Two goitrogenic 1,3-oxazolidine-2-thione derivatives from Brassicales taxa: Challenging identification, occurrence and immunomodulatory effects

Article abstract of DOI:10.1016/j.fct.2017.10.005

1,3-Oxazolidine-2-thione derivatives are glucosinolate-related food constituents known to impart (thyreo)toxic properties to some cruciferous vegetables. In this work, 5,5-dimethyl-1,3-oxazolidine-2-thione and (-)-(R)-5-phenyl-1,3-oxazolidine-2-thione, known goitrogens, were isolated from Draba lasiocarpa Rochel (Brassicaceae) and Reseda luteola L. (Resedaceae), respectively, and were fully spectrally characterized. Subsequently, the occurrence of the two 1,3-oxazolidine-2-thiones was verified in six additional taxa out of in total 78 screened Serbian Brassicales taxa. The stereochemistry of 5-phenyl-1,3-oxazolidine-2-thione was inferred from nuclear magnetic resonance experiments with a chiral lanthanide-shift reagent, employed in this work for the first time for this type of compounds. Unexpectedly, during gas chromatography, 5-phenyl-1,3-oxazolidine-2-thione underwent an unreported thermal core isomerization (1,3-oxazolidine-2-thione to 1,3-thiazolidine-2-one). These goitrogenic volatile glucosinolate products were tested for their effect on rat macrophage viability (three assays) and nitric oxide production. It was shown that the compounds displayed different levels of cytotoxicity. All tested compounds caused a significant lactate dehydrogenase leakage, but only (R)-5-phenyl-1,3-oxazolidine-2-thione statistically significantly reduced macrophage mitochondrial activity, whereas the racemic 5-phenyl-1,3-oxazolidine-2-thione and 5,5-dimethyl-1,3-oxazolidine-2-thione had little or no effect. Again only (R)-5-phenyl-1,3-oxazolidine-2-thione exerted nitric oxide production-inhibiting properties, suggesting the higher immunomodulatory potential of this enantiomer compared with its antipode and racemic mixture.

Full text of DOI:10.1016/j.fct.2017.10.005

Accepted Manuscript
Two goitrogenic 1,3-oxazolidine-2-thione derivatives from Brassicales taxa:
Challenging identification, occurrence and immunomodulatory effects
Niko S. Radulović, Milica M. Todorovska, Dragan B. Zlatković, Nikola M. Stojanović,
Pavle J. Randjelović
PII:
S0278-6915(17)30583-5
10.1016/j.fct.2017.10.005
FCT 9330
DOI:
Reference:
To appear in: Food and Chemical Toxicology
Received Date: 3 September 2017
Revised Date: 1 October 2017
Accepted Date: 5 October 2017
Please cite this article as: Radulović, N.S., Todorovska, M.M., Zlatković, D.B., Stojanović, N.M.,
Randjelović, P.J., Two goitrogenic 1,3-oxazolidine-2-thione derivatives from Brassicales taxa:
Challenging identification, occurrence and immunomodulatory effects, Food and Chemical Toxicology
(2017), doi: 10.1016/j.fct.2017.10.005.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Two goitrogenic 1,3-oxazolidine-2-thione derivatives from Brassicales taxa:
challenging identification, occurrence and immunomodulatory effects
,a
a
a
b
Niko S. Radulović,* Milica M. Todorovska, Dragan B. Zlatković, Nikola M. Stojanović,
c
Pavle J. Randjelović
a
Department of Chemistry, Faculty of Sciences and Mathematics, University of Niš, Višegradska
3, 18000 Niš, Serbia
3
b
Faculty of Medicine, University of Niš, Dr Zoran Ðinđić Boulevard 81, 18000 Niš, Serbia
c
Department of Physiology, Faculty of Medicine, University of Niš, Dr Zoran Ðinđić Boulevard
1, 18000 Niš, Serbia
Corresponding author, Tel.: +381 18 223430; Fax: +381 18 533014. E-mail address:
nikoradulovic@yahoo.com
8
*
1

ACCEPTED MANUSCRIPT
Abstract
,3-Oxazolidine-2-thione derivatives are glucosinolate-related food constituents known to impart
thyreo)toxic properties to some cruciferous vegetables. In this work, 5,5-dimethyl-1,3-
1
(
oxazolidine-2-thione and (-)-(R)-5-phenyl-1,3-oxazolidine-2-thione, known goitrogens, were
isolated from Draba lasiocarpa Rochel (Brassicaceae) and Reseda luteola L. (Resedaceae),
respectively, and were fully spectrally characterized. Subsequently, the occurrence of the two
1
,3-oxazolidine-2-thiones was verified in six additional taxa out of in total 78 screened Serbian
Brassicales taxa. The stereochemistry of 5-phenyl-1,3-oxazolidine-2-thione was inferred from
nuclear magnetic resonance experiments with a chiral lanthanide-shift reagent, employed in this
work for the first time for this type of compounds. Unexpectedly, during gas chromatography, 5-
phenyl-1,3-oxazolidine-2-thione underwent an unreported thermal core isomerization (1,3-
oxazolidine-2-thione to 1,3-thiazolidine-2-one). These goitrogenic volatile glucosinolate
products were tested for their effect on rat macrophage viability (three assays) and nitric oxide
production. It was shown that the compounds displayed different levels of cytotoxicity. All
tested compounds caused a significant lactate dehydrogenase leakage, but only (R)-5-phenyl-1,3-
oxazolidine-2-thione statistically significantly reduced macrophage mitochondrial activity,
whereas the racemic 5-phenyl-1,3-oxazolidine-2-thione and 5,5-dimethyl-1,3-oxazolidine-2-
thione had little or no effect. Again only (R)-5-phenyl-1,3-oxazolidine-2-thione exerted nitric
oxide production-inhibiting properties, suggesting the higher immunomodulatory potential of
this enantiomer compared with its antipode and racemic mixture.
Keywords: 1,3-oxazolidine-2-thione, goitrogenic volatile products, cruciferous foodstuff,
macrophages, 5-phenyl-1,3-oxazolidine-2-thione, 5,5-dimethyl-1,3-oxazolidine-2-thione
2

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1
1
. Introduction
,3-Oxazolidine-2-thiones cause hypothyroidism with an enlargement of the thyroid when
consumed by man or animals (Fahey et al., 2001). Definite evidence of a plant-derived food
goitrogen was first put forward by Chesney and co-workers (1928), who reported greatly
enlarged thyroid glands in rabbits fed mainly with cabbage. Racemic goitrin (5-vinyl-1,3-
oxazolidine-2-thione) caused a growth rate depression of poultry and hyperplasia and
hypertrophy of their thyroids (Clandinin et al., 1966). When eaten extensively by livestock,
cruciferous forage plants which yield 1,3-oxazolidine-2-thione derivatives may cause severe
sheep and cattle poisoning (Holmes, 1965). 5,5-Dimethyl-1,3-oxazolidine-2-thione, isolated
from hare's ear mustard (Conringia orientalis) (Hopkins, 1938), has about the same antithyroid
activity as goitrin (Astwood et al., 1945). (-)-(R)-5-Phenyl-1,3-oxazolidine-2-thione, isolated
from numerous edible Brassicales taxa is about two times less active than goitrin (Greer and
Whallon, 1961). Goitrin is assumed to exert its effect in a similar manner to that of the
chemically related 2-thiouracil and other drugs (Astwood et al., 1945).
Due to strong odors and flavors that their volatile hydrolysis (autolysis) products elicit in some
vegetables, glucosinolates have been a part of human sustenance for thousands of years (Halkier
and Gershenzon, 2006). Sometimes called mustard-oil glucosides, glucosinolates represent β-
thioglucosides of (Z)-N-hydroximinosulfate esters which are hydrolysed by myrosinases,
activated by mechanical stimuli as the first line of defense of the plants (Agerbirk and Olsen,
2
012). More than 130 described glucosinolates upon hydrolysis furnish glucose and an unstable
aglycone that can convert spontaneously to form isothiocyanates, nitriles, 1,3-oxazolidine-2-
3

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thiones, etc. (Fahey et al., 2001). In cases where a hydroxyl group is present in the glucosinolate
side-chain at C-2, the formed isothiocyanates undergo ring closure to form 1,3-oxazolidine-2-
thiones (Fahey et al., 2001).
In the continuation of our interest in the volatile autolysis products of Brassicaceae and
Resedaceae (Radulović et al., 2008, 2011, 2012, 2014), we performed a GC-MS screening of
autolyzates of 78 taxa from the Serbian flora, belonging to these families (for the list of screened
taxa, please, confer to Supplementary data, Table S1). Among 159 analyzed samples, that
included different plant organ autolyzates, and plant material collected in different time periods
from several sites, a limited number of taxa contained volatile 1,3-oxazolidine-2-thione
derivatives. Two derivatives, 5,5-dimethyl-1,3-oxazolidine-2-thione and 5-phenyl-1,3-
oxazolidine-2-thione, were detected in considerable amounts in Draba lasiocarpa Rochel
(Brassicaceae) and Reseda luteola L. (Resedaceae), respectively. This provided us with a means
of obtaining pure samples of the two 1,3-oxazolidine-2-thiones via SiO chromatography, and
2
the opportunity to investigate their chemical, spectral and biological properties.
Identification of glucosinolate autolysis products is difficult due to the fact that their mass
spectra are scarce in the commercially available databases, or the availability of only the
graphical form of such data (Spencer and Daxenbichler, 1980). Additionally, the literature
retention indices (RIs) are largely incoherent; for example, RIs of butyl- (RI range 943-1159), 3-
butenyl- (RI range 1003-1091), 4-methylpentyl- (RI range 1163-1272) and hexyl-isothiocyanates
(RI range 1197-1277) (Afsharypuor et al., 1998; Afsharypuor and Balam, 2005; Afsharypuor and
Jamali, 2006; Blažević and Mastelić, 2008; de Pinho et al., 2009). In the current work, we
encountered another problem with GC-MS identification of 5-phenyl-1,3-oxazolidine-2-thione.
A possible reason for the lack of (reliable) literature data on this compound is a combination of
4

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unexpected artefact formation during GC-MS runs and the similarity in the mass spectra between
the artefactual and natural compounds. Herein, we report on the identification of 5,5-dimethyl-
1
,3-oxazolidine-2-thione and 5-phenyl-1,3-oxazolidine-2-thione, differing in their conduct
during GC-MS analyzes, based on chemical and spectral evidences. Also, we describe the
structural elucidation of the artefact(s) formed during the GC-MS analyzes of 5-phenyl-1,3-
oxazolidine-2-thione by preparative scale thermolysis.
A recent work on the natural enantiomers occurrence of both enantiomers of 5-phenyl-1,3-
oxazolidine-2-thione (Agerbirk et al., 2014) encouraged us to determine the enantiomeric excess
of the sample from R. luteola at hand. For this purpose, we developed an NMR-based
methodology utilizing a chiral chemical shift reagent (Genčić and Radulović, 2015). The method
was demonstrated to enable a resolution of enantiomer signals (more specifically within
diastereomeric complexes of europium(III)) of a racemic mixture of 5-phenyl-1,3-oxazolidine-2-
thione prepared by synthesis. Also, it was successfully applied for the first time to the above
mentioned sample from R. luteola.
Macrophages play a crucial role in innate immunity, inflammation, and host defense (Abbas et
al., 2015). Certain immunomodulatory compounds are capable of altering the fagocidal activity
and/or activation of macrophages, influencing the immune system in this way. New and safe
immunotherapeutic medications, possibly of natural origin, are needed since the currently used
immunomodulators display many adverse effects (Bascones-Martinez et al., 2014; Newman and
Cragg, 2007). Despite the apparent biological and toxicological significance of natural 1,3-
oxazolidine-2-thione derivatives, the effect of these compounds on macrophages has not been
investigated to date. An anti-inflammatory extract of an Isatis sp. (Brassicaceae), containing
epigoitrin ((
D)-5-vinyl-1,3-oxazolidine-2-thione), has been demonstrated to influence
5

ACCEPTED MANUSCRIPT
macrophage viability and their functional characteristics (Xiao et al., 2014). Also, many
glucosinolate-related products are known to possess anticarcinogenic and chemoprotective
properties (Gimsing and Kirkegaard, 2009; Kensler et al., 2013). Prompted by this, another aim
of this study was to investigate the immunomodulatory activity of 5,5-dimethyl-1,3-oxazolidine-
2
-thione, (R)-, and rac-5-phenyl-1,3-oxazolidine-2-thiones, via an assessment of macrophage
viability (three assays revealing different aspects of macrophage function, MTT, LDH, Neutral
red tests), adherence (Methylene-blue test) and their ability to produce nitric oxide (NO).
2
2
. Materials and Methods
.1 Chemicals
All chemicals used in the synthetic work or preparative and analytical chromatographies were
obtained from Sigma-Aldrich (St. Louis, MO, USA) or Carl Roth (Karlsruhe, Germany) and
were used as received with the exception of solvents that were additionally dried and purified by
conventional methods prior to use. The lanthanide shift reagent europium tris[3-
3
(heptafluoropropylhydroxymethylene)-(d)-camphorate] (Eu(hfc) ) for NMR experiments was
purchased from Sigma-Aldrich (St. Louis, MO, USA). All other chemicals were of analytical
grade or better (Merck, Darmstadt, Germany; Sigma-Aldrich, St. Louis, MO, USA; TCI, Tokyo,
Japan). Cell culture medium (RPMI medium, acquired from Sigma-Aldrich, St. Louis, MO,
-3
USA) used for cell-related experiments consisted of RPMI 1640 with 20 mmol dm HEPES and
-1
L
-glutamine, without sodium bicarbonate, containing 5% (w/v) fetal bovine serum, 200 mg ml
-1
streptomycin (AppliChem, Darmstadt, Germany) and 200 IU ml penicillin (AppliChem,
Darmstadt, Germany). Standard cytotoxic drug, cisplatin, was acquired from Teva (Belgrade,
Serbia).
3-(4,5-Dimethylthiazole-2-yl)-2,5-diphenyltetrazolium
bromide
(MTT)
and
6

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lipopolysaccharides from Escherichia coli 0111:B4 (LPS), used for cell viability determination
and stimulation, respectively, were obtained from Sigma-Aldrich (St. Louis, MO, USA).
2
.2 Methods
Optical rotations were measured on an Autopol IV (Rudolph Research Analytical, Flanders,
USA) polarimeter equipped with a sodium lamp (589 nm) and a 10-cm microcell. IR
measurements (neat, ATR-attenuated total reflectance) were carried out using a Thermo Nicolet
model 6700 FT-IR instrument (Waltham, USA). UV spectra (in acetonitrile) were measured
using a UV-1800 Shimadzu spectrophotometer (Tokyo, Japan). Microanalysis of carbon,
hydrogen and nitrogen were carried out with a Carlo Erba 1106 microanalyser (Carlo Erba
Strumentazione, Milan, Italy) and their results agreed favorably with the calculated values. Mass
spectra were recorded on a Hewlett Packard 5975B mass selective detector coupled with a
Hewlett Packard 6890N gas chromatograph equipped with a fused silica capillary column DB-
5
MS (5% phenylmethylsiloxane, 30 m × 0.25 mm, film thickness 0.25 µm, Agilent
Technologies, Palo Alto, CA, USA). The injector and interface were operated at 250 °C and 320
C, respectively. Oven temperature was raised from 70 °C to 315 °C at a heating rate of 5 °C/min
°
-1
and then isothermally held for 10 min. As a carrier gas, He at 1.0 ml min was used. The
samples (1 mg per 1 ml of an appropriate solvent) were injected in a pulsed-split mode (the flow
-1
-1
was 1.5 ml min for the first 0.5 min and then set to 1.0 ml min throughout the rest of the
analysis; split ratio, 40:1). MS conditions: ionization voltage, 70 eV, acquisition mass range, 35–
6
50 amu, scan time, 0.32 s. The linear retention indices were calculated relative to the retention
1
13
9
times of C –C21 n-alkanes on the mentioned DB-5MS column. H and C NMR spectra were
recorded on a BrükerAvance III spectrometer (Brüker, Fällanden, Switzerland) operating at 400
7

ACCEPTED MANUSCRIPT
and 100.6 MHz, respectively. 2D experiments (NOESY, ROESY and gradient HSQC, HMBC
1
1
and H– H COSY), as well as multipulse DEPT-90, DEPT-135 and selective homonuclear
decoupling experiments, were run on the same instrument with the built-in Brüker pulse
sequences. NMR spectra were measured at 25 °C in CDCl , CD SOCD and C D with
3
3
3
6
6
tetramethylsilane (TMS) as an internal secondary reference standard. Chemical shifts (δ) are
1
reported in parts per million and referenced to tetramethylsilane (δ = 0 ppm) in H NMR spectra
H
1
3
and/or to solvent signals (residual CHCl
3
: δ
H
= 7.26 ppm, and CDCl
3
: δ
C
6 5
= 77.16 ppm; C HD :
1
3
13
δ
H
C
= 7.16 ppm, and CC
5
D
6
: δ
C
= 128.06 ppm; CHD
2
SOCD : δ
3
H
= 2.50 ppm, and CD
3
SOCD
3
:
δ
= 39.52 ppm) in heteronuclear 2D spectra. Scalar couplings are reported inhertz (Hz). The
following abbreviations were used to designate multiplicities: br, broad signal; ps, pseudo; s,
singlet; d, doublet; t, triplet; q, quartet; sext, sextuplet; oct, octuplet; dd, doublet of doublets; ddd,
doublet of doublet of doublets, dt, doublet of triplets; td, triplet of doublets; m, multiplet; dm,
doublet of multiplets; tm, triplet of multiplets. Preparative medium-pressure liquid
chromatography (MPLC) was performed with a pump module C-601 and a pump controller C-
6
10 Work-21 pump (Büchi, Flawil, Switzerland) and was carried out on pre-packed column
cartridges (40 mm × 75 mm), Silica-gel 60, particle size distribution 40–63 µm, Büchi (Flawil,
Switzerland). Precoated Al silica gel plates (Kieselgel 60 F , 0.2 mm, Merck, Darmstadt,
2
54
Germany) were used for analytical thin layer chromatography (TLC) analyses. The spots on TLC
plates were visualized by UV light (254 nm) and by spraying with vanillin-sulfuric acid reagent
(
6% vanillin (w/v) and 1% H SO (v/v) in ethanol) followed by a short gentle heating.
2 4
2
.3 Plant material
8

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Varying gram quantities of fresh plant material, and at least one whole individual plant, of all
Brassicacae and Resedaceae taxa screened for the presence of 1,3-oxazoline-2-thiones, given in
Supplementary data, Table S1, was collected in eastern and south-eastern Serbia, in the period of
2
007-2017. Appropriate voucher specimens were deposited with the Herbarium Collection of the
Faculty of Science and Mathematics, University of Niš. Identities of the taxa were confirmed by
the Herbarium curator. Several batches of 100 g each of Draba lasiocarpa Rochel (Brassicaceae)
and Reseda luteola L. (Resedaceae) were collected on the slopes of Suva planina mountain, in
the vicinity of Niš, Serbia in February 2016 and June 2014, respectively (exact locations: D.
lasiocarpa 43°17’N 22°04’E; R. luteola 43°15’N 22°06’E). Voucher specimens were deposited
with the same Herbarium, under the accession numbers MT2016-1 and MT2014-7. The identity
of the plant material was initially determined by one of the authors (N.R.) and corroborated by
the mentioned curator.
2
.4 Glucosinolate autolysis - Isolation of hydrolysis products
Autolysis experiments (three repetitions) were performed using the homogenized fresh material
underground parts, aerial parts, leaves, inflorescence, silique/silicle) which was mixed with a
sufficient quantity of distilled water to make a paste (Radulović et al., 2008). Diethyl ether
sufficient to make a visible layer) was added to each sample, the all-glass vessels sealed, and the
(
(
flasks placed in an incubator shaker set at 25 °C and 200 rpm for 8 h. Following hydrolysis,
sodium chloride was added and mixed thoroughly. The ether layer was then separated from the
brine solution and filtered through a Whatman No. 1 filter paper and the residual plant mass was
extracted an additional three times with excess diethyl ether. The combined crude ether extracts
were dried over anhydrous magnesium sulfate, and evaporated to dryness on a rotary evaporator
9

ACCEPTED MANUSCRIPT
at 30 °C. The obtained autolyzates were either without delay subjected to GC-MS analyses, or
kept at -20 °C until analyses. In the case of the aerial parts of D. lasiocarpa and R. luteola (three
batches of 80-g samples) were treated in an analogous way to yield the following amounts of
autolyzates: D. lasiocarpa–360 mg (0.15%); R. luteola–816 mg (0.34%).
2
.5 Isolation of 5,5-dimethyl-1,3-oxazolidine-2-thione (1) and (R)-5-phenyl-1,3-oxazolidine-2-
thione ((-)2)
Hydrolysis products obtained from D. lasiocarpa and R. luteola were chromatographed on silica
gel (MPLC) by gradient elution with hexane/ethyl acetate (from 90:10 to 0:100, v/v).
Compounds 1 (75 mg, obtained from 360 mg of D. lasiocarpa autolyzate) and (-)2 (266 mg from
8
16 mg of R. luteola autolyzate) were isolated in pure form (according to TLC, NMR and
elemental analysis).
3
-1
-1
3
-1
-1
-
Compound 1: UV (nm): 244.5 (ε = 2484 dm mol cm ), 195 (ε = 5944 dm mol cm ), IR (cm
1
): 3180, 2980, 1690, 1525, m.p. 102.2 °C (lit. 106 °C (Leoni et al., 1999)), retention index
(DB5) = 1471, EI-MS m/z (rel. intensity, %): 41 (11), 43 (13), 55 (16), 56 (42), 70 (12), 73 (13),
+
8
8 (4), 98 (3), 116 (16), 131 (100, M ), 132 (7). Elemental analysis: calculated for C
5
H
9
NOS
(%): C, 45.77; H, 6.91; N, 10.68. Found: C, 45.83; H, 6.89; N, 10.66.
3
-1
-1
20
-1
Compound (-)2: UV (nm): 246.5 (ε = 4925 dm mol cm ). α
= -62° (c = 0.5 mg ml ,
D
-1
MeOH), -68° (c = 1 mg ml , CHCl ) (lit. -69.3° ± 1.5° (c 2.0, MeOH, Kjaer and Gmelin, 1958),
3
-1
-
54.1° (c 0.22, CHCl , Seo et al., 1999), -51°, (c 1.0, CHCl , Leoni et al., 1999), IR (cm ): 3150,
3
3
1
1
520, 1155, m.p. 116.6 °C (lit. 126-127 °C (Kjaer and Gmelin, 1958), 116 °C (Leoni et al.,
999), 124-125 °C (Underhill, 1965)), retention index (DB5) = 2068, EI-MS m/z (rel. intensity,
1
0

ACCEPTED MANUSCRIPT
%): 39 (7), 45 (5), 50 (7), 51 (18), 63 (10), 65 (10), 77 (30), 79 (15), 91 (63), 92 (30), 104 (16),
+
1
07 (29), 118 (59), 119 (23), 123 (29), 135 (15), 136 (13), 146 (2), 147 (1), 179 (100, M ), 180
(
10). Elemental analysis: calculated for C H NOS (%): C, 60.31; H, 5.06; N, 7.81. Found: C,
9 9
6
0.21; H, 5.09; N, 7.83.
Compound 3: retention index (DB5) = 1919, EI-MS m/z (rel. intensity, %): 39 (8), 45 (12), 50
(6), 51 (15), 63 (10), 65 (11), 77 (21), 78 (16), 91 (40), 103 (10), 104 (7), 117 (7), 121 (66), 122
+
(
73), 123 (100), 135 (22), 136 (17), 179 (M , 83), 180 (14).
Compound 4: retention index (DB5) = 1775, EI-MS m/z (rel. intensity, %): 39 (4), 50 (4), 51
(10), 63 (5), 65 (5), 77 (17), 79 (36), 90 (5), 91 (16), 105 (13), 107 (100), 108 (13), 118 (12), 119
+
(
3), 163 (M , 20), 164 (3).
For the NMR data of compounds 1, (-)2, 3 and 4, see Results and Discussion (chemical shifts are
listed in Tables 1 and 2; carbon numbering can be found in Scheme 1).
2
.6 Synthesis of 5,5-dimethyl-1,3-oxazolidine-2-thione (1) and rac-5-phenyl-1,3-oxazolidine-2-
thione ((±)2)
Compounds 1 and racemic 5-phenyl-1,3-oxazolidine-2-thione ((±)2) were prepared according to
the procedure by Radulović et al. (2011).
Compound 1: Mixture of 1-amino-2-methyl-2-propanol (113.8 mg, 1.28 mmol), triethylamine
(177µl, 1.28 mmol) and carbon disulfide (115 µl, 1.90 mmol) was stirred in methanol (2 ml) for
1
20 min at 0 °C and 60 min at room temperature. A solution of H 12% (w/w; 900 µl, 3.175
2 2
O
mmol) was added and the obtained cloudy solution was cooled to 0 °C. One drop of conc. HCl
was added to the solution, the supernatant was removed and methanol was evaporated on a
rotovap. The obtained residue was taken in 50 ml of EtOAc and washed with saline. After drying
1
1

ACCEPTED MANUSCRIPT
with anhydrous MgSO
4
and removal of the solvent, 97.8 mg of white crystalline substance was
obtained. The product was purified on a silica gel column (EtOAc:hexane, 1:1 (v/v), R = 0.3)
f
and 67.9 mg (yield: 40.4%) of pure compound 1 was isolated. Spectral data completely agreed
with the data for compound 1 from D. lasiocarpa.
Compound (±)2, rac-5-phenyl-1,3-oxazolidine-2-thione, was prepared in an analogous manner.
The starting reagents used were: (±)-2-amino-1-phenylethanol (200.0 mg, 1.46 mmol),
triethylamine (202 µl, 1.46 mmol) and carbon disulfide (129 µl, 2.14 mmol) in 4 ml of methanol.
After chromatography (EtOAc:hexane, 1:3 (v/v), R = 0.12,145), 60 mg (23% yield) off-white
f
crystalline (±)2 was isolated. M.p. 126.0 °C (lit. 140°C (Kjaer and Gmelin, 1957)). NMR spectra
were identical to the ones for the (R)-enantiomer.
1
2
.7 Separation of H NMR signals of enantiomers using a chiral lanthanide shift reagent
Firstly, the possibility of enantiomer differentiation of compound 2 by an NMR titration of the
synthetic racemic product ((±)2) with a chiral lanthanide shift reagent (europium(III) tris[3-
(
(
(
3
heptafluoropropylhydroxymethylene)-d-camphorate], Eu(hfc) ) was considered. Compound 2
10 mg) was dissolved in 0.7 ml of C
6
D
6
and standard acquisition parameters were loaded
1
number of points in the time domain=32k, spectral width=10 ppm, O1=6.0 ppm, p1=45° H
transmitter pulse, acquisition time=5s and number of scans=16). After the proton spectrum of 2
was recorded, an exact quantity (2-4 mg) of Eu(hfc) was added and the proton spectrum was run
3
again. Concentration of the shift reagent was incrementally increased and this was accompanied
by a gradual resolution of the signals of H-4a and H-4b protons originating from the two
enantiomers (now diastereomeric complexes; see the Results section 3.4.); the molar ratio of
Eu(hfc) to compound (±)2 was varied from 0 to 0.23 during the course of the experiment.
3
1
2

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Enantiomeric excess was calculated from the ratio of signal integral of H-4a(S) or H-4b(S) and
1
one half of the integral of H-4a+b(R) (see Figure 3, A5) from H shifted spectra for which the
molar ratio of Eu(hfc) : 2 led to the best resolution of the signals. Afterwards, the enantiomeric
3
purity/excess of an underivatized sample of compound 2 isolated from R. luteola was determined
in an analogous manner.
2
.8 Thermal isomerization of compound (-)2
Compound (-)2 (30 mg) was dissolved in 0.5 ml of CD
3
3
SOCD and the solution was heated in a
sealed all-glass vessel for 10 minutes at 190 °C. The obtained solution was analyzed (using
NMR and GC-MS) without any previous work-up and compounds 2-4 were identified. The ratio
1
(
calculated from the relative integral values of H-5 in H NMR) of the mentioned compounds
expressed as percentage was as follows: compound 2: 35%; 5-phenyl-1,3-thiazolidine-2-one (3):
3%; 5-phenyl-1,3-oxazolidine-2-one (4): 22%. The spectral data of the identified compounds
are given below in Tables 1 and 2. GC-retention data are given above.
4
2
.9 Evaluation of acute toxicity against Artemia salina L. (Brine shrimp)
Brine shrimps (Artemia salina) were hatched from cysts in a conical shaped vessel (1 l), filled
with artificial seawater (prepared according to reference (Berges et al., 2001)) under constant
aeration for 48 h and termostated at 25 °C. Most of the cysts hatched after approximately 48 h.
Twenty nauplii were drawn through a glass capillary and placed in Petri dishes containing the
tested compounds dissolved in CH SOCH and diluted with artificial seawater (20 ml in total).
3
3
-1
Final concentrations of the tested samples were in the range 0.0125–0.05 mg ml , whereas the
final concentration of CH SOCH was much less than 1% (v/v). CH SOCH was inactive under
3
3
3
3
1
3

ACCEPTED MANUSCRIPT
the stated conditions as demonstrated by a negative control. Sodium dodecyl sulfate served as the
positive control. Brine shrimps were not fed during the test. The tested samples were not aerated
and the test dishes were left at room temperature, under constant illumination. Dead nauplii were
counted after 24 and 48 h. All tests were performed in duplicate.
2
.10 Animal housing, cell isolation and cultivation
Female Wistar rats, weighting 200–250 g, were obtained from the Vivarium of the Institute of
Biomedical Research, Faculty of Medicine, University of Niš, Serbia. Animals were maintained
under the following standard laboratory conditions: temperature 22 ± 2 °C and humidity 60%,
with food and water freely available. All experimental procedures with the animals were
conducted in compliance with the declaration of Helsinki and European Community guidelines
for the ethical handling of laboratory animals (EU Directive of 2010; 2010/63/EU) and were also
approved by the local Ethics Committee (No. 323-07-06862/2016-05/2). Elicited peritoneal
macrophages (Mφs) were isolated following previously described standard procedures
(Stojanović et al., 2016). The isolated cells were counted after viable staining (trypan blue) and
6
the density of the cell suspension was set to 2.5×10 viable cells per ml, where more than 95% of
cells were viable.
2
.11 Cell viability determination
Mφs were allowed to adhere to 96-well plates for 1.5 h, after which fresh RPMI medium
-8
-6
-3
containing different concentrations of cisplatin (4×10 –7×10 mol dm ), compounds 1, (-)2 or
-8
-4
-3
(
±)2 (10 –10 mol dm ) was added and further incubated for 24 h at 37 °C under 5% (v/v) CO
2
atmosphere. After the incubation period, well contents were aspirated and MTT reagent (5 mg
1
4

ACCEPTED MANUSCRIPT
-1
ml ) was added to each well. The formed formazan crystals, 4 h later, were dissolved in
acidified isopropanol and absorbance of each well was read at 540 nm (Multiscan Ascent,
Labsystems, Finland). Additionally, in separate plates, lactate dehydrogenase (LDH) leakage into
the medium was measured after a 24-h incubation period, following standard protocols (Chan et
al., 2013). The results were expressed as % of cytotoxicity based on both formazan formation
and LDH leakage. All experiments were done in triplicate and repeated three times.
Macrophage lysosomal function
Neutral red (NR) assay was performed as described previously by Radulović et al. (2016).
Briefly, after 24 h of incubation with the test compounds, the medium was removed, cells
washed and incubated with NR. Two hours later excess dye was removed and a destaining
solution (ethanol : water : acetic acid = 5:5:1, v/v/v) was added. The amount of the extracted dye
was measured at 540 nm. The obtained results were presented as % of activity relative to RPMI
medium cultured cells.
2
.12 Microscopic examination of Mφs
After 24 h of incubation of Mφs with compounds 1, (-)2, and (±)2, and cisplatin, the cells were
observed using a light microscope Zeiss Z1 AxioObserver (Carl Zeiss, Göttingen, Germany),
with optical magnifications of 100 and 200. The number, shape, morphological characteristics of
Mφs, and the presence of apoptotic cells/bodies in the cultures were evaluated.
2
.13 Estimation of macrophage function
Adherence ability of isolated macrophages
1
5

ACCEPTED MANUSCRIPT
The adherence of isolated Mφs under the influence of compounds 1, (-)2, and (±)2, and cisplatin
was evaluated using methylene blue-staining protocol (Oliver et al., 1989). Non-adhered Mφs
were treated for 1.5 h with the compounds in the abovementioned concentrations. After this
period, methanol was used for cell fixation, afterwards the cells were stained for 30 min with 1%
-3
(
w/v) methylene blue solution in 0.01 mol dm borate buffer (pH 8.5). Excess dye was removed
by a subsequent wash with borate buffer while the cell-retained dye was eluted using ethanol and
-3
0
.1 mol dm HCl mixture (1:1 (v/v)), and the absorbance of the eluates from each well was
recorded at 620 nm.
2
.14 NO production in cultures of isolated Mφs
6
Macrophages (1×10 cells per ml) were incubated with the abovementioned concentrations of
-4
-3
-1
compounds 1, (-)2, or (±)2, and cisplatin (1×10 mol dm ) in the presence of 1 µg ml of LPS
for 24 h. Subsequently, NO concentration in supernatants was estimated based on the formed
-
NO , using the standard Griess reaction (Radulović et al., 2015). The absorbance was measured
2
at 540 nm using the abovementioned microplate reader (section 2.10.) and the nitrite
concentrations were calculated using a standard curve of sodium nitrite.
+
2
.15 In chemico scavenging of NO
+
The Griess reaction was used to assess the NO scavenging ability of compounds 1 and (±)2, i.e.
+
their potential direct reaction with NO . While keeping the concentration of nitrite constant
-
5
-3
+
(
4×10 mol dm ), as a source of NO , the reactions were performed by varying concentrations
of the two compounds. The reactions were initiated by mixing a solution of phosphoric acid 6%
w/w) and the abovementioned nitrite-test compound solutions, and diluted to a constant volume.
(
1
6

ACCEPTED MANUSCRIPT
The course of the reaction was monitored using the UV-1800 Shimadzu spectrophotometer
(
Tokyo, Japan).
A direct reaction between 0.167 mmol (30 mg) of (±)-5-phenyl-1,3-oxazolidine-2-thione, (±)2,
suspended in 5 ml of 6% (w/w) aqueous phosphoric acid, and a stoichiometric amount of NaNO
0.167 mmol, 11.5 mg in 1 ml of water), added dropwise, was conducted under stirring at room
temperature for 30 min. During the addition of the nitrite solution a short complete dissolution of
±)2 took place, followed by a precipitation of a new white solid. The progress of the reaction
was monitored by thin layer chromatography. Using CHCl as the eluent, (±)2 gave a spot at Rf
.5, while the reaction mixture after 30 min and the newly formed precipitate soluble in CHCl3,
showed no (±)2, but another spot at Rf 0.1.
2
(
(
3
0
2
.16 Statistical analysis
The results of biological assays were expressed as the mean ± S.D. Statistically significant
differences were determined by one-way analysis of variance (One-Way ANOVA) followed by
Tukey's post hoc test for multiple comparisons (Graphpad Prism version 5.03, San Diego, CA,
USA). Probability values (p) less than 0.05 were considered statistically significant.
3
3
. Results and Discussion
.1 Occurrence of 5,5-dimethyl-1,3-oxazolidine-2-thione and 5-phenyl-1,3-oxazolidine-2-thione
in autolyzates of Serbian Brassicaceae and Resedaceae
1
7

ACCEPTED MANUSCRIPT
Plant material of taxa belonging to Brassicaceae and Resedaceae families from Serbia, collected
during a 10-year period (2007-2017), yielded 159 autolyzate samples from in total 78 different
plant taxa (see Supplementary data, Table S1). All autolyzate samples were initially screened for
the presence of glucosinolate-related autolysis products by GC-MS. Mass spectra of several
constituents suggested the presence of heterocyclic products of glucosinolate breakdown, i.e.
derivatives of 1,3-oxazolidine-2-thione. However, mass spectra and available literature data on
the retention behavior of 1,3-oxazolidine-2-thiones did not permit us to positively identify them,
i.e. we were unable to differentiate neither 1,3-oxazolidine-2-thiones from 1,3-thiazolidine-2-
ones, nor isomeric 1,3-oxazolidine-2-thiones among themselves.
Autolyzates of two taxa, specifically Draba lasiocarpa and Reseda luteola, appeared to us to be
convenient sources of two different 1,3-oxazolidine-2-thione derivatives. These were utilized to
isolate pure samples of the constituents in question, whose identity was subsequently inferred by
other (spectral and chemical) means. Details on the structural elucidation and problems
encountered during their analyses are given in the following sections. It turned out that D.
lasiocarpa yielded 5,5-dimethyl-1,3-oxazolidine-2-thione (compound 1), while 5-phenyl-1,3-
oxazolidine-2-thione (compound 2) was isolated from autolyzates of R. luteola. These pure
samples and additional synthetic ones (see below) were used as reference compounds in the
screening of the remaining Serbian Brassicaceae and Resedaceae. Autolyzates of only a limited
number of taxa contained the two mentioned 1,3-oxazolidine-2-thiones. Besides D. lasiocarpa,
5
,5-dimethyl-1,3-oxazolidine-2-thione was detected in autolyzates of 4 additional Brassicaceae
species: Conringia austriaca (Jacq.) Sweet, Conringia orientalis (L.) Dumort., Erophila verna
L.) DC. and Thlaspi praecox Wulfen. 5-Phenyl-1,3-oxazolidine-2-thione was a constituent of
(
1
8

ACCEPTED MANUSCRIPT
autolyzates of another Reseda sp. (Reseda phyteuma L.) and one Brassicaceae taxon (Nasturtium
officinale R.Br.).
Most compounds of natural derivation containing 1,3-oxazolidine-2-thione moiety were first
reported by Kjaer and co-workers in the 1950s and 1960s and, in the latter years, these
compounds were proven to be anti-nutritional, antibiotic, thyreotoxic and enzyme-inhibiting
(
Felker et al., 2016; Seo et al., 1999). In spite of Draba L. being the largest genus of the
Brassicaceae family (Jordon-Thaden et al., 2010), glucosinolate-derived volatiles of D.
lasiocarpa have never been investigated previously. In this paper, for the first time, we report the
presence of 5,5-dimethyl-1,3-oxazolidine-2-thione (1), hydrolysis product of glucoconringiin
(
Kjaer et al., 1956), from the underground parts, leaves, inflorescence and silique of this species.
Conringia orientalis is known to produce the mentioned 5,5-dimethyl-1,3-oxazolidine-2-thione
Fahey et al., 2001), while for the three remaining species listed above the current work
(
represents the first report. It is interesting to note that among the six analyzed samples of E.
verna, including the typical subspecies and ssp. praecox, only one sample contained compound 1
and only ssp. verna. Compound 1 was detected in T. praecox, but it was detected only in the
autolyzate of the underground parts, while the aboveground counterparts were devoid of this
compound. Whereas E. verna and T. praecox possessed relatively low amounts of 1, in
comparison to other present glucosinolate-related products, C. austriaca and D. lasiocarpa
yielded autolyzates rich in 1.
Despite R. luteola being a prolific source of compound 2, R. phyteuma furnished autolyzates
with only trace amounts of 2, more specifically the roots of R. phyteuma; on the other hand, the
flowers and leaves and stems showed no presence of 2. Among the analyzed samples herein, the
only Brassicaceae taxon that was shown to produce 2 was N. officinale, albeit in low amounts
1
9

ACCEPTED MANUSCRIPT
with respect to other volatiles. The specific plant organ distribution, i.e. the frequent root-
association of both 1 and 2, indicates a protecting role of these 1,3-oxazolidin-2-thione
derivatives, since the underground parts are most exposed to pathogen attack. It is not clear,
though, whether they represent a general, broad spectrum line of defense, resembling most
isothiocyanates (Radulović et al., 2012), or a specialized one.
Chemical profiles of families within Brassicales showed a homogeneous production of
secondary metabolites, among which, glucosinolates proved to be useful (chemical) markers
(
Leite and Castilho, 2017). Most of the major clades within the Brassicaceae family are
recognized at the tribal level (Al-Shehbaz et al., 2006). Tribe Arabideae DC. consists of at least
six genera, including Draba L. and Erophila DC, among others. The tribe Thlaspideae DC.
comprises 7 genera, including Thlaspi L. The genus Conringia Heist. ex Fabr. was once included
in Brassiceae DC., but recent studies suggest their exclusion from this tribe (Al-Shehbaz et al.,
2
006). Although the herein investigated plant taxa displayed a great variability for the
biosynthesis of 1,3-oxazolidine-2-thione producing glucosinolates, it is possible to conclude that
,3-oxazolidine-2-thiones could be of taxonomic significance: the genera Draba L. and Erophila
1
DC, belong to the same tribe Arabideae DC. In addition, E. verna was previously known as
Draba verna, and its generic placement still remains ambiguous (Jordon-Thaden et al., 2010).
It should be noted that despite being chemically related, compounds 1 and 2, most probably, do
not share the same biosynthetic derivation, i.e. the necessary β-hydroxyls to the NCS-group have
differing origins. In the case of 2, an enzyme introduces this OH in the benzylic position, while
the tertiary alcohol in the isothiocyanate precursor of 1 is likely predetermined by the amino acid
metabolism. Thus, one should not expect compounds 1 and 2 to have any additional relationships
(with taxonomic implications) other than the obvious glucosinolate one. This appears true since
2
0

ACCEPTED MANUSCRIPT
only one taxon (Brassica juncea, Kim et al., 2016) has been reported to yield both 1 and 2;
however, even this co-occurrence was deemed questionable by the authors of that work.
Compound 1 seems to be plant family non-specific within Brassicales (Supplementary data,
Table S2), while compound 2 was detected only in Resedaceae and Brassicaceae (Supplementary
data, Table S2).
3
.2 Structural elucidation of compounds 1-4
Due to a high level of similarity between the mass spectra of isomeric derivatives of 1,3-
thiazolidin-2-one and 1,3-oxazolidine-2-thione, and the unavailability of reliable gas
chromatographic data for the isomers, we had to turn to other spectral techniques to differentiate
one from the others. NMR appeared to be suitable, but requires pure samples of the compounds
in question. We opted for both chromatographic separation of the compounds from the
autolyzates and a synthetic approach. Synthesis would provide an access to different isomers and
an unambiguous distinction between them. For example, an alleged 5-phenyl-1,3-oxazolidine-2-
thione, identified solely by mass spectral comparison, could in fact be either 4-phenyl-1,3-
oxazolidine-2-thione, or both 4- and 5-phenyl-1,3-thiazolidin-2-ones. To make the situation even
more complicated all of these isomers possess spectra that are of analogous multiplicities
differing only in chemical shifts. Thus, it would be of great convenience to have at hand all of
these for the sake of comparison with the spectra of the isolated natural compounds (after SiO2
column chromatography).
We started the synthetic work by preparing the regioisomeric phenyl-1,3-oxazolidine-2-thiones
from the corresponding commercial racemic amino alcohols. Later on, during the experiments
we serendipitously acquired a sample of 5-phenyl-1,3-thiazolidin-2-one (vide infra). A full NMR
2
1

ACCEPTED MANUSCRIPT
characterization facilitated by 2D experiments was accomplished in a straightforward manner
1
13
and the assignment of all H and C NMR resonances is presented in Tables 1 and 2,
respectively, while the numeration scheme and selected HMBC and NOESY interactions are
given in Figure 1. A perfect match between the spectral data of the isolated compound and one of
the synthesized isomers reasoned that all of the corresponding autolyzates contained 5-phenyl-
1
5
,3-oxazolidine-2-thione. Similarly, the compound, that was later unequivocally identified as
,5-dimethyl-1,3-oxazolidine-2-thione, could have been mistaken for a much larger number of
possible isomers (the analogous ones as for compound 2, and additional ones containing an ethyl
group, or two vicinal methyls, or even with a six-membered ring, etc.). Based on the NMR of the
isolated compound and by analogy with the phenyl counterpart, we also synthesized 5,5-
dimethyl-1,3-oxazolidine-2-thiones to remove any doubt from this tentative structural
assignation. For the purpose of providing as much relevant data as possible in one place, the
NMR spectra (Tables 1 and 2) of the two naturally occurring compounds were recorded in the
three most frequently utilized deuterated solvents (CDCl , CD SOCD and C D ). If in the
3
3
3
6
6
possession of samples containing sufficient amounts of 1,3-oxazolidine-2-thiones, based on
Tables 1 and 2, the most straightforward way to differentiate them from other isomeric
1
3
heterocycles is the existence of C NMR signals at ca. δ 189.
1
13
We noted that there are no published data on the assignment of individual H and C NMR
resonances corresponding to the phenyl group of 5-phenyl-1,3-oxazolidine-2-thione, hence, a
simulation of the phenyl group was performed and thus the identity of each individual atom
definitively confirmed. For the details on the structural elucidation, see Tables 1 and 2 and
Supplementary data.
2
2

ACCEPTED MANUSCRIPT
Figure 1. Carbon numbering scheme and selected HMBC and NOESY correlations of 1, (-)2, 3 and 4.
1
Table 1. H NMR data (at 400 MHz, recorded in CDCl
3
, CD
3
SOCD
3
and C
6
D
6
) for compounds
1
-4.
1
-CDCl
3
1-CD
3
SOCD
3
2-CDCl
3
2-C
6
D
6
2-CD
3
SOCD
3
3-CD
3
SOCD
3
3 3
4-CD SOCD
Position
NH
δH, m (J, Hz)
8.10, br s
3.54, s
δH, m (J, Hz)
9.79, br s
3.41, s
δH, m (J, Hz) δH, m (J, Hz) δH, m (J, Hz)
δH, m (J, Hz)
8.21, s
3.54, dd
δH, m (J, Hz)
7.70, br s
3.33, dd
7.79, br s
3.75, ddd
7.40, br s
2.58, dd
10.11, br s
3.60, dd
4
a
*
J
J
J
4b,4a = -9.7
4b,5 =8.1
4b,NH = 0.8
J
J
4b,4a = - 9.7
4a,5 = 7.98
J
4b,4a = -10.4
4b,5 =7.7
J
4b,4a = -10.5
4b,5 = 7.5
4b,NH = 1.2
J
4b,4a = -9.1
J
J
J4b,5 =7.6
J
4
b
**
-
-
-
-
4.16, ddd
2.77, ps t
4b,4a = - 9.7
4.10, dd
4a,4b =-10.4
3.84, ddd
4a,4b = -10.5
3.88, ps t
4a,4b,5 ≈ 8.80
J
J
J
4a,4b = -9.7
4a,5 = 9.3
4a,NH = 0.8
J
J
J
J
J
4a,5 = 9.4
J
4a,5 =7.4
J4a,NH = 1.2
5.16, br ps t
5
5.89, br ps t
4.90, dd
5.92, br dd
5.59, br dd
J
J
5,4a = 9.3
5,4b = 8.1
5,2´/6 = 0.60
5,3´/5´ = 0.25
J
5,4a = 9.1
J
J
5,4a = 7.7
5,4b = 9.4
J
5,4a,4b ≈ 7.4
J
5,4a = 7.6
J5,4b = 8.1
J5,4b = 8.3
J
J
J5,2´/6´ = 0.5
J
5,4´ = 0.05
1
´, 6´
´
1.56, s (2xCH
-
3
)
1.40, s (2xCH
-
3
)
-
-
-
-
2
3
7.38, m***
≈6.93, m
(6.8-7.0)
≈7.40, m**** ≈7.47, m*****
≈7.45, m**** ≈7.37, m*****
≈7.43, m**** ≈7.40, m*****
≈7.38, m
≈7.39, m
≈7.40, m
J
J
J
J
2´,3´ = 8.05
2´,4´ = 1.30
2´,5´ = 0.60
2´,6´ = 2.00
2´,5 = 0.60
7.43, m***
3´,2´ = 8.05
3´,4´ = 7.55
3´,5´ = 1.20
3´,6´ = 0.60
3´,5 = 0.25
7.41, m***
J
´, 5´
-
-
-
-
J
J
J
J
J
4
´
J
J
J
J
4´,2´ = 1.30
4´,3´ = 7.55
4´,5´ = 7.55
4´,6´ = 1.30
J
4´,5 = 0.05
*
Hydrogen atom “cis” with respect to the phenyl group. Determined from Eu(hfc) titration experiment for 2 in
3
C D . The assignment of the rest of the signals 4-H (in different solvents) was made by analogy with 2 in C D .
6
6
6
6
*
*Hydrogen atom “trans” with respect to the phenyl group. Determined from Eu(hfc) titration experiment for 2 in
3
C D . The assignment of the rest of the signals 4-H (in different solvents) was made by analogy with 2 in C D .
6
6
6
6
2
3

ACCEPTED MANUSCRIPT
*
**Overlapping signals: 7.33-7.48, 5H; the values of coupling constants and chemical shifts were determined by a
1
(
iterative full spin analysis) simulation of H NMR spectrum of this compound.
*
*
***Overlapping signals: 7.37-7.48, 5H; approximate centers of the signals were inferred from HSQC spectrum.
****Overlapping signals: 7.37-7.48, 5H; approximate centers of the signals were inferred from HSQC spectrum.
1
3
Table 2. C NMR data (at 100.6 MHz, recorded in CDCl , CD SOCD and C D ) for
3
3
3
6
6
compounds 1-4.
1
-CDCl
3
1-CD
3
SOCD
3
2-CDCl
3
2-C
6
D
6
2-CD
3
SOCD
3
3-CD
3
SOCD
3
3 3
4-CD SOCD
Position
2
δC, m (J, Hz)
δC, m (J, Hz) δC, m (J, Hz) δC, m (J, Hz) δC, m (J, Hz)
δC, m (J, Hz)
δC, m (J, Hz)
188.8, dt
187.2, m
189.8, m
190.0
50.7
187.8, m
172.6
158.8
2
J
J
CH = 5.9
CH = 3.2
3
4
5
55.6, t ps oct
54.7, t
51.4, ps td
50.5, ps tm
50.1
47.5
1
1
1
1
1
J
CH = 145.0
J
CH = 145.1
JCH = 147.3
J
CH = 146.9
3
2
3
2
J
CH = JCH = 4.4
J
CH = 4.2
J
CH = 3.8
88.7, m
87.1, m
84.1, br d
83.2
82.4, dm
49.0
76.4
1
J
CH = 156.1
J
CH = 156.8
1
´
26.9, q ps sext
26.4 q ps sext
137.1, m
138.0
138.2, m
140.0
139.6
1
1
J
CH = 127.9
JCH = 127.4
3
3
J
CH = 3.7
-
J
CH = 4.0
-
2
3
´, 6´
´, 5´
126.2, dm
126.1
128.9
126.3, dm
JCH = 158.4
128.9, br dd
127.4
128.0
125.9
128.7
1
1
J
CH = 158.4
-
-
-
-
129.2, br dd
1
1
J
CH = 161.9
JCH = 161.1
3
3
J
CH = 7.3
J
CH = 6.9
129.0, br dt
1
4
´
129.6, br dt
129.0
128.7
128.9
1
J
CH = 161.2
JCH = 161.4
3
3
J
CH = 7.1
JCH = 6.9
3
.3 Isomerization
Another important, analytically challenging aspect of 2 was encountered during the GC-MS
analyses of samples containing this compound, and even of its pure samples. The unexpected
occurrence of two, interconnected by an elevated base line, dominant peaks in the TIC
chromatograms was noted (Figure 2 and Supplementary data). Both peaks displayed the same
+
m/z values of M and differed only in the intensities of certain fragment ions. This led us to an
assumption that elevated temperatures during GC-MS, in one or more of the three heated zones,
injection port, column and transfer line, cause a thermal isomerization of this compound (one
should note that this was not observed in the case of compound 1). To investigate this, we
decided to undertake a preparative thermolysis and analyze the contents directly (without prior
1
13
separation) by NMR. H, C and 2D NMR spectra (selected 2D spectra are reproduced on
Figure S29, Supplementary data), unequivocally allowed us to identify the major isomerization
2
4

ACCEPTED MANUSCRIPT
product as 5-phenyl-1,3-thiazolidine-2-one (compound 3). This is an, up to now, unreported
thermal isomerization of the 1,3-oxazolidine-2-thione core to the 1,3-thiazolidine-2-one core
(
Figure 2). In addition to signals from this compound, the spectra contained signals originating
from the starting 5-phenyl-1,3-oxazolidine-2-thione, and another unexpected, minor product: 5-
phenyl-1,3-oxazolidine-2-one (compound 4) (probably formed as a consequence of the presence
of traces of moisture in CH SOCH ). It appears that the newly encountered thermal
3
3
isomerization could take place via a free radical mechanism (Figure 2), mediated by a stable
benzylic radical (cf. the inertness of compound 1 under these conditions, due to lower stability of
a possible tertiary radical) which recombines into the thermodynamically more stable compound
3
containing a C=O group.
Such isomerization, i.e. the artefactual formation of 1,3-thiazolidine-2-one, and possibly 1,3-
oxazolidine-2-one derivatives, only additionally complicates the 1,3-oxazolidine-2-thione
identifying process. A reanalysis of all chromatograms showed that compounds 2-4 could be
detected in all of the three above mentioned taxa in widely varying ratios suggesting that the
remainder of the samples or the current setup of the chromatograph influenced the extent of the
isomerization, and desulphurization. In addition, this makes gas chromatographic techniques
inadequate for the quantification of this particular 1,3-oxazolidine-2-thione derivative, while still
it can be used for its detection. Thus, all samples containing 5-phenyl-1,3-oxazolidine-2-thione
will display TIC chromatograms with two or three peaks related to this analyte at RIs (DB5):
1
775, 1919, and 2068, corresponding to compounds 4, 3, and 2, respectively, with the last two
peaks having very similar mass spectra. Compound 1 showed good GC behavior and excellent
concentration-peak area dependence with both FID and MS detectors.
2
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Figure 2. Thermal isomerization of 1,3-oxazolidine-2-thione core to 1,3-thiazolidine-2-one core, the
formation of 1,3-oxazolidine-2-one (a), and the proposed mechanism of this transformation (b).
3
.4 NMR determination of enantiomeric composition of R. luteola autolyzate
The isolated 5-phenyl-1,3-oxazolidine-2-one from the autolyzate of R. luteola displayed a
o
o
specific optical rotation of -62 (methanol, c = 0.5) and -68 (chloroform, c = 1) which generally
corresponded to the optical rotation of the (R)-(-)-enantiomer of the compound (Kjaer and
Gmelin, 1958; Leoni et al., 1999; Seo et al., 1999). However, the value in methanol was lower
than the reported one (Kjaer and Gmelin, 1958; suggesting that we might have an impure
sample), and the one in chloroform was higher that the both previously published ones (Leoni et
al., 1999; Seo et al., 1999; suggesting that on both preceding instances the investigators dealt
with less enantiomerically pure samples). This compound is known to naturally occur
predominantly as the mentioned R-enantiomer originating from (S)-glucobarbarin; however,
recently epiglucobarbarin ((R)-barbarin) was published to be the dominant glucosinolate in some
samples of watercress (Nastutium officinale L., Brassicaceae) (Agerbirk et al., 2014). This
motivated us to further investigate the enantiomeric nature of the sample at hand. The literature
does not offer any additional previous examples of the discernment of these enantiomers.
2
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We were drawn by the easiness of an NMR titration with chiral lanthanide shift reagents (CSR)
as the means for the determination of enantiomeric excess and encouraged by the fact that we
already have a racemate that we can use for the method development. There are no previous
literature reports on chiral 1,3-oxazolidine-2-one resolution by such reagents. Thus, we supposed
that a chiral paramagnetic lanthanide complex (such as Eu(hfc) ) would bind reversibly, via a
3
heteroatom to the metal center, and form two diastereomeric complexes with proton signals
appearing at different shifts. The thiocarbonyl group is the only significant Lewis base present in
3
the molecule and we expected Eu(hfc) to coordinate to the thiocarbonyl sulfur atom.
Incremental addition of Eu(hfc) to (±)2 resulted in a marked change of δ values for all signals.
3
Separation for all non-aromatic moiety signals was observed even after the addition of the first
amount of CSR. Unfortunately, the separation of signals was not possible for aromatic protons
1
due to the occurrence of overlapping resonances in the H NMR spectra recorded without the
3
lanthanide shift reagent, as well as in the spectra after the addition of Eu(hfc) . H-4 proton
signals of the two enantiomers completely separated when the ratio nCSR/n reached 0.23 (where
S
nCSR is the amount in moles of CSR added and n is the amount of compound (±)2, A5, Figure
S
3
3
). H-4 signals from (-)2 (inferred from later experiments) again coalesced to a single peak (at δ
.30) with an integral value of 2.00, well separated from the H-4a and H-4b signals of (+)2 (sum
integral value = 2.00). Enantiomeric excess could be calculated from the ratio of signal integral
1
of H-4a(S) or H-4b(S) (from (+)2) and one half of the integral of H-4a+b(R) (from (-)2) from H
shifted spectra (Figure 3, A5). The results of these experiments clearly proved the validity of the
method, i.e. that CSR can be used to successfully separate signals of (-) and (+)2. Enantiomeric
purity of compound 2 (isolated from the autolyzate of R. luteola) was studied by utilizing the
same CSR (Eu(hfc) ), where upon close inspection no signal separation was detected. Given that
3
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only one set of signals could be observed in the sample of plant origin, we concluded that
compound 2 isolated from R. luteola indeed consisted of only one enantiomer, (-)2. Probably, the
differences in reported α could be attributed to somewhat different experimental conditions
D
(temperature and concentration) and possibly sample purity (in order of decreasing purity: Kjaer
and Gmelin (1958) > our study > Seo et al. (1999) > Leoni et al. (1999)).
Figure 3. The signal shifts for non-aromatic moiety protons after incremental additions of Eu(hfc) to (±)2
3
(
A) and (-)2 (B), recorded in C
6
D
6
(400 MHz). The molar CSR/substrate (±2) ratio was: 0 (A0 and B0),
.0028 (A1 and B1), 0.0064 (A2 and B2), 0.0111 (A3 and B3), 0.0164 (A4 and B4), 0.0232 (A5 and B5).
designates signals originating from Eu(hfc) proton resonances.
0
*
3
In order to evaluate the europium atom coordination position for both enantiomers, we created a
theoretical model of complexes of Eu(hfc) and compounds (-)2 and (+)2. Strong linear
values and molar ratio of CSR and compounds (-)2 and (+)2 was
3
correlation between δδ
H
2
observed, with R values between 0.987 and 0.998 (Figure S32). In both cases, the highest values
of the shift gradient (slope of the plot; ∆Eu) were observed for the proton nearest to the expected
2
8