Synthesis of a 13C-labelled seed-germination inhibitor (3,4,5-trimethylfuran-2(5H)-one) for the mode of action elucidation

Article abstract of DOI:10.1007/s00706-018-2179-x

Abstract: It has been found that the butenolide 3,4,5-trimethylfuran-2(5H)-one, isolated from plant-derived smoke, efficiently inhibits seed germination and significantly reduces the effect of the highly active germination promotor karrikinolide (KAR

Full text of DOI:10.1007/s00706-018-2179-x

Monatshefte für Chemie - Chemical Monthly
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ORIGINAL PAPER
13
Synthesis of a C-labelled seed-germination inhibitor (3,4,5-
trimethylfuran-2(5H)-one) for the mode of action elucidation
1
2
•
Vilmos Soos Petr Beier1
•
ˇ
´
Martin Posta
Received: 22 November 2017 / Accepted: 25 February 2018
Ó Springer-Verlag GmbH Austria, part of Springer Nature 2018
Abstract
It has been found that the butenolide 3,4,5-trimethylfuran-2(5H)-one, isolated from plant-derived smoke, efficiently
inhibits seed germination and significantly reduces the effect of the highly active germination promotor karrikinolide
(KAR1, 3-methyl-2H-furo[2,3-c]pyran-2-one), another smoke-derived compound. This paper reports the synthesis of
stable isotope-labelled 3,4,5-trimethylfuran-2(5H)-ones containing one and two 13C atoms for the identification of meta-
bolic degradation products in order to provide insight into the mechanism of action.
Graphical abstract
Keywords Butenolide Á Seed-germination inhibitor Á Carbon-13 Á Lactuca sativa Á Lactuca serriola Á Smoke
Introduction
furo[2,3-c]pyran-2-one (1) (KAR1), was isolated and
characterised independently by Flematti et al. [4] and Van
Staden et al. [5]. In 2010, a seed-germination inhibitor
active at micromolar concentrations was isolated from the
smoke and its structure was determined as 3,4,5-
trimethylfuran-2(5H)-one (TMB, 2) [6]. Moreover, this
compound, antagonistic to the KAR1, was found to be able
to suppress significantly the seed germination promoting
effect of KAR1, as was demonstrated on lettuce seeds
(Lactuca sativa, cv. ‘Grand Rapids’) [6].
The composition of smoke released by natural fires is very
complex with nearly 5000 chemical species [1]. Some of
them exhibit a biological activity in the regulation of seed
germination. In 2004, a new class of butenolides (karrikins)
significantly promoting seed germination was identified in
the smoke of burning vegetation [2, 3]. Namely, an
extremely efficient germination stimulant, 3-methyl-2H-
These two types of smoke-derived regulators, karrikins
and TMB, constitute a new class of plant-growth regulators
that not only control seed germination [7] but also play an
important role in the development of young plants [8]
(Fig. 1).
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s00706-018-2179-x) contains supplementary
material, which is available to authorized users.
ˇ
& Martin Posta
posta@uochb.cas.cz
Whereas the germination stimulants karrikins have been
a focus of research for their application in agriculture and
their mode of action is intensively being investigated
[9–16], hardly anything is known about the mode of action
of TMB. Despite the structural similarity between 1 and 2,
1
Institute of Organic Chemistry and Biochemistry, CAS, v.v.i.,
´
Flemingovo nam. 2, 166 10 Prague 6, Czech Republic
2
Department of Applied Genomics, Agricultural Institute,
Centre for Agricultural Research, Hungarian Academy of
´ ´
Sciences, Martonvasar 2462, Hungary
123

M. Pošta et al.
difference of 2 mass units between the molecular weights
of the isotopically labelled and unlabelled TMB and makes
it easy to distinguish between metabolites using mass
spectrometry. Another increase in molecular mass is pos-
sible via deuterium labelling of the TMB molecule, but we
have avoided this method due to difficulties in the NMR
analysis of the isolated metabolites.
Compound 5, a molecule bearing one 13C methyl group,
was prepared following the two-step procedure reported by
Surmont et al. [21] (see Scheme 1), starting from the
commercially available 2,3-dimethylmaleic anhydride (3).
The alkylation of the anhydride with freshly prepared 13C
methyllithium gave hemiacetal 4 in a moderate yield of
54% due to the inefficient conversion of 13CH3I to the
corresponding organolithium reagent. The target racemic
butenolide 5 was then obtained by the reduction of 4 with
sodium borohydride in 74% yield. The individual enan-
tiomers were not separated because it had been demon-
strated that both forms were equally active as germination
inhibitors [6].
Fig. 1 The structures of 1 and 2
´
Soos et al. [17] have demonstrated that these two com-
pounds do not compete for the same receptor. Therefore,
the identification of potential TMB-binding proteins and
the elucidation of the importance of enzymatic butenolide
hydrolysis for biological activity require further
investigation.
In order to fill this gap, we have initiated a research
project aimed at the identification of the protein-binding
site using various types of molecular probes [18]. We have
also prepared a series of TMB analogues for a structure–
activity relationship study [19, 20] in order to reveal the
factors influencing activity and to provide insight into the
mode of action of TMB.
In this paper, we report the synthesis of a 13C-labelled
inhibitor for the detailed identification of TMB metabolic
products and the determination of the metabolic pathway
occurring in this process. For this purpose, we have syn-
thesised TMB labelled with one 13C atom (5) following a
literature procedure [21] and we have also developed a
synthetic protocol for the synthesis of TMB labelled with
two 13C atoms (9).
Whereas 5 was synthesised according to a published
procedure [21], the new three-step synthetic protocol out-
lined in Scheme 2 was developed for the synthesis of
germination inhibitor 9, labelled with two 13C atoms.
The reaction sequence consists of the known reduction
of anhydride to the corresponding 5-hydroxylactone, fol-
lowed by the introduction of the methyl substituent at C-5
as reported by Canonne et al. [22]. Our synthesis started
with the treatment of citraconic anhydride (6) with 1.4
equivalents of the reducing agent LiAlH(t-BuO)3, which
provided hemiacetal 7a after the separation of a regioiso-
mer mixture of 7a and 7b, formed in the ratio 9:1. In the
second step, the alkylation of 7a with freshly prepared
13CH3MgI provided the desired butenolide 8 in a moderate
yield of 57%. Finally, methylation in position 3 was
accomplished by treating 8 with a strong base followed by
the addition of 13CH3I to the preformed enolate. First, the
enolate-forming conditions reported by Hinrichs and
Margaretha [23] were tested for the synthesis of
thiophenones.
Results and discussion
Synthesis
Retrosynthetic analysis has revealed that positions 3 and 5
in the TMB molecule are the only accessible sites for
stable-isotope labelling using relatively cheap 13CH3I. This
synthetic protocol has made it possible to introduce two
13C atoms into the molecule, which produces a desirable
However, the heating of 8 with sodium hydride in
DMSO did not give the desired product 9 after the
Scheme 1
123

Synthesis of a 13C-labelled seed-germination inhibitor (3,4,5-trimethylfuran-2(5H)-one) for…
Scheme 2
Table 1 The optimisation of the
Entry
Conditions
Yield of 9/%a
Yield of 10/%a
Yield of 11/%a
synthesis of 9 (the experiments
were carried out with CH3I prior
to the use of 13CH3I)
1
2
3
4
5
6
7
8
NaH, DMSO, 60 °C
NaH, DMSO, r.t.
0
0
6
9
0
0
NaH, THF, r.t.
2
8
3
LDA, THF, - 70 °C to r.t.
t-BuOK, DMSO, 60 °C
t-BuOK, DMSO, r.t.
t-BuOK, THF, r.t.
8
13
15
11
10
8
21
12
16
14
11
9
14
18
27
t-BuOK, THF, - 30 °C to r.t.
aIsolated yield
treatment with CH3I. The reaction mixture contained
mainly polar compounds, most likely resulting from the
opening of the butenolide ring under very basic conditions,
and traces of product 10, which was isolated. After the
reaction conditions were modified (Table 1), t-BuOK was
identified as a suitable base for enolate formation (Table 1,
entry 8). The target compound 9 was isolated in 24% yield
after the addition of 13CH3I (27% yield using CH3I).
Sideproducts 10 and 11 were also isolated and
characterised.
investigation of TMB applied either alone [6] or in com-
bination with karrikins [14], is only sensitive when the
achenes are germinated in the dark. The last type, the
highly TMB-sensitive wild Lactuca serriola, has been
found to be the most suitable to study TMB, as its achenes
have exhibited more delayed germination (Fig. 2).
Therefore, the sensitive and insensitive accessions and
genetic screens based on Lactuca sativa ‘Salinas’ x Lac-
tuca serriola inbred populations could provide a useful tool
for research into the effect of TMB on germination. Along
with stable isotope-labelled derivatives, these systems can
shed light on the proposed metabolism of TMB as well as
its mode of action.
The evaluation of the biological activity of TMB
(2)
To identify the most suitable model plant for a metabo-
lomics study of the 13C-labelled TMB, we have tested three
lettuce accessions. Our tests have demonstrated that culti-
vated lettuce, Lactuca sativa ‘Salinas’, is highly insensitive
to TMB and exhibits only a small delay in germination.
The second type, the photoblastic lettuce cultivar Lactuca
sativa ‘Grand Rapids’, recently used as a model for the
Conclusion
In conclusion, two types of seed-germination inhibitor
(TMB) labelled with one or two 13C atoms have been
synthesised. Compound 5 with one 13C atom has been
synthesised following the procedure reported in the
123

M. Pošta et al.
All syntheses were carried out under an argon or nitro-
gen atmosphere in anhydrous THF or diethyl ether, which
were dried with sodium and benzophenone and distilled
prior to use. Commercially available 2,3-dimethylmaleic
anhydride, citraconic anhydride. LiAlH(t-BuO)3, NaBH4,
and 13CH3I were purchased from Sigma-Aldrich and used
for the synthesis without further purification.
5-Hydroxy-3,4-dimethyl-5-(methyl-13C)furan-2(5H)-one (4)
[21] Lithium (0.5 g, 72.00 mmol) was stirred and heated
to 210 °C in silicone oil under an argon atmosphere until it
melted and very small particles were formed. The mixture
was allowed to cool slowly under vigorous stirring and
then the oil was removed by a syringe and the lithium
particles were washed with anhydrous Et2O (5 9 10 cm3).
The lithium was suspended in 4 cm3 Et2O and a solution of
1.0 g 13CH3I (7.05 mmol) in 2 cm3 Et2O was added
dropwise. The reaction mixture was left to boil sponta-
neously, after which it was stirred for further 30 min at
45 °C. After cooling, the solution was filtered and used for
the alkylation step.
Fig. 2 The germination inhibitory activity of TMB (2) and the
germination characteristics of Lactuca serriola and Lactuca sativa
‘Salinas’ achenes. Achenes (seeds) were germinated in Petri dishes,
imbibed in 3 cm3 of a 20-lM solution of 2. Germinated achenes were
scored for 7 days and the data were analysed with the OriginPro
software
A solution of 630.0 mg 3 (5.00 mmol) in 20 cm3
anhydrous Et2O under an argon atmosphere was cooled
to - 70 °C. Subsequently, the above solution of 13CH3Li
was added dropwise, the mixture was stirred for 15 min
and quenched with 20 cm3 saturated aqueous solution of
NH4Cl. The mixture was extracted with AcOEt
(3 9 30 cm3) and the combined organic fraction was dried
with MgSO4. The solvent was removed in vacuo. Purifi-
cation by column chromatography (SiO2, 30% AcOEt in
petroleum ether) afforded pure 4 (384 mg, 54%) as a white
solid. TLC: Rf = 0.22 (30% AcOEt in petroleum ether); 1H
NMR (400 MHz, acetone-d6): d = 6.03 (d, J = 5.2 Hz,
1H), 1.97 (q, J = 1.2 Hz, 3H), 1.72 (q, J = 1.2 Hz, 3H),
1.55 (d, J = 128.3 Hz, 3H) ppm; 13C NMR (100 MHz,
acetone-d6): d = 172.00 ([C\), 159.45 (d, J = 1.9 Hz,
[C\), 124.24 ([C\), 106.01 (d, J = 45.7 Hz, [C\),
23.92 (CH3), 10.45 (CH3), 8.37 (CH3) ppm; MS (EI): m/z
(%) = 143.0 ([M?], 3), 127.1 (100), 115.1 (10), 99.1 (87),
83.1 (16), 54.1 (53), 44.1 (61).
literature, while a new synthetic protocol has been devel-
oped and optimised for the preparation of 9 with two 13C
atoms in the molecule. These stable isotope-labelled
molecules may be useful for research into the physiological
processes involved in smoke-induced seed-germination
inhibition as well as in capturing TMB metabolites and for
the assessment their structures and functions. The results
from such studies will be reported in due course.
Experimental
1H and 13C nuclear magnetic resonance (NMR) spectra
were measured using a Bruker Avance I 400 spectrometer
(400 and 100 MHz), and in some cases a Bruker Avance I
500 spectrometer (500 and 125 MHz), in acetone-d6 or in
CDCl3 with the residual solvent signal acting as the
internal standard (acetone-d6: 1H 2.05, 13C 29.84 ppm;
3,4-Dimethyl-5-(methyl-13C)furan-2(5H)-one (5) [21]
A
1
CDCl3: H 7.26, 13C 77.16 ppm). The data are reported as
solution of 179 mg 4 (1.25 mmol) in 48 cm3 H2O and
2 cm3 THF was cooled in an ice bath and 237 mg NaBH4
(6.25 mmol) was added in portions. The reaction mixture
was stirred for 5 h, after which it was quenched by the
addition of 50 cm3 1 M HCl. The product was extracted
with AcOEt (3 9 30 cm3) and the combined organic
fraction was dried with MgSO4. The solvent was removed
in vacuo. Purification by column chromatography (SiO2,
30% AcOEt in petroleum ether) afforded pure 5 (118 mg,
74%) as a colourless oil. TLC: Rf = 0.45 (30% AcOEt in
petroleum ether); 1H NMR (500 MHz, CDCl3):
d = 4.74 - 4.72 (m, 1H), 1.89 (m, 3H), 1.73 - 1.72 (m,
follows: the chemical shift (d) in parts per million (ppm),
multiplicity
(s = singlet,
d = doublet,
t = triplet,
q = quartet, dd = doublet of doublet, m = multiplet), cou-
pling constant (Hertz), integration, and interpretation.
High-resolution mass spectra were recorded using a Waters
GCT Premier bench-top orthogonal acceleration time-of-
flight (OA-TOF) mass spectrometer with chemical ionisa-
tion. TLC analysis was carried out using commercial
Sigma-Aldrich aluminium plates coated with silica gel with
fluorescent indicator F254s. Spots were visualised under
UV and developed in permanganate stain.
123

Synthesis of a 13C-labelled seed-germination inhibitor (3,4,5-trimethylfuran-2(5H)-one) for…
3H), 1.32 (dd, J = 128.8, 6.8 Hz, 3H) ppm; 13C NMR
(125 MHz, CDCl3): d = 174.45 ([C\), 160.52 ([C\),
122.74 (d, J = 1.3 Hz, [C\), 79.53 (d, J = 38.5 Hz, CH),
18.11 (13CH3), 11.74 (CH3), 8.39 (CH3) ppm; MS (EI): m/z
(%) = 127.1 ([M?], 36), 111.1 (12), 83.1 (69), 55.1 (100),
54.1 (16), 44.1 (19).
5.01 - 4.94 (m, 1H), 2.09 (ddd, J = 1.5, 0.8, 0.4 Hz, 3H),
1.38 (ddd, J = 128.8, 6.8, 0.4 Hz, 3H) ppm; 13C NMR
(125 MHz, acetone-d6): d = 172.98 ([C\), 171.60 ([C\),
116.35 (CH), 81.38 (d, J = 38.2 Hz, CH), 18.40 (13CH3),
13.52 (CH3) ppm; MS (EI): m/z (%) = 113.1 ([M?], 26),
97.0 (23), 69.0 (100), 68.0 (19), 44.0 (21), 41.1 (56), 40.1
(25), 39.1 (44).
5-Hydroxy-4-methylfuran-2(5H)-one (7a) and 5-hydroxy-3-
methylfuran-2(5H)-one (7b) A solution of 5.34 g LiAlH(t-
BuO)3 (21.00 mmol) in 40 cm3 anhydrous THF was added
dropwise over a 30-min period to a solution of 1.68 g
citraconic anhydride (6, 15.00 mmol) in 50 cm3 anhydrous
THF under a nitrogen atmosphere at - 30 °C. The tem-
perature was maintained at - 15 °C for 3 h and then the
reaction mixture was warmed to ambient temperature. The
reaction was quenched with 50 cm3 1 M HCl, the solution
was saturated with NaCl, the crude product was extracted
with EtOAc (3 9 50 cm3), and the combined organic
fraction was dried over MgSO4. The solvent was removed
in vacuo. Purification by column chromatography (SiO2,
20% AcOEt in petroleum ether) afforded 7a (1.023 g,
60%) and 7b (116 mg, 7%) as yellow oils. TLC: Rf = 0.16
(for 7a), 0.15 (for 7b) (20% AcOEt in petroleum ether).
7a: 1H NMR (400 MHz, acetone-d6): d = 6.67 (bs, 1H),
6.02 (bs, 1H), 5.87 (p, J = 1.5 Hz, 1H), 2.08 (d,
J = 1.5 Hz, 3H) ppm; 13C NMR (100 MHz, acetone-d6):
d = 171.30 ([C\), 166.65 ([C\), 118.68 (CH), 100.25
(CH), 13.15 (CH3) ppm; MS (EI): m/z (%) = 114.0 ([M?],
2), 113.0 (7), 86.0 (61), 85.0 (13), 69.0 (100), 68.0 (82),
41.1 (50), 40.1 (65), 39.1 (93).
4-Methyl-3,5-di(methyl-13C)furan-2(5H)-one (9)
A
sus-
pension of 348 mg t-BuOK (3.1 mmol) in 6 cm3 anhy-
drous THF was cooled to - 30 °C under a nitrogen
atmosphere and then a solution of 338 mg 8 (3.0 mmol) in
2 cm3 THF was added dropwise. The reaction mixture was
stirred for 30 min, after which 916 mg 13CH3I (6.4 mmol)
in 2 cm3 anhydrous THF was added dropwise. The mixture
was allowed to warm to room temperature, stirred over-
night and poured into 100 cm3 water. The pH was adjusted
to 4 with 1 M HCl and saturated with NaCl. The crude
product was extracted with AcOEt and the combined
organic fraction was washed with water, saturated aqueous
solutions of NaHCO3, Na2S2O3, and brine and then dried
over MgSO4. The solvent was removed in vacuo. Purifi-
cation by column chromatography (SiO2, 30% AcOEt in
petroleum ether) afforded 9 (92 mg, 24%) as a colourless
1
oil. TLC: Rf = 0.45 (30% AcOEt in petroleum ether); H
NMR (500 MHz, CDCl3): d = 4.78 - 4.72 (m, 1H), 1.92
(s, 3H), 1.76 (ddp, J = 128.8, 2.1, 1.1 Hz, 3H), 1.36 (dd,
J = 128.8, 6.8 Hz, 3H) ppm; 13C NMR (125 MHz,
CDCl3): d = 174.56 (d, J = 4.1 Hz, [C\), 160.50 ([C\),
122.92 (dd, J = 48.7, 1.3 Hz, [C\), 79.62 (dd, J = 38.5,
3.6 Hz, CH), 18.21 (13CH3), 11.84 (CH3), 8.50 (13CH3)
ppm; MS (EI): m/z (%) = 128.1 ([M?], 47), 112.1 (15),
84.1 (76), 56.1 (100), 55.1 (18), 44.1 (21).
4-Methyl-5-(methyl-13C)furan-2(5H)-one (8) A solution of
2.29 g 13CH3I (16.02 mmol) in 2 cm3 anhydrous Et2O was
added dropwise at room temperature to a stirred suspension
of 600 mg Mg turnings (24.70 mmol) in 5 cm3 anhydrous
Et2O under nitrogen atmosphere in a two necked flask
fitted with a septum and a condenser. The reaction mixture
was allowed to spontaneously boil and then was stirred for
further 1 h at 40 °C.
Germination bioassay
The germination activity of TMB was checked in three
lettuce accessions: Lactuca sativa’Salinas’ cultivated let-
tuce, the wild-type Lactuca serriola lettuce, and the pho-
toblastic Lactuca sativa ‘Grand Rapids’. Freshly collected
(i.e. stored for \ 2 month in a seed vault) achenes were
germinated on two layers of filter paper placed in Petri
dishes. The dishes were dampened with either distilled
water (mock) or 3 cm3 of a 20 lM solution of TMB (2).
Sealed dishes were placed in a controlled environmental
chamber (22 °C; continuous low-light regime, 8 lE or in
the dark). The germination for the three biological repli-
cates (50 achenes each) was assayed for 7 days.
A solution of 625 mg 7a (5.48 mmol) in 5 cm3 anhy-
drous Et2O was added dropwise to a stirred solution of
13CH3MgI at 0 °C. The reaction mixture was cooled in an
ice bath for 1 h, after which it was allowed to warm to
ambient temperature and stirred overnight. The reaction
was quenched by the addition of 50 cm3 1 M HCl
dropwise, the solution was saturated with NaCl and the
crude product was extracted with AcOEt. The combined
organic fraction was washed with saturated aqueous
solutions of NaHCO3, Na2S2O3, and brine and then dried
over MgSO4. The solvent was removed in vacuo. Purifi-
cation by column chromatography (SiO2, 30% AcOEt in
petroleum ether) afforded 8 (352 mg, 57%) as a yellow oil.
TLC: Rf = 0.25 (30% AcOEt in petroleum ether); 1H NMR
(500 MHz, acetone-d6): d = 5.78 (hept, J = 1.3 Hz, 1H),
Acknowledgments The IOCB research plan RVO: 61388963, OTKA
114567 and Bolyai Fellowship, supporting this work, are
acknowledged.
123

M. Pošta et al.
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