First synthesis of (?)-altenuene-D3 suitable as internal standard for isotope dilution mass spectrometry

Article abstract of DOI:10.3390/molecules24244563

Metabolites from Alternaria fungi exhibit a variety of biological properties such as phytotoxic, cytotoxic, or antimicrobial activity. Optimization of a literature procedure culminated in an efficient total synthesis of (?)-altenuene as well as a stable isotope-labeled derivative suitable for implementation in a LC-MS/MS method for mycotoxin analysis.

Full text of DOI:10.3390/molecules24244563

molecules
Article
First Synthesis of (−)-Altenuene-D3 Suitable as
Internal Standard for Isotope Dilution
Mass Spectrometry
Michael A. Sebald 1,*, Julian Gebauer 2, Thomas Sommerfeld 3 and Matthias Koch 3
1
HPC Standards GmbH, Am Wieseneck 7, D-04451 Borsdorf, Germany
AnalytiCon Discovery GmbH, Hermannswerder Haus 17, D-14473 Potsdam, Germany;
2
j.gebauer@ac-discovery.com
Bundesanstalt für Materialforschung und –prüfung, Abteilung Analytische Chemie, Referenzmaterialien,
3
Richard-Willstätter-Strasse 11, D-12489 Berlin-Adlershof, Germany; thomas.sommerfeld@bam.de (T.S.);
matthias.koch@bam.de (M.K.)
*
Correspondence: sebald@hpc-standards.com; Tel.: +49-(0)34291-337236
Academic Editor: Ari Koskinen
Received: 11 November 2019; Accepted: 10 December 2019; Published: 12 December 2019
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: Metabolites from Alternaria fungi exhibit a variety of biological properties such as
phytotoxic, cytotoxic, or antimicrobial activity. Optimization of a literature procedure culminated
in an efficient total synthesis of ( )-altenuene as well as a stable isotope-labeled derivative suitable
−
for implementation in a LC-MS/MS method for mycotoxin analysis.
Keywords: altenuene; Alternaria mycotoxins; food safety; isotope-labeled; SIDA-LC-MS/MS;
Suzuki coupling
1. Introduction
1.1. Alternaria Mycotoxins
Invading crops at the pre- and post-harvest stage, certain filamentous fungi can contaminate
food and feedstuff by producing a variety of toxic secondary metabolites, which are referred to as
mycotoxins [1]. Derived from the extremely wide-spread genus, Alternaria, the commonly named
Alternaria toxins [2,3] are frequently found in agricultural crops, such as grains, fruits, and vegetables,
as well as soil, wall papers, and textures, and have thus been implicated in several animal and
human health disorders. The European Food Safety Authority (EFSA) assessed the risks for public
health related to the presence of Alternaria toxins in food and feed. Although the toxicological data
on various Alternaria toxins was limited, it recommended the supervision of those toxins in foods and
feeds [
4]. Considering the need for possible future regulation, a suitable LC-MS/MS standard method
for the determination of the most relevant Alternaria metabolites is required (Figure 1).
Figure 1. Relevant Alternaria mycotoxins.
Molecules 2019, 24, 4563; doi:10.3390/molecules24244563
www.mdpi.com/journal/molecules

Molecules 2019, 24, 4563
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With ( )-altenuene (ALT, 1a) [5] being the most acutely toxic in mice (LD50 > 50 mg/kg) [4],
−
the development of new analytical methods for its detection and quantification has become of great
importance for human and animal health risk assessment. Due to its high selectivity, sensitivity, and
multi-analyte suitability, LC-MS/MS has become the method of choice for trace analysis of mid polar
and polar organic contaminants in food and feed. As a major step forward in the improvement of
accuracy (trueness and precision), isotope labeled compounds (mostly 13C-, 2D- or 15N-labeling) are
widely used as internal standards to quantify the target analytes by LC-MS/MS. This so-called stable
isotope dilution assay (SIDA, SIDA-LC-MS/MS) is considered the primary ratio method, representing
a high level of metrology. Thus, the development of isotope labeled standards for mycotoxins and
their implementation in LC-MS/MS methods have received much attention over recent years [6].
Moreover, 1a has recently been reported to exhibit interesting cytotoxic activity against HCT116
cell lines with an IC50 value of 3.13
µM. This makes it a potential lead compound for the development
of new anti-tumor drug candidates [
7
], which further supported our interest in working towards
an efficient and reliable access to ALT (1a) and ALT-D3 (1b) by total synthesis.
1.2. Retrosynthetic Analysis
Based on the only total synthesis of 1a reported to date [
preparation of a deuterated ( )-altenuene derivative (ALT-D3, 1b) following an improved procedure.
To prevent any ‘cross talk’ between the native and the labeled analyte we aimed to synthesize ALT-D3
1b) by coupling the deuterated boronate 4b and halogenated allylic alcohol 5b, which should permit
8], we herein wish to present the first
−
(
a facile isotope incorporation using commercially available reagents in an analogous fashion as
described for the native compound 1a (Scheme 1) [8].
Scheme 1. Retrosynthetic analysis of (-)-altenuene (1a) and (-)-altenuene-D3 (1b).
2. Results and Discussion
2.1. Synthesis of Deuterated Boronate 4b
The synthesis of the deuterated boronic acid derivative 4b was efficiently achieved in analogy to
a literature procedure [9] but starting rather with a regioselective alkylation of the 4-hydroxy group
using commercial iodomethane-D3 (99.5atom% D) as the deuterium source (Scheme 2). The slightly
lower yield compared to the Mitsunobu reaction employed by Podlech et al. is compensated by
the simplicity and the low cost of the reagents used in this protocol.
O
O
O
O
CD3I, K2CO3
O
O
a + b
O
O
O
O
acetone, reflux
60%
D3CO
B
74%
D3CO
OH
O
HO
OH
6
7
4b
◦
a Tf2O, pyridine, 0 C – r.t. (82%),
Scheme 2. Synthesis of boronate 4b. Reagents and conditions:
b PinBH, NEt3, Pd(PPh3)4, dioxane, 80 ◦C (73%).

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2.2. Synthesis of Bromo Alcohol 5b
A major drawback in the original synthesis of 1b is the unfavorable diastereoselectivity of ~1:6
in the Grignard reaction affording the required tertiary alcohol 5a (Scheme 1) [ ]. In order to tackle this
8
issue, we first decided to optimize the key carbonyl addition reaction. With the labile iodo substrate
10a limiting the number of applicable reagents, we envisioned the usage of the much more stable
bromo enone derivative 10b, which should allow us to investigate other organometallic reagents.
Thus, bromination of the known enone
according to the literature, gave bromo enone 10b with an excellent overall yield (Scheme 3).
9
obtained in 4 steps from inexpensive D-( )-quinic acid (8)
−
O
O
HO CO2H
Br
[10]
Br2, NEt3
O
O
77%
HO
OH
DCM
86%
O
OMe
O
OMe
OH
MeO
MeO
D-(-)-quinic acid
8
9
10b
Scheme 3. Synthesis of the bromo enone 10b; for the synthesis of compound 9 see [10].
We then focused on the optimization of the inefficient methylation of halo enones 10a and
10b (Table 1). Applying the original reaction conditions (Table 1, Entry 1) to the bromo enone 10b
provided a ~1:4 mixture of 5b and the undesired isomer epi-5b (Table 1, Entry 2). A further refinement
to a ~1:2 ratio could be achieved by the addition of stoichiometrical amounts of CeCl3 (Table 1,
Entry 3). Interestingly, changing from the methyl Grignard reagent to the more reactive methyllithium,
the diastereoselectivity changed completely with the desired carbinol 5b now being the major isomer
(d.r. ~ 1.4:1, Table 1, Entry 6). While the addition of CeCl3 proved to be disadvantageous in this case
(Table 1, Entry 7), lowering the reaction temperature to –78 ◦C resulted in the best diastereomeric ratio
obtained with ~1.7:1 in favor of 5b (Table 1, Entry 8) but came at the cost of incomplete conversion
of the starting material 10b. Applying the same conditions to iodo enone 10a only nonspecific
decomposition was observed as expected (Table 1, Entry 9), emphasizing the initially proposed
enhanced chemical stability of the bromo derivative 10b.
Table 1. Screening of methylation conditions of halo enones 10a and 10b.
Conditions [a]
d.r. 5/epi-5 [b]
Entry
X
M
1
2
3
4
5
6
7
8
9
10
11
12
13
14
I
MgI
MgI
MgI
MgBr
MgBr
Li
Li
Li
Li
AlMe3
AlMe3
DABAL
ZnMe2
ZnMe2
−40 ◦C / THF
~1:6
~1:4
~1:2
Br
Br
Br
Br
Br
Br
Br
I
Br
Br
Br
Br
Br
−40 ◦C / THF
−40 ◦C / CeCl3 / THF
r.t. / Fe-Josiphos / CuBr-SMe2 / MTBE
−78 ◦C / Fe-Josiphos CuBr-SMe2 / THF
−40 ◦C / THF
only 1,4-addition
1,4-addition + traces of 5b
~1.4:1
−40 ◦C / CeCl3 / THF
~1:1
1.7:1
−78 ◦C / THF
−78 ◦C / THF
decomposition
~1:3
only 1,4-addition
no reaction
no reaction
traces
0 ◦C / THF
0 ◦C / [Rh(cod)Cl2]2 / BINAP (rac.) / THF / n-heptane
0 ◦C / [Rh(cod)Cl2]2 / BINAP (rac.) / THF / n-heptane
r.t. / THF
r.t. / Ti(iPrO)4 / toluene
[a]
[b]
0.1 mmol scale.
determined by GC-MS analysis of the crude mixture; retention times: tR (5a) = 17.20 min,
tR (5b) = 17.05 min (separation conditions are given in Section 3, Materials and Methods).

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Any attempts to further improve the diastereoselectivity of the 1,2-addition by employing
either chiral catalytic systems (e.g., BINAP + Josiphos, which is actually known to promote
1,4-addition [11]) and/or alternative methyl donating reagents (e.g., AlMe3, DABAL, ZnMe2) [12–14]
resulted in the formation of either the 1,4-addition product or only traces of the desired alcohol 5b
(Table 1, Entries 4,5,10–14).
On a preparative more useful scale, the newly established conditions (Table 1, Entry 8) delivered
the requisite bromo alcohol 5b with an essentially improved yield of 38% after chromatographic
separation of both isomers by MPLC. With substantial amounts of the labeled boronate 4b and
the crucial alcohol 5b in hand we started assembling the pieces.
2.3. Suzuki Coupling of Bromo Alcohol 5b and Boronate 4b
Alcohol 5b and boronate 4b were subsequently subjected to the reported Suzuki cross coupling
conditions (Pd(OAc)2, S-Phos, Cs2CO3, dioxane/H2O, 80 ◦C) [
8], approved for the arylation of iodo
alcohol 5a. Unfortunately, this test almost exclusively resulted in homodimerization of the boronic
ester 4b. After thoroughly scouring the literature for alternative catalytic systems, which would
permit the desired hetero coupling to proceed, we were delighted to find that a simple alteration of
catalyst and base (Pd(dppf)Cl2, NEt3, THF/H2O, 70 ◦C) [15] facilitated a smooth conversion, providing
the advanced intermediate 11b with 81% isolated yield (Scheme 4).
Scheme 4. Revised endgame to ALT-D3 (1b): a boronate (R = Me: 4a; R = CD3: 4b), NEt3,
Pd(dppf)Cl2·CH2Cl2, THF/H2O, 70 ◦C (81%).
Interestingly, only traces of concomitant lactonization product 12b were observed illustrating
the remarkable mildness of this adjusted protocol. Cyclization using K2CO3 in methanol and cleavage
of the bisketal protecting group by refluxing in aqueous AcOH finally gave rise to 1b in 81% yield after
two steps and purification by preparative HPLC (purity > 99.9% and > 99atom% D). Lastly, submitting
alcohol 5b and the unlabeled boronate 4a (synthesized analogously using iodomethane [
9]) to the same
reaction sequence delivered ALT (1a) with identical yields.
2.4. Implementation of the ALT-D3 Standard (1b) in a LC-MS/MS Method
There are some recent LC-MS/MS methods available for the quantification of Alternaria toxins
including ALT (1a) in food and feed based on positive or negative ionization mode as well using acidic
or alkaline LC conditions [16
conditions was used to set up/optimize the MS/MS and LC parameters of the synthesized ALT-D3
1b) (Table 2). HPLC: Agilent 1200 with autosampler; column: Eurospher 100-5 C18 P, particle size
m, 250 4 mm (Knauer, Berlin, Germany); Inj.-vol: 10
L; oven temp.: 30 ◦C; flow: 0.5 mL/min;
–
18]. Due to better performance data, ESI(
−
) mode under alkaline LC
(
5
µ
×
µ
eluent A: water with 5 mM ammonium acetate and ammonium hydroxide (pH 8.7), eluent B: Methanol
with 5 mM ammonium acetate. 0–5 min 90% A, 5–22 min 0% A, 22–30 min 90% A. MS/MS: AB-Sciex
QTrap4000; turbo ion spray, single reaction monitoring (SRM; negative polarity); TEM: 500 ◦C, CUR:
50 a.u., CAD: 12 a.u., IS: −2000 V; DP: −60 V; CE: −40 V; CXP: −10 V.

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Table 2. Recorded mass transitions for native (1a) and isotope labeled ALT (1b).
Q3 Mass (Da)
Quantifier
Q3 Mass (Da)
Qualifier
Substance
Q1 Mass (Da)
ALT (1a)
ALT-D3 (1b)
291.0
294.0
203.0
203.0
248.0
248.0
ALT-D3 (1b) does not show any signals for the mass transition of native ALT (1a) and vice
versa with identical retention times (tR (1a/1b) = 16.67 min, Figure 2). This is important for ideal
compensation of ionization effects (mostly matrix suppression effects) and the use of ALT-D3 (1b) as
internal standard. Moreover, the presented SIDA-LC-MS/MS method does not only allow the analysis
of ALT (1a) and ALT-D3 (1b) but is also applicable to other relevant Alternaria toxins, e.g., alternariol
(2a), alternariol monomethyl ether (2b), tentoxin or tenuazonic acid (3).
Figure 2. HPLC-MS/MS runs of (
a) synthesized ALT-D3
(
1b) and (
b) native ALT (1a) standards.
Displayed are the selected ion chromatograms (SRM(
ALT-D3 (red) and ALT (blue).
−)-mode) of the quantifier mass transitions for

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3. Materials and Methods
Commercial chemicals and solvents were used as received without any further purification.
Triethylamine was dried over KOH, distilled in vacuo, and stored under an atmosphere of nitrogen.
All reactions were carried out under an inert gas atmosphere using dry grade reagents and solvents
unless stated otherwise. Reactions were monitored by thin-layer chromatography on Merck TLC Silica
gel 60 F254 sheets with UV-visualization (254 nm and 336 nm) or KMnO4 staining. The diastereomeric
ratios of the compounds
Science column, 25 m
5
×
and epi-5 were determined using a hp Series II 5860 GC device (SGE Analytical
0.22 m, BP 0.25 m, HP Inc., Palo Alto, CA, USA; Trajan Scientific,
µ
×
5
×
µ
Ridgewood, Victoria, Australia) connected to a hp 5971 Series mass selective detector. Melting points
were determined with the MP 90 melting point device by Mettler Toledo (Columbus, OH, USA).
¯
MPLC purification was performed with a Shimadzu MPLC system (Shimadzu Corp., Kyoto, Japan).
The conditions and devices used for LC-MS/MS analysis of compounds 1a and 1b are stated in Section 2.4.
ESI-high resolution mass spectra were recorded with a Bruker Daltonik micrOTOF coupled with a LC
Packings Ultimate HPLC system (Bruker Corp., Billerica, MA, USA; Dionex/LC Packings, Sunnyvale,
CA, USA). NMR spectra were either recorded on a Varian Mecury Plus 300 (300.8 MHz), Varian
Mercury Plus 400 (399.95 MHz), or a Bruker Avance III HD (400.13 MHz) spectrometer (Varian/Agilent
Technologies Inc., Santa Clara, CA, USA; Bruker Corp., Billerica, MA, USA). All signals were referenced
to the respective solvent signals reported in the literature [19]. All coupling constants J refer to
hydrogen–hydrogen interactions unless stated otherwise. The 1H and 13C NMR spectra of all new
compounds as well as UV/Vis- and IR-spectra of the native and labeled natural products can be found
in the Supplementary Materials.
3.1. Synthesis of (2S,3S,4aR,8aR)-7-bromo-2,3,4a,5-tetrahydro-2,3-dimethoxy-2,3-dimethylbenzo[b][1,4]dioxin-
6(8aH)-one (10b)
Enone 9 (2.91 g, 12.0 mmol, 1.00 equiv) was dissolved in 32 mL DCM in a 250 mL round-bottom
◦
flask and cooled to 0 C. A solution of 632
µL (1.96 g, 12.3 mmol, 1.02 equiv) bromine in 32 mL
DCM was added slowly over 1 h with a dripping funnel and the mixture was stirred for another
30 min at the same temperature. After that, 2.85 mL (1.96 g, 20.4 mmol, 1.70 equiv) NEt3 was added
and the resulting blue solution was warmed to room temperature while stirring. After 1 h GC-MS
analysis indicated the complete consumption of the starting material and the reaction was quenched
with NaHCO3 (100 mL) and the phases were separated. The aqueous phase was extracted with
DCM (3
×
75 mL), and the combined organic phases were washed with brine (50 mL), dried over
Na2SO4, and the solvent was evaporated. The crude product was purified by chromatography using
a CH/EE-gradient (15
→
25% EE) yielding 3.3 g (86%) of the title compound 10b as a white solid.
Rf (CH/EE, 4:1) = 0.38. MP = 202 ◦C (decomposition). HR-MS: Calc. for [M + Na]+ = 343.0152, found:
1
343.0161. H-NMR (300 MHz, CDCl3):
δ = 1.32 (s, 3 H), 1.36 (s, 3 H), 2.59 (dd, J = 13.5, 16.4 Hz, 1 H),
2.94 (dd, J = 4.8 Hz, 16.4 Hz, 1 H), 3.26 (s, 3 H), 3.31 (s, 3 H), 4.07 (ddd, J = 4.8, 9.1, 13.5 Hz, 1 H),
4.48 (ddd, J = 0.3, 2.0, 9.1 Hz, 1 H), 7.30 (dd, J = 0.3, 2.0 Hz, 1 H). 13C-NMR (75 MHz, CDCl3):
δ = 17.7,
17.8, 41.3, 48.4, 48.5, 67.6, 70.4, 100.0, 101.1, 124.4, 148.9, 188.8.
3.2. Synthesis of (2S,3S,4aR,6R,8aR)-7-Bromo-2,3,4a,5,6,8a-hexahydro-2,3-dimethoxy-2,3,6-trimethylbenzo[b]
[1,4]dioxin- 6-ol (5b)
To a solution of bromo enone 10b (1.0 g, 3.1 mmol, 1.0 equiv) in 62 mL dry THF (0.05 M) at
78 ◦C
−
was added methyllithium (1.6 M in Et2O, 3.0 mL, 4.8 mmol 1.5 equiv) and the reaction mixture was
slowly warmed to r.t. over 1 h. Saturated NH4Cl-solution (40 mL) was added and the aqueous phase
was extracted with MTB (3
was evaporated. Purification of the crude product by normal-phase chromatography (YMC-Gel, 6 nm
S-15 m) using MTB/heptane (3:7) and evaporation of the solvents yielded 374 mg (38%) of alcohol 5b
as a light yellow solid. Rf (n-Hep/MTBE, 7:3) = 0.18. GC-MS: [M
OMe]+ = 305/307, tR = 17.05 min.
MP = 73.9 ◦C. HRMS (ESI): Calc. [M + Na]+ = 359.0465, found: 359.0475. [α]D22(c = 0.1, CHCl3) = +9.
×
50 mL). The combined organic phases were dried (Na2SO4) the solvent
µ
−

Molecules 2019, 24, 4563
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1H-NMR (400 MHz, CDCl3):
δ
= 1.30 (s, 3 H), 1.33 (s, 3 H), 1.42 (s, 3 H), 1.83 (t, J = 13.1 Hz, 1 H),
2.11 (s, 1 H), 2.22 (dd, J = 3.6, 13.3 Hz, 1 H), 3.26 (s, 3 H), 3.27 (s, 3 H), 3.93 (ddd, J = 3.6, 8.9, 12.7 Hz,
1 H), 4.10 (dd, J = 1.7, 8.9 Hz, 1 H), 6.04 (d, J = 1.8 Hz, 1 H). 13C-NMR (101 MHz, CDCl3):
δ = 17.90,
17.94, 30.5, 40.1, 48.1, 48.2, 65.4, 70.6, 73.5, 100.2, 100.7, 130.5, 131.3.
3.3. Synthesis of 5-Hydroxy-7-methoxy-2,2-dimethyl-4H-benzo[d][1,3]dioxin-4-one-D3 (7)
1.01 g (4.81 mmol, 1.00 equiv) 5,7-dihydroxy-2,2-dimethyl-4H-benzo[d][
731 mg (5.29 mmol, 1.10 equiv) K2CO3 were suspended in 15 mL acetone (0.2 M) in a 100 mL screw-top
flask. After stirring at room temperature for 10 min, the flask was charged with 330 l (767 mg,
1,3]dioxin-4-one 6 and
µ
5.29 mmol, 1.10 equiv) CD3I and the mixture was refluxed for 3 h in the sealed vessel. The suspension
was cooled to room temperature, acetone was removed in vacuo, and the residue suspended in 100 mL
EtOAc. The suspension was washed with water (3
with EtOAc (2 30 mL). The combined organic phases were washed with brine (1
sodium sulfate and the solvent was removed. Normal-phase chromatography with PE/EE 9:1 (v/v)
delivered 804 mg (74%) of compound
as an off-white solid. Rf (CH/EE, 4:1) = 0.36. MP = 102.9 ◦C.
×
50 mL), and the aqueous phase was extracted
×
×
50 mL), dried over
7
1H-NMR (400 MHz, CDCl3):
δ = 1.73, (s, 6 H), 6.00 (d, J = 2.3 Hz, 1 H), 6.14 (d, J = 2.3 Hz, 1 H),
10.44 (s, 1 H). 13C-NMR (101 MHz, CDCl3): δ = 25.8, 93.2, 94.8, 95.9, 107.0, 157.0, 163.3, 165.3.
3.4. Synthesis of 7-Methoxy-2,2-dimethyl-4-oxo-4H-benzo[d][1,3]dioxin-5-yl trifluoromethanesulfonate-D3
476
µl (796 mg, 2.82 mmol, 1.50 equiv) Tf2O was added to an ice-cold solution of 428 mg
(1.88 mmol, 1.00 equiv) of phenol
7
in 3.8 mL dry pyridine (0.5 M) dropwise over 5 min, and
the mixture was stirred at 0 ◦C. After 1 h, the GC-MS analysis indicated the complete consumption of
the starting material. The solvent was removed in vacuo, and the residue was dissolved in 100 mL
EtOAc. The solution was washed with CuSO4 (4% in H2O, 2
×
50 mL), H2O (2
×
50 mL), and brine
(1 50 mL). The combined organic phases were dried over Na2SO4, and the solvent was evaporated.
×
Chromatography with cyclohexane/acetone 7:1 (v/v) yielded 628 mg (82%) of the title compound
as a pale yellow solid upon removal of the solvents. Rf (CH/EE, 4:1) = 0.19. MP = 58.5 ◦C. HRMS
1
(ESI): Calc. [M + Na]+ = 382.0258, found: 382.0282. H-NMR (400 MHz, CDCl3):
δ
= 1.74 (s, 6 H),
6.48 (d, J = 2.4 Hz, 1 H), 6.53 (d, J = 2.4 Hz, 1 H)
.
13C-NMR (101 MHz, CDCl3):
δ
= 25.7, 101.3, 105.5,
106.7, 117.3, 120.5, 150.1, 157.2, 159.0, 165.7.
3.5. Synthesis of 7-methoxy-2,2-dimethyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-4H-benzo[d]
[1,3]dioxin-4-one-D3 (4b)
7-methoxy-2,2-dimethyl-4-oxo-4H-benzo[d][
1.12 mmol, 1.00 equiv) was dissolved in a solution of 470
distilled NEt3 in 10 mL dry dioxane, and the mixture was degassed with N2 for 15 min. Then 65 mg
(56 mol, 5.0 mol%) Tetrakis(triphenylphosphin)palladium and 485 L (432 mg, 3.37 mmol, 3.00 equiv)
1,3]dioxin-5-yl trifluoromethanesulfonate-D3 (404 mg,
µL (341 mg, 3.37 mmol, 3.00 equiv) freshly
µ
µ
4,4,5,5-Tetramethyl-1,3,2-dioxaborolane were added successively, and the mixture was stirred at
80 ◦C for 2 h. After cooling down, the solvent was evaporated, and the crude product was directly
submitted to manual flash chromatography with toluene/acetone 19:1 (v/v) yielding 277 mg (73%)
of the title compound
4 as an orange solid without any impurification by the reduced side product.
Rf (CH/EE, 4:1) = 0.22. MP = 102.1 ◦C. HRMS (ESI): Calc. [M + Na]+ = 360.1668, found: 360.1681.
1H-NMR (400 MHz, CDCl3):
1 H). 13C-NMR (101 MHz, CDCl3):
162.0, 165.7.
δ = 1.42 (s, 12 H), 1.71 (s, 6 H), 6.38 (d, J = 2.3 Hz, 1 H), 6.66 (d, J = 2.4 Hz,
δ = 24.9 (4 C), 26.0 (2 C), 84.6, 101.7, 106.3, 108.8, 113.8, 157.8,
3.6. Synthesis of 5-((2S,3R,4aR,6R,8aR)-2,3,4a,5,6,8a-hexahydro-6-hydroxy-2,3-dimethoxy-2,3,6-
trimethylbenzo[b][1,4]dioxin-7-yl)-7-methoxy-2,2-dimethyl-4H-benzo[d][1,3]dioxin-4-one-D3 (11b)
To a degassed solution of alcohol 5b (250 mg, 0.74 mmol, 1.00 equiv), boronate 4b (312 mg,
0.92 mmol, 1.50 equiv), freshly distilled NEt3 (2.19 mL, 1.59 g, 15.7 mmol, 21.2 equiv) in 8.2 mL (0.09 M)

Molecules 2019, 24, 4563
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THF/H2O 9:1 (v/v), Pd(dppf)Cl2
·
CH2Cl2 (48 mg, 60
µ
mol, 8.0 mol%) was added, and the reaction
mixture was stirred for 2 h at 70 ◦C before the solvents were evaporated. The residue was redissolved
in a small amount of DCM and directly purified by FC using EtOAc/cyclohexane 1:2 (v/v). Evaporation
of the solvents furnished 279 mg (81%) of the deuterated coupling product 11b as a white solid.
Rf (CH/EE, 2:1) = 0.17. MP = 165–185 ◦C (decomposition). HRMS (ESI): Calc. [M + Na]+ = 490.2127,
found: 490.2121. [α]2D2 (c = 0.1, MeOH) = +1.5. H-NMR (300 MHz, DMSO-D6):
1
δ (ppm) = 1.20 (s, 6 H),
1.66 (s, 6 H), 1.69 (s, 1 H), 1.84 (m, 1 H), 3.15 (s, 3 H), 3.19 (s, 3 H), 3.95–4.09 (m, 2 H), 5.04 (s, 2 H),
5.20 (s, 1 H), 6.59 (d, J = 2.5 Hz, 1 H), 6.77–6.70 (m, 1 H). 13C-NMR (75 MHz, DMSO-D6):
δ
= 17.6, 17.8,
24.5, 25.8, 27.2, 42.3, 47.3, 47.4, 65.2, 69.7, 71.0, 99.1, 99.8, 100.8, 104.9, 105.6, 112.6, 112.6, 124.6, 142.1,
144.7, 157.9, 159.0, 162.9.
3.7. Synthesis of (6aR,7aR,9S,10S,11aR)-4-Hydroxy-2,9,10-trimethoxy-7a,9,10-trimethyl-6a,7,7a,9,10,11a-
hexahydro-5H-benzo[c][1,4]dioxino[2,3-g]-chromen-5-one-D3 (12b)
To a solution of the coupling product 11b (240 mg, 0.51 mmol) in MeOH (10 mL), K2CO3 (78 mg,
0.6 mmol, 1.1 equiv) was added, and the reaction mixture was stirred for 1 h at r.t. before the solvent was
evaporated. Saturated NH4Cl solution (15 mL) was added, and the aqueous phase was extracted with
EtOAc (3
×
20 mL). The combined organic phases were dried (Na2SO4), and the solvent was evaporated.
Chromatography with n-hexane/ethyl acetate 8:1 (v/v) provided 217 mg (100%) of the protected ALT
derivative 12b as a white solid. Rf (n-Hex/EE, 8:1) = 0.05. MP = 158–160 C. HRMS (ESI): Calc.
[M + Na]+ = 432.1708, found: 432.1705. [α]D22 (c = 0.1, CHCl3) = +6. 1H-NMR (400 MHz, CDCl3):
◦
δ
= 1.33 (s, 3 H), 1.34 (s, 3 H), 1.48 (s, 3 H), 1.90 (dd, J = 12.9, 14.5 Hz, 1 H), 2.49 (dd, J = 4.5, 14.5 Hz,
1 H), 3.26 (s, 3 H), 3.32 (s, 3 H), 3.87 (ddd, J = 4.5, 8.9, 13.2 Hz, 1 H), 4.25 (dd, J = 1.7, 8.9 Hz, 1 H),
6.14 (d, J = 1.7, 1 H), 6.42 (d, J = 2.4 Hz, 1 H), 6.49 (d, J = 2.4 Hz, 1 H). 13C-NMR (101 MHz, CDCl3):
δ
= 17.90, 17.9, 28.4, 38.9, 48.28, 48.33, 66.2, 69.6, 81.5, 100.0, 100.5, 100.6, 100.9, 103.0, 128.8, 133.9, 139.0,
164.2, 166.3, 169.0.
3.8. Synthesis of (2R,3R,4aR)-2,3,4,4a-Tetrahydro-2,3,7-trihydroxy-9-methoxy-4a-methylbenzo[c]chromen-
6-one-D3 ((−)-altenuene-D3, 1b)
◦
The bisketal protected natural product 12b (150 mg, 0.37 mmol) was heated to 100 C while
stirring in 5.2 mL of a 4:1 mixture of AcOH and H2O for 2 h after which the solvents were evaporated.
Residual acetic acid was removed by successive addition and evaporation of DCM (two or three
times). Then the crude product was purified by RP chromatography on a C18 stationary phase with
H2O/MeOH 4:6, delivering 87 mg (81%) of the deuterium labeled natural product 1b as a white solid.
◦
Rf (DCM/MeOH, 20:1) = 0.18. MP = 117–120 C. LC-MS (neg): [M – H]− = 294, tR = 16.67 min.
1
HRMS (ESI): Calc. [M + Na]+ = 318.1027, found: 318.1031. [α]2D2(c = 0.03, MeOH) =
−
9. H-NMR
(400 MHz, DMSO-D6):
δ = 1.47 (s, 3 H), 1.95 (dd, J = 7.3, 14.0 Hz, 1 H), 2.26 (dd, J = 3.5, 14.0 Hz, 1 H),
3.64–3.76 (m, 1 H), 3.91–4.00 (m, 1 H), 5.13 (d, J = 3.8 Hz, 1 H), 5.29 (d, J = 6.2 Hz, 1 H), 6.30 (d, J = 3.3 Hz,
1 H), 6.50 (d, J = 2.4 Hz, 1 H), 6.74 (d, J = 2.4 Hz, 1 H), 11.29 (s, 1 H). 13C-NMR (101 MHz, DMSO-D6):
δ = 22.5, 38.5, 68.8, 69.5, 81.1, 100.0, 100.9, 102.4, 131.8, 139.2, 163.0, 165.8, 168.2.
3.9. Synthesis of (2R,3R,4aR)-2,3,4,4a-tetrahydro-2,3,7-trihydroxy-9-methoxy-4a-methylbenzo[c]chromen-
6-one ((−)-altenuene, 1a)
The native mycotoxin 1a was synthesized analogously with 374 mg (1.16 mmol, 1.00 equiv) allylic
alcohol 5b and 580 mg (1.74 mmol, 1.50 equiv) boronate 4a, yielding 222 mg (81%) (
as a white solid. Rf (DCM/MeOH, 20:1) = 0.18. MP = 117–120 C. LC-MS (neg): [M
tR = 16.67 min. HRMS (ESI): Calc. [M + Na]+ = 315.0839, found: 315.0836. 1H-NMR (400 MHz,
−
)-altenuene (1a
)
◦
−
H]− = 291,
DMSO-D6):
δ (ppm) = 1.47 (s, 3 H), 1.95 (dd, J = 7.5, 14.1 Hz, 1 H), 2.26 (dd, J = 3.5, 14.0 Hz, 1 H),
3.70 (dd, J = 3.8, 7.6 Hz, 1 H), 3.86 (s, 3 H), 3.95 (dt, J = 4.4, 6.2 Hz, 1 H), 5.13 (d, J = 3.8 Hz, 1 H),
5.29 (d, J = 6.1 Hz, 1 H), 6.30 (d, J = 3.3 Hz, 1 H), 6.50 (d, J = 2.3 Hz, 1 H), 6.75 (d, J = 2.4 Hz, 1 H),

Molecules 2019, 24, 4563
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11.29 (s, 1 H). 13C-NMR (101 MHz, DMSO-D6):
102.3, 131.0, 131.8, 139.2, 163.0, 165.8, 168.2.
δ
(ppm) = 27.4, 38.6, 55.9, 68.8, 69.5, 81.1, 100.0, 100.9,
4. Conclusions
In summary, the successful optimization of a reported ALT (1a) synthesis provides efficient access
to a novel deuterated derivative 1b. Starting from commercially available D-( )-quinic acid ( ) as
−
8
an inexpensive chiral pool compound, the D3-labeled natural product 1b was obtained with an overall
yield of 17% after nine steps. The newly synthesized ALT-D3 (1b) proved to be suitable as an internal
standard for SIDA LC-MS/MS and thus ensures the availability of appropriate methods for the reliable
screening of foods and feeds. Moreover, the optimized procedure may facilitate the development of
new anti-tumor drug candidates with (−)-altenuene (1a) being a possible lead compound.
Supplementary Materials: The following are available online at http://www.mdpi.com/1420-3049/24/24/4563/s1:
1H- and 13C-NMR-Spectra of all new compounds as well as UV/Vis- and IR-spectra of the native and labeled
natural products.
Author Contributions: J.G. designed the project. M.A.S. and J.G. decided which methodology to use. M.A.S. and
J.G. carried out the syntheses. T.S. did the HPLC-MS/MS optimization and analysis. M.A.S., J.G. and M.K. wrote
the original draft.
Funding: The work is funded by the Zentrales Innovationsprogramm Mittelstand (ZIM) of the German Federal
Ministry for Economic Affairs and Energy (BMWi) as well as by the Frankenförder Forschungsgesellschaft.
Acknowledgments: We thank Fabian Hofman with the Schneider Research Group (University of Leipzig) for
the opportunity to determine the specific rotation of selected compounds. We also thank Peter Hahn and Anne
Mehnert of the Kersting Group (University of Leipzig) for the UV/Vis- and IR-measurements.
Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
ALT: (
)-altenuene; BINAP: (2,20-bis(diphenylphosphino)-1,10-binaphthyl) ligand; CH: cyclohexane; DABAL:
−
bis(trimethylaluminum)1,4-diazabicyclo[2.2.2]octane adduct; DCM: dichloromethane; d.r.: diastereomeric ratio;
EE: ethyl acetate; ESI: electrospray ionization; HPLC: high pressure liquid chromatography; LC-MS/MS:
liquid-chromatography-tandem mass spectrometry; LD50: lethal dose, 50%; MPLC: medium pressure liquid
chromatography; MTBE: methyl tert-butyl ether; SIDA: stable isotope dilution assay; SRM: selected reaction
monitoring; THF: tetrahydrofuran.
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Sample Availability: ALT and ALT-D3 are available at HPC Standards (www.hpc-standards.com).
©
2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).