Design of photoaffinity labeling probes derived from 3,4,5-trimethylfuran-2(5H)-one for mode of action elucidation

Article abstract of DOI:10.1016/j.tet.2016.03.096

Herein we report the synthesis of new probes for photoaffinity labeling with the aim of receptor identification and mode of action elucidation of 3,4,5-trimethylfuran-2(5H)-one (TMB), recently identified in the smoke of burning vegetation as an efficient seed germination inhibitor. These photoaffinity probes consist of an ethynyl group that acts as a tag for introduction of an optional detectable marker unit by an appropriate chemoselective ligation method after the photo-cross-linking operation.

Full text of DOI:10.1016/j.tet.2016.03.096

Accepted Manuscript
Design of photoaffinity labeling probes derived from 3,4,5-trimethylfuran-2(5H)-one for
mode of action elucidation
Martin Pošta, Vilmos Soós, Petr Beier
PII:
S0040-4020(16)30237-X
10.1016/j.tet.2016.03.096
TET 27636
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 28 January 2016
Revised Date: 15 March 2016
Accepted Date: 29 March 2016
Please cite this article as: Pošta M, Soós V, Beier P, Design of photoaffinity labeling probes derived
from 3,4,5-trimethylfuran-2(5H)-one for mode of action elucidation, Tetrahedron (2016), doi: 10.1016/
j.tet.2016.03.096.
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ACCEPTED MANUSCRIPT
Graphical Abstract
Design of photoaffinity labeling probes derived from
3,4,5-trimethylfuran-2(5H)-one for mode of action
elucidation
Leave this area blank for abstract info.
Martin Pošta, Vilmos Soós, Petr Beier
Institute of Organic Chemistry and Biochemistry AS CR, v.v.i.
Flemingovo nám. 2.,
16610, Prague 6, Czech Republic
Department of Applied Genomics, Agricultural Institute,
Centre forAgricultural Research, Hungarian Academy of Sciences,
2462, Martonvásár, Hungary

1
ACCEPTED MANUSCRIPT
Tetrahedron
journal homepage: www.elsevier.com
Design of photoaffinity labeling probes derived from 3,4,5-trimethylfuran-2(5H)-one
for mode of action elucidation
Martin Poštaa, , Vilmos Soósb and Petr Beiera
aInstitute of Organic Chemistry and Biochemistry v.v.i. Flemingovo nám. 2, Prague, 16610, Czech Republic
bDepatrtment of Applied Genomics, Agricultural Institute, Centre for Agricultural Research, Hungarian Academy of Sciences, Martonvásár, 2462, Hungary
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Herein we report the synthesis of new probes for photoaffinity labeling with the aim of receptor
identification and mode of action elucidation of 3,4,5-trimethylfuran-2(5H)-one (TMB), recently
identified in the smoke of burning vegetation as an efficient seed germination inhibitor. These
photoaffinity probes consist of an ethynyl group that acts as a tag for introduction of an optional
detectable marker unit by an appropriate chemoselective ligation method after the photo-cross-
linking operation.
Available online
Keywords:
photoaffinity labeling probes
protein cross-linking
smoke
2009 Elsevier Ltd. All rights reserved
.
butenolides
1. Introduction
Smoke from burning vegetation is a very complex mixture
containing close to 5000 chemical species1, many of which
exhibit biological activity. One very important group present in
the smoke is the butenolides with germination regulatory
activity.2 In 2004, the butenolide 3-methyl-2H-furo[2,3-c]pyran-
2-one (1) (reffered to as karrikine, KAR1)3 was identified as an
Figure 1. Structure of 1 and 2.
extremely potent germination promotor, active at 10-9
M
concentrations. Later in 2010, a compound with germination
inhibitory activity was also isolated from the smoke by Van
Staden and co-workers, namely 3,4,5-trimethylfuran-2(5H)-one
(TMB)4 (2). This inhibitor was shown to be active at 10 and 100
µM concentrations, and application together with 0.1 µM KAR1
(1) suppressed significantly the germination of ‘Grand Rapids’
lettuce seeds (Lactua sativa cv. ‘Grand Rapids’) in comparison to
treatments with KAR1 alone.4 Both types of smoke-derived
substances (karrikins and TMB) are related to strigolactones, the
newest class of phytohormones and constitute a new type of
smoke-derived plant growth regulators affecting germination
processes5 and seedling development6.
Although there has been an intensive effort toward elucidation
of the mode of action of butenolide-type molecules, including
strigolactones, KAR1 (1) and other structurally related karrikins,7
relatively little research has been carried out investigating the
mode of action of the germination inhibitor 2. Despite the fact
that TMB and KAR1 share significant structural similarity, their
application results in highly contrasting physiological responses.
For this reason, it was intriguing to consider that these two
compounds exhibit mutually antagonistic effect. However, the
recent study revealed that these two compounds are not direct
competitors sharing the same binding site.8 Thus an identification
of the TMB receptor as well as the determination whether TMB
acts, analogously to strigolactones and karrikins, through the
enzymatic hydrolysis of the butenolide ring7e has to be
investigated.
In order to identify the protein binding site and elucidate the
molecular mechanism involved in the germination inhibition
process, we have developed several efficient chemical probes
———
Corresponding author. Tel.: +420-220-183-283; fax: +420-220-183-578; e-mail: posta@uochb.cas.cz

2
Tetrahedron
ACCEPTED MANUSCRIPT
based on the structural modification of 2. It is known that
chemical modification of the karrikine backbone led to a
significant loss of activity.9 The same effect has been also
observed for a number of synthetic analogues of 2 modified in
the easily chemically accessible position C-5.10 On the other
hand, some substituents in this specific position are well tolerated
without loss of activity. This fact encouraged us to develop TMB
molecular probes bearing the photolabile azido-benzoyl unit for
capturing and identification the target proteins of 2 using
photoaffinity labeling (PAL) method11 and its modification with
an ethynyl group for optional introduction of a detectable marker
via Huigsen 1,3-dipolar cycloaddition12 with an azido group,
generally known as “click” chemistry13. This type of molecular
probe could also potentially find application in molecular
o
Scheme 1. i) LiAlH(tBuO)3, THF, -15 C; ii) Pd/C, H2, MeOH; iii) NaNO2,
H2SO4, NaN3, 0 oC; iv) (COCl)2, DMF (cat.) DCM; v) 8, Et3N, DCM.
tattooing14,
a novel tool in optopharmacology combining
photoaffinity-labeling with two-photon microscopy. This very
promising technique enables localization of drug effects at
subcellular resolution in live biological objects including whole
animals.
Several examples of bifunctional photoaffinity probes are
reported in the literature15, however, their design is based on
functionalization of a benzyl alcohol or benzyl halide moiety
attached to the target molecule under basic conditions. In our
case, compound 2 undergoes opening of the lactone ring in the
presence of base. Application of a milder base such as silver or
potassium carbonate on the other hand, returned only starting
material.
In order to eliminate the potential effect of the aromatic ring
on the activity of 2, probes 12 and 14, were also prepared,
bearing an ethylene glycol or triethylene glycol linker
respectively. Butenolides with ethyleneglycol linkers 11 and 13
were prepared according to Scheme 2, starting from 4, which was
converted to the corresponding chloride 10 in excellent yield by
treatment with excess of SOCl2. Precursors with ethylene glycol
or triethylene glycol linkers were prepared by the addition of
chloride 10 to a dilute solution of monosodium ethylene or
triethylene glycolate prepared in situ. Despite the fact that the
reactions were carried out under basic conditions, they provided
desired products 11 and 13 in good yields (77% and 62%
respectively), while the products of the lactone ring opening were
only minor byproducts in this case. Final PAL molecular probes
12 and 14 were obtained by the esterification of 8 with 11 and 13,
respectively.
For this reason, we have developed a new type of bifunctional
molecular probe 22 derived from benzoic acid since its
esterification with 4, 11 or 13 represents a very convenient way
toward the synthesis of TMB molecular probes.
N3
O
O
O
2. Results and discussion
i
ii
iii
O
O
O
Photoaffinity labeling (PAL) is a remarkably efficient method
to study the interaction of biologically significant compounds
(ligands) with their target macromolecules.11,16 The method
allows identification of the target and also the binding domain
within the target protein. An appropriate photoaffinity probe is
usually prepared by attachment of a photoreactive labeling group
such as an aryl azide, aryl diazirine or benzophenone to the
ligand molecule. We have chosen an aryl azide group from the
above mentioned possibilities for the design of our TMB derived
PAL probes.
Cl
OH
O
O
O
O
O
O
4
10 (92%)
11 (77%)
12 (58%)
HO
N3
O
iv
v
O
O
O
O
O
O
O
O
O
13 (62%)
14 (67%)
O
O
HO
2.1. Synthesis of aromatic azide PAL-TMB probes
Scheme 2. i) SOCl2, pyridine, benzene; ii) ethylene glycol, NaH, THF; iii) 8,
Et3N, DCM; iv) triethylene glycol, NaH, THF; v) 8, Et3N, DCM.
We started with the synthesis of molecular probes 9, 12 and
14, bearing only an azide goup, for the evaluation of the
inhibition activity. Photoaffinity tags used for this type of probe
have been prepared in three steps starting from 4-nitrobenzoic
acid. The synthesis started with the reduction of nitro group with
hydrogen and palladium on charcoal in the first step.17 Next, the
azide group was introduced under Sandmayer reaction condition
and the resulting acid 7 was converted to the appropriate 4-
azidobenzoyl chloride 8 using oxalyl chloride.
2.1.1. Evaluation of biological activity of aromatic azide PAL-
TMB probes
In order to obtain a good model system for TMB research,
different Lactuca accessions from the UC Davis lettuce collection
were screened for TMB responsiveness. The Lactuca sativa
‘Salinas’ cultivated lettuce proved to be hyposensitive to TMB,
while the Lactuca serriola wild type photoblastic lettuce showed
increased sensitivity to TMB. Achenes (seeds) were collected
Final PAL probe 9 was prepared via esterification of 8 with
hydroxylactone 4 prepared from 3,4-dimethylmaleic anhydride
(3) following a literature procedure (Scheme 1).18
o
from a greenhouse-grown population, cleaned and stored at 4 C
in a seed vault and used within two months. To test the
germination activity of compounds, achenes were spread onto a
filter paper (7 cm in diameter), placed in Petri dishes, and then
dampened with distilled water (mock) or 3 mL of 25 µM solution
of 2, 9, 12 (dissolved in water) and 14 (dissolved in DMSO).
Dishes were sealed with parafilm and achenes were germinated

3
under a continuous low light regime (8 µE). Germination for
three biological and three technical replicates each of 50 seeds
was assayed for 7 days, and achenes with ruptured testa were
considered as germinated. The biological replicates (independent
seed batches) showed very similar germination; representative
data is presented here.
Compound 2 slightly delayed germination, while 9, 12 and 14
have less, or even inconspicuous effect on the the gemination of
L. ‘Salinas’ (Figure 2). As expected, 2 strongly delayed the
gemination of L. serriola (Figure 2), similar to the photoblastic
Lactuca sativa ’Grand Rapids’, the model cultivar used to
characterize 2 previously.7f Although less effective, 9 and 12 also
inhibited germination indicating that the azide moiety or a
relatively short linker (ethylene glycol) for azide moieties do not
hamper inhibitory activity. However, the slight decrease in
inhibition effect indicates spatial hindrance of binding of the
modified compounds. This is supported also by the finding that
14 (with a longer triethylene glycol linker) has only a very weak
effect on germination.
diazotization-displacement to afford 18.15a The terminal
ACCEPTED MANUSCRIPT
acetylenic compound 19 was prepared using a Sonogashira-type
reaction for the coupling of 18 with trimethylsilylacetylene under
Pd2(dba)3/CuI catalysis.19 Starting from 19, we have tested two
alternative synthetic pathways (A and B) towards 22 (Scheme 3).
Following the pathway A, deprotection of the terminal alkyne
functionality of 19 as well as its ester hydrolysis was
accomplished in one step in 75% yield under reflux in methanol
with LiOH and the resulting 3-ethynyl-5-nitrobenzoic acid (20)
was reduced to a corresponding amino acid 21 with SnCl2 in the
next step. Although there is reported synthesis of 21 in the
literature19, no efficient purification method exists so far and for
this reason we had to use crude 21 (90% purity by 1H NMR) for
the next reaction step. Amino acid 21 was converted to 22 in
moderate yield 40%, using Sandmayer reaction condition with
NaNO2 and NaN3 under acidic conditions.
In order to improve the yield of 22 and eliminate purification
problems, synthetic pathway B was tested. The nitro group of
compound 19 was reduced with SnCl2 to afford 23 in 79% yield,
the azide group was introduced using Sandmayer reaction
conditions to provide 24 in 84% yield. Finally, treating 24 with
LiOH in boiling methanol provided 22 in 81% yield.
The overall yield of 22 obtained from 19 following pathway B
is 54%. It represents a significant improvement in comparison
with pathway A, where the total isolated yield was only 27%.
Scheme 3. i) MeOH, H2SO4 (cat.); ii) (NH4)2S, MeOH; iii) NaNO2, H2SO4,
KI; iv) Pd2(dba)3, CuI, Ph3P, Et3N, TMSA, 40 oC; v) LiOH, MeOH, 60 oC; vi)
SnCl2, AcOEt/AcOH, reflux; vii) NaNO2, H2SO4, NaN3, 0oC; viii) SnCl2,
AcOEt/AcOH, reflux; ix) NaNO2, H2SO4, NaN3, 0oC; x) LiOH, MeOH, 60
oC.
Figure 2 The germination inhibitory activity of 2 and azide PAL-TMB
probes 9, 12, 14. Germination characteristics of Lactuca serriola and Lactuca
sativa ‘Salinas’ achenes. Achenes (seeds) were germinated in Petri dishes,
imbibed in 3 mL of 25 µM solution of 2, 9, 12 and 14. Germinated achenes
were scored for 7 days and the data has been analysed with the OriginPro
software.
Final esterification of benzoyl chloride 25 with 4 provided
combined TMB-molecular probe 26 in moderate yield (56%).
Two other combined TMB-molecular probes 27 and 28 were
prepared by the same manner by esterification with alcohols 11
and 13 in 42% and 66% yield respectively.
All probes (9, 12, 14, 26, 27 and 28) were synthesized and
used as a racemic mixture of isomers, since it has been
demonstrated, that both enantiomers of 2 are equally active as
germination inhibitors.4
2.2. Synthesis of combined bifunctional TMB probes
Positive results from the evaluation of the biological activity
of probes 9, 12 and 14, encouraged us in the synthesis of new
combined PAL probes. These bifunctional probes 26, 27 and 28
were obtained by esterification of 25 with 4, 11 and 13
respectively. Photolabile 3-azido-5-ethynylbenzoic acid 25 was
prepared from commercial 3,5-dinitrobenzoic acid by two
alternative pathways in seven steps (Scheme 3). First,
esterification of acid 15 with MeOH under acidic conditions gave
methyl ester 16 in almost quantitative yield. Next, the
chemoselective reduction of one of the nitro groups of 16 by
(NH4)2S gave 17 in 88% yield and the amino group was
converted in the next step to an iodo group by in situ

4
Tetrahedron
Figure 3. Germination response of Lactuca serriola and Lactuca sativa
N3
ACCEPTED MANUSCRIPT
O
ii, 4
’Salinas’ achenes to compound 26, 27 and 28. Achenes (seeds) were
O
germinated in Petri dishes, imbibed in 3 ml of 25 µM solution of 2, 26, 27 and
28. Germinated achenes were scored for 7 days and the data has been
analysed with the OriginPro software.
O
O
N3
26 (56%)
O
X
N3
(22) X = OH
(25) X = Cl
3. Conclusion
i
ii, 11
In summary, we have succeeded in developing specific
photoreactive inhibitors of seed germination. Novel molecular
probes for the determination of mode of action of TMB were
synthesized and their biological activity was evaluated. These
probes for photoaffinity labeling would help in the study of
processes involved in smoke-induced seed germination
inhibition, in capturing of TMB metabolites, determination of
their structures and functions. The results from such study will be
reported in due course. Additionally, our photoreactive tag,
derived from benzoic acid represents an excellent alternative for
the convenient synthesis of other molecular probes with target
molecules sensitive to basic conditions.
O
O
O
O
O
27 (42%)
N3
ii, 13
O
O
O
O
O
O
O
28 (66%)
Scheme 4. i) (COCl)2, DMF (cat.) DCM; ii) Et3N, DCM.
2.2.1. Evaluation of biological activity of combined bifunctional
TMB probes
4. Experimental section
4.1 General
In this test, experimental procedures were the same as
described in 2.1.1., except that all compounds were dissolved in
DMSO. Compared to 9, 12 and 14, compounds 27 and 28 caused
an inconspicuous delay in germination (especially in ‘Salinas’).
These compounds have an ethylene glycol or triethylene glycol
linker (as do 12 and 14), which may prevent the molecules from
fitting properly into their respective binding pockets. In contrast,
26 had a significant effect; however, this inhibition is less
relevant compared with 2. Although 26 has a lower activity, the
presence of the azide does not fully hamper the inhibitory effect
of 2, and 26 could be a good starting point for the synthesis of
other bifunctional PAL probes.
1H and 13C nuclear magnetic resonance (NMR) spectra were
measured on a Bruker Avance I – 400 (400MHz for δH and 100.6
MHz for δC) or 500 (500MHz for δH and 125.7 MHz for δC)
spectrometer in CDCl3 with Me4Si as an internal standard or in
acetone-d6 referenced to the signal of residual solvent (1H NMR δ
2.05 ppm,13C NMR 29.84 ppm). Chemical shifts are given in
ppm (δ scale), coupling constants (J) in hertz. Complete
assignment of all NMR signals was performed using a
combination of 2D-NMR (H,H-COSY, H,C-HSQC, and H,C-
HMBC) experiments. GCMS spectra were recorded on an
Agilent 7890A gas chromatograph coupled with a 5975C
quadrupole mass-selective electron impact (EI) detector (70 eV).
High-resolution mass spectra were recorded using Waters GCT
Premier bench-top orthogonal acceleration time-of-flight (OA-
TOF) mass spectrometer with chemical ionization. Commercially
available reagents were purchased from Sigma-Aldrich and were
used without further purification unless stated otherwise.
4.2 Synthesis
4.2.1 5-Hydroxy-3,4-dimethylfuran-2(5H)-one (4)18
A solution of LiAlH(tBuO)3 (2.84 g, 11.17 mmol) in 10 mL of
anhydrous THF was added dropwise over 10 min. to a stirred
solution of 2,3-dimethylmaleic anhydride (3) (1.01 g, 8.01 mmol)
o
in 20 mL of anhydrous THF under argon atmosphere at -15 C.
o
The reaction mixture was stirred for 1 hour at -15 C and then at
room temperature for 1 h. The reaction mixture was then
quenched with 20 mL of 1M HCl and extracted with AcOEt.
Purification by silica gel column chromatography (30% AcOEt in
hexane) afforded pure product 4 (829 mg, 81%) as a white solid.
1H and 13C NMR are in agreement with published data.18
Rf (30% AcOEt/hexane) 0.23.
MS(EI): m/z (%) = 128.0 (1, M+), 127.0 (5), 111.0 (4), 100.1
(100), 99.0 (8), 83.1 (30), 82.1 (11), 69.1 (5), 56.1 (6), 55.1 (61),
54.1 (36), 53.1 (21), 43.1 (10), 39.1 (33).
4.2.2 4-Aminobenzoic acid (6)17
Palladium (10%) on charcoal (300 mg) was added to a stirred
solution of 5 (3.00 g, 17.95 mmol) in methanol (100 mL). The
reaction mixture was stirred at room temperature under positive

5
pressure of H2 for 5 h. The solvent was removed under vacuum.
Purification by silica gel column chromatography (30% acetone
in hexane) afforded pure product 6 (2.36 g, 96%).
1H and 13C NMR are in agreement with published data.17
Rf (30% acetone/hexane) 0.48.
dimethylfuran-2(5H)-one (4) (256 mg, 1.998 mmol) in 10 mL
ACCEPTED MANUSCRIPT
of benzene and pyridine (1.00 mL, 12.41 mmol) and reaction
mixture was stirred at room temperature for 30 min. The solvent
was removed under vacuum. Purification by silica gel column
chromatography (30% AcOEt in hexane) afforded pure product
10 (269 mg, 92 %) as the pale yellow oil.
MS(EI): m/z (%) = 137.1 (91, M+), 120.1 (100), 92.1 (34), 65.1
(30), 63.1 (10).
1H and 13C NMR are in agreement with published data21,
1H NMR (400 MHz, CDCl3) δ: 6.44 – 6.28 (1 H, m, CH), 2.08 (3
H, quint, J 1.1 Hz, C3Me), 1.89 (3 H, quint, J 1.3 Hz, C4Me).
13C NMR (100 MHz, CDCl3) δ: 171.0, 156.6, 125.7, 88.1, 11.7,
8.6.
4.2.3 4-Azidobenzoic acid (7)
Potassium nitrite (1.81 g, 26.24 mmol) was added in several
portions to a solution of 6 (2.00 g, 14.58 mmol) in 250 mL of
35% aqueous hydrochloric acid and 100 mL of water at 0 oC and
reaction mixture was stirred for 1 h. NaN3 (1.90 g, 29.23 mmol)
Rf (30% AcOEt/hexane) 0.58.
MS(EI): m/z (%) = 146.0 (5, M+), 119.0 (15), 117.0 (44), 111.1
(100), 89.0 (7), 55.1 (39), 54.1 (10), 53.1 (25), 39.1 (17).
o
was added and the reaction was maintained at 0 C for 3 h. The
product was extracted with AcOEt (3 x 50 mL) and after
evaporation of the solvent it was pure enough for use in the next
reaction step (1.86 g, 78%).
4.2.6 5-(2-Hydroxyethoxy)-3,4-dimethylfuran-2(5H)-one (11)
A suspension of NaH in mineral oil (60%) (44 mg, 1.100 mmol)
was added in several portions to a solution of ethylene glycol (75
mg, 1.208 mmol) in 25 mL of THF under an argon atmosphere.
The reaction mixture was stirred for 1 h at room temperature and
then a solution of 10 (146 mg, 0.996 mmol) in 5 mL THF was
added dropwise. The reaction mixture was stirred for 16 h and
then the solvent was removed under vacuum. Purification by
silica gel column chromatography (40% acetone in hexane)
afforded pure product 11 (132 mg, 77%) as a colorless oil.
1H NMR (400 MHz, acetone-d6) δ: 5.80 (1 H, m, CH), 4.07 –
4.04 (1 H, m, OH), 3.82 – 3.70 (4 H, m, CH2), 1.97 (3 H, quint, J
1.1 Hz, C3Me), 1.76 (3 H, quint, J 1.2 Hz, C4Me).
1H and 13C NMR are in agreement with published data.20
MS(EI): m/z (%) = 162.0 (1), 137.1 (91), 121.1 (8), 120.1 (100),
92.1 (33), 65.1 (30), 63.1 (10).
HRMS (ESI–): [M-H]- - calculated for C7H4O2N3: 162.0309;
found: 162.0328.
4.2.4 3,4-Dimethyl-5-oxo-2,5-dihydrofuran-2-yl 4-
azidobenzoate (9)
a) Preparation of 4-Azidobenzoyl chloride (8)
A solution of oxalyl chloride (0.200 mL, 2.364 mmol) in 2
mL of dichloromethane was added dropwise to a suspension
of 7 (195 mg, 1.195 mmol) in 5 mL of dichloromethane and a
catalytic amount of DMF (1 drop) under an argon
atmosphere. Reaction mixture was stirred for 45 min. in the
dark and then the solvent was evaporated. The crude product
was dissolved in 3 mL of dichlormethane and used in the next
reaction step.
13C NMR (100 MHz, acetone-d6) δ: 172.6, 155.7, 126.4, 104.1,
71.7, 61.6, 11.4, 8.3.
Rf (40% acetone/hexane) 0.36.
MS(EI): m/z (%) = 173.1 (1, MH+), 143.1 (1), 127.0 (10), 112.1
(84), 111.1 (100), 110.1 (15), 83.1 (21), 55.1 (46), 54.1 (11), 39.1
(13).
HRMS (CI+): [M+H]+ - calculated for C8H13O4 : 173.0814,
found: 173.0819.
A
solution of 4-azidobenzoyl chloride (8) in
3
mL
dichloromethane was added dropwise to a solution of 5-hydroxy-
3,4-dimethylfuran-2(5H)-one (4) (118 mg, 0.921 mmol),
prepared according to the procedure reported by Surmont18, and
Et3N (1.00 mL, 7.17 mmol) in 5 mL of dichlormethane under an
argon atmosphere. The reaction mixture was stirred for 3 h in the
dark at room temperature. The solvent was removed under
vacuum and the crude product was dissolved in 50 mL of AcOEt
and poured into 150 mL of water. The aqueous fraction was
washed with AcOEt (2 x 50 mL) and the combined organic
fractions were treated with saturated aqueous solution of
NaHCO3, brine and dried over MgSO4. Purification by silica gel
column chromatography (30% AcOEt in hexane) afforded pure
product 9 (174 mg, 69%) as a yellow solid.
4.2.7 2-((3,4-Dimethyl-5-oxo-2,5-dihydrofuran-2-yl)oxy)ethyl
4-azidobenzoate (12)
a) 4-Azidobenzoyl chloride (8) was prepared from 7 (173 mg,
1.060 mmol) and oxalyl chloride (0.180 mL, 2.127 mmol) by
the same procedure as reported for the preparation of 9.
A
solution of 4-azidobenzoyl chloride (8) in
3
mL
dichloromethane was added dropwise to a solution of 11 (132
mg, 0.767 mmol), and Et3N (1.00 mL, 7.17 mmol) in 5 mL of
dichloromethane under an argon atmosphere. The reaction
mixture was stirred for 3 h in the dark at room temperature. The
solvent was removed under vacuum and the crude product was
dissolved in 50 mL of AcOEt and poured into 150 mL of water.
The aqueous fraction was washed with AcOEt (2 x 50 mL) and
the combined organic fractions were treated with saturated
aqueous solution of NaHCO3, brine and dried over MgSO4.
Purification by silica gel column chromatography (30% acetone
in hexane) afforded pure product 12 (142 mg, 58%) as an orange
oil.
1H NMR (500 MHz, acetone-d6) δ: 8.11 – 8.08 (2H, m, arom.),
7.26 – 7.24 (2 H, m, arom.), 7.01 (1 H, qq, J 1.4, 0.7 Hz, CH),
2.12 – 2.11 (3 H, m, C3Me), 1.87 (3 H, quint, J 1.2Hz, C4Me).
13C NMR (125 MHz, acetone-d6) δ: 172.1, 164.9, 155.3, 146.9,
132.7, 127.3, 126.0, 120.2, 95.2, 11.4, 8.5.
Rf (30% AcOEt/hexane) 0.54.
MS(EI): m/z (%) = 248.1 (12), 247.1 (37), 208.0 (3), 138.1 (5),
137.1 (25), 121.0 (20), 120.1 (100), 111.1 (31), 110.0 (6), 93.0
(14), 83.0 (10), 65.1 (18), 55.1 (19).
1H NMR (400 MHz, acetone-d6) δ: 8.08 – 8.05 (2 H, m, arom.),
7.22 – 7.18 (2 H, m, arom.), 5.87 (1 H, dq, J 1.3, 0.6 Hz, CH),
4.57 – 4.47 (2 H, m, CH2), 4.14 – 4.05 (2H, m, CH2), 1.96 (3 H,
quint, J 1.1 Hz, C3Me). 1.77 (3 H, quint, J 1.2 Hz, C4Me).
13C NMR (100 MHz, acetone-d6) δ: 172.3, 165.9, 155.2, 145.8,
132.2, 127.5, 126.8, 119.9, 103.9, 68.1, 64.5, 11.4, 8.4.
Rf (30% acetone/hexane) 0.48.
HRMS (ESI): [M+Na]+
296.0642; found: 296.0642.
- calculated for C13H11O4N3Na:
4.2.5 5-Chloro-3,4-dimethylfuran-2(5H)-one (10)
Solution of SOCl2 (0.330 mL, 4.549 mmol) in 2 mL of benzene
was added dropwise to
a
solution of 5-hydroxy-3,4-

6
Tetrahedron
ACCEPTED MANUSCRIPT
MS(EI): m/z (%) = 318.1 (1, MH+), 292.1 (10), 291.1 (11),
To a stirred solution of 3,5-dinitrobenzoic acid (6.00 g, 28.29
165.1 (12), 164.1 (28), 137.0 (38), 121.1 (33), 120.1 (100), 119.1
(10), 111.1 (20), 92.1 (13), 65.1 (13), 55.1 (31).
HRMS (ESI): [M+Na]+ - calculated for C15H15O5N3Na :
340.0904, found: 340.0905.
mmol) in MeOH (100 mL), conc. H2SO4 (3.0 mL) was added
dropwise and reaction mixture was heated at reflux for 5 h.
Completion of the reaction was followed by TLC (30% acetone
in hexane). The solvent was removed under vacuum, and the
crude product was extracted with AcOEt and the combined
organic phases washed with water and then the pH was adjusted
to 4 with NaHCO3. The organic fraction was washed with brine
and dried over MgSO4. Product 16 obtained as a white solid after
evaporation of the solvent was pure and no further purification
was necessary (6.20 g, 97%).
4.2.8 5-(2-(2-(2-Hydroxyethoxy)ethoxy)ethoxy)-3,4-
dimethylfuran-2(5H)-one (13)
A suspension of NaH in mineral oil (60%) (44 mg, 1.100 mmol)
was added in several portions to a solution of triethylene glycol
(180 mg, 1.199 mmol) in 25 mL of THF under an argon
atmosphere. The reaction mixture was stirred for 1 h at room
temperature and then a solution of 10 (146 mg, 0.996 mmol) in 5
mL THF was added dropwise. The reaction mixture was stirred
18 h and then the solvent was removed under vacuum.
Purification by silica gel column chromatography (40% acetone
in hexane) afforded pure product 13 (161 mg, 62 %) as a yellow
oil.
1H and 13C NMR data are in agreement with published data.22
1H NMR (400 MHz, acetone-d6) δ: 9.17 – 9.15 (1 H, m, arom.),
9.08 – 9.05 (2 H, m, arom), 4.05 (3 H, s, OMe).
13C NMR (100 MHz, acetone-d6) δ: 164.0, 149.8, 134.4, 129.8,
123.2, 53.8.
MS(EI): m/z (%) = 226.1 (8, M+), 197.1 (7), 196.1 (72), 195.0
(100), 181.1 (18), 149.0 (31), 133.1 (13), 103.1 (8), 75.1 (48),
74.1 (23), 63.1 (7).
1H NMR (400 MHz, acetone-d6) δ: 5.82 – 5.81 (1 H, m, CH),
3.86 – 3.83 (2 H, m, CH2), 3.68 – 3.66 (2 H, m, CH2), 3.62 – 3.59
(7 H, m, CH2 and OH), 3.53 – 3.50 2 H, m, CH2), 1.98 – 1.97 (3
H, m, C3Me), 1.77 – 1.76 (3 H, m, C4Me).
4.2.11 Methyl 3-amino-5-nitrobenzoate (17)
The solution of (NH4)2S23 used for this reaction was prepared by
adding a solution of Na2S·9H2O (15.00 g, 62.47 mmol) in 100
mL of MeOH to a suspension of NH4Cl (13.40 g, 250.50 mmol)
in 100 mL of MeOH and separating the solid material after 30
minutes of stirring at room temperature. The resulting solution
was added within 40 min to a solution of 16 (6.20 g, 27.42 mmol)
in 250 mL of boiling MeOH and heated at reflux for 5 h. After
cooling down, the resulting precipitate of sulfur was filtered off
and the pH was adjusted to 5 with 1 M HCl and the solvent was
removed under vacuum. The crude product was extracted with
250 mL of AcOEt, washed with water, brine and dried over
MgSO4 and concentrated in vacuo. Purification by silica gel
column chromatography (30% acetone in hexane) afforded pure
product 17 (4.73 g, 88%) as an orange solid.
13C NMR (100 MHz, acetone-d6) δ: 172.4, 155.5, 126.4, 104.1,
73.4, 71.2, 71.0, 70.8, 69.3, 61.9, 11.4, 8.3.
Rf (40% acetone/hexane) 0.39.
MS(EI): m/z (%) = 260.1 (3, M+), 252.9 (5), 231.0 (2), 187.1 (4),
142.0 (6), 127.0 (17), 113.1 (18), 112.1 (92), 110.0 (46), 88.1
(24), 57.1 (10), 55.1 (100), 32.0 (48).
HRMS (CI+): [M+H]+ - calculated for C12H21O6 : 261.1338,
found:. 261.1339
4.2.9 2-(2-(2-((3,4-Dimethyl-5-oxo-2,5-dihydrofuran-2-yl)oxy)
ethoxy)ethoxy)ethyl 4-azidobenzoate (14)
a) 4-Azidobenzoyl chloride (8) was prepared from 7 (130 mg,
0.797 mmol) and oxalyl chloride (0.130 mL, 1.536 mmol) by
the same procedure as reported for the preparation of 9.
1H and 13C NMR data are in agreement with published data.24
1H NMR (400 MHz, acetone-d6) δ: 7.93 (1 H, dd, J 2.1, 1.4 Hz,
arom.), 7.71 (1 H, t, J 2.2 Hz, arom.), 7.67 (1 H, dt J 2.2, 0.8 Hz,
arom.), 5.66 (2 H, s, NH2), 3.91 (3 H, s, OMe).
A
solution of 4-azidobenzoyl chloride (8) in
3
mL
dichloromethane was added dropwise to a solution of 13 (161
mg, 0.619 mmol), and Et3N (1.00 mL, 7.17 mmol) in 5 mL of
dichloromethane under an argon atmosphere. The reaction
mixture was stirred for 3 h in the dark at room temperature. The
solvent was removed under vacuum and the crude product was
dissolved in 50 mL of AcOEt and poured into 150 mL of water.
The aqueous fraction was washed with AcOEt (2 x 50 mL) and
combined organic fractions were treated with a saturated aqueous
solution of NaHCO3, brine and dried over MgSO4. Purification
by silicagel column chromatography (30% acetone in hexane)
afforded pure product 14 (172 mg, 67%) as a yellow oil.
1H NMR (600 MHz, acetone-d6) δ: 8.05 – 8.03 (2 H, m, arom.),
7.20 – 7.18 (2 H, m, arom.), 5.79 (1 H, dq, J 2.3, 1.1 Hz, CH),
4.43 – 4.41 (2 H, m, CH2), 3.83 – 3.81 (4 H, m, 2 x CH2), 3.67 –
3.66 (4 H, m, 2 x CH2), 3.64 – 3.61 (2 H, m, CH2), 1.94 (3 H, dt J
2.2, 1.1 Hz, C3Me), 1.73 (3 H, quint, J 1.2 Hz, C4Me).
13C NMR (150 MHz, acetone-d6) δ: 172.5, 166.0, 155.6, 145.7,
132.1, 127.6, 126.5, 119.9, 104.1, 71.2, 71.2, 70.9, 69.6, 69.4,
65.0, 11.4, 8.3.
13C NMR (100 MHz, acetone-d6) δ: 166.0, 151.1, 150.3, 133.1,
20.9, 112.2, 111.9, 52.8.
Rf (30% acetone/hexane) 0.37.
MS(EI): m/z (%) = 197.1 (10), 196.1 (100, M+), 165.1 (33),
150.1 (20), 138.1 (8), 135.1 (22), 122.1 (35), 107.1 (17), 91.1
(23), 90.1 (10), 79.1 (8), 63.1 (20), 52.1 (9).
4.2.12 Methyl 3-iodo-5-nitrobenzoate (18)
A solution of NaNO2 (1.06 g, 15.37 mmol) in 10 mL of water
was added dropwise to a stirred, ice-bath cooled solution of 17
(2.00 g, 10.20 mmol) in 150 mL of water acidified with 5 mL of
o
conc. H2SO4. The temperature was maintained at 2 C and after
1.5 h, a solution of KI (5.20 g, 31.33mmol) in 15 mL of water
was added dropwise and the reaction mixture was stirred for 1 h
at 2 oC. Then it was allowed to warm to room temperature over 4
h period. The crude product was extracted with AcOEt (3 x 50
mL) and the pH of the aqueous fraction was adjusted to 4 with
NaHCO3 and extracted with AcOEt (3 x 50 mL). The combined
organic fractions were washed with 10% aqueous solution of
Na2S2O3·5H2O, brine and dried over MgSO4 and concentrated in
vacuo. Purification by silica gel column chromatography (20%
AcOEt in hexane) afforded pure product 18 (2.63 g, 84%) as a
pale yellow solid.
Rf (30% acetone/hexane) 0.45.
MS(EI): m/z (%) = 406.2 (1, MH+), 379.2 (13), 181.1 (3), 164.1
(26), 138.1 (7), 137.1 (76), 121.1 (9), 120.1 (100), 111.1 (27),
92.1 (15), 65.1 (7).
HRMS (ESI): [M+Na]+ - calculated for C19H23O7N3Na: 428.1428,
found: 428.1428.
1H data are in agreement with published data.25
4.2.10 Methyl 3,5-dinitrobenzoate (16)

7
1H NMR (400 MHz, acetone-d6) δ: 8.76 (1 H, dd J 2.2, 1.6 Hz,
arom.), 8.69 (1 H, dd J 2.2, 1.4 Hz, arom.), 8.65 (1 H, t, J 1.5 Hz,
arom), 3.98 (3 H, s, OMe).
down to room temperature was poured into 200 mL of water
ACCEPTED MANUSCRIPT
with ice and the pH was adjusted to 2 with 1 M HCl and
extracted with AcOEt (3 x 50 mL). Another extraction with
AcOEt (3 x 50 mL) was done after the pH was adjusted to 6 with
NaHCO3. The combined organic fractions were washed with
brine and dried over MgSO4. Evaporation of the solvent provided
crude product 21 (384 mg, 91%) as an orange solid that was used
for next step without purification (1H NMR purity approx. 90%).
1H NMR (400 MHz, acetone-d6) δ: 7.38 – 7.35 (2 H, m, arom.),
7.01 (1 H, dd, J 2.4, 1.5 Hz, arom.), 3.58 (1 H, s, C≡CH).
13C NMR (100 MHz, acetone-d6) δ: 167.2, 149.7, 132.5, 123.8,
122.0, 121.8, 116.5, 80.0, 78.5.
13C NMR (100 MHz, acetone-d6) δ: 164.2, 149.6, 144.4, 136.8,
134.0, 124.2, 94.1, 53.4.
Rf (20% AcOEt/hexane) 0.54.
MS(EI): m/z (%) = 308.1 (10), 307.1 (100, M+), 276.0 (77),
261.1 (10), 246.0 (21), 230.0 (15), 218.0 (7), 201.0 (8), 75.1 (33),
74.1 (18).
4.2.13 Methyl 3-nitro-5-((trimethylsilyl)ethynyl)benzoate (19)
Substrate 18 (2.50 g, 8.14 mmol) and Ph3P (603 mg, 2.299
mmol) were suspended in 25 mL of Et3N under an argon
atmosphere and stirred until fully dissolved. Then CuI (190 mg,
0.998 mmol) and Pd2(dba)3 (275 mg, 0.300 mmol) were added in
one portion. The reaction mixture was stirred for 30 min and then
trimethylsilylacetylene (4.00 mL, 28.30 mmol) was added
HRMS (EI+): [M]+ - calculated for C9H7O2N : 161.0477, found:
161.0479.
4.2.16 3-Azido-5-ethynylbenzoic acid (22)
From 21:
o
dropwise. Temperature was raised to 40 C and reaction mixture
A solution of NaNO2 (124 mg, 1.797 mmol) in 2 mL of water
was added dropwise to a stirred, ice-bath cooled solution of crude
21 (200 mg) in 100 mL of water acidified with 1 mL of conc.
H2SO4. The temperature was maintained at 0 - 2oC and after 2 h,
a solution of NaN3 (500 mg, 7.691 mmol) in 5 mL of water was
was stirred for 18 h. The reaction was quenched with 100 mL of
saturated aqueous solution of NH4Cl, and extracted with AcOEt
(3 x 50 mL). The combined organic fractions were washed with
brine, dried over MgSO4 and concentrated in vacuo. Purification
by silica gel column chromatography (10% AcOEt in hexane)
afforded pure product 19 (2.19 g, 97%) as a pale yellow solid.
1H NMR (400 MHz, acetone-d6) δ: 8.69 (1 H, dd J 2.2, 1.5 Hz,
arom.), 8.46 (1 H,dd, J 2.3, 1.5 Hz, arom.), 8.35 (1 H, t, J 1.5 Hz,
arom.), 3.99 (3 H, s, OMe), 0.28 (9 H, s, SiMe3).
o
added dropwise, the reaction mixture was stirred 1 h at 0 – 2 C.
Then it was allowed to warm to room temperature over 4 h
period. The crude product was extracted with AcOEt (3 x 50
mL), washed with brine, dried over MgSO4 and concentrated in
vacuo. Purification by silica gel column chromatography (50%
AcOEt in hexane with 0.1% of AcOH) afforded pure product 22
(92 mg, 40%) as a yellow solid.
13C NMR (100 MHz, acetone-d6) δ: 164.8, 149.4, 138.4, 133.2,
130.7, 126.0, 124.5, 102.1, 99.1, 53.3, -0.4.
Rf (10% AcOEt/hexane) 0.55.
From 24:
MS(EI): m/z (%) = 277.1 (7, M+), 263.1 (19), 262.1 (100), 216.1
(15), 201.1 (6), 157.1 (5).
Starting material 24 (250 mg, 0.915 mmol) was dissolved in 25
mL of MeOH and solution of LiOH (120 mg, 5.010 mmol) in 25
mL of water was added. The reaction mixture was stirred for 2 h
HRMS (EI+) [M]+ calculated for C13H15NO4Si : 277.0770, found:
277.0772.
o
at 60 C and after cooling down the pH was adjusted to 3 with 1
M HCl. The crude product was extracted with AcOEt (3 x 50
mL) and the combined organic phases were washed with brine,
dried over MgSO4 and concentrated in vacuo. Purification by
silica gel column chromatography (50% AcOEt in hexane with
0.1% of AcOH) afforded pure product 22 (139 mg, 81%) as a
yellow solid.
4.2.14 3-Ethynyl-5-nitrobenzoic acid (20)19
Starting material 19 (1.00 g, 3.61 mmol) was dissolved in 25 mL
of MeOH and solution of LiOH (432 mg, 18.038 mmol) in 25
mL of water was added. The reaction mixture was stirred for 2 h
o
at 60 C and after cooling down the pH was adjusted to 3 with 1
1H NMR (400 MHz, acetone-d6) δ: 7.87 (1 H, t, J 1.4 Hz, arom.),
7.68 (1 H, dd, J 2.3, 1.4 Hz, arom.), 7.41 1 H, dd, J 2.3, 1.4 Hz,
arom.), 3.86 (1 H, s, C≡CH).
M HCl. The crude product was extracted with AcOEt (3 x 50
mL), combined organic phases were washed with brine, dried
over MgSO4 and concentrated in vacuo. Purification by silica gel
column chromatography (30% acetone in hexane with 0.1% of
AcOH) afforded pure product 20 (517 mg, 75 %) as a white
solid.
13C NMR (100 MHz, acetone-d6) δ: 165.9, 142.1, 133.7, 130.0,
127.0, 125.2, 121.1, 82.2, 81.2.
Rf (50% AcOEt/0.1%AcOH/hexane) 0.51.
MS(CI+): m/z (%) = 189.1 (5), 188.0 (100, M+), 175.1 (52),
162.1 (82), 161.1 (20), 160.0 (48), 147.1 (7), 118.1 (19), 116.1
(22), 100.0 (34).
1H NMR (400 MHz, acetone-d6) δ: 8.75 – 8.74 (1 H, m, arom.),
8.51 – 8.50 (1 H, m, arom.), 8.42 – 8.41 (1 H, m, arom.), 4.07 (1
H, s, C≡CH).
HRMS (CI+): [M+H]+ - calculated for C9H6O2N3 : 188.0460,
found: 188.0466.
13C NMR (100 MHz, acetone-d6) δ: 165.0, 149.4, 138.9, 133.8,
130.9, 125.2, 125.0, 82.9, 81.0.
Rf (30% acetone/0.1%AcOH/hexane) 0.24.
4.2.17 Methyl 3-amino-5-((trimethylsilyl)ethynyl)benzoate (23)
MS(EI): m/z (%) = 191.1 (1,M+), 147.1 (6), 146.1 (60), 145.1
(64), 132.1 (10), 131.1 (100), 115.1 (5), 103.1 (94), 102.1 (11),
77.1 (46), 63.1 (7), 51.1 (26).
Methyl 3-nitro-5-((trimethylsilyl)ethynyl)benzoate (19) (1.00 g,
3.61 mmol) and SnCl2 (3.40 g, 17.93 mmol) were suspended in
50 mL of freshly distilled AcOEt and 0.1 mL of glacial AcOH
under an argon atmosphere. The reaction mixture was heated at
reflux for 4 h and after cooling down to room temperature was
poured into 200 mL of water, the pH was adjusted to 8 with
NaHCO3 and extracted with AcOEt (3 x 50 mL). The combined
organic fractions were washed with brine, dried over MgSO4 and
concentrated in vacuo. Purification by silica gel column
chromatography (30% AcOEt in hexane) afforded pure product
23 (704 mg, 79%) as a yellow oil that crystallized upon standing.
HRMS (EI+): [M]+ - calculated for C9H5NO4 : 191.0219, found:
191.0221.
4.2.15 3-Amino-5-ethynylbenzoic acid (21)19
Starting material 20 (500 mg, 2.616 mmol) and SnCl2 (2.50 g,
13.19 mmol) were suspended in 25 mL of freshly distilled AcOEt
and 0.1 mL of glacial AcOH under an argon atmosphere. The
reaction mixture was heated at reflux for 4 h and after cooling

8
Tetrahedron
1H NMR (500 MHz, acetone-d6) δ: 7.31 (1 H, dd, J 2.4, 1.5 Hz,
13C NMR (100 MHz, acetone-d6) δ: 172.0, 164.2, 155.0, 142.5,
ACCEPTED MANUSCRIPT
arom.), 7.28 (1 H, t, J 1.5 Hz, arom.), 6.98(1 H, dd J 2.4, 1.5 Hz,
arom.), 5.09 (2 H, bs, NH2), 3.84 (3 H, s, OMe), 0.22 (9 H, s,
SiMe3).
131.9, 130.1, 128.0, 127.4, 125.5, 121.4, 95.5, 81.9, 81.7, 11.4,
8.5.
Rf (10% AcOEt/hexane) 0.27.
13C NMR (125 MHz, acetone-d6) δ: 166.9, 149.8, 132.2, 124.6,
121.6, 121.5, 116.1, 105.7, 93.9, 52.3, -0.1.
Rf (30% AcOEt/hexane) 0.55.
MS(EI): m/z (%) = 297.1 (9, M+), 269.1 (6), 158.0 (17), 114.0
(15), 111.0 (100), 87.0 (7), 83.0 (8), 55.1 (16).
HRMS (EI+) [M]+ - calculated for C15H11O4N3: 297.0750, found:
297.0752.
MS(EI): m/z (%) = 248.2 (8), 247.2 (40, M+), 234.2 (6), 233.2
(18), 232.2 (100), 216.1 (4), 158.1 (4).
HRMS (EI+) [M]+ - calculated for C13H17NO2Si : 247.1029,
found: 247.1030.
4.2.20 2-((3,4-Dimethyl-5-oxo-2,5-dihydrofuran-2-yl)oxy)ethyl
3-azido-5-ethynylbenzoate (27)
a) 3-Azido-5-ethynylbenzoyl chloride (25) was prepared from
22 (35 mg, 0.187 mmol) and oxalyl chloride (0.10 mL, 1.182
mmol) by the same procedure as reported for the preparation
of 26.
4.2.18 Methyl 3-azido-5-((trimethylsilyl)ethynyl)benzoate (24)
A solution of NaNO2 (290 mg, 4.204 mmol) in 5 mL of water
was added dropwise to a stirred, ice-bath cooled solution of 23
(700 mg, 2.830 mmol) in 150 mL of water acidified with 5 mL of
A solution of 25 in 2 mL dichloromethane was added dropwise to
a solution of 11 (25 mg, 0.145 mmol), and Et3N (0.400 mL,
2.868 mmol) in 3 mL of dichloromethane under an argon
atmosphere. The reaction mixture was stirred for 5 h in the dark
at room temperature. The solvent was removed under vacuum
and the crude product was dissolved in 20 mL of AcOEt and
poured into 100 mL of water. The aqueous fraction was washed
with AcOEt (2 x 20 mL) and the combined organic fractions
were washed with a saturated aqueous solution of NaHCO3,
brine, dried over MgSO4 and concentrated in vacuo. Purification
by silica gel column chromatography (30% of AcOEt in hexane)
afforded pure product 27 (21 mg, 42 %) as an orange oil.
1H NMR (400 MHz, acetone-d6) δ: 7.88 (1 H, t, J 1.4 Hz, arom.),
7.68 (1 H, dd, J 2.3, 1.4 Hz, arom.), 7.42 (1 H, dd, J 2.3, 1.4 Hz,
arom.), 5.88 (1 H, dt, J 1.9, 1.0 Hz, CH), 4.63 – 4.50 (2 H, m,
CH2), 4.17 – 4.06 (2 H, m, CH2), 3.87 (1 H, s, C≡CH), 1.97 (3 H,
quint,, J 1.1 Hz, C3Me), 1.77 (3 H, quint, J 1.2 Hz, C4Me).
13C NMR (100 MHz, acetone-d6) δ: 172.3, 165.1, 155.2, 142.2,
133.2, 129.8, 127.2, 126.9, 125.2, 121.0, 103.9, 82.1, 81.4, 67.9,
65.1, 11.4, 8.4.
o
conc. H2SO4. The temperature was maintained at 0 – 2 C and
after 2 h, solution of NaN3 (911 mg, 14.013 mmol) in 5 mL of
water was added dropwise, the reaction mixture was stirred for 1
o
h at 0 – 2 C. Then it was allowed to warm to room temperature
over 4 h period. The crude product was extracted with AcOEt (3
x 50 mL), the combined organic fractions were washed with
brine, dried over MgSO4 and concentrated in vacuo. Purification
by silica gel column chromatography (10% AcOEt in hexane)
afforded pure product 24 (650 mg, 84%) as a yellow solid.
1H NMR (400 MHz, acetone-d6) δ: 7.78 (1 H, t J 1.4 Hz, arom.),
7.57 (1 H, dd, J 2.3, 1.4 Hz, arom), 7.33 (1 H, dd, J 2.3, 1.4 Hz,
arom), 3.91 (3 H, s, OMe), 0.26 (9 H, s, SiMe3).
13C NMR (100 MHz, acetone-d6) δ: 165.6, 142.0, 133.2, 129.6,
126.6, 125.9, 120.6, 103.5, 97.0, 52.9, -0.2.
Rf (10% AcOEt/hexane) 0.36.
MS(EI): m/z (%) = 273.1 (1, M+), 249.1 (5), 247.1 (100), 233.1
(49), 232.1 (50), 174.1 (3), 117.0 (4), 86.6 (4), 73.1 (9).
HRMS (EI+) [M]+ - calculated for C13H15N3O2Si : 273.0934,
found: 273.0935.
Rf (30% AcOEt/hexane) 0.38.
MS(EI): m/z (%) = 341.0 (1, M+), 281.0 (2), 213.0 (5), 171.0 (9),
170.0 (100), 158.0 (12), 142.0 (7), 141.0 (99), 125.0 (2), 111.0
(5), 78.0 (11), 77.0 (98), 51.0 (19).
4.2.19 3,4-Dimethyl-5-oxo-2,5-dihydrofuran-2-yl3-azido-5-
ethynylbenzoate (26)
a) Preparation of 3-azido-5-ethynylbenzoyl chloride (25)
A solution of oxalyl chloride (0.200 mL, 2.364 mmol) in 2
mL of dichloromethane was added dropwise to a suspension
of 22 (82 mg, 0.438 mmol) in 5 mL of dichloromethane and a
catalytic amount of DMF (1 drop) under an argon
atmosphere. The reaction mixture was stirred for 45 min in
the dark and then the solvent was evaporated. The crude
product was dissolved in 2 mL of dichloromethane and used
in the next reaction step.
HRMS (ESI): [M+Na]+ - calculated for C17H15O5N3Na :
364.0905, found: 364.0904.
4.2.21 2-(2-(2-((3,4-dimethyl-5-oxo-2,5-dihydrofuran-2-
yl)oxy)ethoxy)ethoxy)ethyl-3–azido-5-ethynyl benzoate (28)
a) 3-Azido-5-ethynylbenzoyl chloride (25) was prepared from
22 (38 mg, 0.203 mmol) and oxalyl chloride (0.100 mL,
1.182 mmol) by the same procedure as reported for the
preparation of 26.
A solution of 3-azido-5-ethynylbenzoyl chloride (25) in 2 mL
dichloromethane was added dropwise to a solution of 4 (51 mg,
0.398 mmol), and Et3N (1.00 mL, 7.17 mmol) in 2 mL of
dichloromethane under an argon atmosphere. The reaction
mixture was stirred for 3 h in the dark at room temperature. The
solvent was removed under vacuum and the crude product was
dissolved in 20 mL of AcOEt and poured into 100 mL of water.
The aqueous fraction was washed with AcOEt (2 x 20 mL) and
the combined organic fractions were washed with a saturated
aqueous solution of NaHCO3, brine, dried over MgSO4 and
concentrated in vacuo. Purification by silica gel column
chromatography (10% AcOEt in hexane) afforded pure product
26 (73 mg, 56%) as a white solid.
A solution of 25 in 2 mL dichloromethane was added dropwise to
a solution of 13 (41 mg, 0.158 mmol), and Et3N (0.400 mL,
2.868 mmol) in 3 mL of dichloromethane under an argon
atmosphere. The reaction mixture was stirred for 5 h in the dark
at room temperature. The solvent was removed under vacuum
and the crude product was dissolved in 20 mL of AcOEt and
poured into 100 mL of water. The aqueous fraction was washed
with AcOEt (2 x 20 mL) and the combined organic fractions
were washed with saturated aqueous solution of NaHCO3, brine,
dried over MgSO4 and concentrated in vacuo. Purification by
silica gel column chromatography (30% of AcOEt in hexane)
afforded pure product 28 (45 mg, 66 %) as a yellow oil.
1H NMR (400 MHz, acetone-d6) δ: 7.91 – 7.90 (1 H, m arom.),
7.71 – 7.70 (1 H, m, arom.), 7.51 – 7.50 (1 H, m, arom.), 7.05 –
7.04 (1 H, m, CH), 3.92 (1 H, s, C≡CH), 2.16 – 2.14 (3 H, m,
C3Me), 1.89 (3 H, quint, J 1.2 Hz, C4Me).
1H NMR (400 MHz, acetone-d6) δ: 7.85 (1 H, t, J 1.4 Hz, arom.),
7.64 (1 H, dd, J 2.3, 1.4 Hz, arom.), 7.41 (1 H, dd, J 2.3, 1.4 Hz,
arom.), 5.78 (1 H, dp, J 2.0, 1.1 Hz, CH), 4.49 – 4.43 (2 H, m
OCH2), 3.87 (1 H, s, C≡CH), 3.86 – 3.81 (4 H, m, OCH2), 3.70 –

9
13. Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem. Int.
3.61 (6 H, m, OCH2), 1.95 (3 H, quint, J 1.0 Hz, C3Me), 1.74 (3
H, quint, J 1.2 Hz, C4Me).
ACCEPTED MANUSCRIPT
13C NMR (100 MHz, acetone-d6) δ: 172.4, 165.1, 155.4, 142.2,
133.3, 129.7, 127.0, 126.5, 125.2, 120.9, 104.1, 82.2, 81.4, 71.3,
71.3, 71.0, 69.5, 69.4, 65.6, 11.4, 8.3.
Ed. Engl. 2001, 40, 2004-2021.
14. Kepiró, M.; Varkuti, B. H.; Rauscher, A. A.; Kellermayer, M.
S.; Varga, M.; Malnasi-Csizmadia, A. Chem. Biol. 2015, 22,
548-558.
15. (a) Hosoya, T.; Aoyama, H.; Ikemoto, T.; Hiramatsu, T.;
Kihara, Y.; Endo, M.; Suzuki, M. Bioorg. Med. Chem. Lett.
2002, 12, 3263-3265; (b) Hosoya, T.; Hiramatsu, T.; Ikemoto,
T.; Aoyama, H.; Ohmae, T.; Endo, M.; Suzuki, M. Bioorg.
Med. Chem. Lett. 2005, 15, 1289-1294; (c) Shimotahira, H.;
Fusazaki, S.; Ikeda, I.; Ozoe, Y. Bioorg. Med. Chem. Lett.
2011, 21, 1598-1600.
Rf (30% AcOEt/hexane) 0.21.
MS(EI): m/z (%) = 429.0 (2, M+), 355.1 (12), 281.0 (8), 221.0
(6), 186.0 (10), 155.0 (28), 147.0 (13), 113.0 (16), 111.0 (100),
73.0 (23), 55.0 (18).
HRMS (ESI): [M+Na]+
452.1428, found: 452.1428.
- calculated for C21H23O7N3Na:
16. Dormán, G.; Prestwich, G. D. Trends Biotechnol. 2000, 18, 64-
77.
Acknowledgments
17. Khan, U.; Baseer, M. A.; Kumar, S. R.; Saravanakumar, M.;
Prasannanjali, A. G.; Gupta, P. B.; Kaushik, V. K.; Handa, V.
K.; Islam, A. Synth. Commun. 2010, 40, 2241-2253.
18. Surmont, R.; Verniest, G.; De Kimpe, N. J. Org. Chem. 2010,
75, 5750-5753.
19. Harmrolfs, K.; Mancuso, L.; Drung, B.; Sasse, F.; Kirschning,
A. Beilstein J. Org. Chem. 2014, 10, 535-543.
20. Kutonova, K.; Trusova, M.; Postnikov, P.; Filimonov, V.;
Parello, J. Synthesis 2013, 45, 2706-2710.
IOCB research plan RVO: 61388963, OTKA 114567 and
Bolyai Fellowship supporting this work is acknowledged.
Supplementary Material
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