Enantioselective synthesis and antioxidant activity of 3-(3,4- dihydroxyphenyl)-glyceric acid - Basic monomeric moiety of a biologically active polyether from Symphytum asperum and S. caucasicum

Article abstract of DOI:10.1002/chir.20823

The racemic and enantioselective synthesis of a novel glyceric acid derivative, namely, 2,3-dihydroxy-3-(3,4-dihydroxyphenyl)-propionic acid as well as the antioxidant activities is described. The virtually pure enantiomers, (+)-(2R,3S)-2,3-dihydroxy-3-(3

Full text of DOI:10.1002/chir.20823

CHIRALITY 22:717–725 (2010)
Enantioselective Synthesis and Antioxidant Activity
of 3-(3,4-Dihydroxyphenyl)-Glyceric Acid—Basic
Monomeric Moiety of a Biologically Active Polyether from
Symphytum asperum and S. caucasicum
1
1
1
MAIA MERLANI,1 VAKHTANG BARBAKADZE, LELA AMIRANASHVILI, LALI GOGILASHVILI,
*
ELINA YANNAKOPOULOU,2 KYRIAKOS PAPADOPOULOS,2 AND BEZHAN CHANKVETADZE3
1Kutateladze Institute of Pharmacochemistry, Laboratory of Plant Biopolymers, 0159 Tbilisi, Georgia
2NCSR ‘‘Demokritos’’, Institute of Physical Chemistry, Laboratory of Chemiluminescence, 153 10 Athens, Greece
3Department of Physical and Analytical Chemistry and Molecular Recognition and Separation Science Laboratory,
School of Exact and Natural Sciences, Tbilisi State University, Tbilisi, Georgia
ABSTRACT
The racemic and enantioselective synthesis of a novel glyceric acid de-
rivative, namely, 2,3-dihydroxy-3-(3,4-dihydroxyphenyl)-propionic acid as well as the
antioxidant activities is described. The virtually pure enantiomers, (1)-(2R,3S)-2,3-dihy-
droxy-3-(3,4-dihydroxyphenyl)-propionic acid and (2)-(2S,3R)-2,3-dihydroxy-3-(3,4-dihy-
droxyphenyl)-propionic acid were synthesized for the first time via Sharpless asymmet-
ric dihydroxylation of trans-caffeic acid derivatives using the enantiocomplementary cat-
alysts, (DHQD)2-PHAL and (DHQ)2-PHAL. The determination of enantiomeric purity of
the novel chiral glyceric acid derivatives was performed by high-performance liquid
chromatographic techniques on the stage of their alkylated precursors. The novel gly-
ceric acid derivatives show strong antioxidant activity against hypochlorite and N,N-di-
phenyl-N-picryl-hydrazyl free radical. Their antioxidant activity is about 40-fold higher
than that of the corresponding natural polyether and three-fold higher of trans-caffeic
C
VV
acid itself. Chirality 22:717–725, 2010.
2010 Wiley-Liss, Inc.
KEY WORDS: asymmetric sharpless dihydroxylation; trans-caffeic acid; 3-(3,4-
dihydroxyphenyl)-glyceric acid; chiral HPLC; enantiomeric analysis;
antioxidant activity
INTRODUCTION
hydroxyl functional groups in their aromatic ring.13
Recently, high-molecular (>1000 kDa) water-soluble prep-
arations with impressive immunomodulatory (anticomple-
mentary) and antioxidant activity were isolated from the
roots and stems of Symphytum asperum and S. caucasi-
cum.14,15 According to NMR and IR spectral data, the main
constituent of these preparations was found to be a regular
caffeic acid-derived polyether, namely, poly-[oxy-1-car-
boxy-2-(3,4-dihydroxyphenyl)-ethylene] (Fig. 1).16–18
This polymer is a representative of a new class of natu-
ral polyethers with a residue of 3-(3,4-dihydroxyphenyl)-
glyceric acid as the repeating unit. To our knowledge, 3-
(3,4-dihydroxyphenyl)-glyceric acid is not yet described in
the literature neither in racemic nor in enantiomerically
Comfrey (Symphytum L) extracts have been used in folk
and traditional medicine for healing of various diseases.1
Their most valuable pharmacological properties relate to
wound healing and treatment of inflammation through
their ability to strengthen regenerative process in injured
tissue.2,3 Generally, these properties are associated with
allantoin. Apart from allantoin, several other chemical con-
stituents of comfrey, such as mucilaginous polysaccha-
rides,4,5 caffeic acid, and its analogues rosmarinic acid6
and lithospermic acid7 as well as pyrrolizidine alkaloids8,9
are considered to possess significant biological activities.
Caffeic acid, phenolic acids, and polyphenols occur fre-
quently in fruits, grains, and dietary supplements10 and ex-
hibit antioxidant, anti-inflammatory, anti-atherosclerotic,
antimutagenic, anticancer, immunomodulatory, neuropro-
tective, or pro-apoptotic properties.11–13 Pronounced anti-
oxidant activity of caffeic acid derivatives is believed to be
associated with their ability to scavenge free radicals, build
chelating complexes with metal ions, or inhibit specific
free radicals or lipid hydroperoxides forming enzymes.
The responsible structural feature of caffeic acid deriva-
tives for such activity is the existence of two adjacent
C
Contract grant sponsor: Georgian Research and Development Foundation
(GRDF); Contract grant number: GEB2-3344-TB-06
Contract grant sponsor: Georgia National Science Foundation (GNSF);
Contract grant numbers: GNSF/ST 08/6-469 and GNSF/ST 06/071
*Correspondence to: Maia Merlani, Laboratory of Plant Biopolymers,
Kutateladze Institute of Pharmacochemistry, 36 P. Sarajishvili street, 0159
Tbilisi, Georgia. E-mail: maiamer@hotmail.com
Received for publication 5 June 2009; Accepted 23 October 2009
DOI: 10.1002/chir.20823
Published online 8 February 2010 in Wiley InterScience
(www.interscience.wiley.com).
VV
2010 Wiley-Liss, Inc.

718
MERLANI ET AL.
mV–10 V) is provided with a photomultiplier tube (HAM
105-21) with side window and spectral range from 300 to
620 nm and connected to a potentiometer chart recorder
(GOW-MAC Instrument, Model 70-150) or personal com-
puter equipped with a home made software program,
which allows the continuous monitoring and analysis of
the output signal. HPLC separation of enantiomers was
performed with Agilent 1200 HPLC system equipped with
auto sampler, column thermostat, and variable wavelength
detector. The data collection and treatment were per-
formed by Agilent Chemstation software (Agilent Inc.
Waldbronn, Germany). Various commercially available
chiral columns of Sepapak (Sepaserve GmbH, Muenster,
Germany) series (Sepapak-1, Sepapak-2, Sepapak-3, and
Sepapak-4 all of 4.6 3 250 mm size packed with 5 lm par-
ticles) were used for the determination of enantiomeric
excess on the stage of their benzylated forms. The detec-
tion was performed at 254 nm.
All commercially available reagents, trans-caffeic acid,
dimethylsulphate, benzyl bromide, citric acid, N-methylmor-
pholine-N-oxide (NMO), potassium osmate, palladium on
carbon (10% Pd/C), 2,2-diphenyl-1-picrylhydrazyl (DPPH),
5-amino-2,3-dihydro-1,4-phthalazinedione (luminol), 1,4-
bis(9-O-dihydroquinine)-phthalazine [(DHQ)2PHAL], 1,4-
bis(9-O-dihydroquinidine)-phthalazine [(DHQD)2PHAL]
were of analytical grade and used without further purifi-
cation (all from Sigma-Aldrich, Schnelldorf, Germany).
Fig. 1. Poly-[oxy-1-carboxy-2-(3,4-dihydroxyphenyl)-ethylene] isolated
from S. asperum and S. caucasicum.
pure form. Therefore, we have estimated that it would be
of interest to elaborate a synthetic method for its prepara-
tion. Moreover, as the molecular structures of the novel
dihydroxylated compounds contain the characteristic dihy-
droxyphenyl moiety of caffeic acid with well-known antiox-
idant activities, we determined their antioxidant activities
and compared them with that of trans-caffeic acid itself
and that of natural polyether isolated from comfrey species
S. asperum and S. caucasicum. After carefully search in the
literature, the most convenient method for the preparation
of the novel glyceric acid derivative, and its corresponding
enantiomers seems to be the asymmetric Sharpless dihy-
droxylation reaction (AD) of trans-caffeic acid.
Synthetic Procedures
Alkylation of trans-caffeic acid. The alkylation of
trans-caffeic acid proceeded according to a known proce-
dure.19 In brief, a mixture containing trans-caffeic acid 1
(1.8 g, 10 mmol), 50 ml acetone, powdered potassium car-
bonate (4.55 g), dimethylsulfate (3.12 ml) or benzyl bro-
MATERIALS AND METHODS
General Methods
1H- and 13C-NMR spectra were obtained on a Brucker mide (3.91 ml) was refluxed overnight under stirring. The
Avance DRX-500 spectrometer (Brucker AG, Karlsruhe, solid residues were filtered and washed three times with
Germany). Chemical shifts are expressed in d (parts per 50-ml portions of acetone. The solvent was evaporated,
million) values relative to tetramethylsilane (TMS) as in- and the resulted viscous yellowish residues were purified
ternal reference and coupling constants (J) are given in by crystallization in diethyl ether—petroleum ether.
Hertz. Electrospray ionization (ESI) Mass spectra were
Methyl 3-(3, 4-dimethoxyphenyl)-propenoate (2a). Yield:
recorded on a Finnigan spectrometer (Thermo, Manches- 2.0 g (90%), mp. 69–708C. 1H NMR (500 MHz, CDCl3,
ter, UK) by direct sample injection with a flow rate of 0.1 308C): d 7.61 (1H, d, J 5 15.9, olefinic proton), 7.08 (1H d,
ml/min using a Harvant Syringe pump. The data are J 5 1.9), 7.02 (1H, dd, J 5 8.02, 1.8), 6.84 (1H, d, J 5 8.4),
reported as m/z of the most important fragments. Desolva- 6.28 (1H, d, J 5 15.9, olefinic proton), 3.88 (6H, s, 2
tion was performed by hot nitrogen (Dominic-Hunter OCH3), 3.77 (3H, s, OCH3,); 13C NMR (125 MHz, CDCl3):
UHPLC MS-10). IR spectra were obtained using Perkin- d 5 167.57 (carbonyl group), 151.15, 149.23, 144.71 (ole-
Elmer 283 FT-IR spectrometer (Waltham, MA). Elemental finic carbon), 127.38, 122.53, 115.50, 111.09 (olefinic
analyses (C, H, and N) were carried out on a Perkin-Elmer carbon), 109.75, 55.93 (OCH3), 55.87 (OCH3), 51.50
CHN 2004 instrument (Norwalk, CT). The melting points (OCH3). IR (KBr), mmax (cm21): 2944, 2857, 1708 (car-
were recorded on an Electrothermal apparatus and are bonyl group), 1620, 1596, 1520, 1440, 1260, 1150, 980. MS
uncorrected (Engineering Ltd., VT). Absorption spectra (ESI): m/z 191 (M1ꢀꢀOCH3), 163 (M1ꢀꢀCOOCH3), 148
were run on a Jasco V-560 spectrophotometer (Jasco Co., (M11ꢀꢀCH2COOCH3).
Tokyo, Japan). Circular dichroism spectra were performed
Benzyl 3-(3,4-dibenzyloxyphenyl)-propenoate (2b). Yield:
on a Jasco J-715 instrument (Jasco Co., Tokyo, Japan) 4.05 g (90%), mp. 80–828C. 1H NMR (500 MHz, CDCl3,
equipped with peltier temperature control system. All reac- 308C): d 5 7.59 (1H, d, J 5 15.9), 7.30–7.45 (15H, m, aro-
tions were monitored by TLC on 0.25 mm precoated silica matic protons), 7.11 (1H, d, J 5 1.8 Hz), 7.06 (1H, dd, J 5
gel plates Merck 60, GF-254 (Merck, Darmstadt, Ger- 8.4, 1.8), 6.92 (1H, d, J 5 8.2), 6.26 (1H, d, J 5 15.9), 5.22
many) and visualized with UV light. Chemiluminescence (2H, s, benzylic protons), 5.19 (2H, s, benzylic protons),
measurements were made on a 1250 Bio-Orbit luminome- 5.16 (2H, s, benzylic protons,). 13C-NMR (125 MHz,
ter (Turku, Finland). The luminometer (output range 1.0 CDCl3): d 5 167.1 (C¼¼O, ester group), 151.5, 149.3, 145.3
Chirality DOI 10.1002/chir

3-(3,4-DIHYDROXYPHENYL)-GLYCERIC ACID
719
(olefinic carbon), 136.8, 136.7, 128.6–127.27 (complex tives 2 were dihydroxylated according to a known proce-
area, aromatic carbons), 127.14, 123.3, 116.1, 114.6, 114.1, dure.21 Briefly, in a 50 ml Erlenmeyer flask containing 20
71.7 (benzylic—CH2), 71.3 (benzylic—CH2), 66.6 (ben- ml solution of compound 2b (2.25 g, 5 mmol; 3:3:1 ace-
zylic—CH2). IR (KBr), mmax (cm21): 3068, 3025, 2907, tone–acetonitrile–water mixture) were added, succes-
2857, 1683 (carbonyl group), 1595, 1516, 1445, 1390, 1272, sively, (DHQ)2PHAL or (DHQD)2PHAL (0.0194 g, 0.025
1130, 1021, 946, 871, 840, 816.
mmol), hydrated potassium osmate (K2OsO4ꢁ2H2O, 0.0038
g, 0.01 mmol) and 1.5 ml NMO (50%). The reaction mix-
ture was stirred at room temperature (238C) for 2 hours,
then solution of sodium sulfite (0.74 g, 5.9 mmol) in 3 ml
water was charged in, the mixture was stirred for 30 min
and allowed to stand for 15 min. The organic phase was
concentrated into half volume under reduced vacuum and
extracted three times with ethyl acetate. The combined or-
ganic phases were washed with 10% NaCl (2 3 45 ml).
The aqueous phase was extracted with ethyl acetate twice
and the combined organic phases were concentrated. The
obtained crude product was purified by column chroma-
tography on silica gel, chloroform–methanol (10:1).
Typical Procedure for Dihydroxylation
a) Racemic dihydroxylation of trans-caffeic acid
derivatives. The protected trans-caffeic acid derivatives
2 were dihydroxylated according to a Sharpless proce-
dure.20 Briefly, the alkylated caffeic acid derivatives 2a
(2.22 g, 10 mmol) or 2b (4.14 g, 10 mmol) and citric acid
(3 g, 7.5 mmol) were dissolved in 10 ml of a 3:3:1 mixture
of acetonitrile–acetone–water in a 100 ml Erlenmeyer
flask. Potassium osmate (3.7 mg, 0.1 mol %) was then
added, followed by 50% water solution of NMO (2.28 ml,
1.1 mmol). The reaction mixture turned bright green. Af-
ter stirring at room temperature for 4 hours the reaction
mixture became nearly colorless. The organic solvents
were removed on a rotary evaporator and the aqueous res-
idue was then acidified with hydrochloric acid (1M, 12 ml)
and extracted with ethyl acetate (2 3 50 ml). The com-
bined organic extracts were dried with sodium sulfate and
concentrated giving colorless oils. The oils solidified at
room temperature as white solids and recrystallized from
ether-petroleum ether.
Methyl syn-2,3-dihydroxy-3-(3,4-dimethoxyphenyl)-propio-
nate (3a). Yield: 2.35 g, 92%, mp. 76–788C (lit.,21 mp. 75–
788C). 1H NMR (500 MHz, acetone-d6, 308C): d 5 7.07 (1H,
d, J 5 1.9), 6.93 (1H, dd, J 5 8.2 and 1.9), 6.88 (1H, d, J 5
8.2), 4.91 (1H, d, J 5 3.4, b-CHOH), 4.50 (1H, brd s, OH),
4.23 (1H, d, J 5 3.4, a-CHOH), 4.14 (1H, s, OH), 3.79 (3H,
s, OCH3), 3.78 (3H, s, OCH3), 3.67 (3H, s, OCH3). 13C NMR
(125 MHz, acetone-d6): d 5 173.11 (C¼¼O, ester), 148.72,
148.53, 132.50, 118.54, 110.78, 109.55, 75.15 (b-CHOH),
74.31 (a-CHOH), 55.78 (OCH3), 55.74 (OCH3), 52.51
(OCH3). IR (KBr), mmax (cm21): 3456, 3001, 2954, 2839,
1736, 1597, 1512, 1450, 1265, 1142, 1026, 910, 864, 810.
Benzyl syn-2,3-dihydroxy-3-(3,4-dibenzyloxyphenyl)-propio-
nate (3b). Yield: 4.45 g, 92%, mp. 126–1288C. 1H NMR
(500 MHz, acetone-d6): d 5 7.5–7.30 (15H, m, aromatic
protons), 7.25 (d, J 5 1.8, 1H), 7.01 (dd, J 5 8.4, 1.8, 1H),
6.97 (d, J 5 8.2, 1H), 5.22 (2H, s, benzylic protons), 5.16
(2H, s, benzylic protons), 5.14 (2H, s, benzylic protons),
4.90 (1H, d, J 5 3.4, b-CHOH), 4.50 (1H, broad s, OH),
4.34 (1H, d, J 5 3.4, a-CHOH), 4.21 (1H, broad s, OH).
13C-NMR (125 MHz, acetone-d6): d 5 172.48 (C¼¼O,
ester), 151.65, 149.36, 133.47, 129.06–127.86 (complex
area, aromatic carbons), 127.67, 119.93, 115.25, 113.90,
75.11 (b-CHOH), 74.58 (a-CHOH), 71.71 (2 benzylic—
CH2), 68.11 (benzylic—CH2). IR (KBr), mmax (cm21): 3508,
3063, 3030, 1720 (carbonyl group), 1595, 1504, 1445, 1384,
1344,1250, 1132, 1120, 1008, 1090, 941, 909, 890, 852, 818.
MS (ESI): m/z 5 507 (M1 1 Na). Elem. Analysis, calcu-
lated for C30H28O6 (484.548), theoretical C 5 74.36, H 5
5.82, found C 5 74.26, H 5 5.80.
Benzyl
(2R,3S)-2,3-dihydroxy-3-(3,4-dibenzyloxyphenyl)-
propionate (5). Yield: 2.17 g, 90%, mp. 97–1008C. 1H
NMR (500 MHz, acetone-d6): d 5 7.45–7.37 (15H, m, aro-
matic protons), 7.25 (d, J 5 1.8, 1H), 7.06 (dd, J 5 8.4, 1.8,
1H), 6.90 (d, J 5 8.2, 1H), 5.22 (2H, s, benzylic protons),
5.19 (2H, s, benzylic protons), 5.15 (2H, s, benzylic pro-
tons), 4.92 (1H, d, J 5 3.4, b-CHOH), 4.34 (1H, d, J 5 3.4,
a-CHOH). 13C-NMR (125 MHz, acetone-d6): d 5 172.56
(C¼¼O, ester), 149.4, 148.91, 137.2, 134.9, 133.1, 128.65–
127.26 (complex area, aromatic carbons), 119.51, 114.8,
113.47, 74.8 (b-CHOH), 74.31 (a-CHOH), 71.29 (2 ben-
zylic—CH2), 67.72 (benzylic—CH2). IR (KBr), mmax
(cm21): 3506, 3060, 3032, 1722 (carbonyl group), 1593,
1502, 1443, 1382, 1342,1253, 1134, 1124, 1006, 1092, 940,
906, 888, 850, 819. ee: 97.18% (HPLC).
Benzyl
(2S,3R)-2,3-dihydroxy-3-(3,4-dibenzyloxyphenyl)-
1
propionate (6). Yield: 2.05 g, 85%, mp. 88–918C. H NMR
(500 MHz, acetone-d6): d 5 7.47–7.37 (15H, m, aromatic
protons), 7.32 (d, J 5 1.8, 1H), 7.06 (dd, J 5 8.4, 1.8, 1H),
6.91 (d, J 5 8.2, 1H), 5.22 (2H, s, benzylic protons), 5.19
(2H, s, benzylic protons), 5.163 (2H, s, benzylic protons),
4.92 (1H, d, J 5 3.4, b-CHOH), 4.35 (1H, d, J 5 3.4, a-
CHOH). 13C-NMR (125 MHz, acetone-d6): d 5 172.48
(C¼¼O, ester), 149.4, 148.9, 137.1, 134.8, 133.0, 128.66–
127.25 (complex area, aromatic carbons), 119.49, 114.8,
113.46, 74.72 (b-CHOH), 74.29 (a-CHOH), 71.29 (2 ben-
zylic—CH2), 67.75 (benzylic—CH2). IR (KBr), mmax
(cm21): 3510, 3066, 3034, 1724 (carbonyl group), 1596,
1507, 1447, 1386, 1343,1252, 1133, 1123, 1007, 1094, 943,
909, 892, 854, 812. ee: 98.2% (HPLC).
Typical Procedure for Removing Benzyl Groups
The best yields for products 4, 7, and 8 were obtained
by using the catalytic hydrogenation procedure described
by Spencer and coworkers.22 In brief, a solution of prod-
ucts 3b, 5 or 6 (2.0 mmol) in a 1:1 mixture of ethanol–tet-
rahydrofuran (30 ml) was added to a stirred suspension of
palladium/carbon (10 mol %) in the same solvent system
(20 ml) that had previously been evacuated, purged with
hydrogen, and stirred for 30 min under a hydrogen atmos-
b) Asymmetric sharpless dihydroxylation of trans- phere. The reaction mixture was stirred overnight and
caffeic acid derivatives. Trans-caffeic acid deriva- then filtered through celite. The organic extracts were
Chirality DOI 10.1002/chir

720
MERLANI ET AL.
concentrated under vacuum, and the resulting residue was centrations ranging from 0.1—250 lg/ml.12 The absorb-
purified by crystallization in ethanol. Products 4, 7, or 8 ance of the samples was measured after the reaction
were obtained as white-yellowish solids.
reached a plateau (about 30 min). All measurements were
done at room temperature (238C) and repeated four times.
The radical scavenging activity of the samples was
expressed in terms of IC50 (concentration in lM required
for a 50% decrease in absorbance of DPPH radical) and cal-
culated using the equation [(Ablank 2 Asample)/Ablank)] 3
100), where Ablank is the absorbance of the control (DPPH
solution without sample) and Asample the absorbance of the
test compound (DPPH solution plus antioxidant). A plot of
absorbance vs. concentration was made to establish the
standard curve, and the linear regression equations from
which the IC50 values were calculated.
rac syn-2,3-dihydroxy-3-(3,4-dihydroxyphenyl)-pro-
pionic acid (4). Yield: 402.3 mg (94%), mp. 152–1588C.
UV (H2O), kmax nm (e): 230 (1470), 278 (5170). H NMR
1
(500 MHz, D2O): d 5 6.86 (1H, d, J 5 1.9 Hz), 6.80 (1H,
dd, J 5 8.2, 1.9 Hz), 6.75 (1H, d, J 5 8.2 Hz), 4.95 (1H, d, J
5 3.4 Hz, b-CHOH), 4.31 (1H, d, J 5 3.4 Hz, a-CHOH).
13C NMR (125 MHz, D2O): d 5 176.23 (C¼¼O, carboxylic
carbon), 144.15, 143.96, 132.99, 119.30, 116.40, 114.68,
75.58 (b-CHOH), 74.25 (a-CHOH). IR (KBr), mmax (cm21):
3445, 3374, 3337 (hydroxyl groups), 1748 (C¼¼O, carbox-
ylic group), 1700, 1602, 1523, 1443, 1406, 1289, 1206, 1106,
1039, 985, 937, 879, 864, 826, 810, 769. MS (CI), m/z: 153
(M11 ꢀꢀCO2, ꢀꢀH2O), 135 (M11ꢀꢀCO2, ꢀꢀ2H2O), 123
(M11ꢀꢀCO2, ꢀꢀ2H2O, ꢀꢀCH2O). Elem. Analysis, calcu-
lated for C9H10O6 3 2H2O (248.187), theor. C 5 43.55, H
5 4.87, found C 5 43.52, H 5 4.42.
b) Chemiluminescence technique. The light reac-
tion of the blank was started by adding sodium hypochlo-
rite (100 ll, 1.0%) into a mixture of alkaline luminol (100
ll, 50 lM) and sodium hydroxide (100 ll, 0.1 M).23 The
obtained light intensities were compared with the light
intensities of samples containing alkaline luminol solutions
(100 lL, 50 lM), test compound solutions of various con-
centrations (100 lL, 1.0–250 lg/ml diluted in 0.1M
NaOH) and sodium hypochlorite (100 ll, 1.0%). The solu-
tions were injected into the reactor cell with a Hamilton sy-
ringe through a septum. The antioxidant activity of the
samples was calculated from the equation: AA (%) 5 (I0 2
I)/I0) 3 100, where I0 and I, are the relative light inten-
sities of the blank and sample solutions, respectively. All
measurements were repeated four times, at room tempera-
ture (238C). The light signals reached their intensity maxi-
mum in less than 0.7 seconds and disappeared after 10
seconds. A plot of CL intensities vs. concentration was
made to establish the standard curve and the linear regres-
sion equations from which the IC50 values were calculated.
(1)-(2R,3S)- 2,3-Dihydroxy-3-(3,4-dihydroxyphenyl)-
propionic acid (7). Yield: 407 mg (95%), mp. 157–1608C.
1H NMR (500 MHz, D2O): d 5 6.90 (1H, d, J 5 1.9 Hz),
6.85 (1H, dd, J 5 8.2, 1.9 Hz), 6.81 (1H, d, J 5 8.2 Hz),
4.92 (1H, d, J 5 3.4 Hz, b-CHOH), 4.32 (1H, d, J 5 3.4 Hz,
a-CHOH). 13C NMR (125 MHz, D2O): d 5 175.6 (C¼¼O,
carboxylic carbon), 144.0, 143.77, 132.58, 119.08, 116.16,
114.46, 75.14 (b-CHOH), 73.96 (a-CHOH). IR (KBr), mmax
(cm21): 3333 (hydroxyl groups), 1750 (C¼¼O, carboxylic
group), 1702, 1605, 1522, 1446, 1410, 1299, 1210, 1105,
1037, 980, 935, 877, 862, 822, 811, 764. [a]D 5 133.38 (C
5 0.48, H2O), CD-spectrum (C 5 0.56 3 1023 M, H2O):
De275 5 20.4, De228 5 12.5, De209 5 14.8.
(2)-(2S,3R)-2,3-Dihydroxy-3-(3,4-dihydroxyphenyl)-
propionic acid (8). Yield: 364 mg (85%), mp. 155–
1
1588C. H NMR (500 MHz, D2O): d 5 6.88 (1H, d, J 5 1.9
RESULTS AND DISCUSSION
Synthesis and Structure Elucidation of
Synthesized Compounds
Hz), 6.82 (1H, dd, J 5 8.2, 1.9 Hz), 6.79 (1H, d, J 5 8.2
Hz), 4.90 (1H, d, J 5 3.4 Hz, b-CHOH), 4.30 (1H, d, J 5
3.4 Hz, a-CHOH). 13C NMR (125 MHz, D2O): d 5 176.0
(C¼¼O, carboxylic carbon), 144.1, 143.89, 132.52, 119.04,
116.12, 114.40. IR (KBr), mmax (cm21): 3332 (hydroxyl
groups), 1753 (C¼¼O, carboxylic group), 1704, 1607, 1523,
1448, 1412, 1297, 1213, 1103, 1036, 957, 937, 880, 864, 826,
810, 769. [a]D 5 232.58 (C 5 0.4, H2O), CD-spectrum (C
A systematic search in the literature and preliminary
test experiments revealed that the most economic way for
the preparation of the monomer, 3-(3,4-dihydroxyphenyl)-
glyceric acid 4, and its corresponding antipodes 7 and 8
is the asymmetric Sharpless dihydroxylation of alkylated
trans-caffeic acid derivatives (Scheme 1).
The hydroxyl groups as well as the carboxylic group of
trans-caffeic acid 1 are appropriately protected by benzyl
or methyl groups19 and the double bond of caffeic acid de-
rivative 2 dihydroxylated according to a modified Sharp-
less asymmetric dihydroxylation (AD) procedure.20,21 At
this point, it must be mentioned that it is of paramount im-
portance the right choice of chiral auxiliaries for the syn-
thesis of both enantiomers of 3-(3.4-dihydroxyphenyl)-gly-
ceric acid 4. Taking into account the mechanistic rules of
5 0.5 3 1023 M, H2O): De275 5 21, De228 5 25, De209
210.5.
5
Typical Procedures of Antioxidant Activities
Measurements
The antioxidant activity (AA%) of novel compounds was
determined spectrophotometrically (DPPH-method) or
with chemiluminescent techniques (luminol-hypochlorite
system).
a) DPPH-method. Briefly, the absorption of a metha- Sharpless24 and using the cinchona alkaloid derivatives
nolic solution of 2.0 ml DPPH (500 lM) and 2.0 ml metha- (DHQ)2-PHAL and (DHQD)2-PHAL as chiral auxiliaries,
nol was measured at 515 nm (blank) and compared with the dihydroxylated products 5 and 6, respectively, could
the absorbance of samples containing 2.0 ml DPPH and be produced in virtually optically pure forms by Sharpless
2.0 ml methanolic solutions of sample compounds in con- asymmetric dihydroxylation reaction. All dihydroxylated
Chirality DOI 10.1002/chir

3-(3,4-DIHYDROXYPHENYL)-GLYCERIC ACID
721
Scheme 1. Synthetic route for the preparation of 3-(3,4-dihydroxyphenyl)-glyceric acid (4) and its antipodes (7) and (8). (a) Dimethyl sulfate, potas-
sium carbonate, acetone, reflux (b) Benzyl bromide, potassium carbonate, acetone, reflux (c) citric acid, potassium osmate, NMO acetonitrile/acetone/
water (3:3:1), room temperature (d) [(DHQ)2PHAL], potassium osmate, NMO, acetonitrile/acetone/water (3:3:1), room temperature (e)
[(DHQD)2PHAL], potassium osmate, NMO, acetonitrile/acetone/water (3:3:1), room temperature (f) 10% Pd/C, H2, ethanol–tetrahydrofuran, room tem-
perature (g) HBr/acetic acid, reflux; trimethylsilyl chloride–potassium iodide, acetonitrile, room temperature.
products show the relative syn-configuration. This has and 209 nm, while the (2S,3R)-isomer of 8 shows positive
been explained by comparison of the measured coupling signs at the same wavelengths.
constants (J 5 3.4 Hz) of protons attached to the a and b-
The enantiomeric excess of enantiomeric novel glyceric
carbons on the glyceric moiety with those of cinnamic and acid derivatives was determined by high performance liq-
caffeic acid derivatives obtained under similar reaction uid chromatography on the stage of their benzylated deriv-
conditions.20 The absolute configurations of dihydroxy- atives 3b, 5 and 6 using sepapak chiral columns (Figs. 2,
lated products 5, 6, 7, and 8 were determined assuming 3 and 4). In Fig. 2 the chiral resolution of 3b is reported.
the same mechanistic approach proposed by Sharpless25 The enantiomeric excess of the corresponding enan-
for the enantioselective dihydroxylation of trans-cinnamic tiomers 5 and 6 was 97.18 and 98.2%, respectively (Figs. 3
acid derivatives, where the chiral auxiliary (DHQ)2-PHAL and 4).
leads to the (2R,3S) configuration, while (DHQD)2-PHAL
At this point it must be noted, that polysaccharide phenyl-
to the opposite configured enantiomer. So to products 5 carbamate derivatives (filling material of sepapak columns)
and 7 configuration was assigned (2R, 3S) [chiral auxiliary represent the most powerful group of chiral stationary
(DHQ)2-PHAL] and (2S,3R) to products 6 and 8 [chiral phases for analytical and preparative scale separations of
auxiliary (DHQD)2-PHAL]. The opposite configuration of enantiomers.26,27 The phenylcarbamate derivatives of cellu-
both enantiomers is also confirmed by measurements of lose and amylose containing electron-donating and electron-
the optical rotation (1)/(2)-values and circular dichroism withdrawing substituents on their phenyl moiety are charac-
spectra of products 7 and 8 as well as by HPLC analysis terized with quite universal chiral recognition ability and can
of products 5 and 6 using chiral columns (Sepapak) be used for separation of enantiomers in normal-, polar or-
(retention times of the enanantiomers). In optical rotation ganic-, and reversed-phase mode.28–30 Several of these new
measurements, the (1)-sign is assigned to the stereoiso- derivatives of polysaccharides have been commercialized
mer (2R,3S) while the (2)-sign to the opposite enantiomer recently and used for solving of various separation problems
(2S,3R). The same results found in their corresponding of pharmaceutical and biomedical relevance.31–33
CD spectra. As given in experimental part, the (2R,3S)-iso-
In frame of this work, five different chiral sepapak col-
mer of 7 shows negative signs (2) at wavelengths 228 umns (Fig. 5) were tested for the evaluation of enantio-
Chirality DOI 10.1002/chir

722
MERLANI ET AL.
separation. This allowed longer retention and eventually
better separation of enantiomers. The longer retention was
actually observed with increasing content of water in the
mobile phase. Nearly/almost baseline enantioseparation
was observed with the mobile phase containing 20% of
water in methanol (v/v) with analysis time of 38.1 and
42.8, respectively. Typical normal phase eluent such as n-
hexane/2-propanol (85/15, v/v) was used as the next in
combination with Sepapak-4 column. A good baseline sep-
aration of two enantiomers was observed, therefore, this
system was used for the enantiomeric excess determina-
tion of benzylated derivatives (Figs. 3 and 4). According to
HPLC analysis data, the enantiomeric excess (ee) for prod-
ucts 5 and 6 was 97.18 and 98.2%, respectively.
At this point, it must also be noted that preliminary stud-
ies on unprotected acidic (free acid) monomers did not
interact/retain with chiral stationary phases sufficiently
and so could not be separated. Therefore, benzyl deriva-
tives were used for the enantiomeric excess determination.
The chemical yields of products 3, 5, and 6 obtained in
acetone–acetonitrile–water mixture (3:1:1) were in the
range of 85–92% and much lower in case of t-butanol–water
(1:1). On the last step the protective (benzyl) groups were
removed successfully by hydrogenation using 10% Pd/C
in tetrahydrofuran–ethanol.22 Using concentrated hydro-
gen bromide–acetic acid or trimethylsylilchloride–potas-
sium iodide in acetonitrile, the reaction proceeded well
but the purification of product 4 was not successful. The
Fig. 2. a. Chromatogram of racemic benzyl-[2,3-dihydroxy-3-(3,4-diben-
zyloxyphenyl)]-propionate on Sepapak-4 column. Mobile phase: metha-
nol/water, 80/20 (v/v). Flow rate 2.0 ml/min. b. Chromatogram of race-
mic benzyl-[2,3-dihydroxy-3-(3,4-dibenzyloxyphenyl)]-propionate on Sepa-
pak-4 column. Mobile phase: n-hexane/2-propanol, 85/15 (v/v). Flow rate
2.0 ml/min. [Color figure can be viewed in the online issue, which is avail-
able at www.interscience.wiley.com.]
desired 3-(3,4-dihydroxyphenyl)-glyceric acid,
4 was
obtained in good yield only by hydrogenation. The struc-
tures of all products were elucidated by UV-Visible, 1H
NMR-, 13C NMR-, FTIR-, and MS-spectral data.
All spectra of monomer 4 were compared with that of
polyether obtained from S. asperum and S. caucasicum. In
the ultra-violet spectrum of glyceric acid derivative 4 the
two characteristic bands at 230 and 278 nm (Fig. 6) are
similar to those of the natural polyether but shifted about
7–8 nm toward lower wavelengths. The absorption bands
of polyether appear at 237 and 286 nm (Fig. 6).
meric excess of enantiopure glyceric acid derivatives 5
and 6 using initially methanol as a mobile phase.
Partial separation of the enantiomers 5 and 6 was
observed using Sepapak-1, Sepapak-3, and Sepapak-4 col-
umns. Sepapak-2 and Sepapak-5 did not provide any meas-
urable separation of the enantiomers (data not shown).
Since Sepapak-4 provided the most promising separation
in pure methanol further optimization of the mobile phase
was performed with this material. Water additives were
considered at first to convert/transform the pure polar or-
ganic mobile phase separation to reversed phase enantio-
The IR spectrum of monomer 4 is also similar to that of
natural polyether and contains all the characteristic bands
corresponding to the hydroxyl groups attached to the aro-
Fig. 3. Chromatogram of virtually enantiopure benzyl (2R,3S)-2,3-dihy-
droxy-3-(3,4-dibenzyloxyphenyl)-propionate. Chiral column: Sepapak-4;
mobile phase: n-hexane/2-propanol, 85/15 (v/v); flow rate 2.0 ml/min.
The major product is obtained using the chiral auxiliary (DHQ)2-PHAL.
(ee 5 97.18%). [Color figure can be viewed in the online issue, which is
available at www.interscience.wiley.com.]
Fig. 4. Chromatogram of virtually enantiopure benzyl (2S,3R)-2,3-dihy-
droxy-3-(3,4-dibenzyloxyphenyl)-propionate. Chiral column: Sepapak-4;
mobile phase: n-hexane/2-propanol (85/15 v/v); flow rate 2.0 ml/min.
The major product is produced using the chiral auxiliary (DHQD)2-PHAL.
(ee 5 98.2%). [Color figure can be viewed in the online issue, which is
available at www.interscience.wiley.com.]
Chirality DOI 10.1002/chir

3-(3,4-DIHYDROXYPHENYL)-GLYCERIC ACID
723
Fig. 5. Structures of filling materials in Sepapak columns. Sepapak-1: cellulose tris-(3,5-dimethylphenylcarbamate); Sepapak-2: cellulose tris-(3-chloro-
4-methylpenylcarbamate); Sepapak-3: amylose tris-(5-chloro-2-methylphenylcarbamate); Sepapak-4: cellulose tris-(4-chloro-3-methylphenylcarbamate);
Sepapak-5: cellulose tris-(3,5-dichlorophenylcarbamate).
matic ring as well as the carboxylic group. No significant nals of carbon atoms were observed in the spectra of both
differences are observed in the 13C NMR spectra of the compounds.16–18 The signals at 74.25 and 75.58 ppm are
monomer and isolated natural polyether. Nine distinct sig- assigned to the aliphatic carbon atoms, whereas the six
signals at 114.68, 116.40, 119.30 (protonated carbons) and
132.99, 143.96, 144.15 ppm (nonprotonated carbons)
belong to the aromatic ones. The signal at 176.23 ppm is
assigned to the carboxylic group. Small differences were
1
observed also in the H NMR spectra of monomer and its
corresponding polymer. In particular, the peaks corre-
sponding to the aromatic protons at 6.89, 6.84, and 6.79
ppm and those to the aliphatic ones at 4.88 and 4.27 ppm
are sharp signals, while those of the polyether are broad-
ened and shifted. Namely, the aliphatic protons appear at
4.88 and 5.33 ppm, while the aromatic ones at 7.13 and
7.24.16–18
Antioxidant Activities of Racemic Derivative 4
and its Enantiomers 7 and 8
The ability of 3-(3,4-dihydroxyphenyl)-glyceric acid to
scavenge free radicals or neutral reactive oxygen species
Fig. 6. UV-Vis spectrum of 3-(3,4-dihydroxyphenyl)-glyceric acid, 4.
was tested against the relatively stable DPPH-radical and
Chirality DOI 10.1002/chir

724
MERLANI ET AL.
TABLE 1. Measured antioxidant activities of racemic 4 and virtually enantiopure products 7 and 8 as well as those of
natural polyether isolated from the roots of S. asperum and trans-caffeic acid against hypochlorite and DPPH-radicals
Antioxidant activity (IC50a, lg/ml)
Compound
DPPH-method
CL-method
rac 2,3-Dihydroxy-3-(3,4-dihydroxyphenyl)-propionic acid
(1)-(2R,3S)-2,3-Dihydroxy-3-(3,4-dihydroxyphenyl)-propionic acid
(2)-(2S,3R)-2,3-Dihydroxy-3-(3,4-dihydroxyphenyl)-propionic acid
Poly-[oxy-1-carboxy-2-(3,4-dihydroxyphenyl)-ethylene]
trans-Caffeic acid
3.8 6 0.3
4.1 6 0.4
3.9 6 0.2
146.5 6 7.6
12.2 6 0.7
2.8 6 0.2
3.2 6 0.3
3.0 6 0.2
120.4 6 9.8
9.4 6 0.5
aIC50 6 standard deviation (n 5 4)
hypochlorite. Sodium hypochlorite was chosen as repre- sepapak columns. The absolute and relative configurations
sentative for reactive oxygen species because of its impor- of the novel enantiopure glyceric acid derivatives 7 and 8
tance in oxidizing reactions in biological systems.34 In liv- were assigned on the stage of their alkylated forms and
ing organisms hypochlorous acid is produced by certain determined by assuming the same mechanistic approach
oxidizers from chloride anions and hydrogen peroxide.35 suggested by Sharpless for dihydroxylated products
The in vitro antioxidant activity estimation of 3-(3, 4-dihy- obtained from trans-cinnamic acid derivatives.
droxyphenyl)-glyceric acid derivatives against hypochlo-
Moreover, it has been shown that the novel racemic gly-
rite ions, could also be used for the estimation of this reac- ceric acid derivative 4 as well as its enantiomeric pure
tive oxygen species in vivo experiments, too. The antioxi- derivatives 7 and 8 possess strong antioxidant activities
dant activities of racemic and vitually enantiopure products against reactive oxygen species, such as hypochlorite or
4, 7, and 8 as well as those of natural polyether isolated free radicals such as DPPH. Such pronounced antioxidant
from the roots of S. asperum and trans-caffeic acid are properties give appropriate background for further deep
given in Table 1.
investigation of the biological activity of this phenolic com-
As shown in Table 1, all phenolic compounds show pound. The use either as food additive or in medicinal
strong antioxidant activities against hypochlorite ions and preparations for treatment of oxidative disorder-related dis-
DPPH free radical. The inhibitory effect of racemic prod- eases can be considered.
uct 4 as well as this of the corresponding enantiomers
appeared to be about 40-fold and three-fold higher than
ACKNOWLEDGMENTS
that of polyether or trans-caffeic acid, respectively. The
unexpected lowest antioxidant activity of polyether may be
explained with the less stabilizing effects of phenoxide rad-
icals via intra- or intermolecular hydrogen bonds observed
in monomeric products. Such stabilizing effects of phenox-
ide radicals in phenolic compounds via intra- or intermo-
lecular hydrogen bonds are reported in reference.36 This
hypothesis is strengthened by the similarity of structural
features of novel 3-(3,4-dihydroxyphenyl)-glyceric acid
derivatives 4, 7, and 8 and trans-caffeic acid. In case of
compounds 4, 7, and 8, the phenoxide radicals produced
during the reaction can be stabilized intramolecularly via
hydrogen bonds, while in case of trans-caffeic acid, over
the extended conjugated system. As shown in Table 1 no
significant differences have been noted regarding the anti-
oxidant activities of pure enantiomers and the racemic
monomer.
The authors would like to thank Dr. Dimitra Benaki and
Dr. Leondios Leondiadis at the NCSR ‘‘Demokritos’’ for
performing the CD and MS spectra measurements,
respectively.
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