An efficient synthesis of optically active [4-13 c] labelled quorum sensing signal autoinducer-2

Article abstract of DOI:10.3390/molecules26020369

A new synthetic route for the quorum sensing signal Autoinducer-2 (AI-2) is described and used for the preparation of [4-¹³ C]-AI-2 starting from [1-¹³ C]-bromoacetic acid. The key step in this process was the enantioselective reduction of an intermediate ketone. This synthesis pro-vides, selectively, both enantiomers of the labelled or unlabelled parent compound, (R) or (S)-4,5-dihydroxypentane-2,3-dione (DPD) and was used for an improved synthesis of [1-¹³ C]-AI-2.

Full text of DOI:10.3390/molecules26020369

molecules
Article
An Efficient Synthesis of Optically Active [4-13C] Labelled
Quorum Sensing Signal Autoinducer-2
Osvaldo S. Ascenso 1, Gonzalo Carrau 1,2 , Karina B. Xavier 3 , M. Rita Ventura 1,
*
and Christopher D. Maycock 1,4,
*
1
Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa,
2780-157 Oeiras, Portugal; oascenso@itqb.unl.pt (O.S.A.); gcarrau@itqb.unl.pt (G.C.)
Facultad de Química, Universidad de la República, Montevideo 11800, Uruguay
Instituto Gulbenkian de Ciência, 2780-156 Oeiras, Portugal; kxavier@igc.gulbenkian.pt
Faculdade de Ciências da Universidade de Lisboa, Departamento de Química e Bioquímica,
1749-016 Lisboa, Portugal
2
3
4
*
Correspondence: rventura@itqb.unl.pt (M.R.V.); maycock@itqb.unl.pt (C.D.M.)
Abstract: A new synthetic route for the quorum sensing signal Autoinducer-2 (AI-2) is described
and used for the preparation of [4-13C]-AI-2 starting from [1-13C]-bromoacetic acid. The key step
in this process was the enantioselective reduction of an intermediate ketone. This synthesis pro-
vides, selectively, both enantiomers of the labelled or unlabelled parent compound, (R) or (S)-4,5-
dihydroxypentane-2,3-dione (DPD) and was used for an improved synthesis of [1-13C]-AI-2.
Keywords: DPD; AI-2; quorum sensing; enantioselective synthesis; 13C-DPD
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
1. Introduction
Citation: Ascenso, O.S.; Carrau, G.;
Xavier, K.B.; Ventura, M.R.; Maycock,
C.D. An Efficient Synthesis of
Optically Active [4-13C] Labelled
Quorum Sensing Signal
Bacterial populations use cell-cell communication to coordinate their behaviour, facili-
tating adaptation to changing environments. Chemical communication among bacteria is
called “quorum sensing”. Examples of quorum sensing-regulated behaviours are biofilm
formation, virulence-factor expression, antibiotic production and bioluminescence [
Autoinducer-2 (AI-2) is produced and detected by many phylogenetically distinct bacte-
ria [ ]. Using AI-2, bacteria are able to detect the presence of other bacterial species in
their vicinity and regulate gene expression according to the species composition in the
environment [ ]. (S)-4,5-dihydroxypentane-2,3-dione (DPD) is the precursor of AI-2
1,2].
Autoinducer-2. Molecules 2021, 26,
369. https://doi.org/10.3390/
molecules26020369
3,4
5
,
6
1
Academic Editor: Herman O. Sintim
Received: 18 December 2020
Accepted: 7 January 2021
that in aqueous medium is an equilibrium mixture of the linear form, two anomeric cyclic
forms 2 and 3, and their hydrated versions 4 and 5 (Scheme 1) [7].
Published: 12 January 2021
Publisher’s Note: MDPI stays neu-
tral with regard to jurisdictional clai-
ms in published maps and institutio-
nal affiliations.
Copyright: © 2021 by the authors. Li-
censee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and con-
ditions of the Creative Commons At-
tribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Scheme 1. AI-2 is an equilibrium mixture of the linear and cyclic forms of DPD 1.
Molecules 2021, 26, 369. https://doi.org/10.3390/molecules26020369
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 369
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It has been shown that many species of Gram-positive and Gram-negative bacteria
can detect different forms of this molecule and thus all these forms are known collectively
as AI-2. Specifically, many Vibrio have the LuxP-type of AI-2 receptor which recognises
borated isomer 6 [8], whereas members belonging to phylogenetically distinct families such
as the human pathogens Salmonella typhimurium and Bacillus anthracis, as well as different
Clostridium and the plant symbiont Sinorhizobium meliloti having the LsrB-type of receptors,
recognise the non-borated diastereoisomer
5 [9,10]. Bacteria that have the LsrB-type of
receptor internalise this signal via an ABC transporter. Once inside the cell the non-borated
form of AI-2 is phosphorylated and in bacteria such as Escherichia coli the phosphorylated
compound is cleaved by a thiolase (LsrF) into metabolites which are channelled into
glycolysis and the citric acid cycle, as means to terminate quorum sensing [11]. This thiolase
is not present in all bacteria that have the LsrB receptor, indicating that AI-2 metabolism
might not be as conserved as the receptors [10].
Ultimately, the understanding of the molecular mechanisms that bacteria use to reg-
ulate their behaviours can lead to the development of new therapies to control bacterial
infections and also to develop biotechnological applications for the control of the indus-
trial scale production of beneficial bacterial products, such as antibiotics or recombinant
proteins.
Even though naturally occurring (S)-DPD (AI-2) is a small molecule, it is highly func-
tionalised, optically active and unstable at high concentrations. This constitutes a challenge
for producing significant amounts of this product. To the best of our knowledge, there have
been described to date three racemic syntheses [12
–14], four syntheses of the enantiop-
ure form starting from the chiral pool [ 15 17], and our group has previously described
7
, –
the synthesis of the natural enantiomer starting from non-chiral starting materials [18].
This route has proven to be efficient, highly reproducible and can be easily modified to
generate new analogues in the optically pure form [19,20].
The asymmetric synthesis of DPD is fundamental since only one enantiomer is pro-
duced and recognised by bacteria. Studies have shown that the unnatural borated (R)-DPD
showed only 1% of the activity of the borated (S)-DPD form
6 (Figure 1) in the V. harveyi
bioassay (LuxP receptor) [ ]. For the assay of the isomer (LsrB receptor), to induce the
7
5
E. coli reporter, six times more (R)-DPD than (S)-DPD was necessary in order to obtain the
same activity [18]. Thus, the obtention of DPD with the highest enantiopurity is important
for accurate biological studies.
The direct detection of AI-2 in biological samples is not easy, due to the complexity of
the samples, low natural abundance and the instability of AI-2. A liquid chromatography-
tandem mass spectrometric method for the quantification of AI-2 in saliva samples has been
described [21]. For this, extracted AI-2 was converted into a corresponding more stable
quinoxaline before being analysed and [1-13C]-DPD was used as the internal standard.
To date, this is the only 13C labelled DPD reported [7].
This work describes a considerable improvement of our previous synthesis of optically
active DPD [18] and has been applied to the synthesis of [4-13C]-DPD.
2. Results and Discussion
[4-13C]-DPD is particularly important for in vivo studies, since it has been shown that
AI-2, when internalised in Escherichia coli, is processed to dihydroxyacetone phosphate
(DHAP) and acetyl-CoA, and these are key metabolites that are then used by the cell into
central metabolism via glycolysis and the citric acid cycle [22]. In this final enzymatic
reaction, the AI-2 molecule is broken: The C-1 is incorporated into acetyl-CoA and the
other carbons remain in DHAP. Thus, [4-13C]-DPD is important as it allows the labelling of
a different metabolic product of AI-2 than from [1-13C]-DPD. The metabolic fate of AI-2
in bacteria that do not have homologues to the LsrF-thiolase, such as several Clostridium,
has yet to be determined [10]. Since the 13C labelled starting material is relatively expensive
and the labelled carbon atom is introduced in the first step of the synthesis, it was important

Molecules 2021, 26, 369
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to improve the enantioselectivity and global yield of the DPD synthesis and avoid time
consuming chromatographic purifications.
Compound 11 was prepared by a four-step sequence starting from [1-13C]-bromoacetic
acid (7
), as illustrated in Scheme 2. [1-13C]-Bromoacetic acid was chosen over [1-13C]-
glycolic acid since it was commercially available and less expensive. For the synthesis of
the unlabelled DPD, bromoacetic acid is economically the best starting material even though
more steps are required. Weinreb amide
described by Hu and coworkers [23]. Acetolysis of the bromide
in 86% yield [24] and the acetate group methanolised to obtain alcohol 10 in 83% yield.
It is noteworthy that attempts to directly hydrolyse bromide were unsuccessful and the
8
was formed in 88% yield using a procedure
8
afforded compound
9
8
expected alcohol 10 was never isolated from these experiments. Finally, the alcohol was
protected with the tert-butyldiphenylsilyl (TBDPS) group, rendering compound 11 in 63%
overall yield. This synthetic route does not require any chromatographic purification until
the last step. From Weinreb amide 11, an aqueous solution of [4-13C]-DPD was obtained
following the same synthetic method reported previously (Scheme 2), except that the
asymmetric reduction was carried out by the Noyori reduction [25] instead of the Midland
reduction described previously [18]. The Noyori procedure afforded alcohol 13 in 95% yield
with an ee of 94%, thus avoiding the recrystallisation step used to enhance the enantiomeric
excess in previous work [18]. Furthermore, the purification of 13 obtained using Noyori’s
chiral Ru catalyst was much more straightforward than when using (S)-Alpine borane as
the chiral reduction agent.
It is also possible to prepare [1-13C]-labelled DPD by this method using [3-13C]-
propyne or by alkylation of the terminal alkyne 24 (Semmelhack intermediate), prepared
as described in Scheme 3, with 13CH3I [7].
Scheme 2. Synthesis of [4-13C]-DPD. Reagents and conditions: ( ) PBr3, MeNH(OMe), toluene, from 0 ◦C to 60 ◦C, 88%;
a
◦
◦
(
b
) AcOH, Et3N, ClCH2CH2Cl, reflux, 86%; (
c
) MeONa, MeOH, 0 C, 83%; (
d
) TBDPSCl, Et3N, DMAP, CH2Cl2, from 0 C
) Ru(p-cymene)[(S,S)-TsDPEN], i-PrOH, rt, 95% (ee = 94%); ( ) TBAF,
) BzCl, (i-Pr)2NEt, DMAP, CH2Cl2, 93%; ( ) 1,1-dimethoxycyclohexane, H2SO4, DMF, 91%; ( ) NaIO4, RuO2,
CCl4/MeCN, rt, 86%; (k) Dowex 50WX8, H2O, pH 3, rt.
◦
to rt, 100%; (
THF, rt, 86%; (
e) n-BuLi, Propyne, THF,
−
78–0 C, 95%; (
f
g
h
i
j

Molecules 2021, 26, 369
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Scheme 3. Synthesis of [1-13C]-DPD. Reagents and conditions: (
a
) LDA, THF, from
−
78 ◦C to 0 ◦C, 74%; (
b
) (S)-Alpine
) 1,1-Dimethoxycyclohexane,
) Dowex
borane, THF, rt, 67%; (
H2SO4, DMF, 78%; (
) LDA, 13CH3I, THF, from
50WX8, H2O, pH 3, rt.
c
) TBAF, THF, rt, 71%; (
d
) BzCl, (i-Pr)2NEt, DMAP, CH2Cl2, 96%; (
e
◦
f
−
78 C to rt, 94%; (
g) NaIO4, RuO2, CCl4/MeCN, rt, 91%; (
h
Figure 1. By-product 27 from the reaction of the Weinreb amide 18 with lithium trimethylsilylacety-
lene (Scheme 3).
Interestingly, reaction of the Weinreb amide 18 with lithium trimethylsilylacetylene
afforded ketone 20 as the major product (62%) along with trimethylsilylacetylenic ketone 19
(12%) and 1,3-dione 27 (16%, Figure 1) [26]. Midland reduction of ketone 20 was chosen for
this synthetic sequence, since terminal alkynes are strong inhibitors of Noyori’s reduction.
Alcohol 21 was obtained with 66% yield and 86% ee. As diol 22 was not crystalline,
the optical. purity of the product could not be enhanced by recrystallisation. Benzoylation
of the diol did not produce a crystalline product.
The formation of the cyclohexylidene acetal 24, alkylation of the terminal alkyne with
13CH3I afforded 25 and oxidation to the diketone 26 was successfully achieved as described
previously [
DPD (Scheme 3).
In conclusion, [4-13C]-AI-2 and [1-13C]-AI-2 were efficiently synthesised and the
corresponding enantiomers obtained using the enantiomeric versions of the reduction
reagents used. Noyori’s asymmetric transfer hydrogenation of -acetylenic ketone 12
7
]. Hydrolysis of the acetal with acidic Dowex resin afforded [1-13C]-labelled
α,β
was employed for the first time in AI-2 synthesis, increasing the enantiomeric excess of the
alcohol obtained and overcoming the difficult separation of pinene from (S)-Alpine borane
previously used.

Molecules 2021, 26, 369
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All reactions were reproducible and afforded stable intermediates in high yields. This
is particularly relevant since 13C labelled compounds are expensive and, on the other hand,
sufficient quantities of AI-2 and 13C labelled AI-2 are needed for biological studies.
AI-2 labelled at different carbon atoms is important to determine the metabolic fate of
this molecule in vivo in bacteria where AI-2 metabolism remains to be discovered and to
quantify potential incorporation of this molecule into central metabolism. Additionally,
[13C]-AI-2 is also useful as a standard for the mass spectrometric quantification of AI-2 and
derivatives in biological samples of in vivo animal studies.
3. Materials and Methods
1H-NMR spectra were obtained at 400 MHz in CDCl3 or D2O with chemical shift
values (δ
) in ppm downfield from tetramethylsilane in the case of CDCl3 and 13C-NMR
spectra were obtained at 100.61 MHz in CDCl3 (see Supplementary Materials). Assignments
are supported by 2D correlation NMR studies. Medium pressure preparative column
chromatography: silica gel 60H. Analytical TLC: Aluminium-backed silica gel 60 F254.
20
Specific rotations ([
α
]
D) were measured using an automatic polarimeter. Reagents and
solvents were purified and dried according to the literature [27]. All reactions were
carried out under an inert atmosphere (argon), except when the solvents were undried.
The enantiomeric excesses were determined by HPLC on a Waters 600E/U6K instrument
using a Daicel Chiralpack AD-H column (see Supplementary Materials).
[1-13C]-2-bromo-N-methoxy-N-methylacetamide (
8
). [1-13C]-2-bromoacetic acid (
7
, 510 mg,
3.64 mmol) was dissolved in dry toluene (18 mL), under argon atmosphere, and the solu-
tion was cooled to 0 ◦C. NHMe(OMe) (805
L, 10.93 mmol) was added, and 10 min later
µ
a solution of PBr3 (173
µ
◦ L, 1.82 mmol) in 4 mL of toluene was added dropwise. After
stirring for 10 min at 0 C, the reaction was warmed to room temperature, left stirring
during 10 min and then warmed up to 60 ◦C. After the starting material was consumed
(TLC, approximately 30 min), a saturated aqueous solution of NaHCO3 (35 mL) was
added, and the aqueous phase was extracted with 3 portions of 18 mL of AcOEt. The com-
bined organic layers were dried with MgSO4, filtered and concentrated under reduced
pressure. The crude was purified by flash column chromatography (7:3 Hexane:AcOEt),
and 586 mg of compound
(400 MHz, CDCl3)
8
was obtained as a colourless oil (Yield = 88%). 1H-NMR
2
δ
(ppm): 4.02 (d, JH-C = 4.0 Hz, 2H, CH2), 3.80 (s, 3H, OMe), 3.24
(
d, 3JH-C = 2.1 Hz, 3H, NMe). 13C-NMR (101 MHz, CDCl3)
δ
(ppm): 167.8 (C1), 61.8 (O
CH3),
32.6 (NCH3), 25.3 (d, 1JC-C = 57.2 Hz
, CH2). FT-IR (neat): 1618.6 (C=O). HRMS: calcd. for
[C313CH8BrNNaO2]+: 204.96642; found: 204.96647.
[1-13C]-2-acetoxy-N-methoxy-N-methylacetamide (
9). To a solution of glacial AcOH
(293
(713
µ
L, 5.12 mmol) in dry 1,2-dichloroethane (2 mL), under argon atmosphere, Et3N
µL, 5.12 mmol) was added dropwise. Then, a solution of (586 mg, 3.20 mmol) in dry
8
1,2-dichloroethane (2 mL) was added, and the reaction was heated at reflux temperature.
Once the starting material was consumed (TLC, approximately 1 h), 50 mL of HCl 5%
was added, and the aqueous phase was extracted with 3 portions of 55 mL of CH2Cl2.
The combined organic layers were dried with MgSO4, filtered and concentrated under
reduced pressure, rendering 446 mg of compound 19 that was used without any further
purification (colourless oil, yield = 86%). 1H-NMR (400 MHz, CDCl3)
δ (ppm): 4.83
3
(
d, 2JC-H = 4.1 Hz, 2H, AcOCH2-), 3.74 (s, 3H, OMe), 3.20 (d, JC-H = 2.1 Hz, 3H, NMe),
2.18 (s, 3H, CH3C(O)O). 13C-NMR (101 MHz, CDCl3)
δ
(ppm): 170.8 (CH3 (O)O), 168.1
C
(
C
(O)N), 61.6 (O
C
H3), 61.1 (d, 1JC-C = 57.0 Hz, AcO
C
H2), 32.4 (N H3), 20.7 (CH3C(O)O).
C
FT-IR (neat): 1743.3 (AcO), 1639.1 (C(O)N). HRMS: calcd for [C513CH11NNaO4]+: 185.06138;
found: 185.06149.
[1-13C]-2-hydroxy-N-methoxy-N-methylacetamide (10). A solution of ester
9 (409 mg,
◦
2.52 mmol) in dry MeOH (5.5 mL) was cooled to 0 C under argon atmosphere, and MeONa
(21 mg, 0.39 mmol) was added in small portions. The reaction was left to stir at 0 ◦C for
2 h, and then Dowex 50WX8 resin was added (until acidic pH). The resin was then filtered
off, washed with methanol, and the solvent was removed under reduced pressure. 252 mg

Molecules 2021, 26, 369
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of compound 10 was obtained as a colourless oil, and it was used without any further
1
purification (yield = 83%). H-NMR (400 MHz, CDCl3)
δ
(ppm): 4.31 (d, 2JH-C = 4.4 Hz,
2H, CH2OH), 3.71 (s, 3H, OMe), 3.24 (d, 3JC-H = 2.0 Hz, 3H, NMe). 13C-NMR (101 MHz,
CDCl3)
δ (ppm): 173.2 (C=O), 61.5 (OC CH2OH), 32.50 (NMe).
H3), 59.8 (d, 1JC-C = 50.1 Hz,
FT-IR (neat): 3434.4 (OH), 1614.8 (C(O)N). HRMS: calcd for [C313CH9NNaO3]+: 143.05082;
found: 143.05097.
[1-13C]-2-(tert-Butyldiphenylsilyloxy)-N-methoxy-N-methylacetic acid (11
).
A solution of
◦
alcohol 10 (213 mg, 1.78 mmol) in dry CH2Cl2 (4 mL) was cooled to 0 C under argon
atmosphere, Et3N (494
µ
L, 3.55 mmol), TBDPSCl (554 µL◦ , 2.13 mmol) and a catalytic
amount of DMAP were added. The reaction was stirred at 0 C during a 10 min period, and
then it was left to stir overnight at room temperature. Then, a saturated aqueous solution
of NH4Cl was added, and the aqueous phase was extracted with 3 portions of CH2Cl2.
The combined organic layers were dried with MgSO4, filtered and concentrated under
reduced pressure. The crude was purified by flash column chromatography (gradient:
5/95
→
10/90
→
20:80 AcOEt/Hexane), and 635 mg of compound 11 was obtained as a
1
white solid (Yield = 100%). M.P. 54–55 ◦C (0.574 g). H-NMR (400 MHz, CDCl3)
δ
(ppm):
7.76–7.70 (m, 4H, Har-m), 7.45–7.35 (m, 6H, Har-o-p), 4.42 (d, 2JC-H = 4.1 Hz, 2H, CH2), 3.43
3
(
s, 3H, OMe), 3.13 (d, JC-H = 2.1 Hz, 3H, NMe), 1.11 (s, 9H, t-Bu). 13C-NMR (101 MHz,
CDCl3)
127.7 (4
(
δ
(ppm): 171.8 (C=O), 135.7 (4
×
Carom), 133.2 (2
×
Carom-quat), 129.8 (2
×
Carom-p),
×
Carom), 62.0 (d, 1JC-C = 56.1 Hz,
C
H2), 61.2 (OMe), 32.5 (NMe), 26.7 (t-Bu), 19.3
t-Bu). FT-IR (neat): 1666.8 (C(O)N). HRMS: calcd. for [C3813C2H54N2NaO6Si2]+: 739.34797;
found: 739.34843.
[2-13C]-(tert-Butyldiphenylsilyloxy)pent-3-yne-2-one (12
in THF, 1.76 mL, 2.5 mmol) was slowly added n-BuLi (1.6 M in hexanes, 1.6 mL, 2.4 mmol)
at
78 ◦C. After 30 min at 78 ◦C, the Weinreb amide 11 (0.748 g, 2.1 mmol) in THF was
added. The reaction mixture was stirred at
78 ◦C for 20 min, and then at 0 ◦C for 1 h.
Saturated NH4Cl solution (10 mL) was added and the mixture extracted with CH2Cl2
). To a solution of propyne (1.52 M
−
−
−
(3
×
10 mL) and the combined extracts dried (MgSO4), concentrated and purified by
flash chromatography (30:70 EtOAc/hexane) to afford 12 as white crystals. M.P. 54–55 ◦C
1
(0.638 g, 85%). H-NMR (400 MHz, CDCl3)
δ
(ppm): 7.69–7.66 (m, 4H), 7.44–7.37 (m, 6H),
4.30 (d, J = 4.1 Hz, 2H), 1.99 (d, J = 1.4 Hz, 3H), 1.10 (s, 9H). 13C-NMR (100 MHz, CDCl3)
(ppm): 186.1, 135.6, 132.8, 129.9, 128.7, 93.3, 78.8, 70.5 (d, J = 49.7 Hz), 26.7, 19.3, 4.2.
δ
(2R)-2-[13C]-(tert-Butyldiphenylsilyloxy)pent-3-yne-2-ol (13
). Ru(p-cymene)[(S,S)TsDPEN]
catalyst was prepared in accordance with the procedure described in the literature [25].
To a degassed solution of ketone 12 (537 mg, 1.59 mmol) in dry i-PrOH (12 mL) was added
Ru(p-cymene)[(S,S)TsDPEN] (72 mg, 0.0955 mmol, 0.06 eq.) in dry and degassed i-PrOH
(3 mL) and the brown solution was left to stir at room temperature under argon atmosphere.
After 2 h, the solvent was removed under reduced pressure and the crude was purified
20
by flash chromatography (5:95 EtOAc/hexane) to afford 13 (513 mg, 95%, 94% ee). [
α
]
D
1
+7.48 (c 2.62, CH2Cl2). H-NMR (400 MHz, CDCl3)
δ
(ppm): 7.70–7.65 (m, 4H), 7.44–7.37
(m, 6H), 4.43 (dm, J = 148.6 Hz, 1H), 3.77 (dd, J = 3.8, 10.2 Hz, 1H), 3.70–3.66 (m, 1H), 1.81
(t, J = 1.6, 3H), 1.07 (s, 9H). 13C-NMR (100 MHz, CDCl3)
δ
(ppm): 135.6, 135.5, 133.0, 132.9,
129.9, 129.8, 127.8, 127.7, 82.0 (d, J = 12.7 Hz), 79.9, 67.9 (1H, J = 39.9 Hz), 79.9, 63.4, 26.8,
19.3, 3.5.
(2R)-2-[13C]-Pent-3-yne-1,2-diol (14
). To alcohol 13 (0.544 g, 1.6 mmol) in THF (3 mL)
was added TBAF 1 M in THF (1.61 mL, 1.6 mmol) at r.t. The reaction mixture was stirred
for 1h. Water (5 mL) was then added, and the aqueous phase was extracted with ethyl
acetate (3 x 5 mL). The combined organic phases were concentrated, and the crude residue
was purified by flash chromatography (70:30 EtOAc/hexane) to afford 14 as white crystals
20
1
M.P. = 67–68 ◦C (0.114 g, 71%). [
δ
α
]
−
21.2 (c 0.99, CH2Cl2). H-NMR (400 MHz, CDCl3)
D
(ppm): 4.42 (dm, J = 148.7 Hz, 1H), 3.71 (ddd, J = 0.64, 3.7, 11.3, 1H), 3.63 (ddd, J = 2.4,
6.64, 11.4, 1H), 1.86 (dd, J = 1.52, 1.88 Hz, 3H). 13C-NMR (100 MHz, CDCl3)
δ (ppm):
81.7 (d, J = 12.4 Hz), 78.9, 66.7 (d, J = 38.5 Hz), 63.4, 3.5. HRMS: calcd. for C4H8O2Na13C
124.0450, found: 124.0453 (M + Na).

Molecules 2021, 26, 369
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(2R)-2-[13C]-Pent-1,2-benzoyloxy-3-yne (15
).
To the diol 14 (0.010 g, 0.1 mmol) in CH2Cl2
◦
(1 mL) at 0 C was added N-ethyldiisopropylamine (0.070 mL, 0.4 mmol), benzoyl chloride
(0.035 mL, 0.3 mmol) and a catalytic amount of DMAP. The mixture was stirred at room
temperature until total consumption of the starting material. A saturated solution of
NaHCO3 was added and the resulting mixture was extracted with CH2Cl2, concentrated
under vacuum and purified by flash chromatography (70:30 EtOAc/hexane) to give 15 as
white crystals (0.028 g, 91% yield) M.P. = 70 ◦C. [
(400 MHz, CDCl3)
(m, 2H), 7.45–7.39 (m, 4H), 5.96 (dm, J = 154 Hz, 1H), 4.65–4.63 (m, 2H), 1.87 (t, J = 1.44 Hz,
3H). 13C-NMR (100 MHz, CDCl3)
(ppm): 166.04 (d, J = 2 Hz), 165.5 (d, J = 2 Hz), 133.2,
α
]
−
46.3 (c 1.35, CH2Cl2). H-NMR
20
1
D
δ
(ppm): 8.07 (d, J = 7.2 Hz, 2H), 8.01 (d, J = 7.1 Hz, 2H), 7.58–7.52
δ
133.1, 129.8, 129.7, 129.63, 129.60, 128.42, 84.0 (d, J = 13 Hz), 79.5, 73.5, 72.7, 65.4 (d, J = 42.2
Hz), 63.0, 3.7. HRMS: calcd. for C18H16O4Na13C 332.0974, found: 332.0972 (M + Na).
(2R)-2-[13C]-1,2-Cyclohexylidenedioxypent-3-yne (16
). To diol 14 (0.059 mg, 0.6 mmol) in
DMF (2.0 mL) at room temperature, was added 1,1-dimethoxycyclohexanone (0.269 mL,
1.8 mmol) and H2SO4 (1 drop), and the reaction mixture was stirred overnight. The reaction
was quenched with NaHCO3 sat. solution (2 mL) (pH
Et2O (3 2 mL), concentrated and purified by flash chromatography (5:95 EtOAc/hexane)
to afford 16 (94 mg, 89%) as a colourless oil. [
(400 MHz, CDCl3) (ppm): 4.67 (dm, J = 153.6 Hz, 1H), 4.10 (dd, J = 1.68, 7.8 Hz, 1H),
3.81 (ddd, J = 2.4, 7.1, 14.8 Hz, 1H), 1.85 (d, J = 1.8 Hz, 3H), 1.74–1.39 (m, 10H). 13C-NMR
≥
8). The mixture was extracted with
×
20
α
]
−
37.6 (c 1.31, CHCl3). 1H-NMR
D
δ
(100 MHz, CDCl3)
δ (ppm): 110.4, 82.1 (d, J = 13.9 Hz), 75.9, 69.6 (d, J = 32.9 Hz), 65.5, 35.7,
35.4, 25.0, 23.8, 23.7, 22.8, 3.6. HRMS: calcd. for C10H15O213C 180.1100, found: 180.1101
(M-H).
(4S)-4-[13C]-4,5-Cyclohexylidenedioxy-2,3-pentadione (17
). To compound 16 (106 mg,
0.6 mmol) in CCl4 (3.2 mL) and MeCN (3.2 mL) was added NaIO4 (286 mg, 1.3 mmol) in
H2O (4.8 mL) and RuO2.H2O (2.0 mg, 0.015 mmol), the reaction mixture was vigorously
stirred until all starting material has been consumed (TLC). The mixture was extracted
with ethyl acetate (3
×
10 mL), filtred by silica gel and concentrated to afford a yellow oil
20
1
17 (103 mg, 82%). [
α
]
−
11.5 (c 0.8, CHCl3). H-NMR (400 MHz, CDCl3)
δ
(ppm): 5.13
D
(ddd, J = 5.3, 7.9, 153.5 Hz, 1H), 4.35 (t, J = 8.4 Hz, 1H), 4.0 (dd, J = 5.3, 8.9 Hz, 1H), 2.39
(s, 3H), 1.74–1.34 (m, 10H). 13C-NMR (100 MHz, CDCl3)
δ (ppm): 198.1, 194.7, 111.9, 76.5,
65.5 (d, J = 34.6 Hz), 35.4, 34.7, 24.9, 24.5, 23.8, 23.7.
(4S)-4-[13C]-DPD (
1)
.
The experimental procedure described in ref. 8 was followed.
1H NMR (500 MHz, CDCl3)
δ
(ppm): 4.05 (ddd, J = 149.6, 7.0, 5.8 Hz, H4, cyclic form),
3.87–3.82 (m), 3.70 (dm, J = 145 Hz, H4, cyclic form), 3.65 (dm, J = 145 Hz, H4, linear form),
3.52–3.47 (m), 3.24 (ddd, J = 10.95, 5.4, 5.4 Hz), 2.04 (CH3, linear form), 1.11 (CH3, cyclic
form), 1.08 (CH3, cyclic form). 13C-NMR (100 MHz, CDCl3)
73.8 (C4, linear form), 73.2 (C4, cyclic form).
δ (ppm): 74.0 (C4, cyclic form),
1-(tert-Butyldiphenylsilyloxy)but-3-yne-2-one (19
(1.25 mL, 8.09 mmol) in THF (13.6 mL), was added n-Buli (1.6M in hexane, 4.23 mL,
6.76 mmol) dropwise at 0 ◦C, and stirred at 0 ◦C over 10 min. At 78 ◦C, it was added
). To a solution of N-diisopropylamine
−
trimethylsilylacetylene (0.99 mL, 7.1 mmol), after 30 min Weinreb amide 18 was added (2.12
g, 5.93 mmol), previously dissolved in THF (17.3 mL). The mixture was stirred at −78 ◦C
and warmed to room temperature. Water was added (20 mL), the resulting mixture was
extracted with CH2Cl2 (3
vacuum and purified by flash chromatography (30:70 AcOEt:hexane) to afford colourless oil
20 (0.850 g, 62%), 19 (0.107 g, 12% yield) and 27 (0.330 g, 16% yield). Data for 19: H-NMR
×
15 mL), dried with anhydrous MgSO4, concentrated under
1
(400 MHz, CDCl3)
δ
(ppm): 7.70–7.67 (m, 4H), 7.44–7.37 (m, 6H), 4.29 (s, 2H), 1.10 (s, 9H),
1
0.22 (s, 9H). Data for 20: H-NMR (400 MHz, CDCl3)
δ
(ppm): 7.68–7.65 (m, 4H), 7.45–7.38
(ppm): 185.3,
C), 1706 (C=O).
(m, 6H), 4.34 (s, 2H), 3.26 (s, 1H), 1.10 (s, 9H). 13C-NMR (100 MHz, CDCl3)
δ
135.5, 132.5, 130.0, 127.9, 81.2, 79.6, 70.6, 26.6, 19.3. FT-IR (film): 2094 (C
≡
1
Data for 27: H-NMR (400 MHz, CDCl3)
δ
(ppm): 7.84 (d, J = 12.9 Hz, 1H), 7.69–7.67 (m,
4H), 7.43–7.34 (6H, m), 5.92 (d, J = 12.9 Hz, 1H), 4.17 (s, 2H), 1.11 (s, 9H). 13C-NMR (100
MHz, CDCl3) δ (ppm): 196.2, 148.3, 135.5, 133.3, 129.7, 127.7, 90.7, 68.9, 26.8, 19.3.

Molecules 2021, 26, 369
8 of 9
(2R)-1-(tert-butyldiphenylsilyloxy)but-3-yn-2-ol (21
).
Acetylenic ketone 19 (1 g, 3.1 mmol)
was dissolved in THF (10 mL) at room temperature. (S)-Alpine Borane (0.5 M in THF,
9.3 mL, 4.65 mmol) was added, the mixture was stirred for 36 h. NH4Cl saturated solution
(15 mL) was added, the resulting mixture was extracted one time with ethyl ether (10 mL)
and then with dichloromethane (2
under vacuum and purified by flash chromatography (30:70 AcOEt:hexane and 1:12:12
×
10 mL), dried with anhydrous MgSO4, concentrated
EtOAc:hexanes:CH2Cl2) to give 21 (0.675 g, 67% yield). Usually, no purification was
1
done as it was very difficult to isolate the product. H-NMR (400 MHz, CDCl3)
δ
(ppm):
7.69–7.65 (m, 4H), 7.44–7.37 (m, 6H), 4.47–4.44 (m, 1H), 3.81 (dd, J = 10.2, 3.9 Hz, 1H),
3.74 (dd, J = 10.2, 6.5 Hz, 1H), 2.41 (d, J = 2.1 Hz, 1H), 1.08 (s, 9H). 13C-NMR (100 MHz,
CDCl3) δ (ppm): 135.6, 135.5, 132.8, 132.7, 129.9, 127.87, 127.85, 81.8, 73.6, 67.4, 63.0, 26.8,
19.3. FT-IR (film): 3427 (O-H). To quantify the enantioselectivity of the reaction, a small
sample of alcohol 21 was treated with (R)-MTPA-Cl (3 eq) and DMAP (3 eq) in CH2Cl2 at
room temperature. Integration of 1H-NMR (400 MHz, CDCl3) peaks at
δ (ppm): 2.50 (d,
J = 2.2 Hz, minor) and 2.43 (d, J = 2.2 Hz, major) ppm indicated a dr of 93:7 corresponding
to 86% ee.
(2R)-But-3-yne-1,2-diol (22). To the alcohol 21 (0.5 g, 1.54 mmol) in THF (5 mL) was
added TBAF (1M in THF, 1.85 mL, 1.85 mmol) at room temperature. The reaction mixture
was stirred until all starting material has been consumed (TLC) and then quenched with
H2O (5 mL). The resulting mixture was extracted with ethyl acetate (3
×
5 mL), concentrated
under vacuum and purified by flash chromatography (70:30 EtOAc:hexane) to give 22 as a
20
1
colourless liquid (0.094 g, 71% yield). [
CDCl3)
α
]
=
−
27.4 (c 1.92, CH2Cl2). H-NMR (400 MHz,
D
δ (ppm): 4.49–4.46 (m, 1H), 3.77 (dd, J = 11.4, 3.7 Hz, 1H), 3.70 (dd, J = 11.4, 6.5 Hz,
1H), 2.49 (d, J = 2.1 Hz, 1H). 13C-NMR (100 MHz, CDCl3)
FT-IR (film): 3291 (O-H), 2117 (C≡C).
δ (ppm): 81.8, 74.1, 66.4, 63.0.
(2R)-1,2-Cyclohexylidenedioxybut-3-yne (24). To the diol 22 (0.094 g, 1.09 mmol) in DMF
(2 mL) at room temperature was added 1,1-dimethoxycyclohexane (0.665 mL, 4.37 mmol)
and one drop of conc. H2SO4 and stirred overnight. NaHCO3 saturated solution was added
(pH
≥
8) followed by H2O (5 mL). The mixture was extracted with ethyl ether (3
×
8 mL),
concentrated under vacuum and purified by flash chromatography (5:95 EtOAc:hexane)
to afford colourless oil 24 (0.141 g, 78% yield). H-NMR (400 MHz, CDCl3)
1
δ
(ppm): 4.71
(ddd, J = 6.3, 6.3, 2.0 Hz, 1H), 4.16 (dd, J = 8.4, 6.3 Hz, 1H), 3.94 (dd, J = 8.0, 6.2 Hz, 1H),
2.48 (d, J = 2.2 Hz, 1H), 1.77–1.59 (m, 8H), 1.42–1.40 (m, 2H). 13C-NMR (100MHz, CDCl3)
(ppm): 111.2, 81.6, 73.7, 69.5, 64.9, 35.6, 35.4, 27.0, 25.0, 23.8. FT-IR (film): 2119 (C≡C).
δ
(2R)-5-[13C]-1,2-Cyclohexylidenedioxypent-3-yne (25
). To a solution of N,N-diisopropy-
lamine (0.298 mL, 2.1 mmol) in THF (3.23 mL), was added n-Buli (1.6 M in hexane, 1.06 mL,
1.7 mmol) at 0 ◦C and stirred for 10 min. At
−
78 ◦C, 24 (0.141 g, 0.848 mmol), previously
dissolved in THF (1.15 mL) was added. After 30 min, 13CH3I (0.133 mL, 2.1 mmol) was
◦
added. The mixture was stirred at
−
78 C and allowed to warm to room temperature.
NH4Cl saturated solution was added (5 mL), the resulting mixture was extracted with
CH2Cl2 (3 5 mL), dried with anhydrous MgSO4, concentrated under vacuum and
purified by flash chromatography (50:50 AcOEt:hexane) to afford colourless oil 25 (0.150 g,
×
1
94% yield). H-NMR (400 MHz, CDCl3)
δ
(ppm): 4.69–4.66 (m, 1H), 4.11 (dd, J = 7.9, 6.2
Hz, 1H), 3.82 (dd, J = 7.8, 7.0 Hz, 1H), 1.85 (dd, J = 131.5, 2.0 Hz, 3H), 1.67–1.58 (m, 8H),
1.41–1.38 (m, 2H).
(4S)-1-[13C]-4,5-cyclohexylidenedioxy-2,3-pentadione (26
)
.
To the alkyne 25 (0.083 g, 0.462
mmol) dissolved in CCl4 (2.5 mL) and MeCN (2.5 mL) was added a solution of NaIO4
(0.224 g, 1.05 mmol) in H2O (3.8 mL) and RuO2 H2O (0.0015 g, 0.0115 mmol) and the
·
reaction mixture was stirred vigorously for 15 min. The mixture was extracted with CH2Cl2,
filtered by a short pad of silica for medium pressure chromatography and concentrated
under vacuum to give the bright yellow oil 26 (0.089 g, 91% yield) with NMR data identical
to that described in the literature[5 δ (ppm): 5.13 (dd, J = 7.9,
].1H-NMR (400 MHz, CDCl3)
5.3 Hz, 1H), 4.35 (dd, J = 8.9, 7.9 Hz, 1H), 4.00 (dd, J = 8.9, 5.3 Hz, 1H), 2.38 (d, J = 129.3 Hz,
3H), 1.77–1.38 (m, 10H).

Molecules 2021, 26, 369
9 of 9
(4S)-1-[13C]-DPD (
The characterisation data are in accordance with those described in ref. [5]
1).
The experimental procedure described in ref. [
8] was followed.
Supplementary Materials: The following are available online: Figures S1 and S2: HPLC chro-
matogram of racemic 15 and HPLC chromatogram of optically active 15. Figures S3–S39: NMR spectra.
Author Contributions: C.D.M. and M.R.V. conceived and supervised the experiments; M.R.V.
and K.B.X. acquired funding; O.S.A. and G.C. performed the experiments; O.S.A., G.C., M.R.V.,
and C.D.M. analysed the data. M.R.V., G.C., and K.B.X. wrote the paper. All authors read and agreed
to the published version of the manuscript.
Funding: This research was funded by Fundação para a Ciência e Tecnologia (FCT), project PTDC/BIA-
BQM/29668/2017, and MostMicro Research Unit (financially supported by LISBOA-01-0145-FEDER-
007660 funded by FEDER funds through COMPETE2020 (POCI) and by national funds through FCT.
Acknowledgments: The NMR data was acquired at CERMAX, ITQB-NOVA, Oeiras, Portugal with
equipment funded by FCT, project AAC 01/SAICT/2016.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Samples of the compounds are available for purchase from the authors.
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