Synthesis of stable isotope-labeled nasturlexins and potential precursors to probe biosynthetic pathways of cruciferous phytoalexins

Article abstract of DOI:10.1002/jlcr.3591

The syntheses of perdeuterated phytoalexins nasturlexins A and C, and putative biosynthetic precursors, including phenylethyl isothiocyanates and phenylethyl dithiocarbamates, using commercially available [2,3,4,5,6-D₅]phenylalanine, [2,3,4,5,6

Full text of DOI:10.1002/jlcr.3591

Pedras and To
Synthesis of stable isotope–labeled nasturlexins and potential
precursors to probe biosynthetic pathways of cruciferous
phytoalexins
M. Soledade C. Pedras* and Q. Huy To
Department of Chemistry, University of Saskatchewan, 110 Science Place,
Saskatoon SK S7N 5C9, Canada
*Corresponding author: M. Soledade C. Pedras
Department of Chemistry, University of Saskatchewan,
110 Science Place, Saskatoon, SK S7N 5C9, Canada
Telephone: (306)966-4772
Fax: (306)966-4730
E-mail: s.pedras@usask.ca
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doi: 10.1002/jlcr.3591
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Pedras and To
Abstract
The syntheses of perdeuterated phytoalexins nasturlexins A and C, and putative
biosynthetic precursors, including phenylethyl isothiocyanates and phenylethyl
dithiocarbamates, using commercially available [2,3,4,5,6-D5]phenylalanine, [2,3,4,5,6-
D5]nitrobenzene and [2,3,4,5,6-D5]benzaldehyde are described. In addition, application of an
efficient deuterium-hydrogen exchange transformation to non-labeled starting materials
allowed access to new deuterated compounds, including 3-hydroxyphenylethyl glucosinolate.
Keywords: 3-hydroxyphenylethyl glucosinolate; 3-hydroxyphenylethyl isothiocyanate;
nasturlexin; (E, Z)-styryl isothiocyanate; cruciferous phytoalexin
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Pedras and To
1. Introduction
A novel group of phytoalexins, the nasturlexins, has been recently discovered in three
related crucifer species: nasturlexins A (1), B (2) and tridentatol C (5) in watercress
(Nasturtium officinale R. Br.),1 nasturlexins C (3), D (6) and the corresponding sulfoxides 4
and 7 in winter cress (Barbarea vulgaris R. Br.) and in upland cress (B. verna (P. Mill.)
Aschers)2 (Fig. 1). Nasturlexin A (1) together with the cruciferous phytoalexins brassinin (8)
and methoxylated derivatives of 8 constitute a unique group of plant natural products due to
an unusual dithiocarbamate functionality.3 Phytoalexins are plant defense metabolites with
antimicrobial activity, produced in response to stress such as pathogen attack, that are not
present in naturally healthy plants.3,4
Our ongoing work on phytoalexins and other chemical defenses of crucifers aims to
uncover metabolic pathways that protect crucifer crops from microbial diseases. While most
of the metabolites involved in the multistep biosynthetic pathway of brassinin (8) have been
identified,3,5 the biosynthetic precursors and intermediates of the nasturlexin group of
phytoalexins (1–7) remain undetermined. Taking into consideration the biosynthetic
precursors of brassinin (8), derived from (S)-tryptophan via indolylmethyl glucosinolate (9),
it was hypothesized that nasturlexin A (1) was derived from (S)-phenylalanine via the
corresponding phenylethyl glucosinolate (10).1 The potential biosynthetic intermediates of
nasturlexin A (1) and phytoalexins 3, 5, and 6 were proposed1 as shown in Scheme 1. By
analogy to brassinin (8) biosynthesis,5 nasturlexin A (1) is anticipated to result from
phenylethyl glucosinolate (10) (step i) via phenylethylisothiocyanate (13) (step vi).1 Similarly,
compounds 16 and 17 could result from the corresponding isothiocyanates 14 and 15,
respectively, or from selective enzymatic hydroxylations of 1.
(5).
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Pedras and To
The key issue resides in determining the intermediates between nasturlexins A (1)
and C (3), for which compounds 18, 21 and 24, steps ii-iv, respectively, are proposed, with
the final step (step v) involving oxidation of 24 to 3. To probe these biosynthetic pathways in
planta and in vitro, administration of isotope-labeled compounds to plant tissues followed by
detection and analyses of isotope-labeled products is necessary. Herein the syntheses of
stable isotope-labeled compounds that are potential precursors/intermediates of the
phytoalexins 1–7 are reported. Towards this end, the syntheses of the following compounds
was undertaken: [2,3,4,5,6-D5]nasturlexins A (1a) and C (3a), [2,4,6-D3,15N ]-3-
hydroxyphenylethyl glucosinolate (12a), [2,3,4,5,6-D5]phenylethyl isothiocyanate (13a),
[2,3,5,6-D4]-4-hydroxyphenylethyl
isothiocyanate
(14a),
[2,3,4,5,6-D5]-(E,Z)-styryl
isothiocyanate (18a), methyl [2,3,5,6-D4]-4-hydroxyphenylethyl dithiocarbamate (16a),
[2,4,6-D3,15N]-3-hydroxyphenylethyl isothiocyanate (15a), [D3CS]methyl [2,4,6-D3,15N ]-3-
hydroxyphenylethyl dithiocarbamate (17a), methyl [2,3,4,5,6-D5]-(E)-styryl dithiocarbamate
(21a), [2,3,4,5,6-D5]dihydronasturlexin C (24a).
2. Results and discussion
Considering the number of compounds necessary to probe the biosynthetic pathway
of nasturlexins 1–4, 6, 7 and tridentatol C (5), it was important to devise synthetic routes that
afforded isotope-labeled compounds efficiently. The stable isotope deuterium (D) was chosen
due to the availability of highly sensitive analytical tools that can reliably measure D
incorporation into each metabolite, namely HPLC-ESI-MS. At the outset, all synthetic routes
were chosen based on the commercial availability of deuterated starting materials (D, ≥98%),
or the possibility of selectively obtaining high percentages of deuteration (≥92%) by
transformation of non-labeled materials. All synthetic procedures were established and
optimized using non-labeled starting materials. The purity and percentage of deuteration of
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Pedras and To
1
labeled compounds was established by H NMR spectroscopy or HR-FD-MS. To
differentiate between non-labeled and isotopically labeled compounds, all structures of
deuterated compounds have a number followed by the letter a or a' e.g. 1a.
2.1 Synthesis of isothiocyanates, dithiocarbamates and nasturlexins
Initially, the syntheses of monosubstituted phenyl compounds 1a and 13a were
carried out, using as key intermediate the corresponding amine 28a. Straightforward
syntheses of [2,3,4,5,6-D5]nasturlexin A (1a) and [2,3,4,5,6-D5]phenylethylisothiocyanate
(13a) starting from commercially available [2,3,4,5,6-D5]phenylalanine (26a) were developed
as shown in Scheme 2. Oxidative decarboxylation of 26a using trichloroisocyanuric acid
(TCICA) afforded the corresponding nitrile 27a,6 which was reduced using ZrCl4 and
7
NaBH4 to yield [2,3,4,5,6-D5]amine 28a. [2,3,4,5,6-D5]Nasturlexin A (1a) was obtained
from amine 28a after addition of CS2 followed by MeI (72% overall yield from 27a).1 To
prepare [2,3,4,5,6-D5]phenylethylisothiocyanate (13a), amine 28a was treated with
thiophosgene8 to afford 13a in 70% yield from 27a. The synthesis of [2,3,4,5,6-
D5]phenylethyl isothiocyanate (13a) reported here (68% overall yield from [2,3,4,5,6-
D5]phenylalanine (26a)) is simpler and more efficient than a previous preparation using
[2,3,4,5,6-D5]bromobenzene as starting material (five steps, 62% overall yield).8
Next, the syntheses of hydroxyphenyl compounds 14a and 16a were carried out.
Similar to the syntheses of 1a and 13a (Scheme 2), [2,3,5,6-D4]-4-hydroxyphenylethyl
isothiocyanate (14a) and methyl [2,3,5,6-D4]-4-hydroxyphenylethyl dithiocarbamate (16a)
were obtained from the corresponding amine, [2,3,5,6-D4]tyramine (33a) (Scheme 3). Amine
33a was prepared from reaction of [2,3,4,5,6-D5]nitrobenzene with 2-
(phenylthio)acetonitrile9 to afford [2,3,5,6-D4]-4-nitrophenylacetonitrile (30a) in 66% yield.
The [2,3,5,6-D4]nitro compound 30a was reduced to [2,3,5,6-D4]-4-aminophenylacetonitrile
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Pedras and To
(31a), which was then converted to [2,3,5,6-D4]-4-hydroxyphenylacetonitrile (32a) via
diazotization and subsequent Cu(I)-catalyzed hydrolysis.10 Next, reduction of nitrile 32a
using ZrCl4 and NaBH4 afforded [2,3,5,6-D4]tyramine (33a),7 which was used for the
synthesis of [2,3,5,6-D4]dithiocarbamate 16a, as described for compound 1a. However, due
to the reactivity of the phenolic hydroxyl, the method used for the preparation of
isothiocyanate 13a afforded 14a in rather low yield (26%). Thus, a method previously
reported for preparation of isothiocyanate 1411 was used to prepare 14a; treatment of 33a
with of CS2 followed by (t-Boc)2O yielded 14a in reasonable yield (63% yield from 32a).
Isothiocyanate 1411 and dithiocarbamate 1612 were previously synthesized from commercially
available tyramine (33), but the corresponding tetradeuterated compounds have not been
reported previously.
Unlike the synthesis of 14a and 16a, no suitable perdeuterated starting material
containing a 3-hydroxyphenyl moiety was readily available to synthesize isothiocyanate 15a
and dithiocarbamate 17a, hence a proton/deuterium exchange transformtion13 was pursued.
Initially, the readily available 3-hydroxyphenylethanoic acid (34) was chosen as starting
material, as selective deuteration at the ortho/para positions to the phenol group was
expected. Preliminary experiments (DCl/D2O, 80–100 °C, 2-5 h) afforded extensive but
undesirable deuterium exchange; however, further modification established a procedure that
afforded high deuterium content at positions C2, C4 and C6 of the phenyl ring (ortho and
para to the hydroxyl group) (Scheme 4). Much to our delight, one-pot deuteration and
esterification of 3-hydroxyphenylethanoic acid (34) in D2SO4/CD3OD at 60 °C afforded the
corresponding [D3]methyl [2,4,6-D3]-3-hydroxyphenylethanoate (35a) in excellent yield
1
(94%). Integration of the H NMR signals of 35a corresponding to the aromatic protons H2
(0.08H, s, H 6.77), H4 (0.07H, d, H 6.74), H5 (0.99H, t, 7.19) and H6 (0.07H, d, H 6.83)
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Pedras and To
and the methylene protons (2.0H, s, H 3.58) indicated ca. 92% of deuterium at positions C2,
C4 and C6 (no unlabeled material was detected by LC-MS). Ester 35a was reduced with
DIBAL/DCM to aldehyde 36a, which was transformed into oxime 37a using 15NH2OH·HCl
15
and NaOAc. The use of N was advantageous as its incorporation into plant metabolites can
confirm that both the deuterium and nitrogen containing moieties of a given structure are
incorporated together (detection of labeled metabolites by HPLC-MS analysis of [M+4]+/-
fragments). Finally, reduction of oxime 37a using NaBH4/NiCl2·6H2O yielded amine 38a,
which was used for the syntheses of isothiocyanate 15a11 and dithiocarbamate 17a,1 as in the
previous syntheses (Scheme 3). [2,4,6-D3,15N]-3-Hydroxyphenylethyl isothiocyanate (15a),
[D3] methyl [2,4,6-D3,15N]-3-hydroxyphenylethyl dithiocarbamate (17a) and methyl 3-
hydroxyphenylethyl dithiocarbamate (17) have not been reported to date, but 3-
hydroxyphenylethyl isothiocyanate (15) was previously synthesized from amine 38.14
The next group of deuterated compounds remaining to be synthesized (3a, 18a, 21a
and 24a) took advantage of a common key intermediate (42a) used previously in the
synthesis of their non-deuterated counterparts (3, 18, 21 and 24, respectively). To prepare
pentadeuterated ammonium salt 42a, commercially available [2,3,4,5,6-D5]benzaldehyde
(39a) was chosen as the starting material (Scheme 5). Addition of nitromethane to aldehyde
39a afforded nitro alcohol 40a in good yield,15 which was then reduced to amine 41a. Next,
amine 41a was treated with HCl followed by SOCl2 to give the ammonium salt 42a.16
Reaction of 42a with CS2 and CH3I gave dihydronasturlexin C (24a), which was further
oxidized with DDQ to [2,3,4,5,6-D5]nasturlexin C (3a).1 Ammonium salt 42a was treated
with thiophosgene to yield the corresponding isothiocyanate 43a,8 which upon heating
afforded [2,3,4,5,6-D5]styryl isothiocyanates 18a/18a' as a mixture of E/Z isomers (ca. 5:1).18
Treatment of 18a/18a' with sodium thiomethoxide17 afforded dithiocarbamates 21a/21a' as a
mixture of E/Z isomers, which were separated by FCC. To date, methyl styryl
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Pedras and To
dithiocarbamate 21 has not been reported, but nasturlexin C (3) and its dihydro relative 24
were previously synthesized from amino alcohol 41,2 whereas the corresponding deuterated
isomers have not been reported. Isothiocyanate 18 was synthesized from isothiocyanate 43,18
but not the deuterated isomer.
2.2 Synthesis of [2,4,6-D3,15N]-3-hydroxyphenylethyl glucosinolate (12a)
Although the synthesis of 3-hydroxyphenylethyl glucosinolate (12) has not been
previously reported, our strategy to prepare a perdeuterated phenyl glucosinolate took
advantage of well established protocols. In brief, the "thiohydroximate disconnection"19 is a
1,3-addition of a thiohydroximate glucoside to a nitrile oxide generated in situ, under basic
conditions, from a hydroximoyl chloride, the key intermediate.
Since no suitable perdeuterated starting material containing a 3-hydroxyphenyl
moiety was readily available, a proton/deuterium exchange13 was pursued, similar to the
preparation of 15a and 17a. The readily available 3-hydroxyphenylpropanoic acid (44) was
chosen as starting material, since selective deuteration at the ortho/para positions to the
phenol group was expected. As above, a one-pot deuteration and esterification of (3-
hydroxyphenyl)propanoic acid (44) in D2SO4/CD3OD afforded ester 45a in excellent yield
(99%), with three deuterium atoms at C2, C4 and C6 positions of the phenyl ring (ortho and
1
para positions of the OH group) (Scheme 6). Integration of the H NMR signals of 45a
corresponding to the aromatic protons H2 (0.08H, s, H 6.680), H4 (0.08H, d, H 6.676), H5
(0.92H, t, 7.15) and H6 (0.08H, d, H 6.76) and to both methylene groups (2.0H, t, H 2.91;
2.0H, t, H 2.63) indicated ca. 92% of deuterium at C2, C4 and C6 positions (no unlabeled
material was detected by LC-MS). Reduction of ester 45a using DIBAL/DCM at -78 ºC
yielded aldehyde 46a, which was treated with acetyl chloride to protect the reactive phenolic
15
group to yield 47a. Treatment of 47a with NH2OH·HCl and NaOAc afforded oxime 48a,
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Pedras and To
which was chlorinated with NCS to yield the corresponding hydroximoyl chloride, followed
by addition of 1--D-thioglucose tetraacetate to yield thiohydroximate 49a. Sulfonation of
49a using pyridine sulfur trioxide complex20 yielded 50a. Deacetylation carried out using
K2CO3/ MeOH21 yielded glucosinolate 12a quantitatively. The ESI-MS of the product
showed the ion peak corresponding to the incorporation of three deuterium atoms and one 15N
atom. 3-Hydroxyphenylethyl glucosinolate (12) has not been isolated previously, neither has
its synthesis been reported. The corresponding desulfated derivative 51 was isolated from leaf
and floral extracts of Barbarea vulgaris.22
3. Experimental
3.1 General
Chemicals were purchased from Sigma-Aldrich Canada Ltd., Oakville, ON, and Alfa
Aesar 26 Parkridge Rd, Ward Hill, USA. Isotope-labeled compounds were purchased from
Cambridge Isotope Laboratories Inc., Andover, MA, and C/D/N Isotopes Inc., Pointe-Claire,
QB. All solvents were HPLC grade and used as such or treated as specified for each case.
Flash column chromatography (FCC) was carried out using silica gel grade 60, mesh size
230–400 Å, or WP C18 prep-scale bulk packing 275 Å (J.T. Baker, NJ, USA).
Nuclear magnetic resonance (NMR) spectra were recorded on Bruker 500 MHz
1
13
Avance spectrometers (for H, 500.3 MHz and for C, 125.8 MHz) and Bruker Avance III
HD 600 MHz spectrometer (for 1H, 600.2 MHz and for 13C, 150.9 MHz). Chemical shifts ()
are reported in parts per million (ppm) relative to TMS; coupling constants (J) are reported to
the nearest 0.5 Hz.
HRMS data were obtained on a Jeol AccuToF GCv 4G mass spectrometer [field
desorption (FD)], or on a VG 70 SE mass spectrometer employing a solids probe [electron
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Pedras and To
impact (EI)], or on a Qstar XL MS/MS System [electrospray ionization (ESI)] by direct
insertion.
HPLC-DAD analysis was carried out with Agilent 1100 system equipped with
quaternary pump, autosampler, diode array detectors (DAD, wavelength range 190–600 nm,
bandwidth 4 nm), degassers and Zorbax Eclipse XDB-C18 columns (5 µL particle size silica,
150 x 4.6 mm I.D.), equipped with an in-line filter. Method A (non-polar metabolites) used
the mobile phase H2O – CH3CN from 75:25 to 25:75, linear gradient for 35 min, with a flow
rate of 1.0 mL/min. Method B (glucosinolates) used the mobile phased H2O (0.1% TFA) –
MeOH (0.1% TFA) from 100:0 to 50:50, linear gradient for 25 min, followed by 50:50 to
0:100, linear gradient for 10 min, with a flow rate of 1.0 mL/min.
3.2 Syntheses and characterization of labeled compounds
3.2.1 [2,3,4,5,6-D5]Nasturlexin A (1a)
Trichloroisocyanuric acid (136 mg, 0.59 mmol) was added in small portions, over a
10 min period, to a solution of (R,S)-[2,3,4,5,6-D5]phenylalanine (D, 99.4%) (26a) (100 mg,
0.59 mmol) and pyridine (95 µL, 1.18 mmol) in MeOH (1.0 mL) at rt. After stirring for 30
min, the reaction mixture was filtered, the precipitate was rinsed with MeOH and the MeOH
wash combined with the filtrate. The filtrate was concentrated and fractionated by FCC
(DCM) to yield [2,3,4,5,6-D5]phenylethanenitrile (27a) (70 mg, 0.57 mmol, 97%) as a liquid.
NaBH4 (179 mg, 4.7 mmol) was added in portions to a vigorously stirred suspension of ZrCl4
(130 mg, 0.56 mmol) in THF (1.5 mL) at rt, followed by a solution of [2,3,4,5,6-
D5]phenylacetonitrile (27a) (57 mg, 0.47 mmol) in THF (0.5 mL). After stirring for 5 h, the
mixture was diluted with cold H2O, basified with NH4OH and extracted with EtOAc. The
organic extract was dried over Na2SO4 and concentrated to give crude [2,3,4,5,6-
D5]phenylethyl amine (28a) (60 mg, 0.47 mmol), which was used without purification. CS2
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Pedras and To
(42 µL, 0.71 mmol) was added to a solution of amine 28a (60 mg, 0.47 mmol) in pyridine
(1.5 mL) at rt, followed by Et3N (195 µL, 1.41 mmol). After stirring for 30 min, CH3I (44 µL,
0.71 mmol) was added and stirring was continued for an additional 30 min. The mixture was
diluted with toluene and concentrated to dryness. The crude mixture was fractionated by FCC
(EtOAc-hexane, 1:9) to give [2,3,4,5,6-D5]nasturlexin A (1a) (73 mg, 0.34 mmol, 72% from
nitrile 27a; D ≥ 99%, determined by HR-FD-MS) as a yellowish solid, mp 44-45 °C.
Compound 1a: HPLC tR = 21.5 min (method A). 1H NMR (500 MHz, CDCl3):  6.92
(1H, br), 4.03-3.99 (2H, m), 2.98 (2H, t, J = 7.0 Hz), 2.61 (3H, s), and a minor rotamer at
2
7.59 (s), 3.72-3.70 (m), 2.96 (t), 2.69 (s). HR-FD-MS m/z [M]+: calc. for C10H8 H5NS2:
216.0803, found 216.0805 (100%). UV (HPLC, CH
3
CN–H O) max (nm): 253, 272.
2
3.2.2 [2,3,4,5,6-D5]Nasturlexin C (3a)
DDQ (32 mg, 0.14 mmol) was added to a solution of 24a (25 mg, 0.12 mmol) in 1,4-
dioxane (0.50 mL) at rt. The mixture was heated to 100 °C and stirred for 6 h in a sealed vial.
H2O was added and the mixture was extracted with DCM. The organic extract was dried over
Na2SO4, concentrated and separated by FCC (EtOAc-hexane, 1:9) to give [2,3,4,5,6-
D5]nasturlexin C (3a) (18 mg, 0.085 mmol, 71%; D ≥ 99%, determined by HR-FD-MS) as a
yellowish solid, mp 30-31 °C.
Compound 3a: HPLC tR = 23.6 min (method A). 1H NMR (500 MHz, CDCl3):  7.81,
2
(1H, s), 2.74 (3H, s). HR-FD-MS m/z [M]+: calc. for C10H4 H5NS2: 212.0490, found
212.0494 (100%). UV (HPLC, CH3CN – H2O) max (nm): 310.
3.2.3 [2,4,6-D3,15N]-3-Hydroxyphenylethyl glucosinolate (12a)
D2SO4 (D, 99%, 0.60 mL) was added to
a
suspension of 3-(3-
hydroxyphenyl)propanoic acid (44) (150 mg, 0.90 mmol) in CD3OD (D, 99.8%, 0.90 mL).
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Pedras and To
The mixture was stirred at 60 °C in a sealed vial, diluted with H2O and extracted with DCM.
The organic extract was dried over Na2SO4 and concentrated to yield ester 45a (165 mg, 0.89
mmol, 99%). DIBAL (1.5 M in toluene) (1.08 mL, 1.62 mmol) was added dropwise to a
solution of ester 45a (150 mg, 0.81 mmol) in dry DCM (4.0 mL) at -78 °C. After stirring for
10 min, the reaction mixture was neutralized with HCl 1M and extracted with DCM. The
organic extract was dried over Na2SO4, concentrated and fractionated by FCC (EtOAc-
hexane, 1:3) to yield aldehyde 46a (92 mg, 0.60 mmol, 74%). Acetyl chloride (25 µL, 0.35
mmol) was added dropwise to a solution of aldehyde 46a (48 mg, 0.31 mmol) and Et3N (43
µL, 0.31 mmol) in DCM (2.0 mL) at rt. After stirring for 30 min, HCl 1M was added and the
reaction mixture was extracted with DCM. The organic extract was dried over Na2SO4,
concentrated and separated by FCC (EtOAc-hexane, 1:3) to give compound 47a (56 mg, 0.29
mmol, 94%).
A solution of 15NH2OH.HCl (15N, 98%, 27 mg, 0.39 mmol) and NaOAc (32 mg, 0.39
mmol) in H2O (1 mL) was added to a solution of aldehyde 47a (50 mg, 0.26 mmol) in EtOH
(1.0 mL) at 0 °C. After stirring for 1 h, the reaction mixture was diluted with H2O and
extracted with EtOAc. The organic extract was dried over Na2SO4 and concentrated to yield
oxime 48a (50 mg, 0.24 mmol, 92%). NCS (35 mg, 0.26 mmol) was added in portions to a
solution of oxime 48a (50 mg, 0.24 mmol) and pyridine (0.15 mL) in DCM (1.5 mL) at rt.
After stirring for 30 min, a solution of 1-β-D-thioglucose tetraacetate (62 mg, 0.17 mmol) and
Et3N (100 µL, 0.72 mmol) in DCM (0.5 mL) was added and stirring was continued for 3 h.
The mixture was diluted with 5% H2SO4 and extracted with DCM. The organic extract was
dried over Na2SO4, concentrated and fractionated by FCC (EtOAc-hexane, 1:1) to yield
thiohydroximate 49a (60 mg, 0.10 mmol, 59%) as a white solid, mp 155-156 °C
Compound 49a: 1H NMR (600 MHz, CDCl3):  7.31 (1H, s), 5.25 (1H, t, J = 9.5 Hz),
5.09-5.04 (2H, m), 5.00 (1H, d, J = 10.0 Hz), 4.16-4.08 (2H, m), 3.74-3.71 (1H, m), 2.99 (2H,
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Pedras and To
t, J = 7.5 Hz), 2.90-2.85 (1H, m), 2.80-2.74 (1H, m), 2.30 (3H, s), 2.06 (3H, s), 2.03 (3H, s),
2
15
2.00 (3H, s), 1.94 (3H, s). HR-ESI-MS m/z [M+H]+: calc. for C25H29 H3 NO12S: 574.1798,
found 574.1790 (78%), 331.10 (100%).
Compound 49: white solid, mp 156-157 °C; 1H NMR (600 MHz, CDCl3):  7.33-7.30
(1H, m), 7.07 (1H, d, J = 7.0 Hz), 6.96-6.95 (2H, m), 5.25 (1H, t, J = 9.5 Hz), 5.09-5.04 (2H,
m), 5.00 (1H, d, J = 10.0 Hz), 4.15 (1H, dd, J = 12.5, 5.5 Hz), 4.09 (1H, dd, J = 12.5, 2.0
Hz), 3.74-3.71 (1H, m), 2.99 (2H, t, J = 7.5 Hz), 2.90-2.85 (1H, m), 2.80-2.74 (1H, m), 2.30
(3H, s), 2.05 (3H, s), 2.02 (3H, s), 2.00 (3H, s), 1.94 (3H, s). 13C NMR (125 MHz, CDCl3): 
170.8, 170.4, 169.8, 169.6, 169.4, 151.5, 151.0, 142.3, 129.8, 126.0, 121.7, 119.8, 80.0, 76.1,
73.9, 70.2, 68.1, 62.3, 34.2, 33.0, 21.4, 20.8, 20.8, 20.7. HR-ESI-MS m/z [M+H]+: calc. for
C25H32NO12S: 570.1640, found 570.1658 (90%), 331.11 (100%).
PySO3 (52 mg, 0.33 mmol) was added to a solution of thiohydroximate 49a (37 mg,
0.065 mmol) in dry DCM (2.0 mL). After stirring at 40 °C for 18 h, the reaction mixture was
diluted with H2O and extracted with MeOH-CHCl3 (1:4). The organic extract was dried over
Na2SO4, concentrated and fractionated by FCC (MeOH-DCM, 5:95) to yield compound 50a
(24 mg, 0.037 mmol, 57%) as a white solid, mp 138-139 °C.
1
Compound 50a: H NMR (600 MHz, CD3OD):  7.32 (1H, s), 5.35 (1H, t, J = 9.0
Hz), 5.28 (1H, d, J = 10.0 Hz), 5.01 (1H, t, J = 9.5 Hz), 4.97 (1H, dd, J = 9.5, 9.0 Hz), 4.13
(1H, dd, J = 12.5, 5.5 Hz), 4.09 (1H, dd, J = 12.5, 2.5 Hz), 3.96-3.93 (1H, m), 3.15-3.01 (4H,
m), 2.27 (3H, s), 2.03 (3H, s), 2.01 (3H, s), 1.97 (3H, s), 1.87 (3H, s). HR-ESI-MS m/z [M-
2
15
H]-: calc. for C25H27 H3 NO15S2: 652.1216, found 652.1236 (100%).
Compound 50: white solid, mp 135-136 °C; 1H NMR (600 MHz, CD3OD):  7.33 (1H,
t, J = 8.0 Hz), 7.20 (1H, d, J = 8.0 Hz), 7.09 (1H, t, J = 2.0 Hz), 6.96-6.94 (1H, m), 5.36 (1H,
t, J = 9.0 Hz), 5.29 (1H, d, J = 10.0 Hz), 5.01 (1H, t, J = 9.5 Hz), 4.97 (1H, dd, J = 9.5, 9.0
Hz), 4.14 (1H, dd, J = 12.5, 6.0 Hz), 4.09 (1H, dd, J = 12.5, 2.5 Hz), 3.97-3.94 (1H, m),
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Pedras and To
13
3.15-3.03 (4H, m), 2.28 (3H, s), 2.03 (3H, s), 2.02 (3H, s), 1.97 (3H, s), 1.87 (3H, s). C
NMR (150 MHz, CD3OD):  172.4, 171.6, 171.4, 171.4, 171.1, 159.1, 152.6, 144.0, 130.7,
127.4, 123.1, 120.8, 80.8, 76.9, 75.1, 71.4, 69.7, 63.6, 35.2, 34.0, 21.1, 20.7, 20.7. HR-ESI-
MS m/z [M-H]-: calc. for C25H30NO15S2: 648.1062, found 648.1061 (100%).
K2CO3 (12 mg, 0.087 mmol) was added to a solution of compound 50a (19 mg, 0.029
mmol) in MeOH (1.0 mL) at rt. After stirring for 30 min, the mixture was neutralized with
AcOH (ca. 1 drop), filtered and concentrated to yield a mixture of glucosinolate 12a (D ca.
92%, determined by 1H NMR) and sodium acetate (quantitative).
1
Compound 12a: HPLC tR = 8.2 min (method B). H NMR (600 MHz, D2O):  7.28
(1H, s), 4.86 (1H, d, J = 9.5 Hz), 3.87-3.85 (1H, m), 3.71-3.68 (1H, m), 3.52-3.43 (2H, m),
2
15
3.10-3.06 (4H, m). HR-ESI-MS m/z [M-K]-: calc. for C15H17 H3 NO10S2: 442.0693, found
442.0698, [M+4]+ (100%); [M+3]+ (20%). UV (HPLC, CH3CN – H2O) max (nm): 220, 275.
Compound 12: HPLC tR = 8.2 min (method B). 1H NMR (600 MHz, D2O):  7.27 (1H,
t, J = 8.0 Hz), 6.92 (1H, d, J = 8.0 Hz), 6.85 (1H, s), 6.80 (1H, d, J = 8.0 Hz), 4.85 (1H, d, J
= 9.5 Hz), 3.85 (1H, d, J = 12.5 Hz), 3.69 (1H, dd, J = 12.5, 5.0 Hz), 3.51-3.43 (2H, m),
13
3.09-3.00 (4H, m). C NMR (125 MHz, D2O):  163.4, 155.6, 142.5, 130.1, 120.8, 115.5,
113.5, 81.6, 80.1, 77.0, 71.8, 69.0, 60.5, 33.7, 32.5. HR-ESI-MS m/z [M-K]-: calc. for
C15H20NO10S2: 438.0534, found 438.0537 (100%). UV (HPLC, CH3CN – H2O) max (nm):
220, 275.
3.2.4 [2,3,4,5,6-D5]Phenylethyl isothiocyanate (13a)
CSCl2 (37 µL, 0.48 mmol) was added to a mixture of amine 28a (20 mg, 0.16 mmol),
Na2CO3 (34 mg, 0.32 mmol) in H2O (0.5 mL) and DCM (0.5 mL) at rt. After stirring for 15
min, the mixture was diluted with H2O and extracted with DCM. The organic extract was
dried over Na2SO4, concentrated, and fractionated by FCC (hexane, 1:9) to give
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Pedras and To
isothiocyanate 13a as an oil (19 mg, 0.11 mmol, 70% from nitrile 27a; D ≥ 99%, determined
by HR-FD-MS). All spectroscopic data was in agreement with reported data.8
Compound 13a: HPLC tR = 24.7 min (method A).1H NMR (500 MHz, CDCl3): 
2
3.73 (2H, t, J = 7.0 Hz), 3.00 (2H, t, J = 7.0 Hz). HR-FD-MS m/z [M]+: calc. for C9H4 H5NS:
168.0770, found 168.0774 (100%). UV (HPLC, CH
3
CN–H O) max (nm): 220, 250.
2
3.2.5 [2,3,5,6-D4]-4-Hydroxyphenylethyl isothiocyanate (14a)
A solution of [2,3,4,5,6-D5]nitrobenzene (29a) (D, 99%, 306 µL, 3.0 mmol) and
(phenylthio)acetonitrile (392 µL, 3.0 mmol) in DMSO (3.0 mL) was added dropwise over a 3
min period to a vigorously stirred suspension of powdered NaOH (1.20 g, 30 mmol) in
DMSO (3.0 mL) at rt.23// 1 After stirring for 30 min, the reaction mixture was poured into cold
HCl 1M (100 mL), extracted with CHCl3 and the organic extract was washed with H2O, dried
over Na2SO4 and concentrated. The crude residue was separated by FCC (Et2O-hexane, 1:1)
to yield [2,3,5,6-D4]-4-nitrophenylacetonitrile (30a) (327 mg, 1.97 mmol, 66%) as the major
product, and [2,3,5,6-D4]-2-nitrophenylacetonitrile (55 mg, 0.33 mmol, 11%). Pd/C (70 mg)
was added (under argon) to a solution of [2,3,5,6-D4]-4-nitrophenylacetonitrile (30a) (200 mg,
1.20 mmol) and acetic acid (345 µL, 6.0 mmol) in MeOH (5.0 mL) at rt. The mixture was
stirred under H2 (balloon) for 1 h and filtered. The filtrate was concentrated, diluted with H2O,
basified with NaOH 1M and extracted with CHCl3. The organic extract was dried over
Na2SO4 and concentrated to yield [2,3,5,6-D4]-4-aminophenylacetonitrile (31a) (150 mg,
1.10 mmol, 92%). A solution of NaNO2 (77 mg, 1.11 mmol) in H2O (0.5 mL) was added
dropwise to a suspension of [2,3,5,6-D4]-4-aminophenylacetonitrile (31a) (100 mg, 0.74
mmol) in H2SO4 25% (1.0 mL) at 0 ºC. After stirring for 10 min, urea (22 mg, 0.37 mmol)
was added and stirring was continued for another 10 min. The reaction mixture was added to
a vigorously stirred solution of CuSO4.5H2O (5.55 g, 22.2 mmol) in H2O (30 mL) at rt,
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Pedras and To
followed by the addition of Cu2O (212 mg, 1.48 mmol). After stirring for 1 h, the mixture
was extracted with EtOAc, dried over Na2SO4 and fractionated by FCC (MeOH-DCM, 2:98)
to yield [2,3,5,6-D4]-4-hydroxyphenylacetonitrile (32a) (71 mg, 0.52 mmol, 70%) as a yellow
solid. NaBH4 (304 mg, 8.0 mmol) was added in portions to a suspension of ZrCl4 (224 mg,
0.96 mmol) in THF (2.0 mL) at rt, followed by a solution of [2,3,5,6-D4]-4-
hydroxyphenylacetonitrile (32a) (110 mg, 0.80 mmol) in THF (2.0 mL). After stirring at rt
for 24 h, the mixture was filtered, the filtrate was concentrated and fractionated by FCC
(MeOH-DCM-NH4OH, 20:80:1) to give [2,3,5,6-D4]tyramine (33a) (104 mg, 0.74 mmol,
92%) as a white solid. CS2 (13 µL, 0.21 mmol) was added to a mixture of [2,3,5,6-
D4]tyramine (33a) (10 mg, 0.071 mmol) in EtOH (0.30 mL) at rt, followed by Et3N (10 µL,
0.071 mmol). After stirring for 30 min, the mixture was cooled to 0 ºC. A solution of (t-
Boc)2O (15 mg, 0.071 mmol) in EtOH (0.20 mL) was added, followed by a catalytic amount
of DMAP (ca. 0.5 mg). After stirring for 4 h, the mixture was concentrated and separated by
FCC (EtOAc-hexane, 1:4) to give isothiocyanate 14a (9.0 mg, 0.049 mmol, 63% from 32a; D
≥ 97%, determined by 1H NMR) as an oil.
1
Compound 14a: HPLC tR = 14.5 min (method A). H NMR (500 MHz, CDCl3): 
4.84 (1H, br), 3.68 (2H, t, J = 7.0 Hz), 2.92 (2H, t, J = 7.0 Hz). HR-FD-MS m/z [M]+ calc.
for C9H5D4NOS: 183.0656, found: 183.0658 (100%). UV (HPLC, CH3CN-H2O) max (nm):
224, 280.
3.2.6 [2,4,6-D3,15N]-3-Hydroxyphenylethyl isothiocyanate (15a)
D2SO4 (D, 99%, 0.40 mL) was added to a suspension of 3-(3-hydroxyphenyl)ethanoic
acid (34) (150 mg, 0.99 mmol) in CD3OD (D, 99.8%, 0.60 mL). The mixture was stirred at
60 °C for 1 h in a sealed vial. H2O was added and the mixture was extracted with DCM. The
organic extract was dried over Na2SO4 and concentrated to give ester 35a (160 mg, 0.93
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Pedras and To
mmol, 94%). DIBAL (25% wt. in toluene, 2.16 mL, 3.80 mmol) was added dropwise to a
solution of ester 35a (260 mg, 1.52 mmol) in dry DCM (5.0 mL) at -78 °C. After 15 min, the
mixture was quenched with HCl 1M and extracted with EtOAc. The organic extract was
dried over Na2SO4, concentrated and fractionated by FCC (EtOAc-hexane, 1:2) to yield
[2,4,6-D3]phenylacetaldehyde 36a (131 mg, 0.94 mmol, 62%) as the major product, plus a
minor product, [2,4,6-D3]-2-phenylethanol (52 mg, 0.37 mmol, 24%), resulting from
15
overreduction. A solution of NH2OH.HCl (15N, 98%, 99 mg, 1.41 mmol) and NaOAc (116
mg, 1.41 mmol) in H2O (2.0 mL) was added to a solution of aldehyde 36a (131 mg, 0.94
mmol) in EtOH (2.0 mL) at rt. After stirring for 1 h, the mixture was concentrated, diluted
with H2O and extracted with EtOAc. The organic extract was dried over Na2SO4 and
concentrated to give oxime 37a (135 mg, 0.87 mmol, 93%). NaBH4 (233 mg, 5.88 mmol)
was added in portions to a mixture of oxime 37a (130 mg, 0.84 mmol) and NiCl2.6H2O (238
mg, 1.0 mmol) in MeOH (3.0 mL) at 0 °C. After stirring for 15 min, the mixture was filtered
through celite, the filtrate was concentrated and fractionated by FCC (MeOH-DCM-NH4OH,
20:80:1) to yield amine 38a (75 mg, 0.53 mmol, 63%). CS2 (33 µL, 0.55 mmol) was added to
a solution of amine 38a (15 mg, 0.11 mmol) in EtOH (0.20 mL) at rt, followed by Et3N (15
µL, 0.11 mmol). After stirring for 1 h, the mixture was cooled to -10 ºC and a solution of (t-
Boc)2O (24 mg, 0.11 mmol) and DMAP (ca. 0.5 mg) in EtOH (0.10 mL) was added. Stirring
was continued for 2 h at -10 ºC, then 2 h at rt. The reaction mixture was concentrated and
fractionated by FCC (EtOAc-hexane, 1:3) to give isothiocyanate 15a (11 mg, 0.060 mmol,
55%; D ≥ 93%, determined by 1H NMR).
1
Compound 15a: HPLC tR = 14.9 min (method A). H NMR (500 MHz, CDCl3): 
7.21 (1H, s), 3.71 (2H, td, J = 7.0, 2.5 Hz), 2.94 (2H, td, J = 7.0, 3.5 Hz). HR-FD-MS m/z
2
15
[M]+: calc. for C9H6 H3 NOS: 183.0564, found 183.0564, [M+4]+ (100%); [M+3]+ (29%);
[M+2]+ (4%). UV (HPLC, CH3CN-H2O) max (nm): 220, 275.
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Pedras and To
3.2.7 Methyl [2,3,5,6-D4]-4-hydroxyphenylethyl dithiocarbamate (16a)
CS2 (10 µL, 0.17 mmol) was added dropwise to a suspension of [2,3,5,6-D4]tyramine
(33a) (20 mg, 0.14 mmol) in pyridine (0.30 mL) at 0 °C, followed by Et3N (40 µL, 0.28
mmol). After stirring at rt for 30 min, CH3I (10 µL, 0.17 mmol) was added and stirring was
continued for additional 15 min. The mixture was diluted with toluene and concentrated to
dryness. The crude mixture was fractionated by FCC (EtOAc-hexane, 1:2) to yield
dithiocarbamate 16a (22 mg, 0.097 mmol, 63%; D ≥ 98%, determined by HR-FD-MS) as a
yellow solid, mp 76-77 °C.
1
Compound 16a: HPLC tR = 11.8 min (method A). H NMR (500 MHz, CDCl3): 
7.06 (1H, br), 5.61 (1H, br), 3.95-3.91 (2H, m), 2.88 (2H, t, J = 7.0 Hz), 2.59 (3H, s), and a
minor rotamer at 7.76 (br), 3.65-3.62 (m), 2.85 (t), 2.68 (s). HR-FD-MS m/z [M]+: calc. for
2
C10H9 H4NOS2: 231.0690, found 231.0683 (100%). UV (HPLC, CH3CN-H2O) max (nm): 220,
250, 270.
4.2.8 [D3CS]Methyl [15N 2,4,6-D3]-3-hydroxyphenylethyl dithiocarbamate (17a)
CS2 (10 µL, 0.16 mmol) was added to a solution of amine 38a (11 mg, 0.078 mmol)
in pyridine (0.30 mL) at rt, followed by Et3N (32 µL, 0.23 mmol). After stirring for 15 min,
CD3I (10 µL, 0.16 mmol) was added and stirring was continued for another 15 min. The
mixture was diluted with 5% H2SO4 and extracted with EtOAc. The organic extract was dried
over Na2SO4, concentrated and fractionated by FCC (EtOAc-hexane, 1:3) to yield
1
dithiocarbamate 17a (15 mg, 0.064 mmol, 82%; D ≥ 92%, determined by H NMR) as a
gummy material.
1
Compound 17a: HPLC tR = 12.9 min (method A). H NMR (500 MHz, CDCl3): 
7.19 (1H, s), 6.90 (1H, td, J = 90, 5.5 Hz), 3.81 (1H, br), 4.01-3.97 (2H, m), 2.95-2.92 (2H,
m) and a minor rotamer at 7.65 (br), 7.47 (br), 3.7-3.69 (m). HR-FD-MS m/z [M]+: calc. for
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Pedras and To
2
15
C10H7 H6 NOS2: 234.0786, found 234.0797; [M+7]+ (100%); [M+6]+ (24%); [M+5]+ (3%).
UV (HPLC, CH3CN-H2O) max (nm): 220, 250, 270.
Compound 17: HPLC tR = 12.9 min (method A). 1H NMR (500 MHz, CDCl3):  7.18
(1H, t, J = 7.5 Hz), 7.06 (1H, br), 6.78-6.68 (3H, m), 5.59 (1H, br), 3.98-3.95 (2H, m), 2.91
(2H, t, J = 7.0 Hz), 2.59 (3H, s) and a minor rotamer at 7.74 (br), 5.70 (br), 3.69-3.65 (m),
13
2.87 (t), 2.68 (s). C NMR (125 MHz, CDCl3)  199.4, 156.1, 140.2, 130.2, 121.3, 115.8,
114.1, 48.0, 34.2, 18.3, and a minor rotamer at 202.2, 156.2, 139.2, 130.4, 121.1, 114.4, 47.4,
34.7, 19.2. HR-FD-MS m/z [M]+: calc. for C10H13NOS2: 227.0439, found 227.0445 (100%).
UV (HPLC, CH3CN-H2O) max (nm): 220, 250, 270.
3.2.9 [2,3,4,5,6-D5]-(E,Z)-Styryl isothiocyanate (18a/18a')
CSCl2 (117 µL, 1.53 mmol) was added to a mixture of compound 42a (100 mg, 0.51
mmol) and Na2CO3 (162 mg, 1.53 mmol) in H2O (2.0 mL) and DCM (2.0 mL) at rt. After
stirring for 1 h, the reaction mixture was diluted with H2O and extracted with DCM. The
organic extract was dried over Na2SO4, concentrated and fractionated by FCC (EtOAc-
hexane, 1:4) to yield isothiocyanate 43a (80 mg, 0.40 mmol, 78%). A solution of
isothiocyanate 43a (30 mg, 0.15 mmol) and Et3N (63 µL, 0.45 mmol) in dioxane (0.30 mL)
was stirred at 100 °C in a pressure tube. After 6 h, the reaction mixture was diluted with H2O
and extracted with DCM. The organic extract was dried over Na2SO4, concentrated and
fractionated by FCC (hexane) to yield β-styryl isothiocyanates (8.0 mg, 0.048 mmol, 48%) as
a mixture of two isomers (18a/18a', E/Z, ca. 5:1; D ≥ 99%, determined by HR-FD-MS) plus
isothiocyanate 43a (10 mg, 0.050 mmol, 33% recovered).
Compounds 18a/18a': HPLC tR = 28.7 min (E isomer), 27.2 (Z isomer) (method A).
1H NMR (500 MHz, CDCl3):  6.68 (1H, d, J = 14.0 Hz), 6.58 (1H, d, J = 14.0 Hz), 6.17 (1H,
2
d, J = 8.5 Hz), 6.14 (1H, d, J = 8.5 Hz). HR-FD-MS m/z [M]+: calc. for C9H2 H5NS:
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Pedras and To
166.0613, found 166.0608 (100%). UV (HPLC, CH3CN – H2O) max (nm): 220, 240, 300, 310.
3.2.10 Methyl [2,3,4,5,6-D5]-(E)-styryl dithiocarbamates (21a)
Sodium thiomethoxide (8.0 mg, 0.12 mmol) was added to a solution of isothiocyanate
18a/18a' (10 mg, 0.060 mmol) in EtOH (1.5 mL) at rt. After stirring for 15 min, the mixture
was diluted with H2O and extracted with EtOAc. The organic extract was dried over Na2SO4,
concentrated and separated by FCC (EtOAc-hexane, 5:95) to give methyl [2,3,4,5,6-D5] -(E)-
styryl dithiocarbamate (21a) (8.0 mg, 0.037 mmol, 62%; D ≥ 99%, determined by HR-FD-
MS) and [2,3,4,5,6-D5] -(Z)-styryl dithiocarbamate (21a´) (2.0 mg, 0.0093 mmol, 16%; D ≥
99%, determined by HR-FD-MS) as a minor product.
Compound 21a (E): white solid, mp 117-118 °C; HPLC tR = 23.1 min (method A).
1H NMR (500 MHz, CDCl3):  8.47 (1H, br), 8.13 (1H, br), 6.28 (1H, d, J = 13.5 Hz), 2.72
(3H, s), and a minor rotamer at 9.13 (br), 7.38 (br), 6.21 (br). HR-FD-MS m/z [M]+: calc. for
2
C10H6 H5NS2: 214.0647, found 214.0650 (100%). UV (HPLC, CH3CN – H2O) max (nm): 220,
290, 340.
Compound 21a´ (Z): HPLC tR = 24.5 min (method A). 1H NMR (500 MHz, CDCl3): 
8.75 (1H, br), 7.55 (1H, br), 5.97 (1H, br), 2.70 (3H, s) and a minor rotamer at 9.25 (br), 6.85
2
(br), 5.87 (br). HR-FD-MS m/z [M]+: calc. for C10H6 H5NS2: 214.0647, found 214.0655
(100%). UV (HPLC, CH3CN – H2O) max (nm): 220, 230, 360.
1
Compound 21 (E): white solid, mp 115-116 °C; HPLC tR = 23.1 min (method A). H
NMR (500 MHz, CDCl3):  8.44 (1H, br), 8.13 (1H, br), 7.37-7.22 (5H, m), 6.28 (1H, d, J =
13
14.0 Hz), 2.72 (3H, s), and a minor rotamer at 9.07 (br), 6.21 (br). C NMR (125 MHz,
CDCl3):  195.9, 135.5, 129.0, 127.6, 126.6, 126.2, 116.1, 18.6. HR-FD-MS m/z [M]+: calc.
for C10H11NS2: 209.0333, found 209.0326 (100%). UV (HPLC, CH3CN – H2O) max (nm):
220, 290, 340.
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Pedras and To
1
Compound 21 (Z): HPLC tR = 24.5 min (method A). H NMR (500 MHz, CDCl3): 
8.74 (1H, br), 7.55 (1H, br), 7.43-7.40 (2H, m), 7.32-7.29 (3H, m), 5.97 (1H, br), 2.70 (3H, s)
and a minor rotamer at 9.25 (br), 6.85 (br), 5.87 (br). HR-FD-MS m/z [M]+: calc. for
C10H11NS2: 209.0333, found 209.0328 (100%). UV (HPLC, CH3CN – H2O) max (nm): 220,
230, 360.
3.2.11 [2,3,4,5,6-D5]Dihydronasturlexin C (24a)
Et3N (25 µL, 0.18 mmol) was added to a solution of [2,3,4,5,6-D5]benzaldehyde (39a)
(D, 99.7%, 184 µL, 1.80 mmol) and CH3NO2 (192 µL, 3.60 mmol) in EtOH (0.40 mL) at rt.15
2 After stirring for 18 h, the reaction mixture was neutralized with AcOH (ca. 1 drop), diluted
with H2O and extracted with Et2O. The organic extract was dried over Na2SO4, concentrated
and fractionated by FCC (EtOAc-hexane, 1:4) to yield compound 40a (275 mg, 1.60 mmol,
89%) as an yellow oil. A solution of 40a (100 mg, 0.58 mmol) in MeOH (1.5 mL) was added
to a flask containing Pd/C (33 mg) (under argon) at rt. The reaction mixture was stirred under
H2 (balloon) for 3 h and filtered. The filtrate was concentrated to give amine 41a (80 mg,
0.56 mmol, 97%), which was used for the next step without further purification. Conc. HCl
(87 µL, 0.87 mmol) was added dropwise to a solution of amine 41a (80 mg, 0.56 mmol) in
isopropanol (1.0 mL) at 0 °C. The mixture was precipitated in Et2O and filtered to give the
corresponding ammonium salt (84 mg, 0.47 mmol). A solution of SOCl2 (65 µL, 0.90 mmol)
and DMF (10 µL) in CHCl3 (0.5 mL) was added to a suspension of the ammonium salt (80
mg, 0.45 mmol) in CHCl3 (2.0 mL) at 0 °C. After stirring at rt for 12 h, the reaction mixture
was concentrated and the residue was rinsed with Et2O to yield compound 42a (74 mg, 0.38
mmol, 68% from 41a) as a white solid. CS2 (46 µL, 0.76 mmol) was added to a suspension of
42a (74 mg, 0.38 mmol) in CHCl3 (1.5 mL) at rt, followed by Et3N (160 µL, 1.14 mmol).
After stirring for 15 min, CH3I (47 µL, 0.76 mmol) was added and the reaction mixture was
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Pedras and To
stirred at 60 °C for 3 h in a sealed vial. H2O was added and the reaction mixture was
extracted with CHCl3. The organic extract was dried over Na2SO4, concentrated, and
fractionated by FCC (EtOAc-hexane, 1:9) to yield nasturlexin 24a (67 mg, 0.31 mmol, 82%;
D ≥ 99%, determined by HR-FD-MS) as a yellow oil.
1
Compound 24a: HPLC tR = 20.6 min (method A). H NMR (500 MHz, CDCl3): 
5.09 (1H, dd, J = 8.5, 6.0 Hz), 4.54 (1H, dd, J = 15.0, 8.5 Hz), 4.35 (1H, dd, J = 15.0, 6.0
2
Hz), 2.59 (3H, s). HR-FD-MS m/z [M]+: calc. for C10H6 H5NS2: 214.0647, found 214.0645
(100%). UV (HPLC, CH
3
CN–H O) max (nm): 220, 260.
2
Compound 24: HPLC tR = 20.6 min (method A). 1H NMR (500 MHz, CDCl3):  7.36-
7.27 (5H, m), 5.10 (1H, dd, J = 8.5, 6.0Hz), 4.56 (1H, dd, J = 15.0, 8.5 Hz), 4.36 (1H, dd, J =
15.0, 6.0 Hz), 2.61 (3H, s). 13C NMR (125 MHz, CDCl3):  166.1, 141.1, 129.0, 128.1, 127.3,
72.3, 57.0, 15.5. HR-FD-MS m/z [M]+: calc. for C10H11NS2: 209.0333, found 209.0331
(100%). UV (HPLC, CH
3
CN–H O) max (nm): 220, 260.
2
4. Conclusion
In conclusion, an efficient one-pot deuterium exchange-esterification reaction was
devised to prepare several labeled intermediates containing a 3-hydroxylphenylethyl moiety.
Additional perdeuterated compounds, isothiocyanates, dithiocarbamates, glucosinolate, and
phytoalexins nasturlexins, were prepared from simple and commercially available
perdeuterated starting materials. All products were obtained in good yields and with high
deuterium content, as summarized in Table 1. These perdeuterated compounds will be used in
future work to probe the various biosynthetic relationships among potential intermediates of
nasturlexins using HPLC-MS techniques.
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Pedras and To
Supporting Information
13
1H and C NMR spectra for all new compounds (1a, 3a, 49a, 49, 50a, 50, 12a, 12,
14a-17a, 17, 21a, 21, 24a, 24, 35a, 45a) are published online as supplementary material
hosted within Wiley Online Library.
Acknowledgements
Financial support for the authors’ work was obtained from the Natural Sciences and
Engineering Research Council of Canada (Discovery Grant to M.S.C.P.), the Canada
Research Chairs program, Canada Foundation for Innovation, the Saskatchewan Government,
and the University of Saskatchewan (graduate assistantship to Q.H.T.). We acknowledge the
technical assistance of K. Thoms (MS) from the Department of Chemistry and Saskatchewan
Structural Sciences Centre.
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Pedras and To
References
1. Pedras MSC, To QH. Non-indolyl cruciferous phytoalexins: Nasturlexins and tridentatols,
a striking convergent evolution of defenses in terrestrial plants and marine animals?
Phytochemistry. 2015;113:57–63.
2. Pedras MSC, Alavi M, To QH. Expanding the nasturlexin family: Nasturlexins C and D
and their sulfoxides are phytoalexins of the crucifers Barbarea vulgaris and B. verna.
Phytochemistry. 2015;118:131–138.
3. Pedras MSC, Yaya EE, Glawischnig E. The phytoalexins from cultivated and wild
crucifers: chemistry and biology. Nat Prod Rep. 2011;28:1381–1405.
4. Bailey JA, Mansfield JW. Phytoalexins. Glasgow, U.K: Blackie and Son; 1982. 334 p.
5. Pedras MSC, Yaya EE. Dissecting metabolic puzzles through isotope feeding: a novel
amino acid in the biosynthetic pathway of the cruciferous phytoalexins rapalexin A and
isocyalexin A. Org Biomol Chem. 2013;11:1149–1166.
6. Hiegel GA, Lewis JC, Bae JW. Conversion of α‐amino acids into nitriles by oxidative
decarboxylation with trichloroisocyanuric acid. Synth Commun. 2004;34:3449–3453.
7. Itsuno S, Sakurai Y, Ito K. Reduction of some functional groups with zirconium
tetrachloride/sodium borohydride. Synthesis. 1988;(12):995–996.
8. Morrison JJ, Botting NP. The synthesis of [phenyl-2H5]gluconasturtiin and its metabolites
for metabolic studies. J Labelled Compd Radiopharm. 2005;48:897–907.
9. Makosza M, Winiarski J. Reaction of organic anions. 96. Vicarious substitution of
hydrogen in aromatic nitro compounds with acetonitrile derivatives. J Org Chem.
1980;45:1534–1535.
10. Boers RB, Randulfe YP, van der Haas HNS, van Rossum-Baan M, Lugtenburg J.
Synthesis and spectroscopic characterization of 1-13C- and 4-13C-plastoquinone-9. Eur J
Org Chem. 2002;(13):2094–2108.
11. Kim T, Kim Y-J, Han I-H, Lee D, Ham J, Kang KS, Lee JW. The synthesis of
sulforaphane analogues and their protection effect against cisplatin induced cytotoxicity
in kidney cells. Bioorg Med Chem Lett. 2015;25:62–66.
12. Ashton WT, Cantone CL, Meurer LC, Tolman RL, Greenlee WJ, Patchett AA, Lynch RJ,
Schorn TW, Strouse JF, Siegl PKS. Renin inhibitors containing C-termini derived from
mercaptoheterocycles. J Med Chem. 1992;35:2103–2112.
13. Atzrodt J, Derdau V, Fey T, Zimmermann J. The renaissance of H/D exchange. Angew
This article is protected by copyright. All rights reserved.

Pedras and To
Chem Int Ed. 2007;46:7744–7765.
14. Spencer ES, Dale EJ, Gommans AL, Rutledge MT, Vo CT, Nakatani Y, Gamble AB,
Smith RAJ, Wilbanks SM, Hampton MB, Tyndall JDA. Multiple binding modes of
isothiocyanates that inhibit macrophage migration inhibitory factor. Eur J Med Chem.
2015;93:501–510.
15. Beebe X, Schore NE, Kurth MJ. Polymer-supported synthesis of cyclic ethers:
Electrophilic cyclization of isoxazolines. J Org Chem. 1995;60:4196–4203.
16. von Reuß SH. Structure elucidation and synthesis of new secondary metabolites from
Liverworts and microorganisms and investigation of their biogenesis. Ph.D. Thesis.
University of Hamburg; 2009. 248 p.
17. Kutschy P, Achbergerová I, Dzurilla M, Takasugi M. Synthesis of indole phytoalexins
brassinin
and
cyclobrassinin
via
[1-(tert-butoxycarbonyl)indol-3-yl]-methyl
isothiocyanate as the key biomimetic intermediate. Synlett. 1997;(3):289–290.
18. Kniežo L, Kristian P. Synthesis, structure, and properties of β-styryl isothiocyanate. Chem
zvesti. 1974;28:848–853.
19. Rollin P, Tatibouët A. Glucosinolates: The synthetic approach. C R Chim. 2011;14:194–
210.
20. Zhang Q, Lebl T, Kulczynska A, Botting NP. The synthesis of novel hexa-13C-labelled
glucosinolates from [13C6]-D-glucose. Tetrahedron. 2009;65:4871–4876.
21. Pedras MSC, To QH, Schatte G. Divergent reactivity of an indole glucosinolate yields
Lossen or Neber rearrangement products: the phytoalexin rapalexin A or a unique β-D-
glucopyranose fused heterocycle. Chem Commun. 2016;52:2505–2508.
22. Agerbirk N, Olsen CE, Heimes C, Christensen S, Bak S, Hauser TP. Multiple
hydroxyphenethyl glucosinolate isomers and their tandem mass spectrometric distinction
in a geographically structured polymorphism in the crucifer Barbarea vulgaris.
Phytochemistry. 2015;115:130–142.
23. Makosza M, Winiarski J. Vicarious nucleophilic substitution of hydrogen in nitroarenes
with α-substituted nitriles and esters. Direct α-cyanoalkylation and α-
carbalkoxyalkylation of nitroarenes. J Org Chem. 1984;49:1494–1499.
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Table 1 Perdeuterated products synthesized in this work and yields.
Deuterated product / starting material
% Yield rT – HPLC method
[2,3,4,5,6-D5]Phenylethyl isothiocyanate (13a) / [2,3,4,5,6-
D5]Phenylalanine (26a)
68
70
27
27
19
28
48
34
22
14
21
24.7 min – A
21.5 min – A
14.5 min – A
11.8 min – A
14.9 min – A
12.9 min – A
20.6 min – A
23.6 min – A
[2,3,4,5,6-D5]Nasturlexin A (1a) / [2,3,4,5,6-
D5]Phenylalanine (26a)
[2,3,5,6-D4]-4-Hydroxyphenylethyl isothiocyanate (14a) /
[2,3,4,5,6-D5]nitrobenzene (29a)
Methyl [2,3,5,6-D4]-4-Hydroxyphenylethyl dithiocarbamate
(16a) / [2,3,4,5,6-D5]nitrobenzene (29a)
[2,4,6-D3,15N]-3-Hydroxyphenylethyl isothiocyanate (15a) /
3-hydroxyphenylethanoic acid (34)
[D3CS]Methyl [2,4,6-D3,15N ]-3-hydroxyphenylethyl
dithiocarbamate (17a) / 3-hydroxyphenylethanoic acid (34)
[2,3,4,5,6-D5]Dihydronasturlexin C (24a) / [2,3,4,5,6-
D5]benzaldehyde (39a)
[2,3,4,5,6-D5]Nasturlexin C (3a) / [2,3,4,5,6-D5]benzaldehyde
(39a)
[2,3,4,5,6-D5]-(E, Z)-Styryl isothiocyanate (18a) / [2,3,4,5,6-
D5]benzaldehyde (39a)
28.7 (E), 27.2 (Z)
min – A
Methyl [2,3,4,5,6-D5]-(E)-styryl dithiocarbamate (21a) /
[2,3,4,5,6-D5]benzaldehyde (39a)
[2,4,6-D3,15N ]-3-Hydroxyphenylethyl glucosinolate (12a) /
3-(3-hydroxy)phenylpropanoic acid (44)
23.1 min – A
8.2 min – B
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Pedras and To
Fig. 1. Chemical structures of the phytoalexins nasturlexins 1–4, 6, 7, tridentatol C (5) and
brassinin (8).
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Pedras and To
Scheme 1. Proposed biosynthetic precursors and intermediates of nasturlexins 1–3, 6
and tridentatol C
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Pedras and To
D
D
D
D
D
D
CO2
NH3
TCICA, py,
MeOH
D
D
CN
D
97%
D
26a
27a
NaBH4, ZrCl4,
THF
D
D
D
CSCl2, Na2CO3
H2O, DCM
D
D
NCS
D
NH2
D
D
D
70% (from 27a)
D
13a
28a
1. CS2, Et3N, py
2. CH3I
72% (from 27a)
S
SCH3
NH
D
D
D
D
D
1a
Scheme 2. Synthesis of [2,3,4,5,6-D5]nasturlexin A (1a) and [2,3,4,5,6-
D5]phenylethyl isothiocyanate (13a).
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Pedras and To
D
D
D
D
D
PhSCH2CN, NaOH
DMSO
Pd/C, H2
MeOH, AcOH
D
D
D
D
CN
CN
O2N
D
H2N
D
66%
92%
O2N
D
D
30a
29a
31a
1. NaNO2, H2SO4
2.CuSO4.5H2O,
Cu2O, H2O
70%
D
D
D
1. CS2, Et3N, EtOH
2. (t-Boc)2O, DMAP
D
NCS
NaBH4, ZrCl4
THF
D
NH2
D
CN
HO
D
D
D
63% (from 32a) HO
HO
D
14a
D
D
33a
1. CS2, Et3N, py
2. CH3I
32a
63% (from 32a)
S
SCH3
NH
D
D
HO
D
D
16a
Scheme 3. Synthesis of [2,3,5,6-D4]-4-hydroxyphenylethyl isothiocyanate (14a) and methyl
[2,3,5,6-D4]-4-hydroxyphenylethyl dithiocarbamate (16a).
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