Stability of benzylic-type isothiocyanates in hydrodistillation-mimicking conditions

Article abstract of DOI:10.1021/jf3041534

Pentadiplandra brazzeana Baillon (Pentadiplandraceae) is known to contain benzyl-, 3-methoxybenzyl-, 4-methoxybenzyl-, 3,4-dimethoxybenzyl-, and indole-type glucosinolates, and the essential oil obtained from its roots is mainly constituted of benzyl isothiocyanate and benzyl cyanide. In a previous study by the authors, it was surmised that partial hydrolytic degradation of 4-methoxybenzyl isothiocyanate, one major expected compound, occurred during the hydrodistillation process of essential oil preparation. To probe this hypothesis, a selection of diversely substituted benzylic-type isothiocyanates was submitted to standard hydrodistillation-mimicking conditions. After extraction with dichloromethane, the reaction mixtures were analyzed using GC-MS. The aqueous phases resulting from liquid-liquid extraction were analyzed by HPLC and GC-MS. 2-Methoxybenzyl, 4-methoxybenzyl, 3,4-dimethoxybenzyl, and 3,4,5-trimethoxybenzyl isothiocyanates underwent conversion into 2-methoxybenzyl, 4-methoxybenzyl, 3,4-dimethoxybenzyl, and 3,4,5- trimethoxybenzyl alcohols, respectively, whereas benzyl, 3-methoxybenzyl, and 4-chlorobenzyl isothiocyanates were converted into the corresponding benzylamines.

Full text of DOI:10.1021/jf3041534

Article
pubs.acs.org/JAFC
Stability of Benzylic-Type Isothiocyanates in Hydrodistillation-
Mimicking Conditions
Gina R. De Nicola,† Sabine Montaut,*,‡ Patrick Rollin,§ Maximilienne Nyegue,∥ Chantal Menut,⊥
Renato Iori,† and Arnaud Tatibouet§
̈
†Consiglio per la Ricerca e la Sperimentazione in Agricoltura, Centro di Ricerca per le Colture Industriali (CRA-CIN), Via di
Corticella 133, 40128 Bologna, Italy
‡Department of Chemistry and Biochemistry, Laurentian University, 935 Ramsey Lake Road, Sudbury, Ontario, Canada P3E 2C6
§Institut de Chimie Organique et Analytique, UMR-CNRS 7311, Universite
́
d’Orlea
Cameroon
⊥IBMM, UMR 5247 UM2-UM1, 15 avenue Charles Flahault, B.P. 14491, 34093 Montpellier, France
́ ́
ns, B.P. 6759, F-45067 Orleans Cedex 2, France
∥Department of Biochemistry, University of Yaounde
́ ́
I, B.P. 812, Yaounde,
ABSTRACT: Pentadiplandra brazzeana Baillon (Pentadiplandraceae) is known to contain benzyl-, 3-methoxybenzyl-, 4-
methoxybenzyl-, 3,4-dimethoxybenzyl-, and indole-type glucosinolates, and the essential oil obtained from its roots is mainly
constituted of benzyl isothiocyanate and benzyl cyanide. In a previous study by the authors, it was surmised that partial hydrolytic
degradation of 4-methoxybenzyl isothiocyanate, one major expected compound, occurred during the hydrodistillation process of
essential oil preparation. To probe this hypothesis, a selection of diversely substituted benzylic-type isothiocyanates was
submitted to standard hydrodistillation-mimicking conditions. After extraction with dichloromethane, the reaction mixtures were
analyzed using GC-MS. The aqueous phases resulting from liquid−liquid extraction were analyzed by HPLC and GC-MS. 2-
Methoxybenzyl, 4-methoxybenzyl, 3,4-dimethoxybenzyl, and 3,4,5-trimethoxybenzyl isothiocyanates underwent conversion into
2-methoxybenzyl, 4-methoxybenzyl, 3,4-dimethoxybenzyl, and 3,4,5-trimethoxybenzyl alcohols, respectively, whereas benzyl, 3-
methoxybenzyl, and 4-chlorobenzyl isothiocyanates were converted into the corresponding benzylamines.
KEYWORDS: Pentadiplandra brazzeana, glucoaubrietin, hydrodistillation, isothiocyanates, glucosinolates, thermal decomposition
essential oil components; the thermal degradation of
carbohydrates and proteins may also result in the formation
of volatile molecules, which affect the acceptability and quality
of freshly distilled oil.5 Distillation over short periods of time
helps in reducing hydrolysis, decomposition, and resinification.
In damaged plant cells, glucosinolates (GLs) are transformed
by the endogenous myrosinase (β-thioglucoside glucohydro-
lase; EC 3.2.1.147) to produce isothiocyanates (ITCs), many of
which can be volatile and pungent. In most cases, ITCs are
sensitive to thermal degradation in aqueous medium.6−9
Indeed, in our continuous program of chemical investigations
on aromatic and medicinal plants from Central and West Africa,
a great chemical variability was observed for essential oils
obtained from Rinorea subintegrifolia O. Ktze (Violaceae family)
roots and Drypetes gossweileri S. Moore (Euphorbiaceae family)
bark.10,11 The content of benzyl isothiocyanate (BITC, 5)
(Figure 1) decreased during the distillation process, whereas
the relative proportion of benzyl cyanide increased, suggesting a
chemical transformation of the ITCs.
INTRODUCTION
■
Natural essential oils, generally obtained from vegetable matter
(flowers, leaves, bark, roots, wood, rhizomes), contain highly
volatile and fragrant organic compounds. Since ancient times,
they have been used for perfumes, flavors, and medicinal
purposes (aromatherapy). Various technologies were developed
to obtain the best aromatic substances to fulfill the needs of the
perfumer and flavorist1 and to respond to the pharmaceutical
industry’s demands. Some authors consider the term “essential
oil” as a generic term that encompasses natural extracts
obtained by various processes (steam distillation, solvent
extraction, supercritical fluid extraction, etc.); however, this
term should be strictly applied to volatile extracts obtained
from a raw material of plant origin by steam distillation,
mechanical processes, or dry distillation.
On the basis of the source material, two major types of
distillation processes are used: water distillation (in which the
plant material is immersed in water and boiled by an external
heat source) and steam distillation (in which steam is pumped
through the plant matrix to extract the oil).2 Improvement in
production technology is an essential element to ameliorate the
overall yield and quality of the product.3 Hydrodistillation,
which is the most “traditional” method for aromatic plant
treatment, is still used for the production of many commercial
oils, especially in tropical countries, because it is a simple and
cheap technology. Nevertheless, chemical changes are invar-
iably associated with water or steam distillations.4 Any
nonoptimized distillation conditions can lead to hydrolysis of
Pentadiplandra brazzeana Baill. (Pentadiplandraceae family)
is a climber from Cameroon, the berries of which are eaten and
used as a sweetener of beverages.12 Recently, the GLs present
in root, seed, and leaf extracts of the plant P. brazzeana were
Received: September 26, 2012
Revised: November 29, 2012
Accepted: December 5, 2012
Published: December 5, 2012
© 2012 American Chemical Society
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Journal of Agricultural and Food Chemistry
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dithiolene, 2-vinyl-4H-1,3-dithiin, 4H-1,2,3-trithiin, 5-methyl-
1,2,3,4-tetrathiane, and N,N′-diallylthiourea.21 In other respects,
the thermal degradation of sulforaphane (4-methylsulfinylbutyl
ITC), in aqueous solution at 100 °C, was shown to produce
dimethyl disulfide, S-methyl methanethiosulfinate, S-methyl
methanethiosulfonate, methyl (methylsulfanyl)methyl disulfide,
1,2,4-trithiolane, 4-isothiocyanato-1-(methylsulfanyl)-1-butene,
3-butenyl ITC, and N,N′-di(4-methylsulfinyl)butyl thiourea.22
It has long been known that indol-3-ylmethyl ITC, resulting
from enzymatic degradation of glucobrassicin, is spontaneously
transformed into indol-3-ylmethanol.23 In addition, approx-
imately 50% of phenylethyl ITC degrades after 4 h in a
phosphate-buffered saline, at pH 7.4 and 37 °C, producing
phenethylamine.24 Finally, 4-hydroxybenzyl ITC, resulting from
enzymatic hydrolysis of glucosinalbin, is unstable in aqueous
media, producing 4-hydroxybenzyl alcohol under release of a
thiocyanate ion.25−27
Figure 1. Hydrolytic degradation of benzylic-type ITCs.
Those observations led us to study and compare the stability
in water at 90 °C of the major 4-methoxybenzyl (2) and benzyl
ITCs (5) corresponding respectively to glucoaubrietin and
glucotropaeolin, present in P. brazzeana root, by mimicking
hydrodistillation extraction conditions. Furthermore, to check
the correlation of substituents on the benzyl moiety with the
transformation of the benzylic-type ITCs under hydrolytic
conditions, we chose to probe the stability of several diversely
substituted ITCs in the same experimental conditions. Our
major objective was to investigate and compare the stability of
benzylic ITCs associated with known naturally occurring
arylaliphatic GLs28 under hydrodistillation-mimicking condi-
tions. Therefore, 2-methoxy (2-MBITC, 1), 3-methoxy (3-
MBITC, 6), and 4-methoxybenzyl (4-MBTIC, 2) ITCs (Figure
1) were tested to check the influence of the substituent’s
location; 3,4-dimethoxy (3,4-DMITC, 3) and 3,4,5-trimethox-
ybenzyl (3,4,5-TMBITC, 4) ITCs (Figure 1) were tested to
evaluate a possible cumulative effect of substituents. Finally,
non-natural 4-chlorobenzyl ITC (4-ClBITC, 7) (Figure 1) was
tested to probe the deactivation effect of a chlorine atom in
comparison with the electron-donating effect of a methoxy
group on the benzyl moiety.
characterized and quantified according to the HPLC analysis of
desulfo-GLs.13 Glucotropaeolin (benzyl GL), glucolimnanthin
(3-methoxybenzyl GL), and glucoaubrietin (4-methoxybenzyl
GL) were shown to be present in the root extract, whereas the
seed mainly contained glucoaubrietin. 3,4-Dimethoxybenzyl
GL, glucobrassicin (indol-3-ylmethyl GL), and traces of
glucotropaeolin were detected in the leaf extract. The
predictions of the GL profile based on desulfo-GL analysis
did not fit the hydrodistillation results. Various extraction and
analysis techniques led to different profiles. In addition, the
above results apparently lacked consistency with data previously
obtained from hydrodistillation experiments.14 In this inves-
tigation, the essential oil, obtained after a 5 h hydrodistillation
of the root, was constituted of 78% of BITC 5, 17% of benzyl
cyanide, 0.1% of 4-methoxybenzyl ITC (4-MBITC, 2), and
0.2% of 4-methoxybenzyl alcohol.14 Therefore, this discrepancy
prompted us to repeat the hydrodistillation process on a
portion of the sample of P. brazzeana root used for the
quantification of GLs.13 The essential oil, obtained after a 5 h
hydrodistillation at pH 5.9, was shown to contain 57% of BITC
5, 10% of benzyl cyanide, and only 5% of 2 (Figure 1).13
Several studies have shown that individual GLs and GLs in
plant extracts are degraded under hydrodistillation-mimicking
conditions.8,15−19 Benzyl cyanide and 5 are expected to result
from the enzymatic decomposition of glucotropaeolin (benzyl
GL).20 Despite the fact that the stability of glucoaubrietin or
the related ITC under hydrodistillation conditions has never
been investigated, it is reasonable to ascribe the decrease of
these compounds to thermal breakdown and leaching into the
heating medium, during hydrodistillation. However, taking into
account that glucoaubrietin is by far the major GL present in P.
brazzeana root and that MBITC 2, the main degradation
product originated from this GL, was found only as a minor
component in the essential oil of P. brazzeana, it had to be
surmised that partial hydrolytic degradation of 2 occurred
during the hydrodistillation process.
MATERIALS AND METHODS
■
Materials. Benzyl ITC (5) was purchased from Fluka Chemie
GmbH (Buchs, Switzerland). PE and EA (analytical grade) were
purchased from Carlo Erba (France). DCM and acetonitrile (HPLC
grade) were purchased from Sigma-Aldrich Chemie GmbH,
(Steinheim, Germany). Ultrapure water (pH 5.0 0.2) was obtained
from a Milli-Q Gradient instrument (Millipore SAS, Molsheim,
France) equipped with a Millipack filter 0.22 μm (Millipore, SAS).
1
CDCl3 was purchased from Euriso-top (St-Aubin, France). H NMR
spectra were recorded at 250 MHz on a Bruker Avance DPX 250
spectrometer, δ values being referenced to residual CHCl3 at 7.26
ppm. Mass spectra were recorded on a Perkin-Elmer Sciex API-300
spectrometer (electrospray, positive mode). Infrared spectra were
recorded on an Attenuated Total Reflectance Thermo-Nicolet Avatar
320 AEK0200713 instrument.
Syntheses of Arylaliphatic Isothiocyanates. ITCs 1−4, 6, and
7 (Figure 1) were prepared from the corresponding amines following
the standard procedure of Goodyer et al.29
2-Methoxybenzyl Isothiocyanate 1. Compound 1 was isolated in
89% yield as a yellow oil. Rf 0.73 (EA/PE 1:3). IR (neat) 2165, 2072
(−NCS). 1H NMR (250 MHz, CDCl3) δ 7.32 (d, 2H, J = 7.5 Hz,
H-4, H-6), 6.98 (td, 1H, J = 7.5, 1.0 Hz, H-5), 6.92 (td, 1H, J = 8.5, 1.0
Hz, H-3), 4.70 (s, 2H, CH2N), 3.86 (s, 3H, OMe). The spectroscopic
data agree with the published values.13,30,31
The stability of some ITCs in aqueous solutions has been
investigated. In distilled water at 37 °C, allyl ITC decomposes
into N,N′-diallylthiourea, allyl allyldithiocarbamate, diallyl
tetrasulfide, and diallyl pentasulfide.6 Allyl ITC isomerizes to
allyl thiocyanate and decomposes into allylamine, allyl
dithiocarbamate, diallylthiourea, carbon disulfide, diallylurea,
and diallyl sulfide in buffer solutions (pH 4, 6, and 8) at 80 °C
for 80 min.7 Within 1 h in boiling water, allyl ITC degrades to
diallyl di-, tri-, and tetrasulfide, allyl thiocyanate, 3H-1,2-
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Figure 2. Gas chromatogram of the DCM fraction obtained after a 10 min degradation of 4-methoxybenzyl ITC (4-MBITC, 2) in water at pH 5.0
0.2 at 90 °C (minor peaks were present in the starting synthesized 2).
3-Methoxybenzyl Isothiocyanate 6. Compound 6 was isolated in
96% yield as a yellow oil. Rf 0.8 (EA/PE 1:3). IR (neat) 2166, 2080
(−NCS). 1H NMR (250 MHz, CDCl3) δ 7.30 (t, 1H, J = 7.8 Hz,
H-5), 6.89 (m, 2H, H-4, H-6), 6.86 (s, 1H, H-2), 4.69 (s, 2H, CH2N),
3.83 (s, 3H, OMe). The spectroscopic data agree with the published
values.13,31
4-Methoxybenzyl Isothiocyanate 2. Compound 2 was isolated in
92% yield as a yellow oil. Rf 0.7 (EA/PE 1:3). IR (neat) 2164, 2073
(−NCS). 1H NMR (250 MHz, CDCl3) δ 7.24 (d, 2H, J = 9.2 Hz,
H-2, H-6), 6.91 (d, 2H, J = 9.2 Hz, H-3, H-5), 4.64 (s, 2H, CH2N),
3.82 (s, 3H, OMe). The spectroscopic data agree with the published
values.13,31,32
analyzed by HPLC and freeze-dried. The resulting residue was
dissolved in DCM and analyzed by GC-MS. Each degradation trial was
performed in triplicate.
GC-MS Analyses of the Dichloromethane and Aqueous
Fractions. The analyses were carried out using a Hewlett-Packard
GCD System model G1800A equipped with a 30 m × 0.25 mm HP-5
ms capillary column. DCM extracts (1 μL) were manually injected in
splitless mode. The flow rate of the carrier gas (He) was 1 mL min−1.
The column temperature was programmed from 60 °C (isocratic 2
min) to 220 °C (isocratic 1 min) at a rate of 10 °C min−1. The
temperatures of the injector and detector were 200 and 280 °C,
respectively. The experiments were carried out with electron impact
ionization (EI) mode at an electron energy of 70 eV. The degradation
products were identified by matching the recorded mass spectra with
the NBS75K.L (NIST, 1992 version) mass spectrum library of the
GC-MS data system. The qualitative analyses of the degradations of
the ITCs were carried out using the normalized area. The detector
response for each eluted compound was set at the default value of 1,
and the peak area of the ITC at time 0 min in the sampling sequence
was assigned the value of 1. The successive peak areas at 10, 20, 30, 40,
50, 60, 120, 180, 240, 300, 360, and 420 min in the degradation trial
were relative to the peak area at 0 min.
HPLC Analyses of the Aqueous Fractions. The analyses were
performed on an HPLC Agilent 1100 system equipped with a
photodiode array detector, on an Inertsil ODS-3 column (250 × 3.0
mm, 5 μm particle size) thermostated at 30 °C. The instrument was
operated with a gradient of water (A) and acetonitrile (B) at a flow
rate of 0.8 mL min−1 using the following program: 1 min, 10% B; 16
min, linear gradient up to 40% B; 3 min, linear gradient down to 10%
B. Eluting peaks were detected by monitoring the absorbance at 226,
240, 254, and 280 nm.
4-Chlorobenzyl Isothiocyanate 7. Compound 7 was isolated in
96% yield as a yellow oil. Rf 0.73 (EA/PE 1:3). IR (neat) 2173, 2073
1
(−NCS). H NMR (250 MHz, CDCl3) δ 7.37 (d, 2H, Jvic = 8.6
Hz, H-3, H-5), 7.26 (d, 2H, Jvic = 8.6 Hz, H-2, H-6), 4.69 (s, 2H,
CH2N).32
3,4-Dimethoxybenzyl Isothiocyanate 3. Compound 3 was isolated
in quantitative yield as a yellowish solid. Rf 0.44 (EA/PE 1:3). IR
(neat) 2163, 2078 (−NCS). 1H NMR (250 MHz, CDCl3) δ 6.86
(m, 2H, Ar−H), 6.82 (m, 1H, Ar−H), 4.64 (s, 2H, CH2N), 3.91 (s,
3H, OMe), 3.89 (s, 3H, OMe). The spectroscopic data agree with the
published values.33
3,4,5-Trimethoxybenzyl Isothiocyanate 4. Compound 4 was
isolated in 94% yield as a yellow oil. Rf 0.32 (EA/PE 1:3). IR (neat)
1
2166, 2080 (−NCS). H NMR (250 MHz, CDCl3) δ 6.52 (s,
2H, Ar−H), 4.65 (s, 2H, CH2N), 3.88 (s, 6H, 2 × OMe), 3.85 (s, 3H,
OMe). The spectroscopic data agree with the published values.34
Stability of Benzylic ITCs in Water. A sample of ca. 20 mg of
ITC was weighed in a 25 mL flask to which 10 mL of ultrapure water
(pH 5.0 0.2) was added. The mixture was vortexed for 2 min, and an
aliquot of 400 μL was transferred into a 1.5 mL glass vial and extracted
with 400 μL of DCM by vortexing for 1 min. The aqueous phase was
analyzed by HPLC and the DCM fraction by GC-MS to have the peak
area at time 0 min. The flask was then fitted with a condenser and the
mixture immediately kept under vigorous stirring in a water bath
heated at 90 2 °C. Aqueous samples of 400 μL were then withdrawn
every 10 min during a 1 h heating period (and every successive hour
over 7 h, in case of slower degradation) and extracted with the same
volume of DCM as described above for the first point at time 0 min.
Each aqueous fraction was then analyzed by HPLC, whereas each
organic fraction was analyzed by GC-MS as described in the following
two sections. After complete degradation, the water phase was
RESULTS AND DISCUSSION
■
The stability of the ITCs 1−7 (Figure 1) in water at 90 °C was
studied according to the protocol described above and
monitored by GC-MS and HPLC. All of the ITCs and
degradation products were detected by GC-MS analysis in the
DCM fractions obtained after liquid−liquid extraction of the
aqueous samples. All tested ITCs were susceptible to hydrolysis
at 90 °C. Each ITC afforded only a single detectable
degradation product in the DCM fractions. Finally, checking
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the aqueous fractions by HPLC and GC-MS analyses allowed
us to rule out the presence of any other degradation product.
Two types of degradation pathways were observed. The first
type displayed hydrolytic conversion of the ITCs into the
corresponding benzyl alcohols, in analogy with the case of
glucosinalbin and 4-hydroxybenzyl ITC previously studied by
Borek and Morra.27 In fact, 4-MBITC (2) was transformed into
4-methoxybenzyl alcohol (Figures 2 and 3) and 2-MBITC (1)
Figure 3. Degradation of 4-methoxybenzyl ITC (4-MBITC, 2) and
concomitant formation of 4-methoxybenzyl alcohol in water at pH 5.0
0.2 at 90 °C.
into 2-methoxybenzyl alcohol at a slower rate compared with 2
(Figure 4) over the first hour under the experimental
conditions used. Figure 3 shows the degradation of 4-MBITC
(2) and the concomitant formation of 4-methoxybenzyl
alcohol. The qualitative analyses of the degradations of the
ITCs were carried out using normalized area as described under
Materials and Methods. The peak area of 2 at time 0 min was
set at the default value of 1, and the peak area of 4-
methoxybenzyl alcohol at time 60 min was assigned the value of
1. The other peak areas in the sampling sequence are relative to
the peak area at 0 min for 2 and to the peak area at 60 min for
4-methoxybenzyl alcohol. As a matter of fact, the initial area
value for 2 and the final one for 4-methoxybenzyl alcohol are
arbitrary. Even though we can exclude from our GC-MS and
HPLC analyses the presence of any other degradation product
apart from 4-methoxybenzyl alcohol, the trends reported in
Figure 3 are only qualitative. The quantification in terms of
percentage of degraded substrate and product formed could be
affected by several factors such as a different solubilization in
water or a different detector response for the two compounds.
3,4-DMBITC (3) was transformed into 3,4-dimethoxybenzyl
alcohol, showing a rate of hydrolysis similar to the one
observed for 2. The more substituted 3,4,5-TMBITC (4)
underwent slower degradation in water at 90 °C (Figure 4).
The second type displayed hydrolytic conversion of the ITCs
into the corresponding benzylamines. Thereby, BITC (5) and
3-MBITC (6) were degraded in water at 90 °C to afford
benzylamine and 3-methoxybenzylamine, respectively (Figure
4).
Figure 4. Time course degradation of benzylic-type ITCs in water at
pH 5.0 0.2 at 90 °C: (a) after 1 h; (b) after 7 h.
Figure 5. The intermediacy of a labile pseudoquinone methide
is anticipated, in analogy with Borek and Morra’s mechanistical
Figure 5. Proposed pathway for the conversion of 4-methoxybenzyl
ITC (4-MBITC, 2) into 4-methoxybenzyl alcohol.
assumption27 for the conversion of 4-hydroxybenzyl ITC into
4-hydroxybenzyl alcohol: similarly to the hydroxyl group in
sinalbin, the marked electron-donating character of the
methoxy group in 2 facilitates the hydrolytic process through
resonance stabilization of the transient pseudoquinone
methide.35 As they both bear a p-methoxy group, 3 and 4 are
assumed to produce the respective alcohols following the same
pathway. However, whereas the hydrolyses of 2 and 3 are
closely comparable, complete conversion of 4 is more sluggish,
owing probably to the conflicting effect of m-methoxy groups
(Figure 4a). Finally, the observed conversion of 1 is slightly
This dramatic difference observed in the behavior of benzylic
ITCs could be rationalized in terms of electron delocalization
within their aromatic frame. A proposed pathway for the
formation of 4-methoxybenzyl alcohol from 2 is shown in
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Journal of Agricultural and Food Chemistry
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slower than the hydrolysis of 2, probably because of a lesser
stabilization in the transient o-quinone methide.35
length of time for P. brazzeana root to modulate the essential
oil composition. The results of that study in progress in our
laboratory will be described in a separate paper.
In contrast, other benzylic ITCs such as 5 and its meta-
substituted derivative 6 are unable to generate or stabilize
exomethylene intermediates to allow nucleophilic substitution
of the ITC group. Therefore, a standard addition of water on
the CN bond occurs to generate an unstable thionocarbamic
species, which degrades to give the primary amine under
elimination of carbon oxysulfide (Figure 6).
AUTHOR INFORMATION
Corresponding Author
*Phone: +1-(705)-675-1151, ext. 2185. Fax: +1-(705)-675-
4844. E-mail: smontaut@laurentian.ca.
■
Funding
We gratefully acknowledge financial support from the Natural
Sciences and Engineering Research Council of Canada
(Discovery grant, S.M.), from the Universite d’Orleans (A.T.
́ ́
and P.R.), and from the Italian Ministry of Agriculture and
Forestry Policies (Research grant, G.R.D.N.)
Notes
The authors declare no competing financial interest.
Figure 6. Proposed pathway for the conversion of benzyl
isothiocyanate (BITC, 5) into benzylamine.
ABBREVIATIONS USED
■
ITC, isothiocyanate; GL, glucosinolate; DCM, dichloro-
methane; EA, ethyl acetate; PE, petroleum ether; BITC, benzyl
isothiocyanate; 2-MBITC, 3-MBITC, 4-MBITC, 2-methoxy-, 3-
methoxy-, 4-methoxybenzyl isothiocyanate; 3,4-DMITC, 3,4,5-
TMBITC, 3,4-dimethoxy-, 3,4,5-trimethoxybenzyl isothiocya-
nate; 4-ClBITC, 4-chlorobenzyl isothiocyanate
Our hypothesis was supported by performing a comparative
hydrolysis experiment on 4-ClBITC (7). Contrary to 2, this
structural analogue displays a deactivating group in the para-
position, which is expected to hamper the formation of the
exomethylene reactive species and favor the nucleophilic
addition of water onto the ITC function. When reacted with
water at 90 °C, 7 was indeed readily converted into 4-
chlorobenzylamine.
The above study clarifies why 2 was not detected in the
essential oil obtained from the hydrodistillation of P. brazzeana
samples: the ITC would be totally hydrolyzed during the
process. The study was extended to a family of methoxylated
benzyl ITCs derived from less common GLs: 2-methoxygluco-
tropaeolin,30 glucolimnanthin,36 3,4-dimethoxyglucotropaeo-
lin,33 and 3,4,5-trimethoxyglucotropaeolin.34 With regard to
hydrolysis of ITCs, the position of the methoxy electron-
donating group on the aromatic ring was shown to be critical.
The related benzylic alcohol is formed only when a methoxy
group is present in ortho- or para-position. In other cases, that
is, when the methoxy group is in the meta-position, or without
any substituent, the reaction shifts toward the formation of the
benzylic amine.
The present study indicates that benzylic ITCs are generally
not stable in water at 90 °C. The sensitivity of those
compounds to hydrolysis has important implications in the
modes of extraction used to obtain essential oil not only from P.
brazzeana but from any other GL-containing plant. In our P.
brazzeana essential oil sample, 4-methoxybenzyl derivatives
were not detected in substantial amounts, whereas benzyl
derivatives were the major compounds in the mixture.13 This is
somehow puzzling, considering that the parent GL glucoau-
brietin is the major GL in the plant in comparison to the minor
glucotropaeolin. Other parameters such as the complexity of
the plant matrix or the harsh hydrodistillation conditions
should probably be considered as well to understand why 4-
methoxybenzyl derivatives can hardly be detected in the
hydrodistillate. It has to be noted that a similar observation has
been made in a previous investigation of the essential oil of R.
subintegrifolia root.11 In addition, the stability of glucoaubrietin
in hydrodistillation-mimicking conditions should be addressed
in complementary studies to evaluate the formation of ITC 2
and other 4-methoxybenzyl derivatives during the process.
Finally, the present paper allows for a better understanding of
our preliminary observations on varying the hydrodistillation
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■
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