New Metal-Free Route towards Imidazole-Substituted Uridine

Article abstract of DOI:10.1002/ejoc.202000563

Nucleosides with a bi(hetero)aryl nucleobase have unique potential applications as antiviral drugs and molecular probes. The need for transition metal catalysis to synthesize these nucleosides from pre-functionalized building blocks and the use of nucleobase protection groups results in expensive and tedious syntheses. Herein we report that 5-imidazolyl-uracil can be obtained by scalable Van Leusen imidazole synthesis and regioselectively introduced on ribose to obtain the desired nucleoside in a 5 step synthesis (total yield 55 percent). The 5-imidazolyl moiety leads to improved fluorescence properties. The only side-product formed was characterized by 2D-NMR and X-ray crystallography and could be suppressed during synthesis in favor of the desired product.

Full text of DOI:10.1002/ejoc.202000563

European Journal of Organic Chemistry
Accepted Article
Accepted Article
Title: New metal-free route towards imidazole substituted uridine
Authors: Henri-Philipppe Mattelaer, Anne-Sophie Van Hool, Flip
de Jong, Mark Van der Auweraer, Luc Van Meervelt, Wim
Dehaen, and Piet Herdewijn
This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Eur. J. Org. Chem. 10.1002/ejoc.202000563
Link to VoR: https://doi.org/10.1002/ejoc.202000563
0
1/2020

European Journal of Organic Chemistry
10.1002/ejoc.202000563
COMMUNICATION
New metal-free route towards imidazole substituted uridine
Henri-Philippe Mattelaer[a,d], Anne-Sophie Van Hool , Flip de Jong , Mark Van der Auweraer , Luc
[a]
[b]
[b]
[
c]
[d]
*[a]
Van Meervelt , Wim Dehaen , Piet Herdewijn
[
a]
Dr. H.P. Mattelaer, A.S. Van Hool, Prof.Dr. P. Herdewijn
Rega Institute for Medical Science, Medicinal Chemistry
KULeuven
Herestraat 49, 3000 Leuven, Belgium
E-mail: Piet.Herdewijn@kuleuven.be
F. de Jong, Prof. Dr. M. Van der Auweraer
Department of Chemistry, Molecular Imaging and Photonics
KULeuven
Celestijnenlaan 200F, 3001 Leuven, Belgium
Prof. Dr. L. Van Meervelt
Department of Chemistry, Biochemistry, Molecular and Structural Biology Section
KULeuven
[b]
[c]
[d]
Celestijnenlaan 200F, 3001 Leuven, Belgium
Prof. Dr. W. Dehaen
Department of Chemistry, Molecular Design and Synthesis
KULeuven
Celestijnenlaan 200F, 3001 Leuven, Belgium
Supporting information for this article is given via a link at the end of the document.
Abstract: Nucleosides with a bi(hetero)aryl nucleobase have unique
potential applications as antiviral drugs and molecular probes. The
need for transition metal catalysis to synthetize these nucleosides
from pre-functionalized building blocks and the use of nucleobase
protection groups results in expensive and tedious syntheses. Herein
we report that 5-imidazolyl-uracil can be obtained by scalable Van
Leusen imidazole synthesis and regioselectively introduced on ribose
to obtain the desired nucleoside in a 5 step synthesis (total yield 55%).
The 5-imidazolyl moiety leads to improved fluorescence properties.
The only side-product formed was characterized by 2D-NMR and X-
ray crystallography and could be suppressed during synthesis in favor
of the desired product.
introduction of imidazoles has led to the functionalisation of DNA
in order to obtain a more stable version of a ribozyme, a
DNAzyme.[7] Retrosynthetically, we aimed at preparing the
substituted base before nucleoside formation, simultaneously
avoiding the use of protection groups where possible; instead of
the classical, transition metal catalysed route where the
heterocycle was coupled to the nucleoside. Literature study only
resulting in one paper by Ressner et al[8] where the desired
imidazole substituted uracil was reported. However after the
Biginelli-condensation to obtain 5-acetyluracil we did not obtain
2
the α-brominated species using several methods (Br , NBS,
HBr/AcOH) to conduct the subsequent imidazole ring formation
with formamide. Therefore another route was investigated starting
from commercial uracil; which was hydroxymethylated at the 5-
position and oxidized to the aldehyde 1 using potassium
peroxodisulfate and a catalytic amount of silver nitrate, both using
Modified nucleobases, nucleosides and nucleotides have
provided a wide range of biologically active compounds such as
antibiotics, anti-cancer and antiviral drugs.[1] Furthermore, they
provide new building blocks to construct and interfere with living
previously reported methods.[9] The aldehyde was then used to
[10]
investigate the Van Leusen imidazole synthesis
using
[
2]
systems, with potential application in synthetic biology.
A
tosylmethylisocyanide (TOSMiC) (Table 1). To the best of our
knowledge, there are no examples of Van Leusen imidazole
synthesis performed on a nucleobase and mostly N-substituted
derivatives are obtained by using primary amines. NH-analogues
are either obtained by deprotection or using ammonium hydroxide
[
3]
particular class of modified nucleosides are fleximers , where the
nucleobase has been modified to a bi(hetero)aryl inspired by
disconnecting purine nucleobases into their respective imidazole
and pyrimidine ring. Nucleosides forming the nucleosidic bond
with the pyrimidine ring (also dubbed "reverse" fleximers[3b]) are
extremely suitable for probing helix and base pair formation.[4]
Additionally, both reverse and conventional fleximers, nucleosidic
bond with the imidazole moiety, have shown promising antiviral
activity, including coronaviruses.[5] However the classical way to
obtain such biheteroaryl systems occurs via palladium-catalysed
reactions such as Suzuki and Stille coupling.[3b, 4-6] Although these
methods access a wide scope of compounds, they pose problems
when scaling up requiring expensive reagents, pre-functionalised
starting materials (mostly halogens) and safety concerns (as is
the case for stannous derivatives).
3
as a base. In our system, using commercially available 7M NH in
methanol as the base in a pressurized reaction vessel results in
the desired product precipitating out of the reaction mixture, which
is isolated in good yield without further need for purification (Table
1, Entry 1-5). Scaling up increases the efficiency of the
precipitation to yield the desired product (Table 1, Entry 1-5).
Using ammonium hydroxide solution as amine source gives a
similar precipitate although less easily filtrated, and much is lost
during work-up (Table 1, Entry 6-7). The amine source was
exchanged for various other bases in order to obtain the oxazole
analogue, however this was unsuccessful (Table 1, Entry 8-11).
Our research group took particularly interest in efficiently
obtaining imidazole substituted uracil and uridine as they embody
the broad applications of (reverse) fleximers[6b] and the
1
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European Journal of Organic Chemistry
10.1002/ejoc.202000563
COMMUNICATION
Table 1. Van Leusen reaction on 5-formyluracil.
Table 2. Optimization of Vorbrüggen conditions.
Entry
Lewis
Acid
Solvent
T
t
Yield[a] (%)
(°C)
(d)
3a
3b
Ratio
a:b)
(
1
2
3
4
5
6
7
8
TMSOTf[b]
TMSOTf[b]
TMSOTf[b]
TMSOTf[b]
ACN
ACN
ACN
DCE
DCE
DCE
THF
DMF
rt
1
1
2
2
2
2
1
1
n.o. [d,e]
ND[f]
3:1
60
60
60
60
60
60
60
34
58
67
73
11
21
5
3:1
13:1
12:1
ND[f]
TMSOTf[c]
9
SnCl [
b]
traces[e]
Inseparable mixture[g]
n.o. [d,e] ND[f]
4
TMSOTf[b]
TMSOTf[b]
Entry
TOSMiC
Base
(eq)
Solvent
Scale
(g)
Yield[a] (%)
(eq)
2a
2b
[
a] Isolated yield, [b] 1 eq, [c] 1.5 eq [d] not observed, [e] monitored by TLC,
[f] not determined, [g] more than 6 spots (TLC)
1
2
3
4
5
6
7
8
9
3
NH
3
3
3
3
3
(6)
MeOH
MeOH
MeOH
MeOH
MeOH
MeOH
0.125
0.125
0.25
0.5
40
-
1.6
1.6
1.6
1.6
1.6
1.6
1.6
1.6
1.6
1.6
NH
NH
NH
NH
(3)
(3)
(3)
(3)
48
-
59
-
Longer reaction time led to increased product formation, with both
products increasing similarly (Table 2, Entry 3). Changing the
solvent to DCE shifted the obtained product ratio towards 3a
(Table 2, Entry 4). However, it was now necessary to reflux the
first silylation step in order to obtain a transparent solution, which
was not necessary using ACN as a solvent. Slightly improved
yields were obtained for both products when increasing the
amount of TMSOTf (Table 2, Entry 5). Another Lewis acid used in
66
-
-
1
85
NH
NH
4
OH (3)
OH (3)
0.25
0.25
0.25
0.25
0.25
0.25
23
-
H
2
O
Deg[b]
-
4
K
2
CO
tBuOK (3)
Et N (3)
DBU (3)
3
(3)
MeOH
MeOH
MeOH
MeOH
-
-
-
-
m.s.[c]
m.s.[c]
m.s.[c]
n.r. [d]
1
0
1
3
1
nucleosidic bond formation (SnCl ), did not show any significant
4
product formation. THF and DMF were not suitable as solvents
[
a] Isolated yield, [b] degradation of starting material, [c] multiple spots,
multiple products (monitored by TLC, MS), [d] no reaction
for this reaction (Table 2, Entry 7-8). The final products 4a and 4b
were obtained by overnight deprotection of the benzoyl groups
using 7M NH in MeOH (Scheme 1). They were analyzed by 2D-
3
Thin layer chromatography (TLC) showed a myriad of products
forming, an intermediate before elimination of the tosyl group
mediated by base. Harsher conditions only resulted in the
degradation of the uracil ring (Table 1, Entry 7-11).
Nucleoside formation with the imidazole substituted uracil was
done via the Vorbrüggen method[11], by transiently protecting the
NMR to ascertain which regioisomers were formed in the earlier
mentioned Vorbrüggen reaction (Supporting Information, SI).
As the designation of the regiochemistry of 4b relied on the
possibly ambiguous absence of some 2D-NMR (HMBC) cross
peaks, the structure was confirmed by x-ray crystallography
nucleobase
bis(trimethylsilyl)acetamide (BSA). This was immediately reacted
one-pot) with 1-O-acetyl-2,3,5-tri-O-benzoyl-D-ribofuranose,
2a
with
trimethylsilyl-groups
using
(
CCDC 1996102, Figure 1, SI). Compound 4b was crystallized as
HCl salt with the imidazole ring being protonated at the N2 atom
(
(
(
SI, Figure 1). The puckering mode of the ribose ring is C3’-endo
pseudorotation phase angle P = 9.8(±0.4)°, puckering amplitude
promoted by the addition of a Lewis acid (Scheme 1). At room
temperature, no reaction products were found on TLC nor isolated
m
=
38.1(±0.3)°). The torsion angle O4-C1-N1-C8 (-
(
Table 2, Entry 1), gentle heating at 60 °C (Table 2, Entry 2)
139.5(±0.4)°), comparable to the  angle used to describe the
showed the formation of two products, a major and a minor one.
orientation about the glycosyl bond, indicates an anti
conformation with the bulk of the substituent pointing away from
the sugar.
Scheme 1. Nucleoside synthesis via Vorbrüggen reaction with 5-imidazolyl uracil. Detailed reaction conditions for the formation of 3a/3b can be found in
Table 2. BSA = Bis(trimethylsilyl)acetamide.
.
2
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European Journal of Organic Chemistry
10.1002/ejoc.202000563
COMMUNICATION
Figure 1. Single crystal X-ray structure of 4b·HCl with thermal
ellipsoids at 50% probability level.
Figure 2. Normalized absorption and emission spectra of 4a
The dihedral angle between the best planes through the imidazole
and uracil ring is 67.4(±0.2)°. The crystal packing is consolidated
through a series of O-H…O/Cl and N-H…O/Cl interactions
between all possible hydrogen bond donors and acceptor, except
sugar atom O4 (Figure S1, Table S2).
-5
(
1×10 M) in acetonitrile (ACN), dichloromethane (DCM),
methanol (MeOH) and phosphate buffered saline (PBS). The
shoulder observed in the emission spectrum at higher energy is
due to Raman scattering. The excitation wavelength for the
emission spectra was 310 nm
Similar DNA fleximers bearing furan and oxazole moieties have
practical applications as fluorescent probe to measure the polarity
at DNA abasic sites.[4a] Therefore the steady-state spectroscopy
of both products (4a and 4b) was investigated in ACN, DCM,
MeOH and phosphate buffered saline (PBS). The results are
presented in Figures 2, S2, S3 and Tables 3, S3, S4. The S0→
S1 band of 4a is very similar in all four solvents, no vibrational
structure is observed and the position of the maximum varies only
slightly from 305 to 308 nm. The latter suggests an apolar ground
state. At higher energy, the onset of a very intense transition is
observed. Having the same regiochemistry as the previously
mentioned fleximers, 4a exhibits similar albeit improved
fluorescent properties. A broad and structureless band is
observed in all solvents, shifting to longer wavelengths with
increasing polarity with similar results for ACN and DCM, in
agreement with literature.[4a] This suggests a slightly polar excited
state. This is no surprise considering the chromophore resembles
a merocyanine moiety. The quantum yield in water (PBS) is
2
The data could be fitted satisfactorily ( = 1.015) with a mono-
exponential model, resulting in a lifetime () of 1.88 ns. This
uncomplicated behaviour is beneficial for a probe and unusual for
a nucleoside.[12] For 4b on the other hand, the S0→S1 transition
is observed at lower wavelengths, with maxima between 270 and
76 nm. No fluorescence could be observed.
Although recent developments have introduced the use of
enzymatic techniques[6b, 6c, 13] in accessing the broad scope of
reverse) fleximers, they still rely on the use of transition metal-
catalyzed reactions. Here we show that two isomers, 4a and 4b
can be obtained without needing to resort to additional protecting
groups and heavy metal catalysis. All this can be achieved in a
simple, scalable reaction scheme of 5 steps which obtains 4a in a
total yield of 55% starting from commercially available uracil and
involving only a single use of chromatographic separation. The
solvent dependency of the regioselective reaction is interesting.
Attempts to increase the yield of 3b/4b by means of a different
Lewis acid or solvent were unfortunately unsuccessful. Further
investigation was beyond the scope of this endeavor but would be
interesting as 4b, belongs to the family of distal fleximers whose
synthesis still remains non trivial.[3a, 6b, 13] The introduction of an
imidazole on the 5-position of uracil (4a), improves the
spectroscopic characteristics when compared to furan, thiophene,
oxazole, thiazole and benzimidazole.[4a, 14] Furthermore, it allows
the possibility of wobble base pair formation[ and catalytic
2
(
increased twofold with respect to
a 5-furane substituted
[
4a]
deoxyuridine, from 0.03 to 0.06. In order to further evaluate its
suitability as a sensor, the excited state dynamics of 4a in PBS
were evaluated. In Figure S3 the fluorescence decay trace and
the corresponding fit are displayed.
Table 3. Summary of absorption and emission properties of 4a in different
solvents.
4b]
[
a]
[b]
[c]
FWHMem[d]
(cm-1)
QY[e]
(%)
Solvent
λ
abs
λ
em
휈
̅
푎푏푠 − 휈
̅
[7]
activity . On the other hand, isomer 4b is a novel fleximer
-
1
(
nm)
(nm)
(cm )
reminiscent of a disconnected xanthine, of which we provide the
first detailed structural determination by 2D-NMR and XRD.
Testing these newly accessed privileged structures against the
current COVID19 pandemic is currently underway.
ACN
DCM
MeOH
PBS
306
305
307
308
399
400
417
442
7610 ± 50
7790 ± 50
8590 ± 50
9840 ± 50
5200 ± 50
5400 ± 50
5460 ± 50
5610 ± 50
2.3 ± 0.2
3.7 ± 0.4
5.7 ± 0.6
6.4 ± 0.6
[
a] Absorption maxima, [b] emission maxima, [c] Stokes' shift, [d] full width
at half maximum of the emission band, [e] fluorescence quantum yield. For
all measurements, the concentration was 1x10-
5
Acknowledgements
M
LVM thanks the Hercules Foundation for supporting the purchase
of the diffractometer (project AKUL/09/ 0035). We thank Jef
Rozenski for recording HRMS; supported by the Hercules
Foundation of the Flemish Government (grant 20100225–7), Luc
3
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European Journal of Organic Chemistry
10.1002/ejoc.202000563
COMMUNICATION
Baudemprez for recording NMR, CA Mattelaer for graphical help
and Chantal Biernaux for editorial help.
Keywords: Nucleobase • Nucleosides • Fluorescent probes •
Fleximer • Green Chemistry
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4
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European Journal of Organic Chemistry
10.1002/ejoc.202000563
COMMUNICATION
Entry for the Table of Contents
Metal-free nucleosides: Bi(hetero)arylic nucleosides have unique potential as antiviral drugs and molecular probes. However their
synthesis is tedious; requiring metal catalysis, pre-functionalized building blocks and nucleobase protection groups. Herein we report
the scalable, metal-free synthesis of 5-imidazolyl-uracil and its regioselective nucleoside formation. Improved fluorescence and
characterization of a new fleximer side-product are reported.
5
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