacological studies of methanol extracts from Euphorbiaceae showed both antimicrobial and antiinflammatory action [17,18]. Antimicrobial monogalactosyl diacylglycerols (MGDGs) have been isolated and characterized from the brown algae Sargassum stolonifolium Phang et Yoshida [7]. However, glycoglycerolipids are usually available from natural sources in only limited quantities and as hardly separable mixtures in terms of their fatty acid compositions so that their versatile synthesis seems to be of importance, especially for their biological investigation. Currently, different synthetic studies of various analogues of natural glycoglycerolipids and their modified forms with unnatural structures are intensively being developed [19e23]. Besides, a method for identifying monoglycosyl diglycerides in lipid extracts by using combined gas chromatographyemass spectrometry was reported [24].

F. Cateni et al. / European Journal of Medicinal Chemistry 43 (2008) 210e221

On the basis of these evidences, which indicate the nature of the lipophilic chain as a crucial structural feature for the activity of many compounds, we decided to prepare glycoglycerolipid analogues using monogalactosyl diglycerides as a starting point in order to study the effect against Gram positive, Gram negative bacteria and fungi. In this paper, we report the synthesis of a series of mono-acid and mixed-acid 1,2-O-diacyl-3-O-b-galactopyranosyl-rac-glycerols 9aeh, 13aec and 14aec with medium to long (from C8 to C18) acyl chains. In particular, as concerning the mixed-acid 1,2-O-diacyl-3-O-b-galactopyranosyl-racglycerols 13aec and 14aec, the effects on antimicrobial activity of long-chain polyunsaturated fatty acids (PUFA) located at the mid-position with medium chain fatty acids (MCFA) at one of the end-positions of the glycerol backbone have been studied. Besides, the compounds obtained in this study, in analogy with the structured lipids, whose biological activity seems directly influenced by the presence of determined fatty acids in predetermined positions of the glycerol moiety, should have an interesting application as nutraceutic compounds [25e27].

OAc

H

Galactosyl diglycerides 9aeh, 13aec and 14aec bearing either saturated or unsaturated fatty acids were synthesized as depicted in Schemes 1 and 2. Glucosidation of D,L-a,b-isopropylideneglycerol 3 under Ko¨enigseKnorr conditions [14] by using tetra-O-acetyl-a-D-galactopyranosyl bromide 2 in dichloromethane in the presence of silver carbonate, gave the desired peracetylated b-glucoside 4 [1H NMR signals of anomeric protons: d 4.61, 4.63 (dd, J ¼ 8 Hz) indicated a 1:1 ratio of diastereomeric products] in 70% yield. Removal of all the acyl protective groups of 4 by alkaline hydrolysis afforded 5 which was subjected to protection of the resulting free hydroxyl groups as levulinate esters 6. Thus, successive treatment of 6 with 70% acetic acid in aqueous solution at 60  C in order to remove the isopropylidene protecting group, provided the diol intermediate 7 in quantitative yield (Scheme 1). The intermediate compound 7 represents the key synthon for the synthesis of the mono-acid and mixed-acid 1,2-O-diacyl-3-O-b-galactosylglycerols 9a, b, 13aec and 14aec (Scheme 2).

OAc H

O a

H AcO

2. Chemistry

OAc

OAc

b HO

H

OAc

H

H

H

Br

1

2

OAc

OAc

3 OH

OH

H

H

O

O

H AcO

O

H

O

c

HO H

H

H

OLev

OLev H

O

O

H LevO

O

O H

H

H

5

OLev

H

d

OH

4

H

O

O H

OAc

H

O

H

O

OLev

O

O +

OAc

H

O

H AcO

OAc H

211

OLev H

6 Lev = -CO(CH2)2COCH3

e

HO

OH

O H

H

O

H LevO H

OLev H

H

7 Lev = -CO(CH2)2COCH3

Scheme 1. Reagent and conditions: (a) HBr, CH3CO2H, r.t.; (b) Ag2CO3, CaSO4, I2, CH2Cl2, r.t.; (c) NaOCH3, CH3OH; (d) [CH3CO(CH2)2CO]2O, Py; (e) CH3CO2H 70%, 60  C.

F. Cateni et al. / European Journal of Medicinal Chemistry 43 (2008) 210e221

212

OLev

OLev

OLev H

O

HO

H LevO

R1O

OR1

O H

H

H

O

H LevO

a

OLev

H

H

OH

O H

OLev

OLev

H

H

H

7

8a-h

Lev = -CO(CH2)2COCH3 b OLev

c

OLev

OH

OH H

O

HO

H LevO

OR1

H

O H

H

H

R1 d

O

R2O

OR1

O H

OLev

H

H

9a-h

OLev

H LevO

OH H

10a : R1= -CO(CH2)6CH3 10b : R1 = -CO(CH2)8CH3

H

OR1

O H

H

10a, b

OLev

R1O

HO

OLev

H

O

H

H

H

11a-c, 12a-c

8a, 9a

-CO(CH2)6CH3

8b, 9b 8c, 9c 8d, 9d 8e, 9e 8f, 9f

-CO(CH2)8CH3 -CO(CH2)10CH3

8g, 9g

-CO(CH2)7(CH=CH-CH2)2(CH2)3CH3

8h, 9h

-CO(CH2)7(CH=CH-CH2)3CH3

-CO(CH2)12CH3 -CO(CH2)16CH3 -CO(CH2)7CH=CH(CH2)7CH3

e OH

OH H

O

R2O

H

OR1

O

HO H

OH

H

H

H

13a-c, 14a-c R1 11a, 13a 11b, 13b 11c, 13c 12a, 14a 12b, 14b 12c, 14c

-CO(CH2)6CH3 -CO(CH2)6CH3 -CO(CH2)6CH3 -CO(CH2)8CH3 -CO(CH2)8CH3 -CO(CH2)8CH3

R2 -CO(CH2)7CH=CH(CH2)7CH3 -CO(CH2)7(CH=CH-CH2)2(CH2)3CH3 -CO(CH2)7(CH=CH-CH2)3CH3 -CO(CH2)7CH=CH(CH2)7CH3 -CO(CH2)7(CH=CH-CH2)2(CH2)3CH3 -CO(CH2)7(CH=CH-CH2)3CH3

Scheme 2. Reagent and conditions: (a) fatty acid, DCCD, DMAP, CH2Cl2, r.t.; (b) NH2NH2 (1 M, PyeCH3CO2H 3:2); (c) M. miehei, vinyl caprylate (vinyl capriate), 37  C; (d) fatty acid, DCCD, DMAP, CH2Cl2, r.t.; (e) NH2NH2 (1 M, PyeCH3CO2H 3:2).

F. Cateni et al. / European Journal of Medicinal Chemistry 43 (2008) 210e221

The mono-acid 1,2-O-diacyl-3-O-(b-D-20 ,30 ,40 ,60 -tetra-Olevulinyl-galactopyranosyl)-rac-glycerols 8aeh were prepared by one step diacylation of 7 with 2 equivalents of the desired fatty acids in the presence of dicyclohexylcarbodiimide (DCCD) and 4-dimethylaminopyridine (DMAP) in dry dichloromethane at room temperature. We selected octanoate, caprate, laurate, myristate and stearate as introduction acyl groups, because they are components of bioactive analogues of natural glycoglycerolipids [19e23], and oleate, linolate and linolenate that are, the major acyl components of MGDGs, MGMGs and DGDGs isolated from Euphorbiaceae [9]. Finally, the levulinyl protective groups of 8aeh were removed by treatment with hydrazine hydrate and mono-acid 1,2-O-diacyl-3-O-(b-D-galactopyranosyl)-racglycerol derivatives with different acyl groups 9aeh were obtained (Scheme 2). On the other hand, the synthesis of mixed-acid 1,2-O-diacyl-3-O-(b-D-galactopyranosyl)-rac-glycerols 13aec and 14aec requires full regioselectivity control and can hardly be undertaken by synthetic organic chemistry methods without multi-step protectionedeprotection processes [16]. Based on their high regioselectivity, lipases are ideally suited as biocatalysts for the synthesis of structured lipids, by acting preferably or exclusively at the primary positions of the glycerol moiety [25e27]. Another important feature offered by lipases is the mild conditions under which they act, which may become crucial in hampering intramolecular acyl-migration side-reactions. A chemoenzymatic approach was developed for the synthesis of 2-O-b-D-glucosylglycerol derivatives through a direct lipase catalyzed monoacylation of the substrate mediated by Pseudomonas cepacia lipase (LPS) [20]. To get the intermediate adducts 10a, b (Scheme 2), compound 7, obtained by standard procedures as depicted in Scheme 1, was directly submitted to transesterification catalyzed by Mucor miehei lipase in the presence of the appropriate octanoate 10a and caprate 10b vinyl esters in dry dichloromethane as solvent. The reactions were completely regioselective and afforded, through the expected acylation of the primary hydroxyl group [25e27], the 1-O-acyl-3O-(b-D-20 ,30 ,40 ,60 -tetra-O-levulinyl-galactopyranosyl)-rac-glycerols 10a and b in good yields (95e98%) (Scheme 2). There were no signs of any acyl-migration taking place and subsequently, in the second step, pure oleic, linoleic and linolenic fatty acids were chemically introduced to the remaining mid-position by DCCD coupling agent to give in excellent yields (58e87%) the intermediate products 11aec and 12aec with the hydroxyl groups of the galactosyl moiety temporarily protected as levulinates. Finally, the levulinyl protective groups of 11aec and 12aec were removed by treatment with hydrazine in acetic acidepyridine mixture at room temperature and 1,2-O-diacyl-3-O-(b-D-galactopyranosyl)-rac-glycerols 13aec and 14aec with medium chain fatty acids (MCFAs) at the end-position and long-chain polyunsaturated fatty acids (PUFA) located at the mid-position of the glycerol back-bone were obtained. The structures of the synthesized compounds were established with the help of spectral analyses.

213

3. Results and discussion All monogalactosyl diglycerides are stable compounds and are found to be homogenous by thin layer chromatography. 3.1. Spectroscopy The mass spectra of all compounds agreed with the proposed structures. The series 9aeh, 13aec and 14aec have the correct chemical shifts for the protons in the 1H NMR spectra and for the carbons in the 13C NMR spectra. The chemical shifts of the target compounds and of all the intermediates are given in Section 5. 4. Pharmacology 4.1. Preliminary antibacterial and antifungal evaluation of monogalactosyl diglycerides 9aeh, 13aec and 14aec All the synthesized 1,2-O-diacyl-3-O-b-D-galactopyranosyl-rac-glycerols 9aeh, 13aec and 14aec were submitted for preliminary evaluation of their in vitro antibacterial activity against Staphylococcus aureus, Enterococcus faecalis, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Mycobacterium tuberculosis, and for antifungal activity against Candida albicans and Candida pseudotropicalis. Antimicrobial activity was always evaluated by reference methods [28e30]. Ciprofloxacin was chosen as a standard in antibacterial activity measurements, as it has excellent activity against most Gram-negative and Gram-positive bacteria, and is known as an antibacterial drug in the treatment of a wide range of infections [31]. Miconazole was chosen as a standard in antifungal activity measurements. MIC was defined as the lowest concentration of compound that inhibited bacterial/fungal growth. Table 1 shows MIC values of the synthesized compounds. No inhibitory activity was exhibited by any compound against Candida spp. As can be seen from the presented data galactosyl diglycerides bearing the same saturated fatty acyls 9aee at C-1 and C-2 displayed no inhibitory activity, except for 9a and b which contain a dicapryl (C8:0) and a dicaprinyl (C10:0) moiety, respectively, and showed interesting MICs against S. aureus and M. tuberculosis strains, respectively. On the other hand, compounds bearing the same unsaturated fatty acyls 9feh had little antimicrobial activity. Most compounds bearing different fatty acyls 13aec and 14aec exhibited an interesting inhibitory activity against mycobacteria 13aec and against S. aureus clinical isolate 13a, c and 14c. A total of six compounds were active against a M. tuberculosis reference and clinical strains with MICs ranging from 64 to 128 mg/ml. The antibacterial activity of the compounds was lost by increasing the length of the saturated acyl moiety 9cee. The presence of a single double bond within the acyl chain (18:1) in 9f did not modify the biological activity. Compound 9g, characterized by the presence of two linolate acyl groups in the positions 1 and 2 of the glycerol moiety displayed

F. Cateni et al. / European Journal of Medicinal Chemistry 43 (2008) 210e221 Miconazole

e e e e e e e e e e e e 8 0.5

Ciprofloxacin

0.5 0.25 1 0.25 0.25 1 >4 0.5 0.5 0.5 0.5 0.5 e e

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inhibitory activity against M. tuberculosis reference and clinical strains, B. subtilis and one clinical strain of S. aureus with MIC of 128 mg/ml. Compound 9h bearing three unsaturations within the acyl moiety showed inhibitory activity against S. aureus (MIC of 128 mg/ml). 5. Experimental protocols

>128 >128 >128 >128 >128 >128 >128 64 32 >128 >128 >128 >128 >128 >128 64 128 128 >128 >128 >128 64 64 >128 >128 >128 >128 >128

14b 14a

128 >128 >128 32 >128 >128 >128 128 >128 >128 >128 >128 >128 >128 >128 >128 >128 128 >128 >128 >128 >128 64 128 128 128 >128 >128

13c 13b

>128 >128 >128 128 >128 >128 >128 >128 128 64 64 128 >128 >128 >128 >128 >128 128 >128 >128 >128 >128 64 128 64 64 >128 >128

13a 9h

128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128 128 >128 >128 >128 >128 128 128 128 128 >128 >128

9g

>128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128

9f 9e 9d 9c

>128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128 32 >128 >128 >128 >128 >128 >128 32 128 128 64 64 >128 >128

9b 9a

32 32 32 32 >128 >128 >128 32 32 128 128 128 >128 >128 S. aureus ATCC 25923 S. aureus ATCC 105487 E. faecalis ATCC 29212 B. subtilis ATCC 6633 E. coli ATCC 25922 P. aeruginosa ATCC 27753 S. aureus 52 S. aureus 85 S. aureus 87 M. tuberculosis H37Rv M. tuberculosis H160 M. tuberculosis H190 C. albicans C. pseudotropicalis

MIC (mg/ml)

Table 1 Antimicrobial activity of compounds 9aeh, 13aec, 14aec against Gram-positive, Gram-negative, tubercular bacteria and against Candida spp.

>128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128

14c

5.1. Chemical analysis 1

H NMR and 13C NMR spectra were recorded with a Varian Gemini 200 MHz spectrometer and a Varian Unity 400 MHz spectrometer. 13C NMR spectra were recorded with 90.5 MHz, Gemini 200 spectrometer. NMR spectra were obtained by using CDCl3 as solvent and chemical shifts are expressed as d units (ppm) relative to tetramethylsilane (TMS) as internal standard. The abbreviations s, d, dd, t, q, m and br s refer to singlet, doublet, doublet of doublet, triplet, quartet, multiplet and singlet broad signal, respectively. The EI-MS spectra were measured with a VG-ZAB 2F spectrometer. The ionizing energy was 70 eV in all cases and compounds were introduced by direct insertion. Electrospray analysis was carried out with API Perkin Elmer (voltage þ 5600 with orifice 90 and/or 120). The column chromatography was performed by using silica ˚ Merck). Analytical gel (Kieselgel 60, 230e400 Mesh, 60 A thin layer chromatography (TLC) was carried out on Merck 60 F254 silica gel plates (0.25 mm thickness) and the spots were detected by spraying 50% aqueous H2SO4 and heating at 110  C. M. miehei (LipozymeÒ, specific activity 42 U/g) was purchased from Fluka, Italy. Infrared spectra were recorded as film on a Jasco FT-IR-200 model spectrophotometer. Reagents (Fluka, Italy) used were AR grade and all solvents for synthesis, extraction and column chromatography were distilled and dried before use. Evaporation under reduced pressure was always effected with the bath temperature maintained below 40  C. The elemental analyses were carried out on a Carlo Erba model 1016 analyzer. 5.1.1. 2,3,4,6-Tetra-O-acetyl-a-D-galactopyranosyl bromide 2 HBr in glacial acetic acid (33%) was added dropwise to a solution of pentaacetylgalactose 1 (2 mmol) in acetic acid (3 ml). The reaction mixture was stirred in the dark overnight at room temperature, then ice-cold water (10 ml) was added and the mixture was extracted with CH2Cl2 (3  20 ml). The combined CH2Cl2 layers were washed with a saturated NaHCO3 solution, H2O and brine, and the filtrate was evaporated in vacuo to give a colorless syrupy mixture 2 recrystallized from Et2O. Yield: 68%. Rf ¼ 0.84 (EtOAcehexane 1:1, v:v). 1H NMR (CDCl3): d 1.97 (s, 3H, CH3CO), 2.0 (s, 3H, CH3CO), 2.09 (s, 3H, CH3CO), 2.15 (s, 3H, CH3CO), 4.10e 4.40 (m, 2H, H2-6), 4.45 (m, 1H, H-5), 5.41 (dd, J1 ¼ 10.2 Hz, J2 ¼ 3.9 Hz, 1H, H-3), 5.27 (dd, J1 ¼ 3.9 Hz, J2 ¼ 10.2 Hz, 1H, H-2), 5.38 (dd, J1 ¼ 3.4 Hz, J2 ¼ 1.2 Hz, 1H, H-4), 6.12 (d, J1 ¼ 3.9 Hz, 1H, H-1). ESI-MS:

F. Cateni et al. / European Journal of Medicinal Chemistry 43 (2008) 210e221

m/z ¼ 434 (M þ Na, 15%)þ. Anal. calcd. for C14H19BrO9: C, 40.87; H, 4.62; Br, 19.46; found: C, 40.91; H, 4.40; Br, 19.90. 5.1.2. General procedure for glycosylation A mixture of 2,2-dimethyl-4-hydroxymethyl-1,3-dioxolan 3 (1.2 mmol), Ag2CO3 (1 mmol) and fine Drierite (2.0 g) in dry dichloromethane (30 ml) was stirred in the dark under N2. After being stirred for half an hour, iodine (0.1 mmol) was added and acetobromo-a-D-galactose 2 (1 mmol) in dry dichloromethane (10 ml) was added dropwise over a period of 1 h. The reaction mixture was stirred overnight at room temperature, filtered on Celite, and the filtrate was evaporated in vacuo to give a colorless syrupy mixture. The mixture was purified by flash chromatography on silica gel, eluted with EtOAcehexane (1:1, v:v) to give the 1:1 mixtures of the two diastereoisomers of 4. 5.1.2.1. 1,2-Isopropylidene-3-O-(b-D-20 ,30 ,40 ,60 -tetra-O-acetylgalactopyranosyl)-rac-glycerol 4. Yield: 68%. Rf ¼ 0.59 (EtOAcehexane 1:1, v:v). 1H NMR (CDCl3): d 1.38, 1.43 (s, 6H, 2  CH3), 1.97, 2.00, 2.09, 2.15 (s, 12H, 4  COCH3), 3.60e4.10 (m, 5H, 5H-glycerol), 4.0e4.35 (m, 2H, H2-60 ), 4.46 (m, 1H, H-50 ), 4.61 (dd, J ¼ 8.0 Hz, 1H, H-10 ), 5.27 (dd, J1 ¼ 3.9, J2 ¼ 10.2 Hz, 1H, H-20 ), 5.38 (dd, J1 ¼ 3.4 Hz, J1 ¼ 1.2 Hz, 1H, H-40 ), 5.40 (dd, J1 ¼ 10.2 Hz, J2 ¼ 3.9 Hz, 1H, H-30 ). ESI-MS: m/z ¼ 485 (M þ Na, 18%)þ. Anal. calcd. for C20H30O12: C, 51.95; H, 6.49; found: C, 51.99; H, 6.52. 5.1.3. General procedure for removal of acetyl protecting groups from 1,2-isopropylidene-3-O-(b-D-20 ,30 ,40 ,60 -tetraO-acetyl-galactopyranosyl)-rac-glycerol 4 CH3ONa (10 mmol) was added to a solution of 4 (1 mmol) in dry methanol (10 ml). The reaction mixture was stirred at room temperature for 2 h, and the solution was neutralized with Amberlist-15, filtered through Celite and concentrated. The mixture was purified by flash chromatography on silica gel, eluted with EtOAceMeOH (1:1, v:v) to give the 1:1 mixtures of the two diastereoisomers of 5. 5.1.3.1. 1,2-Isopropylidene-3-O-(b-D-galactopyranosyl)-racglycerol 5. Yield: 90%. Rf ¼ 0.40 (EtOAceMeOH 5:1, v:v). 1 H NMR (CDCl3): d 1.39, 1.43 (s, 6H, 2  CH3), 3.49e3.52 (m, 2H, H-30 , 50 ), 3.55 (dd, 1H, J20 ,10 ¼ 7.3 Hz, J20 ,30 ¼ 9.5 Hz, H-20 ), 3.53e4.10 (m, 5H, 5H-glycerol), 3.73 (dd, 1H, H-60 b), 3.84 (dd, 1H, J60 a,60 b ¼ 11.7 Hz, H-60 a), 3.90 (d; 1H, J ¼ 3.3 Hz, H-40 ), 4.95 (d, 1H, H-10 ). EI-MS (70 eV): m/ z ¼ 293 (M, 18%)þ, 179 (M  C6H11O2, 28%)þ, 113 (M  C6H11O6, 100%)þ. Anal. calcd. for C12H22O8: C, 48.98; H, 7.48; found: C, 49.01; H, 7.51. 5.1.4. General procedure for preparation of levulinate esters To a solution of 1,2-isopropylidene-3-O-(b-D-galactopyranosyl)-rac-glycerol 5 (2.1 mmol) in dry pyridine (10 ml) at room temperature, under an inert atmosphere, levulinic anhydride (16 mmol) dissolved in dry pyridine (6 ml) was added dropwise. The reaction was stirred for 24 h and ice water

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was added. The mixture was extracted with chloroform (3  30 ml) and the combined organic layers were extracted with a 10% solution of sodium bicarbonate (2  50 ml) first and then with water, dried over Na2SO4 and concentrated in vacuo. 5.1.4.1. 1,2-Isopropylidene-3-O-(20 ,30 ,40 ,60 -tetra-O-levulinyl-bD-galactopyranosyl)-rac-glycerol 6. Yield: 75%. Rf ¼ 0.59 (CH2Cl2eMeOH 1:1, v:v). 1H NMR (CDCl3): d 1.22, 1.27 (2s, 6H, 2  CH3), 2.10 (4s, 12H, 4  CH3CO), 2.60 (br t, 8H, 4  CH2OCO), 2.84 (br t, 8H, 4  CH2COCH3), 3.60e 3.82 (m, 6H, 5H-glycerol, H-50 ), 4.0e4.35 (dd, 2H, J ¼ 12.0 Hz, H2-60 ), 4.54 (d, 1H, J10 ,20 ¼ 7.9 Hz, H-10 ), 5.13 (dd, 1H, J ¼ 10.1, 3.9 Hz, H-20 ), 5.36 (dd, 1H, J ¼ 3.4, 1.3 Hz, H-40 ), 5.41 (dd, 1H, J ¼ 10.1, 3.9 Hz, H-30 ). ESIMS: m/z ¼ 773 (M þ Na, 12%)þ. Anal. calcd. for C32H46O16: C, 55.98; H, 6.70; found: C, 56.02; H, 6.68. 5.1.5. General procedure for deacetonization A solution of 6 (1.90 mmol) in 70% aqueous acetic acid (10 ml) was stirred at 60  C for 4 h. The mixture was concentrated and co-evaporated with toluene four times. The residue was then purified by silica gel flash chromatography, eluted with CHCl3eMeOH (10:1, v:v) to give 7 as a semisolid. 5.1.5.1. 3-O-(b-D-20 ,30 ,40 ,60 -Tetra-O-levulinyl-galactopyranosyl)rac-glycerol 7. Yield: 80%. Rf ¼ 0.36 (EtOAceMeOH 10:1, v:v). 1H NMR (CDCl3): d 2.10 (4s, 12H, 4  CH3CO), 2.60 (br t, 8H, 4  CH2OCO), 2.84 (br t, 8H, 4  CH2COCH3), 3.58e3.69 (m, 4H, H2-1, H2-3), 3.79 (m, 1H, H-2), 3.88 (dd, 1H, H-50 ), 4.0e4.35 (dd, 2H, J ¼ 12.0 Hz, H2-60 ), 4.54 (d, J10 ,20 ¼ 7.9 Hz, 1H, H-10 ), 5.13 (dd, J ¼ 10.1, 3.9 Hz, 1H, H20 ), 5.36 (dd, J ¼ 3.4, 1.3 Hz, 1H, H-40 ), 5.41 (dd, J ¼ 10.1, 3.9 Hz, 1H, H-30 ). ESI-MS: m/z ¼ 669 (M þ Na, 50%)þ. Anal. calcd. for C29H42O16: C, 53.54; H, 7.07; found: C, 53.60; H, 7.02. 5.1.6. General procedure for preparation of mono-acid 1,2O-diacyl-3-O-(b-D-20 ,30 ,40 ,60 -tetra-O-levulinylgalactopyranosyl)-rac-glycerols DCCD (0.4 mmol) was added to a solution of 7 (0.3 mmol), fatty acid (0.7 mmol) and DMAP (0.03 mmol) in dry dichloromethane (10 ml), and stirred for 24 h at room temperature. The solid was removed by filtration and the solvent was evaporated in vacuo. The residue was purified by flash chromatography on silica gel, eluted with EtOAcehexane (8:1, v:v) to give the products 8aeh. All compounds were obtained as the 1:1 mixtures of two diastereoisomers. 5.1.6.1. 1,2-O-Dicaprilyl-3-O-(b-D-20 ,30 ,40 ,60 -tetra-O-levulinylgalactopyranosyl)-rac-glycerol 8a. Yield: 73%. Rf ¼ 0.55 (EtOAcehexane 8:1, v:v). 1H NMR (CDCl3): d 0.90 (br t, 6H, J ¼ 6.7 Hz), 1.22 (br s, 16H, CH2 aliph.), 1.55 (br t, 4H, COCH2CH2), 2.10 (4s, 12H, 4  CH3CO), 2.30 (br t, 4H, COCH2), 2.60 (br t, 8H, 4  CH2OCO), 2.84 (br t, 8H, 4  CH2COCH3), 3.66e3.85 (dd, 2H, J ¼ 10.7, 6.4 Hz, H23), 3.88 (dd, 1H, H-50 ), 4.0e4.13 (dd, 2H, J ¼ 11.9, 6.6 Hz,

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H2-1), 4.30e4.35 (dd, 2H, J60 a,60 b ¼ 12.0 Hz, H2-60 ), 4.55 (d, J10 ,20 ¼ 8.0 Hz, 1H, H-10 ), 5.15 (dd, 1H, J ¼ 10.1, 3.9 Hz, H-20 ), 5.24 (br s, 1H, H-2), 5.37 (dd, 1H, J ¼ 3.4 Hz, H-40 ), 5.41 (dd, 1H, J ¼ 10.1, 3.9 Hz, H-30 ). ESI-MS: m/z ¼ 921 (M þ Na, 30%)þ. Anal. calcd. for C45H70O18: C, 60.13; H, 7.79; found: C, 60.09; H, 7.80. 5.1.6.2. 1,2-O-Didecanoyl-3-O-(b-D-20 ,30 ,40 ,60 -tetra-O-levulinyl-galactopyranosyl)-rac-glycerol 8b. Yield: 70%. Rf ¼ 0.57 (EtOAcehexane 8:1, v:v). 1H NMR (CDCl3): d 0.90 (br t, 6H, J ¼ 6.7 Hz), 1.23 (br s, 24H, CH2 aliph.), 1.55 (br t, 4H, COCH2CH2), 2.10 (4s, 12H, 4  CH3CO), 2.31 (br t, 4H, COCH2), 2.60 (br t, 8H, 4  CH2OCO), 2.84 (br t, 8H, 4  CH2COCH3), 3.66e3.85 (dd, 2H, J ¼ 10.7, 6.4 Hz, H2-3), 3.87 (dd, 1H, H-50 ), 4.03e4.13 (dd, 2H, J ¼ 12.0, 6.6 Hz, H2-1), 4.30e4.37 (dd, 2H, J60 a,60 b ¼ 12.0 Hz, H2-60 ), 4.55 (dd, 1H, J10 ,20 ¼ 8.0 Hz, H-10 ), 5.16 (dd, 1H, J ¼ 10.0, 3.9 Hz, H-20 ), 5.24 (br s, 1H, H-2), 5.37 (dd, 1H, J ¼ 3.4 Hz, H-40 ), 5.42 (dd, 1H, J ¼ 10.1, 3.9 Hz, H-30 ). ESI-MS: m/z ¼ 977 (M þ Na, 26%)þ. Anal. calcd. for C49H78O18: C, 61.63; H, 8.18; found: C, 61.60; H, 8.21. 5.1.6.3. 1,2-O-Dilauroyl-3-O-(b-D-20 ,30 ,40 ,60 -tetra-O-levulinylgalactopyranosyl)-rac-glycerol 8c. Yield: 68%. Rf ¼ 0.59 (EtOAcehexane 8:1, v:v). 1H NMR (CDCl3): d 0.89 (br t, 6H, J ¼ 6.7 Hz), 1.24 (br s, 32H, CH2 aliph.), 1.54 (br t, 4H, COCH2CH2), 2.11 (4s, 12H, 4  CH3CO), 2.31 (br t, 4H, COCH2), 2.60 (br t, 8H, 4  CH2OCO), 2.83 (br t, 8H, 4  CH2COCH3), 3.66e3.85 (dd, 2H, J ¼ 10.6, 6.4 Hz, H23), 3.90 (dd, 1H, H-50 ), 4.02e4.15 (dd, 2H, J ¼ 11.9, 6.6 Hz, H2-1), 4.30e4.37 (dd, 2H, J60 a,60 b ¼ 12.0 Hz, H2-60 ), 4.56 (dd, 1H, J10 ,20 ¼ 8.0 Hz, H-10 ), 5.16 (dd, 1H, J ¼ 9.9, 3.9 Hz, H-20 ), 5.24 (br s, 1H, H-2), 5.38 (dd, 1H, J ¼ 3.4 Hz, H-40 ), 5.43 (dd, 1H, J ¼ 10.0, 3.8 Hz, H-30 ). ESI-MS: m/z ¼ 1033 (M þ Na, 24%)þ. Anal. calcd. for C53H86O18: C, 62.97; H, 8.51; found: C, 63.00; H, 8.49. 5.1.6.4. 1,2-O-Dimyristoyl-3-O-(b-D-20 ,30 ,40 ,60 -tetra-O-levulinyl-galactopyranosyl)-rac-glycerol 8d. Yield: 85%. Rf ¼ 0.65 (EtOAcehexane 8:1, v:v). 1H NMR (CDCl3): d 0.90 (br t, 6H, J ¼ 6.7 Hz), 1.24 (br s, 40H, CH2 aliph.), 1.55 (br t, 4H, COCH2CH2), 2.10 (4s, 12H, 4  CH3CO), 2.31 (br t, 4H, COCH2), 2.60 (br t, 8H, 4  CH2OCO), 2.84 (br t, 8H, 4  CH2COCH3), 3.66e3.85 (dd, 2H, J ¼ 10.7, 6.4 Hz, H2-3), 3.97 (dd, 1H, H-50 ), 4.0e4.15 (dd, 2H, J ¼ 11.9, 6.6 Hz, H2-1), 4.30e4.37 (dd, 2H, J60 a,60 b ¼ 12.0 Hz, H2-60 ), 4.57 (dd, 1H, J10 ,20 ¼ 8.0 Hz, H-10 ), 5.18 (dd, 1H, J ¼ 9.8, 3.9 Hz, H-20 ), 5.24 (br s, 1H, H-2), 5.38 (dd, 1H, J ¼ 3.4 Hz, H-40 ), 5.44 (dd, 1H, J ¼ 10.0, 3.9 Hz, H-30 ). ESI-MS: m/z ¼ 1089 (M þ Na, 48%)þ. Anal. calcd. for C57H94O18: C, 64.16; H, 8.82; found: C, 64.19; H, 8.76. 5.1.6.5. 1,2-O-Distearoyl-3-O-(b-D-20 ,30 ,40 ,60 -tetra-O-levulinyl-galactopyranosyl)-rac-glycerol 8e. Yield: 80%. Rf ¼ 0.68 (EtOAcehexane 8:1, v:v). 1H NMR (CDCl3): d 0.89 (br t, 6H, J ¼ 6.6 Hz), 1.24 (br s, 56H, CH2 aliph.), 1.59 (br t, 4H, COCH2CH2), 2.12 (4s, 12H, 4  CH3CO), 2.40 (br t, 4H,

COCH2), 2.60 (br t, 8H, 4  CH2OCO), 2.83 (br t, 8H, 4  CH2COCH3), 3.66e3.85 (dd, 2H, J ¼ 10.7, 6.4 Hz, H2-3), 4.01 (dd, 1H, H-50 ), 4.0e4.15 (dd, 2H, J ¼ 12.0, 6.6 Hz, H2-1), 4.30e4.37 (dd, 2H, J60 a,60 b ¼ 12.0 Hz, H2-60 ), 4.58 (dd, 1H, J10 ,20 ¼ 8.0 Hz, H-10 ), 5.19 (dd, 1H, J ¼ 9.5, 3.8 Hz, H-20 ), 5.24 (br s, 1H, H-2), 5.39 (dd, 1H, J ¼ 3.5 Hz, H-40 ), 5.44 (dd, 1H, J ¼ 10.0, 3.9 Hz, H-30 ). ESI-MS: m/z ¼ 1201 (M þ Na, 41%)þ. Anal. calcd. for C65H110O18: C, 66.21; H, 9.34; found: C, 66.20; H, 9.37. 5.1.6.6. 1,2-O-Dioleoyl-3-O-(b-D-20 ,30 ,40 ,60 -tetra-O-levulinylgalactopyranosyl)-rac-glycerol 8f. Yield: 71%. Rf ¼ 0.66 (EtOAcehexane 8:1, v:v). 1H NMR (CDCl3): d 0.91 (br t, 6H, 2  CH3 term.), 1.24 (br s, 40H, CH2 aliph.), 1.62 (t, 4H, COCH2CH2), 2.05 (m, 8H, 4  CH2CH]CH), 2.10 (4s, 12H, 4  CH3CO), 2.40 (br t, 4H, COCH2), 2.61 (br t, 8H, 4  CH2OCO), 2.83 (br t, 8H, 4  CH2COCH3), 3.66e3.86 (dd, 2H, J ¼ 10.6, 6.3 Hz, H2-3), 4.01 (dd, 1H, H-50 ), 4.0e 4.15 (dd, 2H, J ¼ 11.9, 6.6 Hz, H2-1), 4.30e4.36 (dd, 2H, J60 a,60 b ¼ 12.0 Hz, H2-60 ), 4.58 (dd, 1H, J10 ,20 ¼ 8.0 Hz, H-10 ), 5.18 (dd, 1H, J ¼ 9.5, 3.9 Hz, H-20 ), 5.24 (br s, 1H, H-2), 5.40 (dd, 1H, J ¼ 3.7 Hz, H-40 ), 5.42 (m, 4H, 2  CH]CH ), 5.44 (dd, 1H, J ¼ 10.0, 3.9 Hz, H-30 ). ESI-MS: m/z ¼ 1198 (M þ Na, 83%)þ. Anal. calcd. for C65H106O18: C, 66.44; H, 9.03; found: C, 66.46; H, 9.00. 5.1.6.7. 1,2-O-Dilinoleoyl-3-O-(b-D-20 ,30 ,40 ,60 -tetra-O-levulinyl-galactopyranosyl)-rac-glycerol 8g. Yield: 54%. Rf ¼ 0.65 (EtOAcehexane 8:1, v:v). 1H NMR (CDCl3): d 0.91 (br t, 6H, 2  CH3 term.), 1.32 (br s, 28H, CH2 aliph.), 1.63 (br t, 4H, COCH2CH2), 2.02 (m, 8H, 4  CH2CH]CH), 2.12 (4s, 12H, 4  CH3CO), 2.35 (br t, 4H, COCH2), 2.60 (br t, 8H, 4  CH2OCO), 2.83 (br t, 8H, 4  CH2COCH3), 2.89 (m, 4H, 2  ]CHCH2CH]), 3.65e3.84 (dd, 2H, J ¼ 10.6, 6.3 Hz, H2-3), 4.0 (dd, 1H, H-50 ), 4.0e4.14 (dd, 2H, J ¼ 11.8, 6.5 Hz, H2-1), 4.30e4.36 (dd, 2H, J60 a,60 b ¼ 12.0 Hz, H2-60 ), 4.59 (dd, 1H, J10 ,20 ¼ 8.0 Hz, H-10 ), 5.17 (dd, 1H, J ¼ 9.5, 3.9 Hz, H-20 ), 5.23 (br s, 1H, H-2), 5.40 (dd, 1H, J ¼ 3.8 Hz, H-40 ), 5.41 (m, 8H, 4  CH]CH ), 5.44 (dd, 1H, J ¼ 10.0, 3.9 Hz, H-30 ),. ESI-MS: m/z ¼ 1194 (M þ Na, 70%)þ. Anal. calcd. for C65H102O18: C, 66.66; H, 8.72; found: C, 66.91; H, 8.69. 5.1.6.8. 1,2-O-Dilinolenoyl-3-O-(b-D-20 ,30 ,40 ,60 -tetra-O-levulinyl-galactopyranosyl)-rac-glycerol 8h. Yield: 45%. Rf ¼ 0.62 (EtOAcehexane 8:1, v:v). 1H NMR (CDCl3): d 0.97 (br t, 6H, 2  CH3 term.), 1.32 (br s, 16H, CH2 aliph.), 1.63 (br t, 4H, COCH2CH2), 2.07 (m, 8H, 4  CH2CH]CH), 2.11 (4s, 12H, 4  CH3CO), 2.41 (br t, 4H, COCH2), 2.62 (br t, 8H, 4  CH2OCO), 2.83 (br t, 8H, 4  CH2COCH3), 2.90 (m, 8H, 2  ]CHCH2CH]), 3.67e3.85 (dd, 2H, J ¼ 10.5, 6.2 Hz, H2-3), 4.0 (dd, 1H, H-50 ), 4.0e4.14 (dd, 2H, J ¼ 11.9, 6.6 Hz, H2-1), 4.30e4.36 (dd, 2H, J60 a,60 b ¼ 12.0 Hz, H2-60 ), 4.60 (dd, 1H, J10 ,20 ¼ 8.0 Hz, H-10 ), 5.18 (dd, 1H, J ¼ 9.6, 3.9 Hz, H-20 ), 5.24 (br s, 1H, H-2), 5.40 (dd, 1H, J ¼ 3.9 Hz, H-40 ), 5.43 (m, 12H, 6  CH]CH ), 5.45 (dd, 1H, J ¼ 10.0, 3.9 Hz, H-30 ),. ESI-MS: m/z ¼ 1190 (M þ Na, 72%)þ. Anal.

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calcd. for C65H98O18: C, 66.89; H, 8.40; found: C, 66.92; H, 8.44. 5.1.7. General procedure for preparation of mixed-acid 1,2O-diacyl-3-O-(b-D-20 ,30 ,40 ,60 -tetra-O-levulinylgalactopyranosyl)-rac-glycerols To a mixture of 7 (1 mmol) and vinyl caprylate (or vinyl capriate) (1.25 mmol) in dry dichloromethane (3.0 ml) was added immobilized M. miehei lipase (0.06 g). The resulting mixture was stirred at 37  C for 8 h. Then, additional lipase (0.01 g) was added to the reaction mixture which was stirred for additional 40 h at the same temperature. The lipase was separated off by filtration and the solvent was removed in vacuo on a rotary evaporator. The residue was purified by flash chromatography on silica gel column, eluted with EtOAce hexane (8:1/v:v) to give the galactosyl monoglycerides 10a, b which were subsequently reacted with equimolar amounts of the second fatty acid, DCC and a little amount of DMAP at room temperature overnight. The solid was filtered off and the solvent was evaporated in vacuo. The residue was purified by flash chromatography on silica gel column, eluted with EtOAcehexane (8:1, v:v) to give the 1:1 mixture of two diastereoisomers 11aec and 12aec. 5.1.7.1. 1-O-Caprylyl-2-O-oleoyl-3-O-(b-D-20 ,30 ,40 ,60 -tetra-Olevulinyl-galactopyranosyl)-rac-glycerol 11a. Yield: 39%. Rf ¼ 0.52 (EtOAcehexane 8:1, v:v). 1H NMR (CDCl3): d 0.95 (br t, 6H, 2  CH3 term.), 1.28 (br s, 28H, CH2 aliph.), 1.62 (br t, 4H, COCH2CH2), 2.00 (m, 4H, 2  CH2CH]CH), 2.10 (4s, 12H, 4  CH3CO), 2.40 (br t, 4H, COCH2), 2.62 (br t, 8H, 4  CH2OCO), 2.82 (br t, 8H, 4  CH2COCH3), 3.67e 3.84 (dd, 2H, J ¼ 10.5, 6.2 Hz, H2-3), 4.02 (dd, 1H, H-50 ), 4.10e4.14 (dd, 2H, J ¼ 11.9, 6.6 Hz, H2-1), 4.30e4.36 (dd, 2H, J60 a,60 b ¼ 12.0 Hz, H2-60 ), 4.60 (dd, 1H, J10 ,20 ¼ 8.0 Hz, H-10 ), 5.18 (dd, 1H, J ¼ 9.6, 3.9 Hz, H-20 ), 5.23 (br s, 1H, H-2), 5.40 (dd, 1H, J ¼ 3.9 Hz, H-40 ), 5.43 (m, 2H, CH]CH ), 5.45 (dd, 1H, J ¼ 10.0, 3.9 Hz, H-30 ),. ESI-MS: m/z ¼ 1060 (M þ Na, 80%)þ. Anal. calcd. for C55H88O18: C, 63.71; H, 8.49; found: C, 63.75; H, 8.47. 5.1.7.2. 1-O-Caprylyl-2-O-linoleoyl-3-O-(b-D-20 ,30 ,40 ,60 -tetraO-levulinyl-galactopyranosyl)-rac-glycerol 11b. Yield: 38%. Rf ¼ 0.52 (EtOAcehexane 8:1, v:v). 1H NMR (CDCl3): d 0.93 (br t, 6H, 2  CH3 term.), 1.28 (br s, 24H, CH2 aliph.), 1.61 (br t, 4H, COCH2CH2), 2.01 (m, 4H, 2  CH2CH]CH), 2.10 (4s, 12H, 4  CH3CO), 2.40 (br t, 4H, COCH2), 2.63 (br t, 8H, 4  CH2OCO), 2.82 (br t, 8H, 4  CH2COCH3), 2.90 (m, 2H, ]CHCH2CH]), 3.67e3.84 (dd, 2H, J ¼ 10.5, 6.2 Hz, H2-3), 4.02 (dd, 1H, H-50 ), 4.10e4.14 (dd, 2H, J ¼ 11.9, 6.6 Hz, H2-1), 4.30e4.36 (dd, 2H, J60 a,60 b ¼ 12.0 Hz, H2-60 ), 4.64 (dd, 1H, J10 ,20 ¼ 8.0 Hz, H-10 ), 5.19 (dd, 1H, J ¼ 9.6, 3.8 Hz, H-20 ), 5.23 (br s, 1H, H-2), 5.39 (m, 4H, CH]CH ), 5.40 (dd, 1H, J ¼ 3.9 Hz, H-40 ), 5.45 (dd, 1H, J ¼ 10.0, 3.9 Hz, H-30 ),. ESIMS: m/z ¼ 1058 (M þ Na, 80%)þ. Anal. calcd. for C55H86O18: C, 63.83; H, 8.32; found: C, 63.85; H, 8.30. 5.1.7.3. 1-O-Caprylyl-2-O-linolenoyl-3-O-(b-D-20 ,30 ,40 ,60 -tetraO-levulinyl-galacto-pyranosyl)-rac-glycerol 11c. Yield: 40%.

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Rf ¼ 0.51 (EtOAcehexane 8:1, v:v). 1H NMR (CDCl3): d 0.96 (br t, 6H, 2  CH3 term.), 1.28 (br s, 16H, CH2 aliph.), 1.60 (br t, 4H, COCH2CH2), 1.98 (m, 4H, 2  CH2CH]CH), 2.11 (4s, 12H, 4  CH3CO), 2.40 (br t, 4H, COCH2), 2.61 (br t, 8H, 4  CH2OCO), 2.82 (br t, 8H, 4  CH2COCH3), 2.90 (m, 4H, 2  ]CHCH2CH]), 3.66e3.85 (dd, 2H, J ¼ 10.5, 6.2 Hz, H2-3), 4.02 (dd, 1H, H-50 ), 4.10e4.14 (dd, 2H, J ¼ 11.9, 6.6 Hz, H2-1), 4.30e4.36 (dd, 2H, J60 a,60 b ¼ 12.0 Hz, H2-60 ), 4.65 (dd, 1H, J10 ,20 ¼ 8.0 Hz, H-10 ), 5.18 (dd, 1H, J ¼ 9.6, 3.9 Hz, H-20 ), 5.23 (br s, 1H, H-2), 5.39 (m, 6H, 3  CH]CH ), 5.42 (dd, 1H, J ¼ 3.9 Hz, H-40 ), 5.45 (dd, 1H, J ¼ 10.0, 3.9 Hz, H-30 ). ESI-MS: m/z ¼ 1055 (M þ Na, 90%)þ. Anal. calcd. for C55H84O18: C, 63.95; H, 8.14; found: C, 63.91; H, 8.00. 5.1.7.4. 1-O-Caprinyl-2-O-oleoyl-3-O-(b-D-20 ,30 ,40 ,60 -tetra-Olevulinyl-galactopyranosyl)-rac-glycerol 12a. Yield: 36%. Rf ¼ 0.51 (EtOAcehexane 8:1, v:v). 1H NMR (CDCl3): d 0.96 (br t, 6H, 2  CH3 term.), 1.28 (br s, 32H, CH2 aliph.), 1.62 (br t, 4H, COCH2CH2), 1.99 (m, 4H, 2  CH2CH]CH), 2.10 (4s, 12H, 4  CH3CO), 2.40 (br t, 4H, COCH2), 2.63 (br t, 8H, 4  CH2OCO), 2.83 (br t, 8H, 4  CH2COCH3), 3.67e 3.85 (dd, 2H, J ¼ 10.5, 6.2 Hz, H2-3), 4.01 (dd, 1H, H-50 ), 4.10e4.14 (dd, 2H, J ¼ 11.9, 6.7 Hz, H2-1), 4.30e4.36 (dd, 2H, J60 a,60 b ¼ 12.0 Hz, H2-60 ), 4.64 (dd, 1H, J10 ,20 ¼ 8.0 Hz, H-10 ), 5.19 (dd, 1H, J ¼ 9.6, 3.9 Hz, H-20 ), 5.23 (br s, 1H, H-2), 5.39 (m, 2H, CH]CH ), 5.42 (dd, 1H, J ¼ 3.9 Hz, H-40 ), 5.45 (dd, 1H, J ¼ 9.8, 3.9 Hz, H-30 ). ESI-MS: m/ z ¼ 1087 (M þ Na, 100%)þ. Anal. calcd. for C57H92O18: C, 64.28; H, 8.64; found: C, 64.32; H, 8.66. 5.1.7.5. 1-O-Caprinyl-2-O-linoleoyl-3-O-(b-D-20 ,30 ,40 ,60 -tetraO-levulinyl-galacto-pyranosyl)-rac-glycerol 12b. Yield: 39%. Rf ¼ 0.50 (EtOAcehexane 8:1, v:v). 1H NMR (CDCl3): d 0.95 (br t, 6H, 2  CH3 term.), 1.28 (br s, 28H, CH2 aliph.), 1.60 (br t, 4H, COCH2CH2), 2.00 (m, 4H, 2  CH2CH]CH), 2.10 (4s, 12H, 4  CH3CO), 2.40 (br t, 4H, COCH2), 2.63 (br t, 8H, 4  CH2OCO), 2.82 (br t, 8H, 4  CH2COCH3), 2.90 (m, 2H, ]CHCH2CH]), 3.66e3.84 (dd, 2H, J ¼ 10.5, 6.2 Hz, H2-3), 4.0 (dd, 1H, H-50 ), 4.10e4.14 (dd, 2H, J ¼ 11.8, 6.5 Hz, H2-1), 4.30e4.36 (dd, 2H, J60 a,60 b ¼ 12.0 Hz, H2-60 ), 4.65 (dd, 1H, J10 ,20 ¼ 8.0 Hz, H-10 ), 5.20 (dd, 1H, J ¼ 9.7, 3.9 Hz, H-20 ), 5.24 (br s, 1H, H-2), 5.40 (m, 4H, 2  CH]CH ), 5.43 (dd, 1H, J ¼ 3.9 Hz, H-40 ), 5.45 (dd, 1H, J ¼ 9.8, 3.9 Hz, H-30 ). ESI-MS: m/z ¼ 1086 (M þ Na, 75%)þ. Calcd. for C57H90O18: C, 64.41; H, 8.47; found: C, 64.38; H, 8.51. 5.1.7.6. 1-O-Caprinyl-2-O-linolenoyl-3-O-(b-D-20 ,30 ,40 ,60 -tetraO-levulinyl-galacto-pyranosyl)-rac-glycerol 12c. Yield: 42%. Rf ¼ 0.51 (EtOAcehexane 8:1, v:v). 1H NMR (CDCl3): d 0.93 (br t, 6H, 2  CH3 term.), 1.29 (br s, 20H, CH2 aliph.), 1.61 (br t, 4H, COCH2CH2), 2.08 (m, 4H, 2  CH2CH]CH), 2.10 (4s, 12H, 4  CH3CO), 2.36 (br t, 4H, COCH2), 2.64 (br t, 8H, 4  CH2OCO), 2.83 (br t, 8H, 4  CH2COCH3), 2.90 (m, 4H, 2  ]CHCH2CH]), 3.66e3.84 (dd, 2H, J ¼ 10.5, 6.2 Hz, H2-3), 4.02 (dd, 1H,

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H-50 ), 4.10e4.14 (dd, 2H, J ¼ 11.7, 6.4 Hz, H2-1), 4.30e 4.36 (dd, 2H, J60 a,60 b ¼ 12.0 Hz, H2-60 ), 4.64 (dd, 1H, J10 ,20 ¼ 8.0 Hz, H-10 ), 5.19 (dd, 1H, J20 ,30 ¼ 9.8, 3.7 Hz, H-20 ), 5.24 (br s, 1H, H-2), 5.37 (m, 6H, 3  CH]CH ), 5.42 (dd, 1H, J ¼ 3.9 Hz, H-40 ), 5.45 (dd, 1H, J ¼ 9.8, 3.9 Hz, H-30 ). ESI-MS: m/z ¼ 1084 (M þ Na, 87%)þ. Calcd. for C57H88O18: C, 64.53; H, 8.30; found: C, 64.58; H, 8.33. 5.1.8. General procedure for selective removal of levulinyl protecting groups from 1,2-O-diacyl-3-O-(b-D-20 ,30 ,40 ,60 tetra-O-levulinyl-galactopyranosyl)-rac-glycerols To a solution of 8aeh, 11aec and 12aec (0.1 mmol) in dry pyridine, 1 M NH2NH2$H2O in pyridineeglacial acetic acid (3:2, v:v) was added. Each mixture was stirred for 1 h at room temperature, under inert atmosphere, the mixture was poured into ice-cold water, extracted with CH2Cl2 (3  50 ml) and washed with 10% NaHCO3 and brine. The combined CH2Cl2 layers were dried (anhydrous Na2SO4) and individually evaporated to give a syrupy mixture. Each mixture was purified by ‘flash chromatography’ on silica gel column, eluted with CH2Cl2eMeOH (10:1) to give 1:1 mixtures of two diastereoisomers 9aeh, 13aec and 14aec. 5.1.8.1. 1,2-O-Dicaprylyl-3-O-(b-D-galactopyranosyl)-racglycerol 9a. Yield: 82%. Rf ¼ 0.48 (CH2Cl2eMeOH 10:1, v:v). 1H NMR (400 MHz, CDCl3): d 0.90 (br t, 6H, J ¼ 6.7 Hz, CH3 term.), 1.25 (br s, 16H, CH2 aliph.), 1.55 (br t, 4H, COCH2CH2), 2.30 (br t, 4H, J ¼ 7.0 Hz, COCH2), 3.52 (m, 1H, H-50 ), 3.57 (m, 1H, H-30 ), 3.60 (m, 1H, H-20 ), 3.63, 3.66 (dd, 1H, J ¼ 10.6, 6.3 Hz, Ha-3), 3.81 (br s, 2H, H2-60 ), 3.87 (br d, 1H, J ¼ 10.6 Hz, Hb-3), 3.96 (m, 1H, H40 ), 4.11, 4.15 (dd, 1H, J ¼ 12.0, 3.3 Hz, Ha-1), 4.22 (d, 1H, J ¼ 7.8 Hz, H-10 ), 4.38 (br d, 1H, J ¼ 12.0 Hz, Hb-1), 5.22 (br s, 1H, H-2). 13C NMR (100 MHz, CDCl3): d 173.3, 173.2 (COO), 104.2 (C-10 ), 74.9 (C-50 ), 73.6 (C-30 ), 71.5 (C-20 ), 70.5 (C-2), 69.2 (C-40 ), 68.4 (C-3), 63.0, 62.9 (C-1), 61.8 (C-60 ), 33.8, 33.7 (COCH2), 32.3e23.2 (CH2 aliph.), 25.5, 25.4 (COCH2CH2), 14.5 (CH3). ESI-MS: m/z ¼ 529 (M þ Na, 25%)þ. Anal. calcd. for C25H46O10: C, 59.29; H, 9.09; found: C, 59.33; H, 9.12. 5.1.8.2. 1,2-O-Dicaprinyl-3-O-(b-D-galactopyranosyl)-racglycerol 9b. Yield: 79%. Rf ¼ 0.48 (CH2Cl2eMeOH 10:1, v:v). 1H NMR (400 MHz, CDCl3): d 0.90 (br t, 6H, J ¼ 6.7 Hz, CH3 term.), 1.24 (br s, 24H, CH2 aliph.), 1.54 (br t, 4H, COCH2CH2), 2.34 (br t, 4H, J ¼ 7.0 Hz, COCH2), 3.52 (m, 1H, H-50 ), 3.58 (m, 1H, H-30 ), 3.61 (m, 1H, H-20 ), 3.62, 3.65 (dd, 1H, J ¼ 10.7, 6.4 Hz, Ha-3), 3.80 (br s, 2H, H2-60 ), 3.85 (br d, 1H, J ¼ 10.5 Hz, Hb-3), 3.97 (m, 1H, H40 ), 4.08, 4.12 (dd, 1H, J ¼ 12.0, 3.3 Hz, Ha-1), 4.21 (d, 1H, J ¼ 7.5 Hz, H-10 ), 4.38 (br d, 1H, J ¼ 10.4 Hz, Hb-1), 5.23 (br s, 1H, H-2). 13C NMR (100 MHz, CDCl3): d 173.6, 173.1 (COO), 104.3 (C-10 ), 74.8 (C-50 ), 73.6 (C-30 ), 71.6 (C-20 ), 70.4 (C-2), 69.4 (C-40 ), 68.4 (C-3), 63.2, 63.1 (C-1), 61.9 (C-60 ), 33.8, 33.7 (COCH2), 32.4e23.0 (CH2 aliph.), 25.3, 25.2 (COCH2CH2), 14.6 (CH3). ESI-MS: m/z ¼ 585

(M þ Na, 26%)þ. Anal. calcd. for C29H54O10: C, 61.92; H, 9.61; found: C, 61.87; H, 9.58. 5.1.8.3. 1,2-O-Dilauroyl-3-O-(b-D-galactopyranosyl)-rac-glycerol 9c. Yield: 79%. Rf ¼ 0.49 (CH2Cl2eMeOH 10:1, v:v). 1H NMR (400 MHz, CDCl3): d 0.90 (br t, 6H, J ¼ 6.7 Hz, CH3 term.), 1.23 (br s, 32H, CH2 aliph.), 1.55 (br t, 4H, COCH2CH2), 2.36 (br t, 4H, J ¼ 6.9 Hz, COCH2), 3.53 (m, 1H, H-50 ), 3.58 (m, 1H, H-30 ), 3.60 (m, 1H, H-20 ), 3.62, 3.65 (dd, 1H, J ¼ 10.7, 6.4 Hz, Ha-3), 3.81 (br s, 2H, H2-60 ), 3.86 (br d, 1H, J ¼ 10.6 Hz, Hb-3), 3.98 (m, 1H, H-40 ), 4.09, 4.14 (dd, 1H, J ¼ 12.0, 3.3 Hz, Ha-1), 4.21 (d, 1H, J ¼ 7.8 Hz, H-10 ), 4.36 (br d, 1H, J ¼ 10.4 Hz, Hb-1), 5.24 (br s, 1H, H-2). 13C NMR (100 MHz, CDCl3): d 173.6, 173.1 (COO), 104.2 (C-10 ), 74.8 (C-50 ), 73.6 (C-30 ), 71.4 (C-20 ), 70.6, 70.5 (C-2), 69.2 (C-40 ), 68.4 (C-3), 63.1, 62.9 (C-1), 61.9 (C-60 ), 33.8, 33.7 (COCH2), 32.3e23.0 (CH2 aliph.), 25.3, 25.2 (COCH2CH2), 14.5 (CH3). ESI-MS: m/z ¼ 641 (M þ Na, 24%)þ. Anal. calcd. for C33H62O10: C, 64.08; H, 10.03; found: C, 63.99; H, 10.01. 5.1.8.4. 1,2-O-Dimyristoyl-3-O-(b-D-galactopyranosyl)-racglycerol 9d. Yield: 80%. Rf ¼ 0.49 (CH2Cl2eMeOH 10:1, v:v); 1H NMR (400 MHz, CDCl3): d 0.89 (br t, 6H, J ¼ 6.6 Hz, CH3 term.), 1.23 (br s, 40H, CH2 aliph.), 1.55 (br t, 4H, COCH2CH2), 2.33 (br t, 4H, J ¼ 7.0 Hz, COCH2), 3.53 (m, 1H, H-50 ), 3.57 (m, 1H, H-30 ), 3.61 (m, 1H, H-20 ), 3.62, 3.65 (dd, 1H, J ¼ 10.7, 6.4 Hz, Ha-3), 3.80 (br s, 2H, H2-60 ), 3.85 (br d, 1H, J ¼ 10.5 Hz, Hb-3), 3.98 (m, 1H, H-40 ), 4.09, 4.13 (dd, 1H, J ¼ 12.0, 3.3 Hz, Ha-1), 4.22 (d, 1H, J ¼ 7.5 Hz, H-10 ), 4.35 (br d, 1H, J ¼ 10.5 Hz, Hb-1), 5.22 (br s, 1H, H-2). 13 C NMR (100 MHz, CDCl3): d 173.4, 173.3 (COO), 104.2 (C-10 ), 74.9 (C-50 ), 73.7 (C-30 ), 71.5 (C-20 ), 70.6, 70.5 (C-2), 69.4 (C-40 ), 68.6 (C-3), 63.3, 63.2 (C-1), 62.0 (C-60 ), 33.8, 34.2 (COCH2), 32.4e23.1 (CH2 aliph.), 25.4, 25.3 (COCH2CH2), 14.6 (CH3). ESI-MS: m/z ¼ 697 (M þ Na, 48%)þ. Anal. calcd. for C37H70O10: C, 65.87; H, 10.38; found: C, 65.90; H, 10.40. 5.1.8.5. 1,2-O-Distearoyl-3-O-(b-D-galactopyranosyl)-racglycerol 9e. Yield: 81%. Rf ¼ 0.51 (CH2Cl2eMeOH 10:1, v:v). 1H NMR (400 MHz, CDCl3): d 0.88 (br t, 6H, J ¼ 6.7 Hz, CH3 term.), 1.25 (br s, 56H, CH2 aliph.), 1.54 (br t, 4H, COCH2CH2), 2.39 (br t, 4H, J ¼ 7.0 Hz, COCH2), 3.52 (m, 1H, H-50 ), 3.57 (m, 1H, H-30 ), 3.61 (m, 1H, H-20 ), 3.62, 3.65 (dd, 1H, J ¼ 10.7, 6.4 Hz, Ha-3), 3.80 (br s, 2H, H2-60 ), 3.87 (br d, 1H, J ¼ 10.6 Hz, Hb-3), 3.97 (m, 1H, H40 ), 4.08, 4.13 (dd, 1H, J ¼ 11.9, 4.4 Hz, Ha-1), 4.21 (d, 1H, J ¼ 7.5 Hz, H-10 ), 4.35 (br d, 1H, J ¼ 11.2 Hz, Hb-1), 5.25 (br s, 1H, H-2). 13C NMR (100 MHz, CDCl3): d 173.8, 173.7 (COO), 104.1 (C-10 ), 74.9 (C-50 ), 73.6 (C-30 ), 71.5 (C-20 ), 70.6, 70.5 (C-2), 69.4 (C-40 ), 68.5 (C-3), 63.2, 63.1 (C-1), 62.0 (C-60 ), 34.2, 34.1 (COCH2), 32.3e23.0 (CH2 aliph.), 25.4, 25.3 (COCH2CH2), 14.6 (CH3). ESI-MS: m/ z ¼ 809 (M þ Na, 41%)þ. Anal. calcd. for C45H86O10: C, 68.70; H, 10.94; found: C, 68.73; H, 10.88. 5.1.8.6. 1,2-O-Dioleoyl-3-O-(b-D-galactopyranosyl)-rac-glycerol 9f. Yield: 75%. Rf ¼ 0.50 (CH2Cl2eMeOH 10:1, v:v).

F. Cateni et al. / European Journal of Medicinal Chemistry 43 (2008) 210e221 1

H NMR (400 MHz, CDCl3): d 0.91 (br t, 6H, J ¼ 6.6 Hz, CH3 term.), 1.24 (br s, 40H, CH2 aliph.), 1.56 (br t, 4H, COCH2CH2), 2.0 (br, 8H, 4  CH2CH]CH), 2.35 (br t, 4H, J ¼ 6.9 Hz, COCH2), 3.52 (m, 1H, H-50 ), 3.58 (m, 1H, H-30 ), 3.60 (m, 1H, H-20 ), 3.62, 3.65 (dd, 1H, J ¼ 10.7, 6.4 Hz, Ha-3), 3.81 (br s, 2H, H2-60 ), 3.88 (br d, 1H, J ¼ 10.6 Hz, Hb-3), 3.97 (m, 1H, H-40 ), 4.10, 4.14 (dd, 1H, J ¼ 12.0, 4.4 Hz, Ha-1), 4.21 (d, 1H, J ¼ 7.8 Hz, H-10 ), 4.35 (br d, 1H, J ¼ 12.1 Hz, Hb-1), 5.24 (br s, 1H, H-2), 5.25e 5.40 (m, 4H, 2  CH]CH ). 13C NMR (100 MHz, CDCl3): d 173.9, 173.4 (COO), 130.3, 130.1 (CH]CH), 104.2 (C10 ), 74.8 (C-50 ), 73.7 (C-30 ), 71.6 (C-20 ), 70.5, 70.4 (C-2), 69.3 (C-40 ), 68.6 (C-3), 63.0, 62.9 (C-1), 61.9 (C-60 ), 33.8, 33.7 (COCH2), 32.3e22.9 (CH2 aliph.), 27.7, 27.5 (CH2CH]CH), 25.5, 25.4 (COCH2CH2), 14.5 (CH3). ESIMS: m/z ¼ 806 (M þ Na, 100%)þ. Anal. calcd. for C45H82O10: C, 69.05; H, 10.48; found: C, 68.99; H, 10.52. 5.1.8.7. 1,2-O-Dilinoleoyl-3-O-(b-D-galactopyranosyl)-racglycerol 9g. Yield: 70%. Rf ¼ 0.50 (CH2Cl2eMeOH 10:1, v:v). 1H NMR (400 MHz, CDCl3): d 0.91 (br t, 6H, J ¼ 6.7 Hz, CH3 term.), 1.22 (br s, 28H, CH2 aliph.), 1.56 (br t, 4H, COCH2CH2), 1.99 (br, 8H, 4  CH2CH]CH), 2.33 (br t, 4H, J ¼ 6.9 Hz, COCH2), 2.82 (br t, 4H, J ¼ 5.5 Hz, 2  ]CHCH2CH]), 3.52 (m, 1H, H-50 ), 3.58 (m, 1H, H-30 ), 3.60 (m, 1H, H-20 ), 3.63, 3.66 (dd, 1H, J ¼ 10.7, 6.4 Hz, Ha-3), 3.80 (br s, 2H, H2-60 ), 3.89 (br d, 1H, J ¼ 10.6 Hz, Hb-3), 3.96 (m, 1H, H-40 ), 4.09, 4.13 (dd, 1H, J ¼ 12.0, 3.3 Hz, Ha-1), 4.22 (d, 1H, J ¼ 7.8 Hz, H-10 ), 4.34 (br d, 1H, J ¼ 12.0 Hz, Hb-1), 5.23 (br s, 1H, H-2), 5.30e5.38 (m, 8H, 4  CH]CH ). 13C NMR (100 MHz, CDCl3): d 173.8, 173.4 (COO), 130.5, 130.4, 128.4, 128.3 (CH]CH), 104.3 (C-10 ), 74.8 (C-50 ), 73.7 (C-30 ), 71.6 (C-20 ), 70.5 (C-2), 69.2 (C-40 ), 68.6 (C-3), 63.3, 63.2 (C-1), 61.7 (C-60 ), 33.8, 33.7 (COCH2), 30.3e21.1 (CH2 aliph.), 27.5 (CH2CH]CH), 26.0 (]CHCH2CH]), 25.5, 25.4 (COCH2CH2), 14.4 (CH3). ESI-MS: m/z ¼ 802 (M þ Na, 100%)þ. Anal. calcd. for C45H78O10: C, 69.41; H, 10.02; found: C, 69.37; H, 10.05. 5.1.8.8. 1,2-O-Dilinolenoyl-3-O-(b-D-galactopyranosyl)-racglycerol 9h. Yield: 70%. Rf ¼ 0.50 (CH2Cl2eMeOH 10:1, v:v). 1H NMR (400 MHz, CDCl3): d 0.97 (br t, 6H, J ¼ 6.8 Hz, CH3 term.), 1.23 (br s, 16H, CH2 aliph.), 1.54 (br t, 4H, COCH2CH2), 2.10 (br, 8H, 4  CH2CH]CH), 2.35 (br t, 4H, J ¼ 6.9 Hz, COCH2), 2.90 (br t, 8H, J ¼ 5.6 Hz, 4  ]CHCH2CH]), 3.53 (m, 1H, H-50 ), 3.57 (m, 1H, H-30 ), 3.61 (m, 1H, H-20 ), 3.63, 3.66 (dd, 1H, J ¼ 10.6, 6.3 Hz, Ha-3), 3.81 (br s, 2H, H2-60 ), 3.87 (br d, 1H, J ¼ 10.6 Hz, Hb-3), 3.96 (m, 1H, H-40 ), 4.10, 4.13 (dd, 1H, J ¼ 11.9, 3.3 Hz, Ha-1), 4.21 (d, 1H, J ¼ 7.5 Hz, H-10 ), 4.38 (br d, 1H, J ¼ 12.0 Hz, Hb-1), 5.22 (br s, 1H, H-2), 5.30e5.41 (m, 12H, 6  CH]CH ). 13C NMR (100 MHz, CDCl3): d 173.8, 173.4 (COO), 132.2, 130.4, 130.1, 128.6, 128.1, 127.5 (CH]CH), 104.2 (C-10 ), 74.9 (C-50 ), 73.7 (C-30 ), 71.5 (C-20 ), 70.5 (C-2), 69.3 (C-40 ), 68.7 (C-3), 63.2, 63.1 (C-1), 61.8 (C-60 ), 34.3, 34.1 (COCH2), 30.3e21.1

219

(CH2 aliph.), 27.7, 27.6, 27.5 (CH2CH]CH), 26.1, 26.0 (]CHCH2CH]), 25.5, 25.4 (COCH2CH2), 14.7 (CH3). ESI-MS: m/z ¼ 798 (M þ Na, 100%)þ. Anal. calcd. for C45H74O10: C, 69.77; H, 9.56; found: C, 69.91; H, 9.19. 5.1.8.9. 1-O-Caprylyl-2-O-Oleoyl-3-O-(b-D-galactopyranosyl)rac-glycerol 13a. Yield: 71%. Rf ¼ 0.49 (CH2Cl2eMeOH 10:1, v:v). 1H NMR (400 MHz, CDCl3): d 0.95 (br t, 6H, J ¼ 6.8 Hz, CH3 term.), 1.23 (br s, 28H, CH2 aliph.), 1.56 (br t, 4H, COCH2CH2), 1.99 (br, 4H, 2  CH2CH]CH), 2.39 (br t, 4H, J ¼ 7.0 Hz, COCH2), 3.52 (m, 1H, H-50 ), 3.58 (m, 1H, H-30 ), 3.60 (m, 1H, H-20 ), 3.62, 3.65 (dd, 1H, J ¼ 10.7, 6.4 Hz, Ha-3), 3.80 (br s, 2H, H2-60 ), 3.88 (br d, 1H, J ¼ 10.6 Hz, Hb-3), 3.96 (m, 1H, H-40 ), 4.10, 4.15 (dd, 1H, J ¼ 12.0, 3.4 Hz, Ha-1), 4.22 (d, 1H, J ¼ 7.5 Hz, H-10 ), 4.35 (br d, 1H, J ¼ 12.1 Hz, Hb-1), 5.24 (br s, 1H, H-2), 5.20e5.40 (m, 2H, CH]CH ). 13C NMR (100 MHz, CDCl3): d 174.2, 174.1 (COO), 130.4, 130.1 (CH]CH), 104.3 (C-10 ), 74.8 (C-50 ), 73.6 (C-30 ), 71.4 (C-20 ), 70.6, 70.5 (C-2), 69.4 (C-40 ), 68.5 (C-3), 63.0, 62.9 (C-1), 61.8 (C-60 ), 34.6, 34.5 (COCH2), 32.3e23.2 (CH2 aliph.), 27.6, 27.5 (CH2CH]CH), 25.3, 25.2 (COCH2CH2), 14.5 (CH3). ESIMS: m/z ¼ 668 (M þ Na, 100%)þ. Anal. calcd. for C35H64O10: C, 65.22; H, 9.94; found: C, 65.19; H, 10.00. 5.1.8.10. 1-O-Caprylyl-2-O-linoleoyl-3-O-(b-D-galactopyranosyl)-rac-glycerol 13b. Yield: 72%. Rf ¼ 0.50 (CH2Cl2eMeOH 10:1, v:v). 1H NMR (400 MHz, CDCl3): d 0.93 (br t, 6H, J ¼ 6.8 Hz, CH3 term.), 1.25 (br s, 24H, CH2 aliph.), 1.55 (br t, 4H, COCH2CH2), 2.0 (br, 4H, 2  CH2CH]CH), 2.30 (br t, 4H, J ¼ 7.0 Hz, COCH2), 2.87 (br t, 2H, J ¼ 5.6 Hz, ]CHCH2CH]), 3.52 (m, 1H, H-50 ), 3.58 (m, 1H, H-30 ), 3.61 (m, 1H, H-20 ), 3.63, 3.66 (dd, 1H, J ¼ 10.7, 6.4 Hz, Ha-3), 3.81 (br s, 2H, H2-60 ), 3.89 (br d, 1H, J ¼ 10.5 Hz, Hb-3), 3.96 (m, 1H, H-40 ), 4.09, 4.14 (dd, 1H, J ¼ 12.0, 3.4 Hz, Ha-1), 4.21 (d, 1H, J ¼ 7.8 Hz, H-10 ), 4.34 (br d, 1H, J ¼ 12.0 Hz, Hb-1), 5.23 (br s, 1H, H-2), 5.20e5.40 (m, 4H, 2  CH]CH ). 13 C NMR (100 MHz, CDCl3): d 173.5, 173.2 (COO), 130.6, 130.4, 128.4, 128.3 (CH]CH), 104.1 (C-10 ), 74.9 (C-50 ), 73.6 (C-30 ), 71.6 (C-20 ), 70.6, 70.5 (C-2), 69.3 (C-40 ), 68.5 (C-3), 63.3, 63.2 (C-1), 61.8 (C-60 ), 34.3, 34.1 (COCH2), 32.4e23.2 (CH2 aliph.), 27.6 (CH2CH]CH), 26.0 (]CHCH2CH]), 25.3, 25.2 (COCH2CH2), 14.7 (CH3). ESI-MS: m/z ¼ 666 (M þ Na, 100%)þ. Anal. calcd. for C35H62O10: C, 65.42; H, 9.65; found: C, 65.91; H, 9.69. 5.1.8.11. 1-O-Caprylyl-2-O-linolenoyl-3-O-(b-D-galactopyranosyl)-rac-glycerol 13c. Yield: 69%. Rf ¼ 0.50 (CH2Cl2e MeOH 10:1, v:v). 1H NMR (400 MHz, CDCl3): d 0.96 (br t, 6H, J ¼ 6.8 Hz, CH3 term.), 1.25 (br s, 16H, CH2 aliph.), 1.57 (br t, 4H, COCH2CH2), 2.10 (br, 4H, 2  CH2CH]CH), 2.31 (br t, 4H, J ¼ 7.0 Hz, COCH2), 2.90 (br t, 4H, J ¼ 5.5 Hz, 2  ]CHCH2CH]), 3.53 (m, 1H, H-50 ), 3.57 (m, 1H, H-30 ), 3.60 (m, 1H, H-20 ), 3.62, 3.65 (dd, 1H, J ¼ 10.7, 6.4 Hz, Ha-3), 3.80 (br s, 2H, H2-60 ), 3.85 (br d, 1H, J ¼ 10.5 Hz, Hb-3), 3.97 (m, 1H, H-40 ), 4.10, 4.13 (dd, 1H, J ¼ 11.9, 3.3 Hz, Ha-1), 4.21 (d, 1H, J ¼ 7.5 Hz, H-10 ), 4.35 (br d, 1H, J ¼ 10.5 Hz, Hb-1),

220

F. Cateni et al. / European Journal of Medicinal Chemistry 43 (2008) 210e221

5.22 (br s, 1H, H-2), 5.23e5.39 (m, 6H, 3  CH]CH ). 13C NMR (100 MHz, CDCl3): d 173.6, 173.5 (COO), 132.3, 130.4, 130.2, 128.5, 128.0, 127.6 (CH]CH), 104.2 (C-10 ), 74.9 (C-50 ), 73.7 (C-30 ), 71.5 (C-20 ), 70.6 (C-2), 70.0 (C-60 ), 69.4 (C-40 ), 68.6 (C-3), 63.1, 62.9 (C-1), 34.3, 34.2 (COCH2), 32.3e23.1 (CH2 aliph.), 27.7, 27.6, 27.5 (CH2CH]CH), 26.2, 26.0 (]CHCH2CH]), 25.5, 25.4 (COCH2CH2), 14.8 (CH3). ESI-MS: m/z ¼ 662 (M þ Na, 100%)þ. Anal. calcd. for C35H60O10: 65.62; H, 9.37; found: C, 65.69; H, 9.40. 5.1.8.12. 1-O-Caprinyl-2-O-oleoyl-3-O-(b-D-galactopyranosyl)-rac-glycerol 14a. Yield: 75%. Rf ¼ 0.50 (CH2Cl2e MeOH 10:1, v:v). 1H NMR (400 MHz, CDCl3): d 0.94 (br t, 6H, J ¼ 6.8 Hz, CH3 term.), 1.24 (br s, 32H, CH2 aliph.), 1.55 (br t, 4H, COCH2CH2), 1.99 (br, 4H, 2  CH2CH]CH), 2.40 (br t, 4H, J ¼ 6.9 Hz, COCH2), 3.53 (m, 1H, H-50 ), 3.58 (m, 1H, H-30 ), 3.61 (m, 1H, H-20 ), 3.62, 3.65 (dd, 1H, J ¼ 10.7, 6.4 Hz, Ha-3), 3.80 (br s, 2H, H2-60 ), 3.85 (br d, 1H, J ¼ 10.5 Hz, Hb-3), 3.98 (m, 1H, H-40 ), 4.09, 4.13 (dd, 1H, J ¼ 12.0, 3.4 Hz, Ha-1), 4.22 (d, 1H, J ¼ 7.8 Hz, H-10 ), 4.38 (br d, 1H, J ¼ 10.4 Hz, Hb-1), 5.23 (br s, 1H, H-2), 5.20e 5.40 (m, 2H, CH]CH ). 13C NMR (100 MHz, CDCl3): d 173.5, 173.4 (COO), 130.3, 130.2 (CH]CH), 104.1 (C-10 ), 74.9 (C-50 ), 73.6 (C-30 ), 71.6 (C-20 ), 70.5, 70.4 (C-2), 69.2 (C-40 ), 68.4 (C-3), 63.3, 63.2 (C-1), 61.9 (C-60 ), 34.1, 33.9 (COCH2), 32.3e23.0 (CH2 aliph.), 27.6, 27.5 (CH2CH]CH), 25.5, 25.4 (COCH2CH2), 14.5 (CH3). ESIMS: m/z ¼ 696 (M þ Na, 100%)þ. Anal. calcd. for C37H68O10: C, 66.07; H, 10.12; found: C, 66.10; H, 10.09. 5.1.8.13. 1-O-Caprinyl-2-O-linoleoyl-3-O-(b-D-galactopyranosyl)-rac-glycerol 14b. Yield: 69%. Rf ¼ 0.50 (CH2Cl2eMeOH 10:1, v:v). 1H NMR (400 MHz, CDCl3): d 0.95 (br t, 6H, J ¼ 6.7 Hz, CH3 term.), 1.24 (br s, 28H, CH2 aliph.), 1.54 (br t, 4H, COCH2CH2), 2.0 (br, 4H, 2  CH2CH]CH), 2.33 (br t, 4H, J ¼ 7.0 Hz, COCH2), 2.88 (br t, 2H, J ¼ 5.6 Hz, ]CHCH2CH]), 3.52 (m, 1H, H-50 ), 3.58 (m, 1H, H-30 ), 3.60 (m, 1H, H-20 ), 3.62, 3.65 (dd, 1H, J ¼ 10.7, 6.4 Hz, Ha-3), 3.81 (br s, 2H, H2-60 ), 3.89 (br d, 1H, J ¼ 10.6 Hz, Hb-3), 3.97 (m, 1H, H-40 ), 4.10, 4.14 (dd, 1H, J ¼ 12.0, 4.4 Hz, Ha-1), 4.22 (d, 1H, J ¼ 7.8 Hz, H-10 ), 4.35 (br d, 1H, J ¼ 10.5 Hz, Hb-1), 5.22 (br s, 1H, H-2), 5.22e5.40 (m, 4H, 2  CH]CH ). 13C NMR (100 MHz, CDCl3): d 173.6, 173.4 (COO), 130.6, 130.4, 128.4, 128.3 (CH]CH), 104.3 (C-10 ), 74.8 (C-50 ), 73.6 (C-30 ), 71.5 (C-20 ), 70.6 (C-2), 70.0 (C-60 ), 69.2 (C-40 ), 68.5 (C-3), 63.2, 63.1 (C-1), 34.3, 33.6 (COCH2), 32.4e23.2 (CH2 aliph.), 27.6 (CH2CH]CH), 26.0 (]CHCH2CH]), 25.3, 25.2 (COCH2CH2), 14.7 (CH3). ESI-MS: m/z ¼ 694 (M þ Na, 100%)þ. Anal. calcd. for C37H66O10: C, 66.27; H, 9.85; found: C, 66.31; H, 9.81. 5.1.8.14. 1-O-Caprinyl-2-O-linolenoyl-3-O-(b-D-galactopyranosyl)-rac-glycerol 14c. Yield: 66%. Rf ¼ 0.50 (CH2Cl2e MeOH 10:1, v:v); 1H NMR (400 MHz, CDCl3): d 0.96 (br t, 6H, J ¼ 6.8 Hz, CH3 term.), 1.24 (br s, 20H, CH2 aliph.), 1.56 (br t, 4H, COCH2CH2), 2.10 (br, 4H, 2  CH2CH]CH),

2.35 (br t, 4H, J ¼ 6.9 Hz, COCH2), 2.91 (br t, 4H, J ¼ 5.5 Hz, 2  ]CHCH2CH]), 3.53 (m, 1H, H-50 ), 3.57 (m, 1H, H-30 ), 3.60 (m, 1H, H-20 ), 3.62, 3.65 (dd, 1H, J ¼ 10.7, 6.4 Hz, Ha-3), 3.80 (br s, 2H, H2-60 ), 3.88 (br d, 1H, J ¼ 10.6 Hz, Hb-3), 3.97 (m, 1H, H-40 ), 4.09, 4.15 (dd, 1H, J ¼ 12.0, 3.4 Hz, Ha-1), 4.21 (d, 1H, J ¼ 7.5 Hz, H-10 ), 4.35 (br d, 1H, J ¼ 12.1 Hz, Hb-1), 5.24 (br s, 1H, H-2), 5.20e5.40 (m, 6H, 3  CH]CH ). 13C NMR (100 MHz, CDCl3): d 173.6, 173.5 (COO), 132.3, 130.4, 130.2, 128.5, 128.0, 127.6 (CH]CH), 104.2 (C-10 ), 74.9 (C-50 ), 73.6 (C-30 ), 71.6 (C-20 ), 70.6, 70.5 (C-2), 70.0 (C-60 ), 69.4 (C-40 ), 68.5 (C-3), 63.3, 63.2 (C-1), 34.4, 34.2 (COCH2), 32.3e23.0 (CH2 aliph.), 27.7, 27.6, 27.5 (CH2CH]CH), 26.2, 26.0 (]CHCH2CH]), 25.4, 25.3 (COCH2CH2), 14.8 (CH3). ESI-MS: m/z ¼ 692 (M þ Na, 100%)þ. Anal. calcd. for C37H64O10: C, 66.47; H, 9.58; found: C, 66.50; H, 9.62. 5.1.9. The antibacterial and antifungal activity of compounds 9a, b, 13aec and 14aec All the synthesized 1,2-O-diacyl-3-O-b-D-galactopyranosylrac-glycerols 9aeh, 13aec, 14aec have been evaluated for their in vitro activity against a number of Gram positive and Gram negative reference strains: S. aureus ATCC 25923, S. aureus ATCC 105487, E. faecalis ATCC 29212, B. subtilis ATCC 6633, E. coli ATCC 25922, P. aeruginosa ATCC 27753, M. tuberculosis H37Rv and against four different S. aureus, clinical strains SA52, SA85, SA87 and two M. tuberculosis clinical isolates, H160 and H190. The antifungal activity of all compounds was evaluated against two strains, clinical isolates, of C. albicans and C. pseudotropicalis. Antibacterial activity was evaluated by a reference agar dilution method [28]. MICs were determined twice in duplicate experiments. Antitubercular activity was evaluated by a standard agar dilution method and by a recently developed colour assay[29]. MIC was defined as the lowest drug concentration that prevented Resazurin colour change and was determined twice in duplicate experiments. Ciprofloxacin was chosen as a standard in antibacterial activity measurements. Antifungal activity has been tested against Candida spp. clinical isolates measuring MICs by a microdilution RPMI reference method, in comparison with miconazole [30]. 6. Conclusions In conclusion, a series of 1,2-O-diacyl-3-O-b-D-galactopyranosyl glycerols were synthesized and characterized, and some of them proved to be antibacterial agents. For all obtained compounds, we have examined the antimicrobial and antifungal activities and some structureeactivity relationships have been explored. It was observed that the antimicrobial activity in this class of compounds is dependent on the nature of fatty acids. The octanoyl chain is the proper structural feature for the maximum activity in 9a. This observation is confirmed by the results showing that compounds containing the octanoyl fatty acyl moiety at C-1 and unsaturated fatty acids at C-2 positions of glycerol 13aec displayed significant activity against B. subtilis, S. aureus 87 and M. tuberculosis reference and

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