Derivatization of isothiocyanates and their reactive adducts for chromatographic analysis

Article abstract of DOI:10.1016/j.phytochem.2015.06.004

Isothiocyanates form adducts with a multitude of biomolecules, and these adducts need analytical methods. Likewise, analytical methods for hydrophilic isothiocyanates are needed. We considered reaction with ammonia to form thiourea derivatives. The hydrophilic, glycosylated isothiocyanate moringin, 4-(α-l-rhamnopyranosyloxy)benzyl isothiocyanate, was efficiently derivatized to the thiourea derivative by incubation with ammonia. The hydrophobic benzyl isothiocyanate was also efficiently derivatized to the thiourea derivative. The thiourea group provided a UV absorbing chromophore, and the derivatives showed expectable sodium and hydrogen adducts in ion trap mass spectrometry and were suitable for liquid chromatography analysis. Reactive dithiocarbamate adducts constitute the major type of reactive ITC adduct expected in biological matrices. Incubation of a model dithiocarbamate with ammonia likewise resulted in conversion to the corresponding thiourea derivative, suggesting that a variety of matrix-bound reactive isothiocyanate adducts can be determined using this strategy. As an example of the application of the method, recovery of moringin and benzyl isothiocyanate applied to cabbage leaf discs was studied in simulated insect feeding assays. The majority of moringin was recovered as native isothiocyanate, but a major part of benzyl isothiocyanate was converted to reactive adducts.

Full text of DOI:10.1016/j.phytochem.2015.06.004

Phytochemistry xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Derivatization of isothiocyanates and their reactive adducts
for chromatographic analysis
b
a
c
b
Niels Agerbirk a, , Gina Rosalinda De Nicola , Carl Erik Olsen , Caroline Müller , Renato Iori
⇑
a Copenhagen Plant Science Center and Plant Biochemistry Laboratory, Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40,
1871 Frederiksberg C, Denmark
b Consiglio per la ricerca in agricoltura e l’analisi dell’economia agraria, Centro di ricerca per le colture industriali (CRA-CIN), Via di Corticella 133, 40128 Bologna, Italy
c Chemical Ecology, Bielefeld University, Universitätsstr. 25, 33615 Bielefeld, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Isothiocyanates form adducts with a multitude of biomolecules, and these adducts need analytical
methods. Likewise, analytical methods for hydrophilic isothiocyanates are needed. We considered reac-
tion with ammonia to form thiourea derivatives. The hydrophilic, glycosylated isothiocyanate moringin,
Received 8 April 2015
Received in revised form 29 May 2015
Accepted 2 June 2015
Available online xxxx
4-(a-L-rhamnopyranosyloxy)benzyl isothiocyanate, was efficiently derivatized to the thiourea derivative
by incubation with ammonia. The hydrophobic benzyl isothiocyanate was also efficiently derivatized to
the thiourea derivative. The thiourea group provided a UV absorbing chromophore, and the derivatives
showed expectable sodium and hydrogen adducts in ion trap mass spectrometry and were suitable for
liquid chromatography analysis. Reactive dithiocarbamate adducts constitute the major type of reactive
ITC adduct expected in biological matrices. Incubation of a model dithiocarbamate with ammonia
likewise resulted in conversion to the corresponding thiourea derivative, suggesting that a variety of
matrix-bound reactive isothiocyanate adducts can be determined using this strategy. As an example of
the application of the method, recovery of moringin and benzyl isothiocyanate applied to cabbage leaf
discs was studied in simulated insect feeding assays. The majority of moringin was recovered as native
isothiocyanate, but a major part of benzyl isothiocyanate was converted to reactive adducts.
Ó 2015 Elsevier Ltd. All rights reserved.
Keywords:
Isothiocyanate
Dithiocarbamate
Thiourea
Liquid chromatography
Recovery
UV-detection
Tandem mass spectrometry
MS
NMR
1. Introduction
derived from oxidative scission of disulfide bonds (Kawakashi
and Kaneko, 1985). In addition, more stable adducts with amines
Glucosinolates are characteristic constituents of plants of the
order Brassicales, including cabbages and many other wild and
cultivated plants (Agerbirk and Olsen, 2012). Upon tissue disrup-
tion, e.g. due to chewing by animals, glucosinolates are converted
to isothiocyanates (or sometimes other products) due to endoge-
nous enzymes, myrosinases (b-thioglucoside glucohydrolases;
E.C. 3.2.1.147). Isothiocyanates (ITCs) are reactive with nucle-
ophiles such as cysteine and proteins (Nakamura et al., 2009;
Brown and Hampton, 2011; Hanschen et al., 2012), and this
reactivity is thought to be a major reason for their toxicity to many
organisms (Holst and Williamson, 2004; Winde and Wittstock,
2011). Some ITC adducts are themselves reactive with
nucleophiles, and might be equally toxic (Nakamura et al., 2009;
Brown and Hampton, 2011). This heterogeneous group is
collectively called ‘‘reactive adducts’’ in the following, and would
be expected to mainly include thiol adducts and similar adducts
can be formed (Nakamura et al., 2009; Brown and Hampton,
2011), while biological adducts with alcohols are mainly known
from intramolecular reactions (Agerbirk et al., 2014; Agerbirk
and Olsen, 2015). A final type of matrix binding is non-covalent
binding of lipophilic ITCs to, e.g., serum proteins (Ji et al., 2005).
Insects are potential targets of ITCs (Winde and Wittstock,
2011). There is some uncertainty in actual levels of ITCs offered
in various published insect feeding tests either topically applied
to other leaf disks or otherwise incorporated into diet, as discussed
elsewhere (Müller et al., submitted for publication), due to the pos-
sibility of loss by evaporation and loss by chemical reaction with
matrix components. In order to be able to quantify remaining ITCs
in insect feeding assays after solvent evaporation, a non-volatile
ITC would be useful for comparison with usually tested volatile
ITCs like allyl ITC and benzyl ITC. We expected the rare class of side
chain-glycosylated glucosinolates (Kjær et al., 1979; Olsen and
Sørensen, 1979; Olsen et al., 1981; Gueyrard et al., 2000; Bennett
et al., 2003; Kim et al., 2004) to form particularly non-volatile ITCs.
⇑
Corresponding author.
E-mail address: nia@plen.ku.dk (N. Agerbirk).
A
representative glucosinolate from this structural class is
http://dx.doi.org/10.1016/j.phytochem.2015.06.004
0031-9422/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Agerbirk, N., et al. Derivatization of isothiocyanates and their reactive adducts for chromatographic analysis. Phytochem-
istry (2015), http://dx.doi.org/10.1016/j.phytochem.2015.06.004

2
N. Agerbirk et al. / Phytochemistry xxx (2015) xxx–xxx
4-(
a
-
L
-rhamnopyranosyloxy)benzylglucosinolate (1) (Kjær et al.,
but significant amounts of free ITC and reactive adducts remain on
cabbage leaf discs even after extended evaporation in simulated
feeding assays.
1979; Gueyrard et al., 2000), also known as glucomoringin
(Fig. 1). Glucomoringin occurs naturally in species of the tropical
plant genus Moringa and in at least one other plant species (De
Graaf et al., 2015; Förster et al., 2015a,b). The corresponding ITC
2. Results and discussion
is 4-(a-L-rhamnopyranosyloxy)benzyl ITC (2), with the suggested
common name moringin (Müller et al., submitted for publication).
As a representative volatile ITC, we chose benzyl ITC (3), the
natural hydrolysis product of benzylglucosinolate (2) that occurs
naturally in garden cress (Lepidium sativum), white mustard (Sina-
pis alba), maca (Lepidium meyenii) and many wild plants from the
Brassicales order (Burow et al., 2007; Agerbirk et al., 2008, 2010;
Esparza et al., 2015) (Fig. 1). Recently, benzylglucosinolate was also
transferred to a non-Brassicales, tobacco, by metabolic engineering
(Møldrup et al., 2012).
In order to test experimentally the expected higher recovery of
the hydrophilic moringin compared to benzyl ITC, and whether any
native benzyl ITC or corresponding reactive adducts would remain
after solvent evaporation, a suitable analysis method was needed.
In addition to direct analysis of the native ITC (Müller et al.,
submitted for publication), another option was the classical deriva-
tization with dilute ammonia in alcohol known to give thiourea
derivatives detectable by UV spectroscopy or paper chromatogra-
phy (e.g. Appelquist and Josefsson, 1967; Harborne, 1973; Olsen
and Sørensen, 1979). A seemingly realistic hope was to include
reactive adducts in analysis based on thiourea derivatives. In case
of a hydrophobic ITC, adaption to contemporary liquid chromatog-
raphy with MS/MS detection had been demonstrated (Ji and
Morris, 2003).
The aims of this work were (1) to examine the conversion of
ITCs and dithiocarbamates to thiourea derivatives, (2) to character-
ize and quantitate the thiourea derivatives using NMR, ion trap
MS/MS and liquid chromatography, and (3) to critically examine
the recovery of ITCs and any reactive adducts under simulated
insect feeding assay conditions. We show that both ITCs and a
dithiocarbamate are converted to thioureas at mild conditions,
and characterize the thioureas. Applying this derivatization, we
show that a non-volatile ITC including reactive adducts can be
recovered after incubation at feeding assay conditions, and that
the majority is present as the intact ITC. In contrast, the vast
majority of a volatile ITC spiked to leaf discs is lost by evaporation,
2.1. Isothiocyanates react quantitatively with ammonia to form
thiourea derivatives
Conversion of moringin to the thiourea derivative 5 under mild
conditions (5% NH3 in 80% aq. MeOH at room temperature over-
night) was tested. Analysis of the crude remnant by 1H NMR and
HPLC after evaporation of the solvent showed complete conversion
essentially to a single product with good chromatographic peak
shape in HPLC-MS and HPLC-PDA: moringin thiourea (5). The UV
spectrum of 5 showed the expected thiourea chromophore with
an absorption band near 240 nm (Appelquist and Josefsson,
1967), suitable for routine detection in HPLC-PDA.
Benzyl ITC (4) also gave essentially a single reaction product
under these derivatization conditions, benzyl thiourea (6) (Sec-
tion 3.7), which likewise showed good chromatographic peak
shape and a characteristic and useful UV absorbance band around
240 nm. In general, this adaptation of the classical ‘thiourea’
method for ITC analysis by liquid chromatography is attractive:
all ITCs can be expected to react and as the UV absorptivity of
the thiourea group will be the same, quantification is possible by
UV detection and comparison with a commercially available ITC
as standard (except if the remaining part of the molecule contains
conjugated systems with overlapping UV absorbance). Although
volatile ITCs are well suited for gas chromatographic determina-
tion, inclusion of polar, non-volatile ITCs would be possible using
the thiourea method.
The identity of moringin thiourea (5) was further confirmed by
MS/MS and NMR spectroscopy after HPLC isolation. Ion trap mass
spectra of the sodium and hydrogen adducts of 5 were characteris-
tic and in agreement with the expected structure (Fig. 2). In 1H
NMR, a sharp singlet (2H) at 4.7 ppm from the benzylic CH2-group
was observed for moringin but was absent from the spectrum of
the thiourea 5. Instead, a broad signal with three maxima between
4.2 and 4.7 ppm was observed from the benzylic CH2-group in 5,
interpreted as a result of tautomerism in the thiourea moeity with
a rate of conversion in the ‘‘NMR time scale’’. A similar signal from
the benzylic CH2 group was observed for the simpler benzyl
thiourea 6, suggesting that this signal shape is a general feature
of a monosubstituted thiourea. The chemical shift of the thiourea
carbon in 5 could not be observed in ordinary 13C NMR. Neither
could C1 (identified in HMBC), in both cases lack of detection
was probably because of their quaternary nature. The thiourea C
was expected near 180 ppm) (Lu et al., 2015). Unfortunately, at
the time of the measurement of the HMBC spectrum, the chemical
shift range was set at 175 ppm, so it is uncertain whether the
chemical shift of the thiourea C could be identified in this spectrum
by correlation with the CH2 protons, or whether the broad shape of
the latter signal would interphere. However, the presence of the
thiourea functional group was obvious from the m/z value and an
observed loss of 76 (thiourea) in MS2 (Fig. 2b + c). Indeed, loss of
thiourea in MS/MS is known for N-substituted thioureas (Ji and
Morris, 2003). Comprehensive NMR analysis provided the remain-
ing NMR spectral characteristics (Section 3.6).
Glucomoringin (1)
5’
3
2
MYR
1’
Moringin (2)
MYR
2.2. A dithiocarbamate also reacts quantitatively with ammonia to
form the thiourea derivative
Benzylglucosinolate (3)
Benzyl ITC (4)
Fig. 1. Investigated glucosinolates (benzylglucosinolate and glucomoringin) and
their enzymatic conversion to isothiocyanate products (unbalanced). The NMR-
numbering system of moringin is indicated. MYR: myrosinase.
The dithiocarbamate adduct 7 was synthesized, characterized
by NMR and ion trap MS/MS (Section 3.8) and subjected to the
Please cite this article in press as: Agerbirk, N., et al. Derivatization of isothiocyanates and their reactive adducts for chromatographic analysis. Phytochem-
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N. Agerbirk et al. / Phytochemistry xxx (2015) xxx–xxx
3
Intens.
+MS, 6.5min #705
[M+H]+
8
a (Moringin thiourea, 5)
x10
[M+Na]+
329
2
1
0
351
6
- H2S+MS2(351), 6.4min #702
x10
- ahRha
b
MS2 of
205
317
2
[ahRha+Na]+
[M+Na]+
- thiourea
1
0
169
275
7
x10
+MS2(329), 6.5min #708
- ahRha
c 107
- thiourea
cwl
cwl
MS2 of
[M+H]+
1.0
0.5
0.0
217
cwl
161
183
199
253
149
150
235
133
171
100
200
250
300
350
m/z
Fig. 2. Ion trap mass spectrometry of moringin thiourea (a–c). a: ESI mass spectrum of moringin thiourea at the HPLC-MS peak apex showing hydrogen and sodium adducts at
approximately equal intensity from NaCl doped eluent. b + c: MS2 ion trap spectra of the sodium adduct (b) and the hydrogen adduct (c). Losses of either H2S, thiourea or
anhydroRha (ahRha) from the molecular adducts explaining the formation of each fragment ion is indicated. In addition, some other fragments in panel c are explained by
consecutive water loss (cwl) and a quinone-like proton adduct.
NH3
a
2
7
UV
of 7
aq. MeOH
RT, ON
Moringin thiourea (5)
NH3
aq. MeOH
RT, ON
Benzyl thiourea (6)
Benzyl ITC (4)
NH3
6
aq. MeOH
6
b
RT, ON
UV
of 6
‘Dithiocarbamate’ (7)
Fig. 3. Tested reactants and observed products at the investigated derivatization
conditions. ITC, isothiocyanate; RT, room temperature; ON, over night.
7
same mild derivatization conditions over a range of concentra-
tions. Analysis by HPLC showed dominant conversion of this
expected type of adduct under the derivatization conditions
(Fig. 4): of six replicates in two blocks, ranging from 0.1 to 1 lmo-
Fig. 4. HPLC-PDA data showing conversion of the dithiocarbamate (7) to benzylth-
iourea (6). Chromatograms (240 nm) are shown with UV spectra of major peaks as
inserts. a: Dithiocarbamate (7) incubated in MeOH (control), dried and redissolved
in 80% aq. MeOH (1.00 mL). b: Products of dithiocarbamate (7) (same amount as in
a) incubated in 5% NH3 in 80% aq. MeOH, dried and re-dissolved in 80% aq. MeOH
(1.00 mL), showing the thiourea (6) as the dominating product.
l/assay, the disappearance of 7 was 98–100% in five experiments
and 52% in the remaining (outlier) experiment. In accordance with
the disappearance of 7 upon incubation with 5% NH3, 6 was formed
with HPLC peak area comparable to the missing peak area of 7 in
each case. Hence, the ITC analysis method based on derivatization
with ammonia at mild conditions would include intact ITCs as well
as other reactive adducts such as dithiocarbamates (Fig. 3).
closed Petri dishes with moist filter paper but without insect larvae
present.
At both high and intermediate levels of spiked moringin, the
corresponding thiourea 5 was the dominant peak in the HPLC-
PDA chromatograms at 240 nm after derivatization with NH3,
while no significant interfering peaks were observed in blanks
(Fig. S1). A clear difference in the recovery was observed for the
non-volatile moringin versus the volatile benzyl ITC (Figs. 5 and
6). There was no systematic decline in moringin levels with time
(results not shown), in contrast to the situation with the volatile
benzyl ITC (Fig. 6). Notably, the major loss of benzyl ITC happened
during the 30 min solvent evaporation, i.e. before or close to ‘‘time
zero’’ in any insect feeding experiment (Fig. 6).
2.3. Recoveries of isothiocyanates and reactive adducts in simulated
feeding assays
With the purpose of measuring the combined recovery of ITCs
and a range of potentially bioactive ITC adducts, the devised
‘thiourea derivative analysis method’ (Fig. 3) was applied at realis-
tic feeding assay-like conditions used in tests of insect feeding.
Therefore, ITCs were applied on cabbage leaf discs (that neither
contain glucomoringin nor benzylglucosinolate) and the solvent
was allowed to evaporate, followed by immediate incubation in
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4
N. Agerbirk et al. / Phytochemistry xxx (2015) xxx–xxx
compared to recoveries from glass vials after solvent evaporation
n.s.
100
80
60
40
20
0
and 3 h incubation. Mean recoveries from glass vials were also
consistently higher, as expected if leaf disc enzymes were involved
in the reduced, variable recovery.
*
2.4. Recovered moringin (2) and benzyl isothiocyanate (4) equivalents
include the free compound
To test whether recovered moringin equivalents, measured
after derivatization with ammonia, included the free ITC, a recov-
ery experiment with determination of the free moringin by direct
HPLC was carried out using the usual evaporation of solvent fol-
lowed by incubation at feeding assay-like conditions for 3 h
(Müller et al., submitted for publication). Two levels tested, 1200
and 120 nmol per leaf disc, did not give significantly different rel-
ative recoveries, and the mean relative recovery for the combined
experiment was 63% (SD 21%, N = 11). Comparison with the, at
most, moderately higher recovery measured after derivatization
with ammonia (Fig. 5) allowed us to conclude that moringin was
relatively stable after spiking to leaf discs and that the majority
of recovered moringin-equivalents were accounted for by free,
non-conjugated moringin.
12
120
1200
Moringin amount (nmol/disc)
Fig. 5. Recovery of moringin after application on Brassica oleracea leaf discs at
simulated feeding assay conditions at either 0 h, 1 h, 2 h, 3 h, 4 h or 5 h (N = 1 per
time point) after the complete evaporation of the solvent. Means ( 1 SD) are given
for each amount of spiked moringin, as there was no systematic time dependency.
The analysis method, based on thiourea derivatives, included free isothiocyanates
and reactive adducts. Statistical significance: n.s., not significantly different (t-test,
P = 0.64); ⁄, significantly different from remaining recoveries (t-test, P < 0.05).
In order to test whether residual benzyl ITC (4) in simulated
feeding assays was present in the free form or as a reactive adduct
(such as a dithiocarbamate like 7), a variation of the analysis aimed
at distinction of apolar 4 and polar adducts of 4 was carried out:
extraction in apolar solvent (hexane) before derivatization of the
extracted ITC (Appelquist and Josefsson, 1967; Ji and Morris,
2003). A moderate reduction (ca. 30%) in yield of the derivative 6
due to extraction and inclusion of hexane was observed in control
experiments. Taking this reduction into account, there was still a
90
Start of feeding assays
80
70
60
50
40
30
20
10
0
1200 nmol
120 nmol
lower recovery of hexane-extractable
4-equivalents including polar adducts: spiking with 1.2
resulted in recovery after 2 h in the simulated feeding assay of
4
compared to total
lmol 4
0.23
0.08
l
mol of total
4
equivalents (SD 0.11
l
mol, N = 5) and
l
mol hexane extractable (intact) 4 (SD 0.06
l
mol, N = 5),
-1
0
1
2
3
4
which was significantly different by a t-test (P = 0.02). This result
suggested that approximately half of the recovered 4-equivalents
after 2 h was actual intact 4 despite the considerable time allowed
for evaporation and possible reaction with the leaf disc matrix,
while another half might be in the form of reactive adducts with
nucleophilic plant constituents.
Time (h)
Fig. 6. Recovery of benzyl isothiocyanate at different time-points after application
on Brassica oleracea leaf discs at simulated feeding assay-conditions. Amounts
applied per leaf disc were either 1200 nmol (N = 2–3, 1 SD) or 120 nmol (N = 1) as
indicated. The analysis method, based on thiourea derivatives, included free
isothiocyanates and reactive adducts. Time ‘zero’ indicates the time when the
solvent had disappeared allowing start of insect feeding assays as indicated with an
arrow.
2.5. Perspectives
An analysis method capable of quantifying the sum of free ITCs
and their reactive adducts was devised, including dithiocarba-
mates and possibly other reactive adducts. Preliminary results
suggest that a significant part of recovered volatile benzyl ITC
was in the form of reactive adducts (possibly with macro-
molecules), while the majority of the non-volatile, hydrophilic
moringin was in the free form even after extended incubation on
leaf discs. Hence, classical and recent investigations of deterrence
and/or repellence of ITCs to glucosinolate-adapted insects
(Li et al., 2000; Agrawal and Kurashige, 2003; Bejai et al., 2012)
should be reconsidered, as reactive adducts may be responsible
for the effects, at least in part.
If ITC reaction products rather than the ITCs as such are respon-
sible for some effects of spiking ITCs to experimental diets, this
would not be unprecedented. For example, an aphid antifeedant
glucosinolate product was identified as a conjugate with Cys
(Kim et al., 2008). On the other hand, common ITC detoxification
products with glutathione likewise constitute reactive adducts as
defined here, but may still serve to detoxify the ITCs by rendering
them prone to secretion (Vanhaelen et al., 2001; Francis et al.,
2005; Schramm et al., 2012; Angelino and Jeffery, 2014).
The mean total recovery of moringin and reactive adducts in
simulated bioassays was generally high at the high and intermedi-
ate level tested: 74% (SD 15%, N = 12) for the pooled 120 nmol and
1200 nmol recoveries, that were not significantly different (Fig. 5).
At the low level tested, 12 nmol/leaf disc, the recovery was lower:
46% (SD 21%, N = 6).
During the actual simulated bioassay, the recovered mean total
of benzyl ITC (4) and reactive adducts varied between 38% and 6%
of the applied amount, with a decreasing trend (Fig. 6). Since the
analysis included the most likely type of reaction product in plant
tissue (dithiocarbamate adducts), the different recovery of the two
ITCs was attributed to the difference in volatility due to the
presence or absence of a rhamnosyloxy group.
A quite large natural variation in recovery of moringin was
apparent (Fig. 5), perhaps due to biochemical differences between
the leaf discs. The variation was mainly attributed to biological
rather than technical variation, because two subsequent control
experiments on separate days showed 2.1 and 5.6-fold higher
absolute standard deviations of recoveries from leaf discs
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N. Agerbirk et al. / Phytochemistry xxx (2015) xxx–xxx
5
The surprising recovery of significant amounts of benzyl ITC and
3.3. Determination of isothiocyanate adducts
activated adducts after prolonged evaporation from leaf surfaces
opens the possibility of testing the related phenethyl ITC in simple
leaf disc feeding assays (although formation of adducts is a
problem for interpretation). Contrasting deterrence of this ITC
and oxazolidine-2-thione type products of related glucosinolates
have been proposed to explain differences in generalist insect
resistance among chemotypes of the crucifer Barbarea vulgaris
(van Leur et al., 2008; Agerbirk and Olsen, 2015), which is an
emerging eco-model plant (Wei et al., 2013), but testing of the
proposed active compounds is lacking.
Recently, antibiotic properties as well as promising physiologi-
cal activities in mammals have been reported for moringin
(Brunelli et al., 2010; Park et al., 2011; Galuppo et al., 2013;
Waterman et al., 2014; Giacoppo et al., 2015). Diverse predicted
reactive adducts with peptides and proteins in serum and cell
extracts (Nakamura et al., 2009; Brown and Hampton, 2011;
Angelino and Jeffery, 2014) may be quantifiable as a group using
the devised derivatization with ammonia.
Adducts were identified and quantitated by HPLC-PDA adapted
for the specific analytes. In all cases the column was a Luna phenyl-
hexyl column (250 mm ꢀ 4.6 mm, 5
lm) (Phenomenex, Torrance,
CA) and the detection wavelength for quantitation was 240 nm
with additional collection of PDA data at 210–370 nm. For morin-
gin thiourea (5) and benzyl thiourea (6), the solvents were H2O
(A) and MeOH (B). The gradient program for 5 was 0–2 min, iso-
cratic 100% A, 2–30 min, linear gradient from 0% to 60% B, followed
by a brief wash with B and 7 min equilibration with water. The tR
for 5 was 21 min (Fig. S1). The gradient program for 6 was 0–2 min,
isocratic 20% B, 2–30 min, linear gradient from 20% to 100% B, fol-
lowed by a brief wash with B and 7 min equilibration with 20% B.
The tR for 6 was 18 min. For combined analysis of the carboxylic
acid 7 and the neutral 6, the gradient program for 6 was slightly
modified by spiking eluent A with TFA to 0.1% (vol.), which secured
a sharp peak of 7 at 25 min and did not affect the tR, peak shape or
area of 6 (Fig. 4). Concentrations of 7 were tentatively calculated
using the same response factor as for
from conversion experiments suggested this approximation to be
reasonable. The injection volume was in general 10 L but was
increased to 100 L for dilute samples with 5 in H2O.
6 at 240 nm, data
l
3. Experimental
l
3.1. Chemicals and general conditions
3.4. Recovery of moringin and activated adducts
The HPLC-MS/MS instrument was an Agilent 1100 Series HPLC
(Agilent Technologies, Germany) coupled to a Bruker HCT-Ultra
ion trap mass spectrometer (Bruker Daltonics, Bremen, Germany)
White cabbage (Brassica oleracea) was obtained at a Copen-
hagen grocery store. Leaf discs (1 cm diameter) were treated with
40 lL of moringin solution in 80% aq. MeOH, either 30 mM, 3 mM,
fitted with a Zorbax C18 column (Agilent; 1.8
The oven temperature was maintained at 35 °C, eluents were
H2O with 0.1% (v/v) HCOOH and 50 M NaCl (A) and acetonitrile
l
m, 2.1 ꢀ 50 mm).
0.3 mM or 0 mM (blank). Calculated concentrations (1200, 120 and
12 nmol, respectively) were based on moringin mass. Recovery-
analysis of moringin equivalents (moringin and reactive conju-
gates) based on derivatization with ammonia (Fig. 3), was carried
out (Fig. 5). After evaporation of the solvent (completed in
80 min, judged visually), one leaf disc from each concentration ser-
ies was immediately taken for analysis, while the remaining were
transferred to simulated feeding assays in glass Petri dishes (9 cm
diameter, 1.6 cm inner height) containing a small piece of moist fil-
ter paper. One leaf disc from each concentration series was
removed at 1, 2, 3, 4 and 5 h after the start of the simulated bioas-
say, and immediately taken for derivatization with NH3 and subse-
quent HPLC-PDA analysis of the thiourea derivative (Fig. S1). For
quantification, standard curves were also produced, using 0, 10,
l
with 0.1% (v/v) HCOOH (B), and the gradient program was 0 to
0.5 min, isocratic 2% B; 0.5–7.5 min, linear gradient 2–40% B;
7.5–8.5 min, linear gradient 40–90% B; 8.5–11.5 isocratic 90% B;
11.60–17 min, isocratic 2% B. The flow rate was 0.2 mL/min but
increased to 0.3 mL/min in the interval 11.2–13.5 min. The mass
spectrometer was run in positive electrospray mode. Ion trap
MS/MS was carried out using the SmartFrag procedure with instru-
ment default settings (isolation width 4 m/z, MS/MS fragmentation
amplitude 1V, start amplitude 30%, end amplitude 200%, acquisi-
tion time 40 ms). Instruments for HPLC-PDA and NMR were as pre-
viously described (Agerbirk and Olsen, 2012). The HPLC-PDA
instrument was equipped with
a Luna phenylhexyl column
20, 30 and 40 lL of each moringin solution, which were immedi-
(5
l
m, 250 ꢀ 4.5 mm) (Phenomenex) with PDA detection at
ately taken for derivatization and analysis. In order to compare
recoveries from leaf discs and an inert surface, recoveries of
1200 nmol aliquots (N = 2 + 2) and 120 nmol aliquots (N = 3 + 3)
were compared after solvent evaporation and 3 h incubation on
either a leaf disc or at the bottom of an open, relatively wide glass
vial. In addition, recovery of native moringin was measured
(Müller et al., submitted for publication).
For statistical analysis of recoveries (using Microsoft Excel
2010), variance homogeneity was confirmed by F-tests and differ-
ence of means were estimated by pairwise t-tests (two-tailed) of
the 120 nmol and 1200 nmol series (not different) and of the
12 nmol series compared to the 120 nmol series, the 1200 nmol
series, and both combined (P < 0.05 in all cases).
210–370 nm, gradient programmes and detection wavelength for
quantitation (band width 8 nm) depended on the specific analyte
as detailed in the relevant sections. Chemical shifts in NMR for
D2O as solvent are relative to those of dioxane (dH = 3.75,
dC = 67.4) and for CD3OD as solvent are relative to those of TMS
at 0.0 ppm. Glucomoringin and moringin were isolated and
identified as described elsewhere (Müller et al., submitted for
publication). Benzyl isothiocyanate (98%) was from Aldrich
(Schnelldorf, Germany) and N-acetylCys (p.a.) was from Merck
Eurolab (Darmstadt, Germany).
3.2. Derivatization with ammonia to form thiourea derivatives
Samples (spiked or control leaf discs as well as the pure com-
pounds 2, 4 and 7) were added to 1 mL of 5% NH3 in 80% aq. MeOH
(MeOH: 25% aq. NH3 20/80). The mixture was incubated at room
temperature over-night (16–20 h), any leaf disk removed with a
clean needle, and remaining solvent and reagent removed by evap-
oration under a gentle air-stream. The residue was dissolved in
1.00 mL H2O (5) or 1.00 mL 80% aq. MeOH (6) and subjected to
HPLC analysis.
3.5. Recovery of benzyl isothiocyanate
This experiment was in principle similar to the experiment with
moringin, except that solvent polarities were adjusted to suit the
apolar nature of 4: solutions for application to leaf discs were pre-
pared in 96% aq. EtOH, the time for evaporation was only 30 min,
leaf discs were sampled at the time of application, after solvent-
evaporation for 30 min (time zero), and after a further 1, 2, 3 and
Please cite this article in press as: Agerbirk, N., et al. Derivatization of isothiocyanates and their reactive adducts for chromatographic analysis. Phytochem-
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6
N. Agerbirk et al. / Phytochemistry xxx (2015) xxx–xxx
4 h in the simulated bioassay. The experiment was carried out with
1200 nmol per leaf disc in duplicate or triplicate at each time point
and with 120 nmol/leaf disc at each time point without replicates.
The assymetrical replication is due to pooling data from two exper-
iments as follows. In the first experiment, recovery at both levels
was studied at application and at 0 h, 1 h and 2 h after the com-
plete evaporation of the solvent. Using 2 h as the last data point
was based on a (wrong) expectation of all ITC being gone by then.
As detectable amounts of ITC were observed at 2 h, a second exper-
iment (in duplicate but only considering the highest ITC concentra-
tion) studied recovery at application and at hourly intervals
between 0 h and 4 h after complete solvent evaporation in
simulated bioassays. As the results were similar, data from the
two experiments were pooled, resulting in N = 2–3 for the various
data points for the high level and N = 1 for the low level.
An additional experiment was aimed at distinction of free (apo-
lar) 4 and hypothetic polar or insoluble (macromolecule-bound)
adducts of 4. Aliquots (1200 nmol) of 4 were applied to leaf discs
as in the 2 h experiment above. At 2 h, half of the leaf discs
(N = 3) were transferred to 5% NH3 as above, while the remaining
(N = 3) were extracted in hexane (3 mL). The hexane was
partitioned against H2O and quantitatively transferred (using an
additional 2 ꢀ 2 mL hexane) to the usual 5% NH3 reagent. All sam-
ples, either with or without hexane, were incubated over-night,
with proper controls for testing the combined effect of hexane
extraction and presence of hexane in the reagent solution.
3.83 (30, 1H, dd, 9.3/3.5), 3.62 (50, 1H, m), 3.45 (40, 1H, ‘tr’, 9.5),
1.21 (60, 3H, d, 6.4). 13C NMR (100 MHz), d: 157.2 (4), ca. 134 (1
detected in HMBC), 130.0 (2 + 6), 117.7 (3 + 5), 99.9 (10), 73.9 (40),
72.3 (20 or 30), 72.1 (20 or 30), 70.7 (50), ca. 49 (Ar-CH2 detected in
HMBC), 18.1 (60). Assignments of glycosidic protons were
confirmed by COSY, informative HMBC correlations included 10 to
4 and 2 to Ar-CH2. HPLC-MS/MS: see Fig. 2. NMR-spectra: see
Fig. S2.
3.7. Benzyl thiourea (6)
Benzyl ITC was derivatized with NH3 as described above and
subjected to preparative HPLC followed by 1H NMR in CD3OD. 1H
NMR (400 MHz), d (assignment, #H, peak type): 7.1–7.35 (Ar-H,
5H, m), 4.3–4.75 (br. irregular peak with three maxima at ca.
4.7 ppm (major), 4.6 ppm (minor) and 4.4 ppm (minor)).
HPLC-UV (kmax/nm): 239 (Fig. 4b). MS (ESI) 177 [M+H]+.
3.8. N-Acetyl-S-(N-benzylthiocarbamoyl)-cysteine (7)
Benzyl ITC (4) (0.25 g, 1.68 mmol) was dissolved in 1.68 mL
MeOH, N-acetylCys (0.29 g, 1.76 mmol) was added and dissolved,
and the mixture allowed to react for 3 days at room temperature.
HPLC-analysis after 3 days revealed a dominant UV-absorbing
product which was isolated by preparative HPLC. The combined
fraction (3 mL) was neutralized by addition of 30 lL 1 M sodium
bicarbonate, dried under a gentle air stream and subjected to
NMR in CD3OD. From the MS and NMR (1H, 13C, DEPT,
HSQC, HMBC), the isolated product (Fig 4a) was identified as the
expected dithiocarbamate-type adduct with the thiol group of N-
acetylCys (7, N-acetyl-S-(N-benzylthiocarbamoyl)-cysteine = 2-
(acetylamino)-3-[(benzylcarbamothioyl)sulfanyl]propanoic acid).
HPLC-UV (kmax/nm): 251, 270 (sh) (Fig. 4a). 1H NMR (400 MHz)
(D2O), d (assignment, #H, multiplicity, J/Hz): 7.2–7.35 (20–60, 5H,
m), ca. 4.9 (Ar-CH2a detected in 2D spectra), 4.86 (Ar-CH2b, d,
14.6), 4.64 (2, 1H, dd, 8.5/4.7), 3.97 (3a, 1H, dd, 14.0/4.7), 3.54
(3b, 1H, dd, 14.0/8.5), 1.93 (200, 3H, s). 13C NMR (100 MHz), d:
198.8 (C@S), 174.4 (100), 173.2 (1), 138.6 (10), 129.5 (30 + 50), 129.1
(20 + 60), 128.5 (40), 54.4 (2), 51.4 (70), 37.4 (3), 22.6 (200). Informa-
tive HMBC correlations included Ar-CH2 with C@S and 3a/b with
C@S. Ion trap MS: see Fig. 7.
3.6. Moringin thiourea (5)
Moringin was converted to the thiourea derivative in 5% NH3 in
80% aq. MeOH over-night. The liquid was removed under a gentle
air stream and the solid remnant dissolved in CD3OD and subjected
to 1H NMR, showing moringin thiourea as dominant constituent.
Analysis by HPLC revealed a dominant product at tR = 21 min
(93% of total area at 240 nm) which was isolated by preparative
HPLC for spectroscopic characterization. HPLC-UV (kmax/nm): 220
(sh), 238, 279 (sh) (Fig. S1). 1H NMR (400 MHz) (CD3OD), d (assign-
ment, #H, multiplicity, J/Hz): 7.26 (2 + 6, 2H, d, 8.5), 7.03 (3 + 5, 2H,
d, 8.5), 5.40 (10, 1H, d, 1.7), 4.25–4.65 (Ar-CH2, 2H, br. irregular
peak with three maxima at ca. 4.63 ppm (major), 4.58 ppm
(intermediate) and 4.33 ppm (minor)), 3.98 (20, 1H, dd, 3.5/1.8),
+H: 183
-H: BzITC, 149
Intens.
+MS, 9.9min #1192
9
a (dithiocarbamate, 7)
x10
[M+H]+
313
335
[M+Na]+
-H: 129
0.5
0.0
+H: N-Ac-Cys, 163
184
164
8
x10
+MS2(335), 9.9min #1195
+MS2(313), 9.8min #1189
[M+Na-183]+
b
152
1.5
1.0
0.5
0.0
[M+Na-149]+
[M+Na-CSO]+
186
275
8
x10
[M+H-129]+
184
c
[M+H-149]+
1.0
164
0.5
0.0
122
125
146
150
100
175
200
225
250
275
300
325
m/z
Fig. 7. Ion trap MS of the dithiocarbamate (7), showing contrasting MS2 spectra of sodium and hydrogen adducts due to different cleavages and ion-affinities. a: ESI mass
spectrum of 7 at the HPLC-MS peak apex. b: MS2 of the sodium adduct, showing dominant cleavage at one side of the bridging S and formation of adducts with the Cys
moiety. Depicted adducts are m/z 152 and m/z 275. The minor loss of 60 was tentatively attributed to elimination of CSO, possibly originating from reaction between the
thiocarbonyl and the carboxylic acid functionalities. c: MS2 of the proton adduct, showing cleavage at both sides of the bridging S with the proton following the former
bridging S. The neutral losses matched 2-(acetylamino)prop-2-enoic acid (129) and the ITC, 4 (149). The depicted adduct is m/z 184.
Please cite this article in press as: Agerbirk, N., et al. Derivatization of isothiocyanates and their reactive adducts for chromatographic analysis. Phytochem-
istry (2015), http://dx.doi.org/10.1016/j.phytochem.2015.06.004

N. Agerbirk et al. / Phytochemistry xxx (2015) xxx–xxx
7
attenuates secondary damage in an experimental model of spinal cord injury.
Bioorg. Med. Chem. 23, 80–88.
Acknowledgement
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glycosylated glucosinolate isolated from Moringa oleifera. Tetrahedron Lett. 41,
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L.W., 2012. Characterization of products from the reaction of glucosinolate-
derived isothiocyanates with cysteine and lysine derivatives formed in either
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Harborne, J.B., 1973. Phytochemical Methods. Chapman and Hall, London, UK.
We thank four anonymous reviewers for helpful comments that
improved this paper. Torben og Alice Frimodts Fond supported this
research.
Appendix A. Supplementary data
Holst, B., Williamson, G., 2004.
A critical review of the bioavailability of
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.phytochem.2015.
06.004. These data include MOL files and InChiKeys of the most
important compounds described in this article.
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istry (2015), http://dx.doi.org/10.1016/j.phytochem.2015.06.004