Synthesis of [3,3-2H2]-Dihydroartemisinic Acid to Measure the Rate of Nonenzymatic Conversion of Dihydroartemisinic Acid to Artemisinin

Article abstract of DOI:10.1021/acs.jnatprod.9b00686

Dihydroartemisinic acid is the biosynthetic precursor to artemisinin, the endoperoxide-containing natural product used to treat malaria. The conversion of dihydroartemisinic acid to artemisinin is a cascade reaction that involves C-C bond cleavage, hydroperoxide incorporation, and polycyclization to form the endoperoxide. Whether or not this reaction is enzymatically controlled has been controversial. A method was developed to quantify the nonenzymatic conversion of dihydroartemisinic acid to artemisinin using LC-MS. A seven-step synthesis of 3,3-dideuterodihydroartemisinic acid (23) was accomplished beginning with dihydroartemisinic acid (1). The nonenzymatic rates of formation of 3,3-dideuteroartemisinin (24) from 3,3-dideuterodihydroartemisinic acid (23) were 1400 ng/day with light and 32 ng/day without light. Moreover, an unexpected formation of nondeuterated artemisinin (3) from 3,3-dideuterodihydroartemisinic acid (23) was detected in both the presence and absence of light. This formation of nondeuterated artemisinin (3) from its dideuterated precursor (23) suggests an alternative mechanistic pathway that operates independent of light to form artemisinin, involving the loss of the two C-3 deuterium atoms. copy; 2019 American Chemical Society and American Society of Pharmacognosy.

Full text of DOI:10.1021/acs.jnatprod.9b00686

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2
Synthesis of [3,3‑ H ]‑Dihydroartemisinic Acid to Measure the Rate
2
of Nonenzymatic Conversion of Dihydroartemisinic Acid to
Artemisinin
Kaitlyn Varela, Hadi D. Arman, and Francis K. Yoshimoto*
Department of Chemistry, The University of Texas at San Antonio, San Antonio, Texas 78249-0698, United States
*
S Supporting Information
ABSTRACT: Dihydroartemisinic acid is the biosynthetic precursor to
artemisinin, the endoperoxide-containing natural product used to treat
malaria. The conversion of dihydroartemisinic acid to artemisinin is a
cascade reaction that involves C−C bond cleavage, hydroperoxide
incorporation, and polycyclization to form the endoperoxide. Whether or
not this reaction is enzymatically controlled has been controversial. A
method was developed to quantify the nonenzymatic conversion of
dihydroartemisinic acid to artemisinin using LC-MS. A seven-step
synthesis of 3,3-dideuterodihydroartemisinic acid (23) was accomplished
beginning with dihydroartemisinic acid (1). The nonenzymatic rates of
formation of 3,3-dideuteroartemisinin (24) from 3,3-dideuterodihydro-
artemisinic acid (23) were 1400 ng/day with light and 32 ng/day without
light. Moreover, an unexpected formation of nondeuterated artemisinin
(
3) from 3,3-dideuterodihydroartemisinic acid (23) was detected in both
the presence and absence of light. This formation of nondeuterated artemisinin (3) from its dideuterated precursor (23)
suggests an alternative mechanistic pathway that operates independent of light to form artemisinin, involving the loss of the two
C-3 deuterium atoms.
rtemisinin is the endoperoxide-containing natural product
treating dihydroartemisinic acid with singlet oxygen, i.e.,
irradiating a CH Cl solution of dihydroartemisinic acid in
the presence of methylene blue and O2. This resulting
1
A
used to treat malaria. Dihydroartemisinic acid is the
2
2
2
12
biosynthetic precursor to artemisinin. The conversion of
dihydroartemisinic acid to artemisinin has been controversial
in regard to whether or not the transformation is enzymatic in
Artemisia annua, with evidence supporting both an enzymatic
hydroperoxide has been shown to convert to artemisinin (2 to
12
3) over 4 days in 17% yield. In a mechanistic study related to
the conversion of dihydroartemisinic acid hydroperoxide to
artemisinin (2 to 3), in trifluoroacetic acid/petroleum ether
3
−5
6,7
process
and a nonenzymatic process.
Our results
13
confirmed the spontaneous, nonenzymatic conversion of
dihydroartemisinic acid to artemisinin (Figure 1, 1 to 3).
Subsequently, a method was developed to quantify the rate of
conversion of dihydroartemisinic acid to artemisinin using an
internal standard to quantify product formation via an LC-MS
method.
medium yielded 25% of artemisinin. Both dihydroartemisinic
acid and its hydroperoxide have been shown to convert to
artemisinin (1 to 3 and 2 to 3) in CDCl when each starting
3
material was stored in an NMR tube over extended periods of
6
time (“several weeks”). The rate of formation of artemisinin
1
was reported as a percentage through integration of the H
2
NMR signals. Additionally, no raw NMR spectroscopic data of
the compounds have been reported of this time course
monitoring the conversion of dihydroartemisinic acid into
Dihydroartemisinic acid, which was isolated from A. annua,
is believed to be the biosynthetic precursor to artemisinin (3),
the endoperoxide-containing natural product used to treat
malaria (Figure 1). Dihydroartemisinic acid (1) was previously
converted to artemisinin in the presence and absence of
chlorophyll A; the production of artemisinin was reported as a
6
artemisinin.
Major efforts have been made to identify the endoperoxide
4
,5
forming enzyme in A. annua. Artemisinin is found primarily
4
2
in the glandular trichomes of A. annua. Proteomic and
percentage of conversion. The endoperoxide of artemisinin is
5
transcriptomic studies have identified the abundance of a
what confers its antimalarial activity, and therefore, many
peroxidase protein and mRNA, suggesting that this peroxidase
is the enzyme that converts dihydroartemisinic acid to
artemisinin. Other studies using recombinant expression of
studies have developed methods to efficiently convert
8
−10
dihydroartemisinic acid to artemisinin.
The isolation of
dihydroartemisinic acid hydroperoxide (2) from A. annua has
1
1
been reported, suggesting that this hydroperoxide is an
intermediate to endoperoxide formation (2 to 3). Dihydroar-
temisinic acid hydroperoxide (2) can also be formed by
Received: July 26, 2019
©
XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.9b00686
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Figure 1. Formation of artemisinin (3) from dihydroartemisinic acid (1) through an allylic hydroperoxide intermediate (2). The numbering shown
14
in structure 3 is the numbering system based on the carbon backbone in dihydroartemisinic acid (2), as has been previously reported, and shown
13
in parentheses is the alternative numbering for artemisinin. To avoid confusion, the results from this article (Figure 4) will refer to the numbering
14
system shown in the former structure.
Figure 2. Dihydroartemisinic acid is converted to its hydroperoxide in the presence of singlet oxygen (A, 1 to 4). The mechanisms of endoperoxide
13
formation from dihydroartemisinic acid hydroperoxide (B−F). Mechanisms B and C were ruled out in a previous study when dihydroartemisinic
1
8
acid hydroperoxide (4) in the presence of O gas confirmed that the oxygens in the endoperoxide bridge did not come from singlet oxygen.
2
Instead, the source of these oxygens is triplet oxygen.
peroxidase of A. annua have shown the inability to convert
endoperoxide oxygen comes from molecular oxygen reacting
with the hydroperoxide intermediate (Figures 2D, E, and F).
This observation rules out mechanisms B and C (Figures 2B
and C), where the oxygens in the endoperoxide bridge come
from the hydroperoxide oxygens (Figure 2, 8). Mechanisms D,
E, and F (Figure 2D, E, and F) show that the oxygen atoms in
the endoperoxide bridge (Figure 2D−F, 15 and 19) come
from molecular oxygen when starting with dihydroartemisinic
acid hydroperoxide (Figure 2D−F, 4). Mechanism D shows a
ring expansion of the hydroperoxide intermediate (Figure 2D,
artemisinic acid to artemisinin on its own, but this reaction was
3
enabled when plant tissue was added. Although these studies
suggest the possibility of the existence of an endoperoxide
forming enzyme (i.e., conversion of 1 to 3, Figure 1), the
6
,7,13
nonenzymatic formation
of the endoperoxide in artemi-
sinin has also been reported (vide infra).
Because the endoperoxide moiety of artemisinin confers its
antimalarial properties, how the endoperoxide bridge is formed
from dihydroartemisinic acid has been a topic of interest for
decades. The first step is thought to involve an ene reaction
with singlet oxygen to form dihydroartemisinic acid hydro-
peroxide (Figure 2A, 1 to 4). The subsequent steps involve the
C−C bond cleavage step and reaction with triplet oxygen to
form the peroxide intermediate, which cyclizes to form
artemisinin (shown in Figures 2B−F). In a study that involved
15
4) to form an initial oxocarbocation (11 via Hock cleavage ),
which is hydrated to form a hemiketal 12. The hemiketal is in
equilibrium with the acyclic enolic carbonyl isomer (13). This
intermediate is reacting with triplet oxygen to form the
hydroperoxide 14, which cyclizes to form the endoperoxide 15.
Mechanism E shows a homolytic cleavage of the C-4−C-5
bond to directly form intermediate 16. Mechanism F shows
the formation of a dioxetane intermediate (Figure 2F, 18) and
1
8
the use of O -gas and the dihydroartemisinic acid hydro-
2
13
13
peroxide in the presence of acid in Et O,
C NMR
2
1
3
spectroscopy was used to analyze the carbon signals of the
simultaneous attack of triplet oxygen. The dioxetane
formed artemisinin. The significant upfield shift of C-3 and C-
rearranges to intermediate 17, which cyclizes to form
endoperoxide 19. Based on previous O2 studies with
1
8
1
2a (C-4 and C-6 of 3 in Figure 1) confirmed that the
B
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1
8
Figure 3. Explanation of the observation of a mixture of unlabeled and mono- O-incorporated artemisinin products consistent with the
1
8
mechanisms shown in Figure 2E and F. (A) Formation of the gem-diol at the ketocarbonyl group (20) to result in the loss of its O atom group.
1
8
(
B) Formation of the gem-diol at the formyl group (21) to result in the loss of its O atom (14 to 22).
1
3
dihydroartemisinic acid hydroperoxide, the resulting artemi-
sinin product had a distribution of nonlabeled, monolabeled,
RESULTS AND DISCUSSION
■
NMR Experiment to Qualitatively Monitor the Rate of
18
and dilabeled products (with O atoms) in a ratio of
Conversion from Dihydroartemisinic Acid to Artemisi-
nin. In order to firmly establish conditions of spontaneous
conversion of dihydroartemisinic acid to artemisinin (1 to 3), a
time course experiment was performed (Figure 4). Dried
aliquots of dihydroartemisinic acid in either clear glass vials or
amber glass vials were stored and open to air for different
periods of time. The clear glass vials were exposed to sunlight,
and the amber vials were stored in a dark cabinet.
The time course experiments using NMR spectroscopy
confirmed the spontaneous formation of artemisinin from
dihydroartemisinic acid when dihydroartemisinic acid was
stored in vials open to air (Figure 4). Interestingly, the
expected chemical shift for dihydroartemisinic acid hydro-
5
6
3:100:43 as determined by mass spectrometry and
1
3
5:100:48, which was determined by C NMR spectrosco-
13
18
py. The observation that two O atoms are incorporated
into artemisinin only supports mechanisms E and F (Figure 2).
The formation of monolabeled and unlabeled artemisinin
products could be explained by the fact that the carbonyl
oxygens of intermediates 17 and 14 (Figure 3) exchange with
water in the medium (Figure 3A and B), resulting in the loss of
1
8
the O atoms in the carbonyl functional groups.
Despite extensive mechanistic studies of endoperoxide
formation in artemisinin from dihydroartemisinic hydro-
peroxide, the biosynthetic conversion of dihydroartemisinic
acid to artemisinin (Figure 1, 1 to 3) has remained
controversial (vide supra). Although singlet oxygen has been
shown to be the source of hydroperoxide functionality (Figure
A, 1 to 4) and is subsequently transformed into artemisinin
in acidic medium, there has also been evidence of direct
conversion of dihydroartemisinic acid to artemisinin (Figure 1,
to 3) after storage of dihydroartemisinic acid in the freezer
−20 °C) in the absence of light for six months.
The purpose of this investigation was to develop a method
to quantify the rate of spontaneous formation of artemisinin
from dihydroartemisinic acid (Figure 1, 3 from 1). Initial pilot
studies involved monitoring artemisinin production through
H NMR spectroscopy (Figure 4). Monitoring the reaction by
H NMR spectroscopy resulted in the observation of
11
peroxide (H-5 proton should appear at δ 5.26) possibly
appeared at the 10-day time point (δ 5.24, slightly upfield of
the satellite peak of H-5 proton of dihydroartemisinic acid).
However, the area of this peak was insignificant relative to the
proton of artemisinin (δ 5.88) and did not exactly match the
expected chemical shift of dihydroartemisinic acid hydro-
peroxide. Furthermore, a proton with a chemical shift of δ 5.64
was present (Figure 4B, circled peak at the 7-day time point).
This proton with a chemical shift of δ 5.64 matches the
chemical shift of the vinyl proton of dihydro-epi-deoxyartean-
1
3
2
1
(
7
16
nuin B (Figure 4D, X). Furthermore, in a previous study,
1
which involved the long-term storage of a solution of
dihydroartemisinic acid (1) in CDCl , dihydroartemisinic
1
3
6
acid (1) was converted to dihydro-epi-deoxyarteannuin B (X).
artemisinin formation through the appearance of the C-5-
acetal proton at δ 5.88 ppm, but the signal-to-noise ratio was
too low to measure an accurate amount of artemisinin (Figure
and Supporting Information). Therefore, we synthesized a
,3-dideuterodihydroartemisinic acid isotopologue (Figure 5,
3) and monitored the formation of 3,3-dideuteroartemisinin
6
As suggested previously, dihydro-epi-deoxyarteannuin B (X) is
formed from an intramolecular S 2′ displacement of hydrogen
N
4
3
2
peroxide by the carboxylic acid moiety in dihydroartemisinic
acid hydroperoxide (2). The mechanism of the intramolecular
S 2′ displacement of hydrogen peroxide to form dihydro-epi-
deoxyarteannuin B (X) is shown in Figure 4D.
N
(
24) while using nondeuterated artemisinin as an internal
standard. The use of an internal standard with a known
amount of artemisinin permitted the quantification of
artemisinin formation through LC-HRMS data.
In this time course (Figure 4), the detection of artemisinin
(3) was confirmed by the presence of the hemiacetal proton at
δ 5.88. Although other diagnostic peaks of artemisinin were
C
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level of the methine proton of artemisinin (δ 5.88) made it
difficult to accurately quantify the formation of artemisinin
(
i.e., Figure 4B and C). Nonetheless, this set of time course
1
experiments using H NMR spectroscopy was helpful in
approximating the time to detect a significant amount of
artemisinin formation when a dried sample of dihydroartemi-
sinic acid was left open to air. Artemisinin formation from
dihydroartemisinic acid (3 from 1) was clearly detected at 7
days with the appearance of the C-5-methine proton at δ 5.88
(
Figure 4B). Moreover, another time course was performed,
which involved dissolving dihydroartemisinic acid (1) in
1
CDCl and acquiring the H NMR spectra at various time
3
points (Supporting Information). Artemisinin was eventually
formed but at a slower rate (i.e., no artemisinin was detected
after 11 days). When artemisinin was detected (day 32 and day
35), a number of other peaks were also detected, suggesting
decomposition of the parent compound (1) to a complex
mixture (Supporting Information). In contrast, the dry
conditions used in Figure 4 showed primarily artemisinin as
the main product without decomposition to other products.
However, the signal-to-noise level of the peak corresponding to
artemisinin was not high enough to quantitatively measure the
rate of artemisinin production (Figure 4B and C).
Therefore, in order to quantify the conversion of
dihydroartemisinic acid to artemisinin (1 to 3), we were
interested in developing a new method to measure the rate of
formation of artemisinin from dihydroartemisinic acid using an
LC-MS method and an internal standard (Figure 5). This
strategy involved the synthesis of dideuterated dihydroartemi-
sinic acid (Figure 5, 23). To measure the rate of conversion
from dihydroartemisinic acid to artemisinin, the 3,3-dideu-
terated dihydroartemisinic acid isotopologue would undergo
(i) a time course involving a dried sample of dideuterated
dihydroartemisinic acid (23) open to air in a vial to allow for
spontanenous formation of dideuterated artemisinin (24), (ii)
an extraction protocol involving the addition of an internal
standard with a known amount of nondeuterated artemisinin
Figure 4. (A) Time course of spontaneous conversion of
dihydroartemisinic acid (1) to artemisinin (3). H NMR spectra
overlay of time course experiment in (B) the light and (C) the dark.
The hemiacetal proton of artemisinin appears at δ 5.88. The vinylic
1
proton of dihydroartemisinic acid appears at δ 5.12 in CDCl solvent
3
at 500 MHz. A time point of zero days and no artemisinin was
detected at δ 5.88 (the NMR sample was prepared immediately after
the dried compound was dissolved in 0.7 mL of CDCl ) (Supporting
3
Information). (D) Mechanism of the possible formation of dihydro-
epi-deoxyarteannuin B (X) from dihydroartemisinic acid hydro-
peroxide (2) through an intramolecular S 2′ displacement of H O
(
3), and (iii) analysis of d - and d -artemisinin by an LC-
2 0
HRMS method (Figure 5).
N
2
2
Chemical Synthesis of 3,3-Dideuterodihydroartemi-
sinic Acid (23) from Dihydroartemisinic Acid (1). The
retrosynthetic analysis of 3,3-dideuterodihydroartemisinic acid
(Figure 6, 23) involves the incorporation of the two
deuteriums at the C-3-position of an enone intermediate
(Figure 6, 25). This enone intermediate (25) would be derived
from commercially available dihydroartemisinic acid (Figure 6,
1), where the C12-carboxylic acid would be masked with a
protecting group and the C-3-position would be oxidized at the
C-3-position to yield the C-3-ketone (25).
by the carboxylic acid nucleophile, which could explain the
observation of the chemical shift at δ 5.64 (circled peak in Figure 4B).
present (e.g., multiplet at δ 3.5), the integration of these
proton signals relative to dihydroartemisinic acid was small
(
i.e., the ratio of the integrals of the hemiacetal proton of
artemisinin to the vinylic proton of dihydroartemisinic acid,
which appears at δ 5.12, was 1 to ∼100). The significant
difference in relative peak areas between artemisinin and
dihydroartemisinic acid coupled with the low signal-to-noise
Figure 5. Schematic showing the use of 3,3-d -dihydroartemisinic acid (23) to measure the rate of nonenzymatic conversion to d -artemisinin (24)
2
2
using d -artemisinin (3) as the internal standard.
0
D
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Use of 3,3-Dideuterodihydroartemisinic Acid (23) to
Quantitatively Measure 3,3-Dideuteroartemisinin (24)
Formation. In order to measure the formation of 3,3-
dideuteroartemisinin (24) from 3,3-dideuterodihydro-
artemisinic acid (23), a dried sample of 3,3-dideuterodihydro-
artemisinic acid (23) was left in a vial open to air either in the
presence of sunlight (Figure 9) or in complete darkness
(Figure 10) (Table 1). Surprisingly, although the amount of
d -artemisinin (24, m/z 285, retention time (t ) ≈ 3.6 min)
Figure 6. Retrosynthetic analysis of 3,3-dideuterodihydroartemisinic
acid (23) from dihydroartemisinic acid (1).
2
R
was significantly more in the light than in the dark, the amount
of d -artemisinin (3, m/z 283, t ≈ 3.6 min) was formed in
0
R
equal amounts under both conditions (Table 1, entries 1 and
The allylic oxidation at C-3 of artemisinic acid to yield 3-
3
). This formation of d -artemisinin (3) was not due to the
14
0
hydroxyartemisinic acid is known. In the previous study,
lack of deuterium incorporation in the synthesized starting
material (23 by LC-HRMS, see Supporting Information),
which is confirmed from the fact that the ratio of d2-
Acton reported the use of SeO to incorporate the (3R)
2
absolute configuration in 3-hydroxyartemisinic acid. The (3R)
configuration was confirmed through comparison of a known
compound from a microbial transformation of artemisinic
artemisinin to d -artemisinin (24 to 3) is not the same under
0
1
7
the light and in the dark conditions (i.e., ∼200:1 with light vs
acid, which resulted in the formation of both (3R)- and (3S)-
hydroxyartemisinic acid and assigned through 2D NOESY
experiments that showed a correlation between H-3 and H-10
∼
5:1 without light, Table 1, entry 1 vs entry 3). Instead, the
formation of d -artemisinin (3) is likely due to a different
0
17,18
mechanism of endoperoxide formation that involves the loss of
the deuterium atoms at C-3.
in the (3S)-hydroxyartemisinic acid.
Nonetheless, we were
interested in synthetically accessing the C-3-allylic alcohol
derivative to subsequently oxidize and form an enone
Additionally, monodeuterated artemisinin (Figure 9, 35)
was also detected in the presence of light (Table 1, entry 1, m/
z 284.1603). Notably, the use of a high-resolution mass
intermediate. The resulting enone would react with LiAlD
and AlCl3 to potentially yield the dideuterated compound
4
1
9
13
spectrometer was essential in distinguishing C-artemisinin
(
i.e., Figure 6, 25 to 23). Initially, dihydroartemisinic acid was
20
and d -artemisinin. In other words, the use of an LTQ Orbitrap
1
treated with LiAlH to afford dihydroartemisinic alcohol
4
XL with 100 000 resolving power enabled the resolution of the
(
Scheme 1, 1 to 26), which was protected as the tert-
1
3
artemisinin isotopologues with one C atom and one
butyldimethylsilyl (TBDMS) ether 27 (Scheme 1). Subse-
quent oxidation at the C-3 allylic methylene by refluxing alkene
deuterium atom ([M + H]+ of m/z 284.1574 and m/z
2
84.1603), which requires a minimum of 97 986 resolving
2
7 with SeO in EtOH−H O (9:1, v/v) resulted in both
2
2
power (M/ΔM) to distinguish these isotopologues with a 10.2
ppm mass difference.
cleavage of the TBDMS ether and allylic oxidation to afford
diol 28. Diol 28 was crystallized, which allowed for assignment
of the (3R) absolute configuration (Figure 7A, crystal structure
of diol 28).
The fact that the amount of d -artemisinin (3) was detected
0
at about the same levels both in the light and in the dark
(
Table 1, entries 1 and 3, areas of 1 860 388 and 1 437 710,
Since the TBDMS ether protecting group was labile during
respectively) suggests that the conversion of 3,3-d -dihydroar-
the allylic oxidation conditions with SeO (Scheme 1, 27 to
2
2
temisinic acid to d -artemisinin (23 to 3) occurs independent
2
8), the primary hydroxy group of dihydroartemisinic alcohol
0
of light. Interestingly, because the formation of d -artemisinin
was protected as the acetate (Scheme 2, 26 to 29). The
0
from 3,3-d -dihydroartemisinic acid (3 from 23) does not
resulting acetate (29) was oxidized at C-3 using SeO , which
2
2
depend on light, its detection could be used to determine the
kept the 12-acetoxy group intact. The resulting allylic alcohol
relative rates of conversion of 3,3-d -dihydroartemisinic acid to
(
30) was oxidized with Dess−Martin periodinane to afford
2
3
,3-d -artemisinin (23 to 24) with and without light. When no
enone 31. Enone 31 was reduced with AlCl and LiAlD to
2
3
4
yield a mixture of the elimination product 32 (Figure 7B,
crystal structure of 32) and 3,3-dideuterodihydroartemisinic
alcohol 33. Alcohol 33 was oxidized with Dess−Martin
periodinane to yield 3,3-dideuterodihydroartemisinic aldehyde
internal standard was added, the ratios of d -artemisinin to d -
2 0
artemisinin (24 to 3) in the presence and absence of light were
120:1 and 3.4:1 (Figures 9C and 10C), a ∼40-fold difference
between d
of d
quantitation of d
-artemisinin and d -artemisinin. In addition, the use
2
0
(
34). The aldehyde 34 was oxidized under Pinnick oxidation
0
-artemisinin as an internal standard allowed for the
-artemisinin formation both with and without
conditions to afford 3,3-dideuterodihydroartemisinic acid (23).
The H NMR spectra overlay of synthesized 3,3-dideuter-
2
1
light (cf. Table S1 for calculation of artemisinin formation over
time). Using an internal standard, the rates of conversion of
3,3-d -dihydroartemisinic acid to 3,3-d -artemisinin (23 to 24)
odihydroartemisinic acid (23) and commercially available
dihydroartemisinic acid (1) is shown in Figure 8 to confirm the
deuterium incorporation at C-3 (boxed δ 1.8−1.9 region).
2
2
with and without light were determined to be 1400 ng/day and
Scheme 1. Preliminary Studies Using TBDMS Ether 27 for the Allylic Oxidation with SeO to Give Diol 28
2
E
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Scheme 2. Synthesis of 3,3-Dideuterodihydroartemisinic Acid (23) from Alcohol 26
Figure 7. Crystal structures of diol 28 and diene 32.
1
Figure 8. H NMR (500 MHz, CDCl solvent) spectra overlay of synthesized 3,3-dideuterodihydroartemisinic acid (23) and commercially
3
available dihydroartemisinic acid (1). Boxed at δ 1.8−1.9 is the C-3 proton region.
3
2 ng/day (Figures 9B and 10B), also a ∼40-fold difference.
to 1, a 40-fold difference) is the same as (ii) the relative rates
of conversion of 23 to 24 with and without light (1400 ng/day
and 32 ng/day, also a 40-fold difference) confirms that the
The fact that (i) the relative amounts of d -artemisinin to d -
artemisinin (24 to 3 with light 120 to 1 and without light 3.4
2
0
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Figure 9. (A) Vials containing d -dihydroartemisinic acid (23) were exposed to light. The samples were treated under either condition after 13
2
days: (B) with internal standard (d -artemisinin, m/z 283.1540, 3) or (C) without internal standard (d -artemisinin, m/z 283.1540, 3) and
0
0
analyzed via LC-HRMS data. Extracted ion chromatogram: d -artemisinin (24):d -artemisinin (35):d -artemisinin (3) (m/z 285.1666, 284.1603,
2
1
0
2
83.1540). Mass spectrum shown below each chromatogram of peak with retention time (t ) of ∼3.6 min (range of m/z 282.0−287.1).
R
Electrospray ionization (ESI) positive mode, 10 ppm mass tolerance window.
formation of d -artemisinin from d -dihydroartemisinic acid (3
the same way as the prior experiments shown in Figures 9 and
10. The LC-HRMS data analysis revealed production of
artemisinin in these samples presumably due to trace amounts
of oxygen in the vial or oxygen permeating through the threads
of the cap and the vial (Supporting Information).
0
2
from 23, both 9 ng/day) is independent of light. These results
suggest that the conversion of d -dihydroartemisinic acid to d -
2
0
artemisinin (23 to 3) occurs at the same rate both with and
without light. Importantly, there is likely a kinetic isotope effect
at C-3, and this minor mechanistic pathway probably occurs at
The mechanism of conversion of 3,3-d -dihydroartemisinic
2
21
a faster rate when the C-3 deuterium atoms are not present.
acid to 3,3-d -artemisinin (23 to 24) is shown in Figure 11.
2
Furthermore, a parallel set of time course experiments with
another set of vials, the solvent of which was removed with a
The retention of the two C-3 deuterium atoms is consistent
with the mechanisms shown in Figure 2. Moreover, there was
stream of N gas and immediately capped, were also analyzed
also detection of 3-d -artemisinin (Figure 9C, m/z 284, 35),
2
1
for artemisinin production. Other than the caps, these vials,
which could be explained by the tautomerization of the methyl
ketone intermediate (Figure 11, 13 to 14 to 15).
containing 3,3-d -dihydroartemisinic acid (23), were treated in
2
G
DOI: 10.1021/acs.jnatprod.9b00686
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Figure 10. (A) Vials containing d -dihydroartemisinic acid (23) were kept in darkness, and the samples were treated under the following
2
conditions after 13 days: (B) with internal standard (d -artemisinin, m/z 283.1540) and (C) without internal standard (d -artemisinin, m/z
0
0
2
2
83.1540, 3) and analyzed by LC-HRMS. Extracted ion chromatogram: d -artemisinin (24):d -artemisinin (35):d -artemisinin (3) (m/z 285.1666,
2 1 0
84.1603, 283.1540). Mass spectrum shown below each chromatogram of peak with retention time (t ) of ∼3.6 min (range of m/z 282.0−287.1).
R
Ten ppm mass tolerance window. Electrospray ionization (ESI) positive mode.
The mechanism for the loss of the two C-3 deuterium atoms
spontaneous decomposition of dihydroartemisinic acid hydro-
6
in the conversion of 3,3-d -dihydroartemisinic acid to d -
peroxide (2) in CDCl in an NMR tube. An alternative endo-
2
0
3
artemisinin (23 to 3) is proposed through an alkyne
intermediate (Figure 11, 42). Alkyne 42 is formed through
the elimination of the C-3 deuterium of dihydro-oxepine 41,
resulting in the regiospecific loss of the two C-3-deuterium
atoms (Figure 11). The alkyne intermediate (42) is converted
to artemisinin as shown in Figure 11b (42 to 3). The alkyne
intermediate 42 forms the dihydro-oxepine 43 through an
endo-dig cyclization. The dihydro-oxepine structure (43) has
been isolated and reported in an experiment that involved the
dig cyclization from the peroxide intermediate 47 is also shown
in Figure 12A to form the endoperoxide 48, which undergoes
cyclization to form artemisinin (3). However, this endo-dig
cyclization (Figure 11, 42 to 43 or Figure 12A, 47 to 48) does
not have any literature precedence. Therefore, alternative
mechanisms for the loss of the two C-3 deuterium atoms are
proposed in Figure 12B and C. Figure 12B shows the
formation of an alternative allylic hydroperoxide (50) from
dihydroartemisinic acid (23), which undergoes C−C bond
H
DOI: 10.1021/acs.jnatprod.9b00686
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Journal of Natural Products
Article
Table 1. LC-HRMS Results for 13-Day Time Point (Data
from Figures 9 and 10) Summarized
form the oxocarbocation 57 through protonation, which can
re-form the nondeuterated dihydro-oxepine 43 (Figure 12C).
The resulting nondeuterated dihydro-oxepine can undergo
endoperoxide formation as shown in Figure 11 (43 to 3).
In conclusion, a dried sample of 3,3-dideuterodihydroarte-
misinic acid (23) that was open to air spontaneously converted
to 3,3-dideuteroartemisinin (24) with and without sunlight at
rates of 1400 and 32 ng/day, respectively (∼44-fold difference
in rate). The formation of artemisinin from dihydroartemisinic
a
internal
std
285.1666
(m/z)
284.1603
(m/z)
283.1540
(m/z)
entry light
1
2
3
4
yes
yes
no
no
yes
no
227 610 515
323 413 830
4 869 817
7 367 620
11 756 727
N.D.
1 860 388
23 322 650
1 437 710
22 008 172
no
yes
7 304 669
252 006
a
Internal standards: 90 μL of a solution of artemisinin (12.7 μg/mL,
:1 MeOH to CH Cl , v/v) and 10 μL of a solution of
1
acid was detected through H NMR spectroscopy to roughly
2
2
2
determine the rate of formation of artemisinin from
dihydroartemisinic acid (Figure 4). In developing this new
technique to quantify the rate of artemisinin formation from
dihydroartemisinic acid, a new synthesis of 3,3-dideuterodihy-
droartemisinic acid (23) was developed (Scheme 2).
Furthermore, a method using the synthesized 3,3-dideutero-
dihydroartemisinic acid isotopologue (23) and an internal
standard of artemisinin (3) enabled the quantification of
artemisinin production through LC-HRMS (Figure 5).
Unexpectedly, nondeuterated artemisinin (3) was also formed
from the 3,3-dideuterodihydroartemisinic acid (23) starting
material both with and without light (Figures 9 and 10),
dihydroartemisinic acid (5.7 mg/mL in CH Cl ). Area of peaks
corresponding to indicated masses are reported in the table: d2-
2
2
artemisinin (24) (m/z 285.1666), d -artemisinin (35) (m/z
1
2
84.1603), and d -artemisinin (3) (m/z 283.1540). N.D.: none
0
detected. Entries 1 and 2: Figure 9B and C, entries 3 and 4: Figure
0B and C.
1
scission to yield the enolic aldehyde 51. The resulting enol 51
tautomerizes to lose the deuterium, giving the keto aldehyde
5
4, which in turn is converted into artemisinin (3). In another
mechanistic proposal, as shown in Figure 12C, the
monodeuterated dihydro-oxepine 41 (from Figure 11) can
Figure 11. Mechanisms of formation of dideutero-, monodeutero-, and nondeuterated artemisinin isotopologues (24, 35, and 3) from 3,3-
dideuterodihydroartemisinic acid (23). Pathway (a) is dominated by the conversion promoted by sunlight to form 3,3-dideuteroartemisinin (24).
Pathway (b) is the minor pathway that occurs both in the light and in the dark to result in the formation of the loss of the two C-3 deuterium atoms
to form nondeuterated artemisinin (3).
I
DOI: 10.1021/acs.jnatprod.9b00686
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Journal of Natural Products
Article
Figure 12. Alternative pathways (vs Figure 11b) leading to the formation of nondeuterated artemisinin (3) from (A) alkyne intermediate 42, (B)
,3-d -dihydroartemisinic acid 23, and (C) dihydro-oxepine 41.
3
2
suggesting that a minor mechanistic pathway is operative to
form the endoperoxide ring of artemisinin, which involves the
loss of the two C-3 deuterium atoms. This minor pathway
likely occurs at a faster rate when there are no C-3 deuterium
atoms present in dihydroartemisinic acid due to a kinetic
isotope effect (i.e., stronger C−D bond vs weaker C−H bond).
In the presence of sunlight, the formation of the endoperoxide
from 3,3-dideuterodihydroartemisinic acid (23) retains the two
USA). NMR data were analyzed on Topspin software (Bruker).
HRMS data were acquired on an LTQ Orbitrap XL instrument
(Thermo Fisher Scientific) connected to a Waters Acquity UPLC
system (Waters Corp, Milford, MA, USA). MS data were analyzed on
Qualbrowser software (Thermo Fisher Scientific). The UPLC column
used was a Phenomenex (Torrance, CA, USA), Synergi 4 μm, Fusion-
RP (reverse-phase) 80 Å. TLC plates with 254 nm fluorescent
indicator were used.
LC-MS Conditions. Samples were run on an Acuity UPLC
connected to an LTQ Orbitrap XL mass spectrometer. The liquid
chromatography conditions were set as follows: mobile phase A was
0.01% formic acid in water, v/v, and mobile phase B was 0.01% formic
acid in MeCN, v/v. The flow rate was 0.6 mL/min. The gradient was
as follows (over 10 min): from 0 to 1 min, 98% mobile phase A; 1 to
C-3 deuterium atoms to yield 3,3-d -artemisinin (24) (Figure
2
9
). Although an enzymatic conversion of dihydroartemisinic
acid to artemisinin is not ruled out, the mechanisms proposed
for this nonenzymatic transformation of dihydroartemisinic
acid to artemisinin may reflect how the endoperoxide of
artemisinin is formed in nature.
3
min, the gradient shifted from 98% A to 50% A; 3 to 6 min, 50% A
to 2% A; from 6 to 7.9 min held at 2% A; 7.9 to 8 min, from 2% A to
8% A; 8 to 10 min, 98% A. The mass spectrometer was tuned with a
9
EXPERIMENTAL SECTION
■
solution of 1 mM artemisinin in MeOH. The tuning conditions were
as follows: sheath gas flow rate 35, aux gas flow rate 8, sweep gas flow
rate 0, spray voltage (kV) 5.00, capillary temperature 250 °C, capillary
voltage 14 V, tube lens 125 V. Before mass spectrometry samples were
run on the instrument, the mass spectrometer was calibrated using a
Pierce LTQ ESI positive calibration ion solution (Thermo Fisher,
catalog number: 88322); see Supporting Information for calibration
results. Each injection was 10 μL of volume for data shown in Figures
9 and 10. Samples were analyzed under electrospray ionization
positive mode (ESI-positive mode).
General Experimental Procedures. Melting points were
recorded on a melting point apparatus (Global Medical and Lab
Solutions, India, or Optimelt MPA100/Stanford Research Systems,
Sunnyvale, CA, USA). Optical rotations were recorded on an Autopol
IV polarimeter (Rudolph Research, Hackettstown, NJ, USA). IR data
were acquired on an FTIR system (Nicolet iS50 FT-IR spectrometer,
Thermo Fisher Scientific, Waltham, MA, USA). IR data were analyzed
on OMNIC software (Thermo Fisher Scientific). NMR spectra were
recorded on a Bruker 500 MHz spectrometer (Bruker, Billerica, MA,
J
DOI: 10.1021/acs.jnatprod.9b00686
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Journal of Natural Products
Article
Time Course of Dihydroartemisinic Acid to Artemisinin (1
to 3) Monitored by 1H NMR Spectroscopy. A solution of
dihydroartemisinic acid (1) in CH Cl (1.0 g in 50 mL) was divided
3H), 1.47−1.37 (m, 1H), 1.22−1.13 (m, 2H), 0.94 (d, J = 6.4 Hz,
3H), 0.91 (s, 3H), 0.90 (s, 9H), 0.86 (d, 6.7 Hz, 3H), 0.03 (br s, 6H);
1
3
2
2
C NMR (125 MHz, CDCl ) δ 135.0, 121.3, 67.0, 42.9, 42.3, 37.7,
3
into 40 clear glass vials and 40 amber glass vials at 0.5 mL per vial.
3
6.9, 35.9, 27.9, 26.9, 26.6, 25.8, 24.0, 20.0, 18.5, 15.4, −3.43, −5.26.
After drying the solution first with a stream of N and then under
2
HRMS run on ESI positive mode, but ion not found due to small
molecule not ionizing.
house vacuum in a desiccator, the clear glass vials were let to stand by
the window sill open to air while the amber glass vials were placed in a
black box in a cabinet in total darkness. Various time points were
Synthesis of (R)-2-((1R,4R,4aS,6R,8aS)-1,2,3,4,4a,5,6,8a-Oc-
tahydro-6-hydroxy-4,7-dimethylnaphthalen-1-yl)propan-1-ol
taken by dissolving the dried samples in CDCl (0.7 mL) to monitor
(28). SeO (0.31 g, 2.8 mmol, 1 molar equiv) was added to a solution
3
2
the formation of artemisinin (3).
of TBDMS ether 27 (0.93 g, 2.8 mmol) in a mixture of EtOH and
water (30 mL, 9:1, v/v). The reaction mixture was heated under reflux
for 6 h. The reaction mixture was diluted with EtOAc (200 mL) and
washed with water (2 × 50 mL). The organic layer was concentrated
by reduced pressure and purified by column chromatography (silica
gel, 100% hexanes to 10% hexanes in EtOAc, v/v) to yield diol 28 as a
solid (0.20 g, 0.84 mmol, 30%). The solid was dissolved in EtOAc and
hexanes (3 mL, 1:1 EtOAc and hexanes, v/v) and left in the hood for
Time Course of 3,3-d -Dihydroartemisinic Acid (23) to
2
Artemisinin (3, 24, and 35) Monitored by HRMS. A solution of
3
,3-d -dihydroartemisinic acid (23) (2.7 mg) in CH Cl (2 mL) was
2
2
2
aliquoted into 20 2 mL clear glass vials and 20 2 mL amber glass vials
with the addition of 50 μL for each vial. The solvent was evaporated
under house vacuum in a desiccator. The clear glass vials containing
23 were placed in a clear plastic vial rack on the window sill. The
amber glass vials with 23 were placed in a black plastic box, which was
stored in a cabinet. At different time points, the compounds were
extracted through two different methods: [i] with internal standards
2
days to afford block-shaped crystals. R 0.42 (hexanes−EtOAc, 1:1,
f
20
v/v); [α]
3
+7.0 [c 3.4 mg/mL in CHCl ]; IR (neat) 3373.02,
D
3
−1
1
315.64, 2924.65, 2865.61, 1705.31, 1661.08 cm ; H NMR (500
(
e.g., Figure 9B) and [ii] without internal standards (e.g., Figure 9C).
MHz, CDCl ) δ 5.35 (br s, 1H), 4.14−4.07 (m, 1H), 3.74 (dd, J =
3
1
The solvents in four vials in each set were blown down with a stream
1
0 Hz, J = 3.1 Hz, 1H), 3.53 (dd, J = 10.4 Hz, J = 6.0 Hz, 1H),
2
1
2
of N and quickly sealed with a cap (Supporting Information for LC-
2
2.59−2.54 (m, 1H), 2.40 (ddd, J = 12 Hz, J = 5.7 Hz, 1H, J = 2.4
1
2
3
MS traces).
Hz, 1H), 1.76 (br s, 3H), 1.75−1.55 (m, 4H), 1.51−1.34 (m, 4H),
[
i] With internal standards: 90 μL of solution B and 10 μL of
solution D were added. Solution B was 12.7 μg of artemisinin
in 1 mL of a MeOH−CH Cl (2:1, v/v) mixture, and solution
1.25−1.16 (m, 2H), 1.00 (d, J = 6.8 Hz, 1H), 0.91 (d, J = 5.7 Hz,
1
3
1H); C NMR (125 MHz, CDCl ) δ 136.9, 124.6, 68.9, 66.8, 44.9,
3
42.6, 38.2, 37.06, 36.95, 35.7, 29.0, 26.6, 19.9, 19.8, 15.1; HRMS (m/
2
2
+
D was 5.65 mg of dihydroartemisinic acid in 1 mL of CH Cl .
z) calculated for C H O [M + H] , 239.2006; found, 239.1987 (Δ
2
2
15 27
2
[
ii] Without internal standards: 100 μL of MeOH was added.
When analyzing for d -artemisinin (3), d -artemisinin (35),
7.94); mp 107.2−107.5 °C.
Synthesis of (R)-2-((1R,4R,4aS,8aS)-1,2,3,4,4a,5,6,8a-Octahy-
dro-4,7-dimethylnaphthalen-1-yl)propyl Acetate (29). Ac O
0
1
and d -artemisinin (24), the following masses were searched
2
2
for through extracted ion chromatography: m/z 283.1540,
(14 mL, 150 mmol, 2.0 molar equiv) was added to a solution of
alcohol 26 (16.0 g, 72.3 mmol, 1 molar equiv) in pyridine (100 mL,
0.7 M). The reaction mixture was stirred for 30 min, and the resulting
2
84.1603, and 285.1666 with a 10 ppm mass tolerance
1
3
window. The mass for C-labeled artemisinin was m/z
84.1574.
2
solution diluted with water and extracted with Et O (3 × 100 mL).
2
The organic layer was concentrated under reduced pressure, and the
Synthesis of 2R-(1R,4R,4aS,8aS)-4,7-Dimethyl-
,2,3,4,4a,5,6,8a-octahydronaphthalen-1-yl)propan-1-ol (26).
resulting oil was filtered through a short pad of silica gel with 200 mL
1
of 50/50 EtOAc−hexanes, v/v, to afford acetate 29 as a colorless oil
Dihydroartemisinic acid (1, 30.0 g, 127 mmol) in Et O (150 mL) was
2
20
(
−
2
11.5 g, 43.5 mmol, 60%): R 0.76 (hexanes−EtOAc, 4:1, v/v); [α]
added to a suspension of LiAlH (19.3 g, 508 mmol, 4 molar equiv) in
f
D
4
72.2 [c 2.3 mg/mL in CHCl ]; IR (neat) 2959.61, 2908.35,
867.57, 2851.13, 1738.20 cm ; H NMR (500 MHz, CDCl ) δ 5.18
Et O (100 mL) at −78 °C under an atmosphere of N . The reaction
3
2
2
−
1 1
was stirred for 30 min, warmed to room temperature, and left stirring
3
(
s, 1H), 4.19 (dd, J = 11 Hz, J = 3.2 Hz, 1H), 3.89 (dd, J = 11 Hz,
for 3 h at room temperature. The reaction mixture was cooled to −78
1
2
1
J = 6.9 Hz, 1H), 22.48 (s, 1H), 2.05 (s, 3H), 1.95 (m, 1H), 1.94−
°
C, and acetone (100 mL) was added dropwise, followed by
2
1
2
0
3
.91 (m, 2H), 1.83- 1.77 (m, 2H), 1.63 (br s, 3H), 1.59−1.52 (m,
H), 1.50−1.35 (m, 1H), 1.26−1.17 (m, 3H), 1.07−0.98 (m, 1 H),
.97 (d, J = 7.1 Hz, 3H), 0.91 (d, J = 3.0 Hz, 1H), 0.86 (d, J = 6.9 Hz,
Rochelle’s salt (30 g, 110 mmol) in water (100 mL) (added
dropwise) by an addition funnel at −78 °C. The reaction mixture was
filtered using a fritted filter funnel to afford dihydroartemisinic alcohol
1
3
H); C NMR (125 MHz, CDCl ) δ 171.5, 135.5, 120.5, 68.70,
(
26) as a white solid (28.2 g, 128 mmol, 94%). No further purification
3
2
0
43.32, 42.15, 37.55, 35.70, 33.97, 27.78, 26.79, 26.44, 25.94, 23.94,
was done; R 0.56 (hexanes−EtOAc, 4:1, v/v); [α]
mg/mL in CHCl ]; IR (neat) 3401.44, 3328.95, 2962.73, 2919.27,
2
3
+10.2 [c 5.6
f
D
+
2
2
1.12, 19.89, 15.46; HRMS (m/z) calculated for C H O [M + H] ,
65.2162; found, 265.2138 (Δ 9.05 ppm).
17 29 2
3
−
1 1
864.28, 2842.95 cm ; H NMR (500 MHz, CDCl ) δ 5.21 (s, 1 H),
3
Synthesis of (R)-2-((1R,4R,4aS,6R,8aS)-1,2,3,4,4a,5,6,8a-Oc-
.74 (dd, J = 14 Hz, J = 7.4 Hz, 1H), 3.52 (dd, J = 16.8 Hz, J = 4.3
1
2
1
1
tahydro-6-hydroxy-4,7-dimethylnaphthalen-1-yl)propyl Ace-
Hz, 1H), 2.47 (s, 2H), 1.65−1.50 (m, 6H), 1.63 (br s, 3H), 0.99 (m,
H); 1 C NMR (125 MHz, CDCl ) δ 135.1, 120.6, 66.73, 42.62,
3
tate (30). SeO (12.65 g, 114.0 mmol, 1 molar equiv) was added
2
4
4
2
2
3
to a solution of EtOH (200 mL), water (20 mL), and acetate 29 (30.0
g, 114.0 mmol, 1 molar equiv). The reaction mixture was refluxed at
2.01, 41.73, 37.44, 36.58, 36.36, 35.59, 27.62, 26.62, 26.29, 25.77,
+
3.77, 19.73, 14.92; HRMS (m/z) calculated for C H O [M + H] ,
1
5
27
90 °C for 10 h, cooled to rt, and diluted with EtOAc (200 mL). The
23.2056; found, 223.2039 (Δ 7.62 ppm); mp 72.3−72.8 °C.
Synthesis of (R)-2-((1R,4R,4aS,8aS)-1,2,3,4,4a,5,6,8a-Octahy-
resulting solution was washed with water (200 mL), and the organic
layer was concentrated by reduced pressure. The crude material was
purified by silica gel column chromatography (90% hexanes in EtOAc,
v/v, to 10% hexanes in ethyl acetate, v/v) to afford allylic alcohol 30
dro-4,7-dimethylnaphthalen-1-yl)propyl tert-Butyldimethyl-
silyl Ether (27). Imidazole (5.0 g, 73.4 mmol, 4.7 molar equiv)
and TBDMSCl (3.1 g, 20.6 mmol, 1.3 molar equiv) were added to a
solution of alcohol 26 (3.5 g, 15.8 mmol) in MeCN (200 mL). After
the reaction mixture was stirred for 1 h, EtOAc (500 mL) was added,
and the reaction mixture was diluted with water (200 mL). The
organic layer was concentrated under reduced pressure to afford
as a yellow oil (22.85 g, 81.5 mmol, 76%): R
0.38 (hexanes−EtOAc,
f
2
0
4:1, v/v); [α]
+31.1 [c 5.1 mg/mL in CHCl ]; IR (neat) 3390.21,
D
3
−
1
1
2910.02, 2867.15, 2850.37, 1736.83, 1722.03 cm ; H NMR (500
MHz, CDCl ) δ 5.33 (s, 1H), 4.18 (dd, J = 11 Hz, J = 3.4 Hz, 1H),
4.13−4.07 (m, 1H), 3.90 (dd, J = 11 Hz, J = 7.0 Hz, 1H), 2. 57 (s,
3
1
2
TBDMS ether 27 as a clear oil (3.7 g, 11 mmol, 70%): R 0.61 (100%
1
2
f
hexanes); [α]20 −30.0 [c 1.0 mg/mL in CHCl ]; IR (neat) 2953.45,
1H), 2.06 (s, 3H), 1.80−1.75 (m, 1H), 1.76 (br s, 3H), 1.56 (s, 1H),
1.50−1.45 (m, 1H), 1.38 (s, 2H), 1.20−1.14 (m, 2H), 1.02- 0.97 (m,
D
3
−1
1
2
925.01, 2905.90, 2854.10, 1707.34 cm ; H NMR (500 MHz,
1
3
CDCl ) δ 5.22 (br s, 1 H), 3.68 (dd, J = 9.7 Hz, J = 3.2 Hz, 1H),
2H), 0.97 (d, J = 6.9 Hz, 3H), 0.91 (d, J = 6.1 Hz, 3H); C NMR
3
1
2
3
1
.41 (dd, J = 9.7 Hz, J = 4.5 Hz, 1H), 2.49−2.43 (m, 1H), 1.96−
(125 MHz, CDCl ) δ 171.5, 137.1, 124.3, 68.83, 68.51, 44.82, 43.15,
38.04, 36.99, 35.60, 34.16, 28.93, 26.49, 21.14, 19.83, 19.77, 15.45;
1
2
3
.85 (m, 2H), 1.84−1.75 (m, 1H), 1.65−1.51 (m, 4H), 1.63 (br s,
K
DOI: 10.1021/acs.jnatprod.9b00686
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Journal of Natural Products
Article
+
HRMS (m/z) calculated for C H O [M + H] , 281.2111; found,
3H), 0.99 (d, J = 6.8 Hz, 3H), 0.97- 0.89 (m, 2H), 0.86 (d, J = 6.8 Hz,
1
7
29
3
1
3
2
81.2085 (Δ 9.25 ppm).
3H); C NMR (125 MHz, CDCl ) δ 135.3, 120.9, 66.97, 42.84,
3
Synthesis of (R)-2-((1R,4R,4aS,8aS)-1,2,3,4,4a,5,6,8a-Octahy-
42.23, 37.68, 36.81, 35.82, 27.85, 26.51, 25.80, 23.97, 19.96, 15.14,
+
dro-6-keto-4,7-dimethylnaphthalen-1-yl)propyl Acetate (31).
Dess−Martin periodinane (19.45 g, 45.9 mmol, 1 molar equiv) was
added to a solution of allylic alcohol 30 (12.85 g, 45.9 mmol) in
CH Cl (250 mL). The resulting solution was diluted with CH Cl
1.16; HRMS (m/z) calculated for C H D O [M + H] , 225.2182;
1
5
25
2
found, 225.2160 (Δ9.77 ppm); mp 64.5−65.5 °C. *This reaction is
water sensitive, and oven-dried glassware, dried under N atmosphere,
2
2
2
2
2
were used. Although the diene 32 was visible on the TLC plate under
(
100 mL) and washed with saturated Na S O (aqueous, 100 mL).
The organic layer was washed with saturated NaHCO (aqueous, 100
mL). The organic layer was concentrated under reduced pressure and
purified by silica gel chromatography (90% CH Cl to 10% EtOAc).
The crude residue was washed with water and NaHCO3 and
concentrated under reduced pressure to obtain enone 31 as a yellow
solid (9.4 g, 33.8 mmol, 73%): R 0.51 (hexanes−EtOAc, 4:1, v/v);
α] +92.5 [c 5.4 mg/mL in CHCl ]; IR (neat) 2955.34, 2941.67,
a UV lamp, the R values of the monoalkene 33 and diene 32 were
2
2
4
f
3
identical by TLC. When the reaction conditions were not dry (i.e.,
water condensed during the reaction), the reaction mixture turned
pink in color, and the major product was diene 32.
Synthesis of (R)-2-((1R,4R,4aS,8aS)-6,6-Dideutero-
1,2,3,4,4a,5,6,8a-octahydro-4,7-dimethylnaphthalen-1-yl)-
propan-1-al (34). Dess−Martin periodinane (52.0 mg, 0.12 mmol,
1.1 molar equiv) was added to a solution of alcohol 33 (24.0 mg, 0.11
2
2
f
20
[
2
D
3
−
1
1
920.93, 2905.21, 2878.09, 2851.31, 1731.93, 1666.76 cm ;
H
mmol, 1 molar equiv) in CH Cl (50 mL) at room temperature. The
2 2
NMR (500 MHz, CDCl ) δ 6.49 (s, 1H), 4.22 (dd, J = 11 Hz, J =
3
1
2
3
2
.4 Hz, 1H), 3.95 (dd, J = 11 Hz, J = 6.8 Hz, 1H), 2.91 (br s, 1H),
1 2
.75 (dd, J = 17 Hz, J = 2.6 Hz,, 1H), 2.44 (dd, J = 17 Hz, J = 4.9
1
2
1
2
Hz, 1H), 2.07 (s, 3H), 1.85−1.82 (m, 1H), 1.77 (br s, 3H), 1.49−
1
.44 (m, 1H), 1.36−1.30 (m, 1H), 1.05 (m, 3H), 1.01 (d, J = 7.2 Hz,
1
3
1
H), 0.97−0.92 (m, 1H), 0.85 (d, J = 6.2 Hz, 1H); C NMR (125
MHz, CDCl ) δ 199.9, 171.4, 144.1, 136.9, 68.08, 46.14, 43.08, 39.31,
3
3
5.33, 34.24, 28.79, 26.96, 21.11, 19.65, 16.21, 15.55; HRMS (m/z)
+
calculated for C H O [M + H] , 279.1955; found, 279.1929 (Δ
17
27
3
9
.31 ppm); mp 98.0−99.4 °C.
*
To remove an aldehyde impurity (see Supporting Information),
which coeluted with the enone product (9) derived from the
formation of the primary allylic alcohol in the SeO oxidation, the
2
purified enone was subjected to Pinnick oxidation conditions to
convert the aldehyde impurities to corresponding carboxylic acids,
which would be removed upon basic aqueous workup: A solution of
NaH PO (4.15 g, 34.61 mmol, 0.83 molar equiv) in water (25 mL)
2
4
and NaOCl (3.77 g, 20.9 mmol, 0.5 molar equiv) were added to a
solution of enone 31 (9.69 g, 41.7 mmol) in t-BuOH (4.38 mL) and
2
-methyl-2-butene (5.43 mL). The mixture was washed with
NaHCO (saturated aqueous solution, 100 mL) and extracted with
3
EtOAc (3 × 50 mL). The organic layer was concentrated by reduced
pressure. The crude material was purified by silica gel column
chromatography (90% hexanes in EtOAc, v/v, to 10% hexanes in
EtOAc, v/v) to yield pure enone 31 (7.1 g, 30.6 mmol, 73%) as a
white solid (see Supporting Information for structures).
Synthesis of (R)-2-((1R,4R,4aS,8aS)-6,6-Dideutero-
1
,2,3,4,4a,5,6,8a-octahydro-4,7-dimethylnaphthalen-1-yl)-
propan-1-ol (33). LiAlD (0.68 g, 16.19 mmol, 4.5 molar equiv) was
4
added to a solution of AlCl (8.63 g, 64.7 mmol, 18 molar equiv) in
3
Et O (100 mL) at −78 °C. The reaction flask was evacuated and
2
backfilled with N . After 5 min, enone 31 (1.00 g, 3.59 mmol, 1.5
2
molar equiv) dissolved in Et O (100 mL) was added. The reaction
27.83, 27.58, 25.72, 23.95, 19.85, 15.27; HRMS (m/z) calculated for
2
+
mixture was gradually warmed to room temperature and stirred for an
additional 10 h. The mixture was cooled to −78 °C and quenched
with water (100 mL). The resulting solution was diluted with EtOAc
C H D O [M + H] , 239.1975; found, 239.1971 (Δ1.7 ppm); mp
1
5
23
2
2
2
110−120 °C. H NMR spectroscopy was performed by dissolving
compound 23 (12 mg) in 1 mL of a solution of CDCl −CHCl (0.5
3
3
(
200 mL). The organic layer was concentrated under reduced
to 4.5 mL) and two broad peaks were observed corresponding to the
two deuteriums incorporated at C-3 with the following chemical
shifts: δ 1.89 and 1.79.
pressure. The crude material was purified by silica gel column
chromatography (100% hexanes to 10% EtOAc in hexanes, v/v, to
2
0% EtOAc in hexanes, v/v) to afford a mixture of alcohol (33) and
X-ray Crystallography of Compound 28. Single crystals of
compound 28 were prepared by slow evaporation of 2 mL of a
solution of 28 in 1:1 EtOAc−hexanes, v/v, which was left to stand
overnight. Suitable colorless plate-like crystals for compound 28 with
dimensions of 0.30 mm × 0.24 mm × 0.17 mm were mounted in
Paratone oil onto a nylon loop. All data were collected at 98(2) K,
using a Rigaku AFC12/Saturn 724 CCD fitted with Mo Kα radiation
(λ = 0.710 75 Å). Data collection and unit cell refinement were
diene 32 as a white solid. Diene 32 was slightly more polar than
monoalkene 33 (i.e., when the compounds eluted off of the column,
the mixture was collected in 10 test tubes (20 mL volume each tube),
and the solvent in each tube was evaporated; the resulting compound
was characterized by H NMR spectroscopy). The mixture of 33 (59
mg, 0.26 mmol, 7%) and 32 (98 mg, 0.44 mmol, 12%) was further
separated through a second column. *All compound characterization
1
2
0
22
is for monoalkene (33): R 0.49 (hexanes−EtOAc, 4:1, v/v); [α]
performed using CrysAlisPro software. The total number of data
f
D
−
54.6 [c 1.7 mg/mL in CHCl ]; IR (neat) 3398.21, 3328.32,
were measured in the range 5.57° < 2θ < 55.0°, using ω scans. Data
processing and absorption correction, giving minimum and maximum
transmission factors (0.9635, 1.000), were accomplished with
3
2
2
962.93, 2919.63, 2863.37, 2842.91, 2160.84, 2127.28, 2082.21,
−1
1
023.07, 1716.14, 1661.06, 1558.32 cm ; H NMR (500 MHz,
2
3
23
CDCl ) δ 5.21 (s, 1H), 3.74 (dd, J = 11 Hz, J = 2.7 Hz, 1H), 3.52
CrysAlisPro and SCALE3 ABSPACK, respectively. The structure,
24 25
3
1
2
(
(
m, 1H), 2.47 (s, 1H), 1.91 (dd, J = 14 Hz, J = 3.6 Hz,, 1H), 1.62
br s, 3H), 1.56−1.49 (m, 1H), 1.48−1.36 (m, 1H), 1.25−1.18 (m,
using Olex2, was solved with the ShelXT structure solution
26
1
2
2
program using direct methods and refined (on F ) with the ShelXL
L
DOI: 10.1021/acs.jnatprod.9b00686
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
refinement package using full-matrix, least-squares techniques. All
non-hydrogen atoms were refined with anisotropic displacement
parameters. All carbon-bound H atom positions were determined by
geometry and refined by a riding model. The oxygen-bound H atom
position was determined by electron density plot.
(5) Soetaert, S. S. A.; Van Neste, C. M. F.; Vandewoestyne, M. L.;
Head, S. R.; Goossens, A.; Van Nieuweburgh, F. C. W.; Deforce, D. L.
D. BMC Plant Biol. 2013, 13, 220.
(6) Sy, L.-K.; Brown, G. D. Tetrahedron 2002, 58, 897−908.
(7) Brown, G. D.; Sy, L.-K. Tetrahedron 2004, 60, 1125−1138.
(8) Levesque, F.; Seeberger, P. H. Angew. Chem., Int. Ed. 2012, 51,
1706−1709.
X-ray Crystallography of Compound 32. Single crystals of
C H O (compound 32, diene) was prepared by slow evaporation of
15
24
2
mL of a solution of 32 in 1:1 MeOH−acetone, v/v, which was left
to stand overnight. Suitable colorless plate-like crystals for compound
32) with dimensions of 0.33 mm × 0.30 mm × 0.13 mm were
mounted in Paratone oil onto a nylon loop. All data were collected at
00(2) K, using a Rigaku AFC12/Saturn 724 CCD fitted with Mo Kα
radiation (λ = 0.710 75 Å). Data collection and unit cell refinement
(9) Triemer, S.; Gilmore, K.; Vu, G. T.; Seeberger, P. H.; Seidel-
Morgenstern, A. Angew. Chem., Int. Ed. 2018, 57, 5525−5528.
(10) Amara, Z.; Bellamy, J. F. B.; Horvath, R.; Miller, S. J.; Beeby, A.;
Burgard, A.; Rossen, K.; Poliakoff, M.; George, M. W. Nat. Chem.
2015, 7, 489−495.
(
1
(11) Wallaart, T. E.; Pras, N.; Quax, W. J. J. Nat. Prod. 1999, 62,
1160−1162.
22
were performed using CrysAlisPro software. The total number of
data were measured in the range 4.46° < 2θ < 52.0°, using ω scans.
Data processing and absorption correction, giving minimum and
maximum transmission factors (0.9733, 1.000), were accomplished
(12) Roth, R. J.; Acton, N. J. Nat. Prod. 1989, 52, 1183−1185.
(13) Acton, N.; Roth, R. J. J. Org. Chem. 1992, 57, 3610−3614.
(
(
14) Acton, N. J. Nat. Prod. 1999, 62, 790−793.
2
3
23
with CrysAlisPro and SCALE3 ABSPACK, respectively. The
15) Turconi, J.; Griolet, F.; Guevel, R.; Oddon, G.; Villa, R.; Geatti,
24
25
structure, using Olex2, was solved with the ShelXT 2 structure
A.; Hvala, M.; Rossen, K.; Goller, R.; Burgard, A. Org. Process Res. Dev.
014, 18, 417−422.
16) Dudley, G. B.; Engel, D. A.; Ghiviriga, I.; Lam, H.; Poon, K. W.
C.; Singletary, J. A. Org. Lett. 2007, 9, 2839−2842.
17) Elmarakby, S. A.; El-Feraly, F. S.; Elsohly, H. N.; Croom, E. M.;
Hufford, C. D. Phytochemistry 1988, 27, 3089−3091.
18) Zhu, J.-H.; Yu, R.-M.; Yang, L.; Hu, Y.-S.; Song, L.-Y.; Huang,
Y.-J.; Li, W.-M.; Guan, S.-X. Process Biochem. 2010, 45, 1652−1656.
19) Cunningham, I. M.; Overton, K. H. J. Chem. Soc., Perkin Trans.
1 1974, 21, 2458−2462.
20) Schwertz, G.; Zanetti, A.; de Oliveira, M. N.; Fernandez, M. A.
solution program using direct methods and refined (on F ) with the
2
(
26
ShelXL refinement package using full-matrix, least-squares techni-
ques. All non-hydrogen atoms were refined with anisotropic
displacement parameters. All carbon-bound H atom positions were
determined by geometry and refined by a riding model. The oxygen-
bound H atom position was determined by electron density plot.
(
(
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jnatprod.9b00686.
(
■
*
S
(
G.; Dioury, F.; Cossy, J.; Amara, Z. Tetrahedron 2019, 75, 743−748.
(
(
21) Westheimer, F. H. Chem. Rev. 1961, 61, 265−273.
22) CrysAlisPro 1.171.38.41; Rigaku, 2015.
1
D NMR and IR spectra of 26−34 and 23, HRMS
spectra of 26, 28−34, and 23, crystallographic data for
diol 28 and diene 32, and optimization conditions for 23
to 24 (time course, Table S1) (PDF)
(23) SCALE3 ABSPACK; Oxford Diffraction Ltd., 2005.
(24) Dolomanov, O. V.; Bourhis, L. J.; Gildea, R. J.; Howard, J. A.
K.; Puschmann, H. J. Appl. Crystallogr. 2009, 42, 339.
(
25) Sheldrick, G. M. Acta Crystallogr., Sect. A: Found. Adv. 2015,
A71, 3−8.
26) Sheldrick, G. M. Acta Crystallogr., Sect. A: Found. Crystallogr.
008, A64, 112−122.
X-ray data for diol 28 (CIF)
X-ray data for diene 32 (CIF)
(
2
AUTHOR INFORMATION
Corresponding Author
E-mail: francis.yoshimoto@utsa.edu.
■
*
ORCID
Francis K. Yoshimoto: 0000-0002-2308-2999
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This research was generously supported by the Bill & Melinda
Gates Foundation [OPP1188432] and startup funds from
UTSA. The research was also supported from the UTSA NIH
RISE Program (GM060655). A National Science Foundation
Major Research Instrumentation grant (award number:
1
625963) to support NMR facilities is acknowledged. We
are grateful to Dr. Trevor Laird for helpful advice and
comments.
REFERENCES
■
(
(
1) Wang, J.; Xu, C.; Liao, F. L.; Jiang, T.; Krishna, S.; Tu, Y. N.
Engl. J. Med. 2019, 380, 2087−2089.
2) Wallaart, T. E.; Van Uden, W.; Lubberink, H. G. M.;
Woerdenbag, H. J.; Pras, N.; Quax, W. J. J. Nat. Prod. 1999, 62,
30−433.
3) Hang, Y.-S.; Liu, B.-Y.; Li, Z.-Q.; Ye, H.-C.; Wang, H.; Li, G.-F.;
Han, J.-L. Acta Bot. Sin. 2004, 46, 1338−1346.
4) Bryant, L.; Flatley, B.; Patole, C.; Brown, G. D.; Cramer, R. BMC
Plant Biol. 2015, 15 (175), 1−13.
4
(
(
M
DOI: 10.1021/acs.jnatprod.9b00686
J. Nat. Prod. XXXX, XXX, XXX−XXX