Synthesis and characterization of various 5′-dye-labeled ribonucleosides

Article abstract of DOI:10.1039/c8ob01606b

Hitherto unknown chromophoric nucleosides are reported. This novel set of visibly coloured dye-labeled 5′-nucleosides, including 1,2,4,5-tetrazine, dicyanomethylene-4H-pyran, benzophenoxazinone, 9,10-anthraquinone and azobenzene chromophores, were prepared mainly under Cu-catalyzed azide-alkyne cycloaddition (CuAAC). The design criteria are outlined. Several derivatives possess in supplement a fluorescence property. The absorption and fluorescence spectra of all coloured nucleosides were recorded to study their potential as visible-range probes. Such nucleodyes are of great interest for future competitive lateral flow test MIP-based strips.

Full text of DOI:10.1039/c8ob01606b

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Chem., 2018, DOI: 10.1039/C8OB01606B.
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January 2016 Pages 1–372
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Takeharu Haino et al.
Solvent-induced emission of organogels based on tris(phenylisoxazolyl)
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Synthesis and Characterization of various 5’‐Dye‐labeled
Ribonucleosides
a
a
a
b
b
Coralie De Schutter, Vincent Roy, Patrick Favetta, Corentin Pavageau, Stéphane Maisonneuve,
Received 00th January 20xx,
Accepted 00th January 20xx
b
b
a
Nicolas Bogliotti, Juan Xie and Luigi A. Agrofoglio*
DOI: 10.1039/x0xx00000x
Hitherto unknown chromophoric nucleosides are reported. This novel set of visibly coloured dye‐labeled 5’‐nucleosides
including 1,2,4,5‐tetrazine, dicyanomethylene‐4H‐pyran, benzophenoxazinone, 9,10‐anthraquinone and azobenzene
chromophores, were prepared mainly under Cu‐catalyzed azide‐alkyne cycloaddition (CuAAC). Design criteria are outlined.
Several derivatives possess in supplement a fluorescence property The absorption and fluorescence spectra of all coloured
nucleosides were recorded to study their potential as visible‐range probes. Such nucleodyes are of great interest for future
competitive lateral flow test MIP‐based strips.
www.rsc.org/
study, we have prepared several hitherto unknown uridine and
adenosine dye analogues and we turn our attention to visibly
coloured nucleoside probes with intense dyes such as 1,2,4,5‐
tetrazine (or s‐tetrazine), dicyanomethylene‐4H‐pyran (DCM),
benzophenoxazinone, 9,10‐anthraquinone and azobenzene. The 5’‐
position of nucleoside analogues was chosen for the introduction of
the dye. Their photophysical features were characterized.
Introduction
Nucleosides are structural subunits of nucleic acids and the
biochemical precursors of nucleotides, which play an important role
in cell metabolism. Their analogues are largely used to combat
cancer and viral infections and some nucleoside metabolites of RNA,
1
are well known potential biomarkers of cancer. Labeled
nucleos(t)ides using fluorescence polarization are critical elements
for various applications, including study of nucleic acid functions
2
Results and Discussion
and structure, sequence detection, nucleoside and nucleotide
cellular interaction, nucleotide‐binding proteins or for competitive Synthesis of 5’‐dye labelled nucleoside
nucleotide binding assay. The first fluorescent nucleotide, reported
3
by
Hiratsuka
and
Uchida,
was
the
2’,3’‐O‐(2,4,6‐
First of all, we turned our attention to the introduction of various
chromophoric 1,2,4,5‐tetrazines at C5’ of uridine in order to obtain
coloured uridines ranging from purple to orange to red. According
to the nature of the s‐tetrazine substituent, fluorescence properties
can also be displayed.11 All s‐tetrazine derivatives were prepared
starting from commercially available uridine (1), which, after
protection of the 2'‐and 3'‐hydroxyls by isopropylidene group (to 2),
was reacted with 3,6‐dichloro‐s‐tetrazine12 in presence of 2,4,6‐
trinitrocyclohexadienylidene) adenosine 5’‐triphosphate. The site of
incorporation of the dye on the nucleoside is chosen in order to
minimize the effect on the structures of the corresponding
oligonucleotides. Thus, the dye is generally introduced by
conjugation through linkers to the sugar hydroxyl groups or more
generally to the nucleobase at the C5 position of pyrimidines or C8
4
5
4
position of purines.
Recently we have reported the quantitative detection of urinary collidine as a non‐nucleophilic base (ratio 2/s‐tetrazine/2,4,6‐
modified nucleoside biomarkers by new sensors based on collidine = 1/1/1) . The monosubstituted derivative 3 was obtained in
molecularly imprinted polymer (MIP).6‐8 In order to explore high yield (90%). Removal of the isopropylidene moiety by a dilute
9
competitive lateral flow test MIP‐based strips (Figure S1), a rapid
growing strategies for qualitative and quantitative analysis, the
development of 5’‐dye‐labeled nucleosides is now mandatory.
solution of 0.5 M hydrochloric acid in methanol at 55°C for 3h13
unfortunately led also to the cleavage of the 6‐chloro‐s‐tetrazine
moiety. The C‐O bond between the uridine‐ and tetrazine‐moiety
was similarly cleaved when the reaction was carried out in HCl 0.5 M
in dioxane at room temperature, and with 10% acetic acid at 90°C for
If fluorescent nucleosides have been largely developed,
1
0
chromophoric nucleoside analogues have been less explored (and
only for azo dye) but offer often advantage of a high degree of
stability under most application conditions. In the current
1
0 min, degradation products were observed.14 We thus explored
some Lewis acid reagents which have been fairly successful in
selective removing isopropylidene ketal in the presence of other
acid‐sensitive moieties.
a.
Univ. Orléans, CNRS, ICOA, UMR 7311, F‐45067 Orléans, France
E‐mail : luigi.agrofoglio@univ‐orleans.fr
b.
PPSM, ENS Paris‐Saclay, CNRS, Univ. Paris‐Saclay, F‐94253 Cachan, France
Electronic Supplementary Information (ESI) available: [details of any supplementary
information available should be included here]. See DOI: 10.1039/x0xx00000x
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Scheme 1. Synthesis of nucleosides bearing 1,2,4,5‐tetrazine moiety
The obtained s‐tetrazine‐uridine 3 was treated, under mild aprotic obtained in 36% yield. Finally, deprotection in presence of ZnBr
condition, with a large excess of anhydrous ZnBr (8 equiv.) in to 5’‐O‐(6‐methoxy‐1,2,4,5‐tetrazin‐3‐yl)‐uridine (8) with a good
After 21h stirring, the yield.
2
led
2
1
5
dichloromethane at room temperature.
deprotected s‐tetrazine‐uridine 4 was obtained in 64% yield.
Substitution of the remaining chlorine atom of the s‐tetrazine‐
uridine 3 by alkyl‐ or aromatic‐thiol proceeded readily in presence of
We faced similar difficulties to obtain other asymmetric
disubstituted s‐tetrazines and we failed to obtain the 3‐morpholino‐
6‐uridine‐s‐tetrazine. Thus, in order to circumvent these difficulties
triethylamine in acetonitrile to give thioethers
5
and 6,
1
6
and to obtain desired 5’‐dye‐labeled ribonucleosides, we focused our
respectively. Unfortunately, all our attempts to selectively remove
the isopropylidene group, using either the previous Lewis acid attention on the Huisgen 1,3‐dipolar cycloaddition between 5’‐azido‐
conditions, a Dowex‐H resin (50Wx8‐100) in methanol at 40°C for uridine derivative 1720 and various alkynes 11, 15 and 16, bearing a
+
1
7
1
1
6h. or deionized water under microwave irradiation (10 min, various chromophoric group.
1
8
20°C), failed. We observed either no reaction either the released
of the substituted s‐tetrazine‐moiety.
(i) The DCM fluorophore 11, an analogue of 4‐dicyanomethylene‐2‐
t‐butyl‐6‐methyl‐4H‐pyran, can be readily prepared from the
commercially available aldehyde 9, by O‐propargylation to 10,
followed by Knoevenagel condensation with the tert‐butyl‐DCM
A second nucleophilic substitution of chorine atom of 3 by sodium
methoxide didn’t lead to compound 7 but the starting material was
recovered. As reported by Clavier and Audebert,12 such
(
Scheme 2).
unsymmetrical disubstitution of s‐tetrazines is often achieved with
poor to moderate yields (25% average) and required the use of an
alcoolate. Thus, using the previous conditions, 3,6‐dichloro‐s‐
tetrazine was treated with sodium methoxide to afford 3‐chloro‐6‐
1
9
methoxy‐s‐tetrazine in a 71% yield. Under initial conditions (ratio
/s‐tetrazine/2,4,6‐collidine = 1/1/1), the desired s‐tetrazine 7 was
isolated after 32h stirring in 3% yields. After an optimization (ratio
/s‐tetrazine/2,4,6‐collidine = 1/2/4), the s‐tetrazine‐uridine 7 was
2
2
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Compounds 18, 20 and 22 were then dissolved in a 90% TFA in water
mixture and stirred at room temperature for 10 min to afford the
corresponding deprotected tagged‐nucleosides dicyanomethylene‐
4H‐pyran 19, naphthooxazine 21 and 9,10‐anthracenedione 23,
respectively, in high yields. This method can be applied to a large
range of coloured tag with other 5’‐azido‐5’‐deoxy nucleosides
(
Scheme 4).
Product
Scheme 2. Synthesis of alkyne‐functionalized DCM 11
11
1
8, R=C(CH3)2 99%
(
(
ii) The benzophenoxazinone 1521a,b was obtained from the 2‐
diethylamino)‐phenol (12) through an optimized one‐pot
19, R=H
99%
procedure. Compound 12 was first converted into its nitroso
derivative 13 with amyl nitrite, the latter being reacted with 1,6‐
dihydroxynaphtalene at refluxed DMF21c and submitted to a
Williamson ether synthesis to its propargyl ether analogue (Scheme
3).
15
2
0, R=C(CH3)2 78%
1, R=H
74%
2
16
2
2, R=C(CH3)2 67%
3, R=H
85%
2
Scheme 4. Synthesis of various coloured nucleosides through click chemistry
The synthesis of azo dye Disperse Red 13 uridine derivative 27
started from commercially available Disperse Red 13 which has a
deep dark red color. We first tried the direct substitution of 2’,3’‐O‐
isopropylidene‐5’‐O‐toluenesulfonyluridine by Disperse red 13 in
presence of a large excess of sodium hydride in THF. Unfortunately,
the degradation of the reagents was observed; likewise, the
nucleophilic substitution, under these conditions, of 4‐bromobut‐1‐
Scheme 3. Synthesis of alkyne‐functionalized benzophenoxazinone 15
(
iii) The 1‐amino‐4‐(but‐3‐ynylamino)anthracene‐9,10‐dione (Tag 16) yne (for CuAAC) with the OH of this tag failed. The difficulties met to
was easily obtained from a solution of commercially available 1,4‐ introduce the alkyne group on the red‐tag led us to investigate
diaminoanthraquinone and K
2
CO
3
in ACN with 4‐bromobutyne.
another pathway than the [3+2] cycloaddition reaction. The
introduction of the azo dye at 5’‐OH of uridine was thought to be
easily done through a nucleophilic substitution of a halo azo dye.
Copper‐catalyzed azide‐alkyne Huisgen cycloaddition (CuAAC)
between those coloured tags 11, 15 and 16 and 5′‐azido‐5′‐ Thus, the hydroxyl group of Disperse Red 13 dye was reacted with
deoxy‐2′,3′‐O‐isopropylidene‐uridine was then explored, 1,5‐dibromopentane in presence of potassium hydroxide and 18‐
O (8:2) mixture crown‐6 to afford the bromo derivative 2423 in 59% yields, (Scheme
(
Scheme 4). No reaction was observed in a tBuOH/H
2
2
2
5). The nucleophilic displacement of bromine of 24 by the 5’‐hydroxy
group of protected uridine 2 with sodium hydride or potassium
hydroxide failed. Based on the work reported by Yao, compound 26
was obtained by reacting the 5’‐amino‐5’‐deoxy‐2’,3’‐O‐
isopropylideneuridine (25) with azo dye 24 using potassium
at 28°C for 16h. An increase of CuSO4 (from 5 to 10 mol%), of
sodium ascorbate (from 10 to 20 mol%), and in DMSO at 60°C led to
the desired compounds 18 (98% yield) and 20 (74% yield),
2
4
respectively.
Similar protected uridines 18, 20 and 22,
respectively, were better obtained in high yields after 3 minutes
under microwave irradiation in presence of copper(I) sulfate catalyst
and sodium ascorbate.
carbonate in DMF with
a moderate yield. Isopropylidene
deprotection was performed in presence of TFA in water to afford
the red azo‐uridine derivative 27 in 70% yields.
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a
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&
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Br
5
introduction of the uridine group on the 3,6‐dichloro‐s‐tetrazine
increased only slightly λ (501 to 509 nm, entries 1 and 2) but the
change was more significant on λ (303 to 323 nm, entries 1 and 2).
(
O
1
,5-dibomopentane
KOH, 18-crown-6
1
N
N
2
Disperse Red 13 dye
O N
N
2
THF
The same pattern is observed when the chlorine of 3 is replaced by a
methoxy group (entry 6; Figure 1, purple line). This bathochromic
effect was more pronounced when chlorine is replaced by a
thioether moiety (entries 4 and 5; Figure 1, blue lines). This shifting
allowed the s‐tetrazine nucleosides to range from pink to orange as
illustrated in Figure 2 (a) and (b). This range of colour could be very
useful in the case where the coloured derivatives are immobilized on
a urinary strip used in the semi‐quantitative analysis of nucleoside
biomarkers by displacement method. No modification of the three
wavelengths has been observed after deprotection of the sugar ring
even when ethanol was used for s‐tetrazine 4 to solve solubility
problems. This bathochromic shift was still present for the emission
5
9%
Cl
2
4
O
N
NH
K CO
H2
N
O
2
3
O
DMF
3%
4
O
O
2
5
O
N
NH
H
N
O
O
(
O
5
N
N
RO
OR
O
2
N
N
em
maxima of 3 and 7 (or 4 and 8) but weaker (Δλ ≈ 10 nm). Thus, both
Cl
2
6, C(CH3)
2
TFA, H O, 70%
compounds appeared yellow under UV‐light (Figure 2, c). The major
difference between these two s‐tetrazines was the significant drop
of fluorescence, to its complete disappearance for the thioethers s‐
tetrazines 5 and 6.
2
2
7, R=H
Scheme 5. Synthesis of red azo dye uridine derivative 27
Finally, yellow‐coloured adenosine nucleoside 30 was prepared by
2
5
CuAAC of 5’‐azido‐5’‐deoxy‐2’,3’‐O‐isopropylideneadenosine 28,
with the alkyne yellow‐11, under microwave irradiation to 29 in 81%
yields, and subsequent deprotection of the sugar moiety by TFA,
(
Scheme 6).
NH2
N
N
N
N
N
3
O
O
O
28
1
1
CuSO , Na
4
ascorbate
NH2
N
H O/EtOH
2
N
N
N
N
MW (80°C, 3 min)
N
N
N
N
O
Figure 1. Absorbance (solid line) and Fluorescence (dotted line) spectra for s‐
O
tetrazine compounds in CH
uridine 1 (black), 3,6‐dichloro‐s‐tetrazine (green), 3 (red), 4 (orange), 5 (blue),
(dark blue), 7 (purple) and 8 (pink).
3
CN, except compound 4 in EtOH, at 0.1 mM:
O
RO OR
N
6
2
9, C(CH3)
TFA/H O
2
2
These observations were correlated with the results obtained by
Audebert, Clavier and co‐workers with non‐nucleosides‐s‐
3
0, R=H
8
2%
1
3
Scheme 6. Synthesis of yellow adenine derivative
tetrazines. Modifications of the para positions of the tetrazine from
Cl‐Cl (3,6‐dichloro‐s‐tetrazine) to Ur‐Cl (3, 4), or Ur‐OR (7, 8), or Ur‐
SR (5, 6) results in a bathochromic shift for the λ¬2 absorption
wavelength (303 to 393 nm) thus changing the colour from orange to
pink. In addition of colours from s‐tetrazine derivatives, the visible
colours panel available was increased with tags 11, 13, 15 and 16,
from blue to red, passing by yellow‐orange and pink. For example,
the colours given by the dicyanomethylene‐4H‐pyran derivative 18
and naphthooxazine derivative 20, were showed in Figure 3 (a and
c). These both tagged‐uridines possessed also a high intensity
emission fluorescence (Figure 3, b and c). This property could help to
decrease the detection limit of the urinary nucleosides by strip type
sensors coupled to spectrofluorometric reading.
Photophysical Properties
The absorption and fluorescence spectra of all coloured nucleosides
were recorded in acetonitrile unless otherwise stated. The
absorption and fluorescence (when relevant) spectra of the s‐
tetrazine derivatives 3 to 8 are displayed in Figure 1 and the data are
collected in Table 1. All absorption spectra of compounds 3 to 8
3
showed an important signal around 260 nm (λ ). The absence of this
band in the 3,6‐dichloro‐s‐tetrazine (green line) spectrum clearly
indicates that this signal belong to the uridine moiety (black line) of
the compounds. The s‐tetrazine moiety presents two smaller bands
1 2
(λ and λ ), which are approximately at 350 and 515 nm. The
4
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Table 1: Absorption and fluorescence maxima for s‐tetrazine derivatives recorded in CH
3
CN
O
NH
N
N
N
N
N
O
X
O
O
RO OR
Substituent
X
Compd
UV/Vis absorption
Fluorescence
abs
abs
abs
‐1
‐1 [a]
λem [b] (nm)
R
λ
1
(nm)
λ
2
(nm)
λ
3
(nm)
ε (L.mol .cm )
φ
f
1
2
3
4
5
6
7
3,6‐dichloro‐s‐tetrazine
501
509
512
521
522
519
518
303
323
323
393
382
342
342
_
_
551; 567[c]
0.14[c]
0.15
0.17
_
Cl
C(CH
H
3
)
2
3
4 [d]
256
261
256
256
256
260
569
320
486
430
492
219
551
555
_
Cl
S(CH
SPh
2
)
7
CH
3
C(CH
C(CH
C(CH
H
3
3
3
)
)
)
2
2
2
5
6
_
_
OCH
OCH
3
7
562
562
0.10
0.08
3
8
[
a] molar absorption coefficient determined at λ
1
abs. [b] λex = 509 nm for 3 and 4; 519 nm for 7 and 8. [c] From ref.13a [d] UV/Vis absorption and fluorescence
spectra were recorded in EtOH.
The photophysical properties evaluation of the other chromophore
substituents is listed in Table 2. For a clearer reading, the absorbance
intensities of tagged‐nucleosides and the emission intensities of
fluorescent tagged‐nucleosides were plotted according wavelength
in Error! Reference source not found.2, and in Error! Reference
source not found., respectively. The absorbance capacities of the s‐
tetrazines chromophore tags were lower than the other ones.
(a)
(c)
(b)
Figure 2. Pictures of two s‐tetrazine compounds at 0.2 mM in CH
b) 3, (c) 3 under 365 nm UV‐light
3
CN. (a) 7,
(
Table 2: Absorption and fluorescence maxima recorded in CH
3
CN
a]
Product
UV/Vis absorption
Fluorescence[
abs
abs
abs
abs
‐1
‐1 [b]
em
λ
1
(nm)
λ
2
(nm)
λ
3
(nm)
λ
4
(nm)
ε (L.mol .cm )
λ
(nm)
φ
f
1
2
11
465
372
373
352
352
290[c]
262
3.95.104
3.03.104
597
0.13
0.14
UrPrBV 18
466
466
535
535
535
608
611
614
507
507
507
467
466
599
599
607
607
607
_
3
4
5
6
7
8
9
1
1
1
1
1
UrBVOH 19
15
373
266
264
265
566
568
570
288
352
_
265
_
3.31.104
3.04.104
3.35.104
2.46.104
1.19.104
1.28.104
0.82.104
3.54.104
2.85.104
2.09.104
3.80.104
3.12.104
0.14
0.24
0.24
0.24
_
UrPrCP 20
UrCPOH 21
16
UrPrAQ 22 [d]
UrAQOH 23 [d, e]
24
_
_
_
_
250
251
253
_
_
_
_
_
_
_
_
0
1
2
3
4
_
_
_
UrPrRed 26
UrRedOH 27
AdPrBV 29
AdBVOH 30
288[f]
288[f]
373
261
263
352
352
_
_
_
_
_
_
262
262
599
599
0.13
0.14
373
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SpectraManager software was used for changing the format of the
recorded spectra to ASCII. For all spectroscopic measurements a 1
cm four‐sided Hellma quartz cuvette was used.
Absorption spectra were measured on a Shimadzu UV‐1800 UV‐Vis
spectrophotometer (Shimadzu France, Noisiel) with 1 nm resolution
and corrected for the blank with the second cell containing only
®
solution solvent. Hellma absorption cuvettes, pathlength 10 mm,
were used for all absorption spectra.
Here, the quantum yield was measured by relative method, where
the relative quantum yield of synthesized molecules was calculated
according to a standard fluorophore with a well‐known quantum
yield. So, standards fluorophores were fluorescein in ethanol ( =
0.79) and rhodamine B in ethanol (f = 0.5) using dilute sample
solutions using the following equation:
Figure 3. Picture of compounds: (a) 18 and (c) 20 at 0.05 mM in ACN; (b) 18
and (d) 20 at 0.05 mM in ACN under 365 nm UV‐light.
ꢄꢁ
ꢊꢅꢆꢇ
ꢊꢁ
ꢌꢁ
ꢍꢁ
ꢀꢁ
ꢂ ꢃ
ꢈ ∙ ꢉ
ꢋ ∙ ꢃ
ꢈ ∙ ꢃ
ꢈ ∙ ꢀꢅꢆꢇ
ꢄꢅꢆꢇ
ꢌꢅꢆꢇ
ꢍꢅꢆꢇ
Where f, I, A, n and D stand for quantum yield, integrated emission
intensity, absorbance at lexc, refractive index of solvent, and dilution
ratio, respectively. Sample and reference are denoted by ꢎ and ref,
respectively. The exc is in a very close proximity for the sample and
reference solutions to circumvent correction of the difference in
excitation energy at different wavelengths.
Conclusion
We present herein hitherto unknown visibly coloured chromophoric
nucleoside analogues in which the dye has been introduced at the 5’
position of the sugar moiety. Absorption and fluorescence spectra
(when relevant) of all coloured nucleosides are presented to study
their potential as visible‐range probes. The complementarity of these
coloured derivatives of nucleoside, possibly being a urinary modified
nucleoside biomarker of human cancer, and a selective recognition
strip would open the road to user‐friendly bedside sensors.
Spectroscopic grade solvents were obtained from Sigma Aldrich,
Saint Quentin Fallavier, France. Aqueous samples were prepared
with ultra‐high pure water from Elga apparatus. The spectroscopic
samples were prepared from concentrated acetonitrile or Ethanol
stock solutions, hence, all samples contain 200 µM.
5
(
’‐O‐(6‐Chloro‐1,2,4,5‐tetrazin‐3‐yl)‐2’,3’‐O‐isopropylideneuridine
3). To a mixture of 2’,3’‐O‐isopropylideneuridine (100 mg, 0.35
mmol) and 3,6‐dichloro‐s‐tetrazine (58 mg, 0.39 mmol) in CH Cl (10
Experimental
2
2
Materials and methods
mL) was introduced dropwise over 30 min 2,4,6‐collidine (50 µL, 0.35
mmol). After 16 h stirring at RT, the mixture was washed with water,
brine, dried over MgSO , filtrated and concentrated under reduced
4
pressure. The crude product was purified by flash column
chromatography (hexanes/AcOEt 2:8) to obtain 3 (119 mg, 90%) as
an orange‐pink oil. H NMR (400 MHz, CDCl
(d, J = 8.0 Hz, 1H, H‐6), 5.72 (d, J = 8.0 Hz, 1H, H‐5), 5.62 (s, 1H, H‐1’),
5.12 (d, J = 6.2 Hz, 1H, H‐2’), 5.04 (dd, J = 6.2, 4.2 Hz, 1H, H‐3’), 4.92
Commercially available chemicals were of reagent grade and used as
received. The reactions were monitored by thin layer
chromatography (TLC) analysis using silica gel plates (Kieselgel
6
0F254, E. Merck). Compounds were visualized by UV irradiation.
1
) δ 9.81 (s, 1H, NH), 7.28
Column chromatography was performed on Silica Gel 60 M (40‐63
3
1
13
13
µm, E. Merck). H and C NMR spectra were recorded at 250 nm ( C,
1
3
6
2.9 MHz) or at 400 nm ( C, 100.62 MHz). Chemical shifts are given
(
dd, J = 11.5, 6.5 Hz, 1H, H‐5’a), 4.87 (dd, J = 11.5, 4.0 Hz, 1H, H‐5’b),
in parts per million using tetramethylsilane (TMS) as internal
standard. Coupling constants (J) are reported in Hertz (Hz) and
multiplicities are reported as s (singlet), d (doublet), t (triplet), q
1
3
4
.62 – 4.58 (m, 1H, H‐4’), 1.57 (s, 3H, CH
) δ 166.7 (C‐Cl), 164.7 (C‐O), 163.6 (C‐4), 150.4 (C‐2),
43.2 (C‐6), 114.8 (C(CH ), 103.0 (C‐5), 96.2 (C‐1’), 85.7 (C‐4’), 84.5
3 3
), 1.36 (s, 3H, CH ). C NMR
(
101 MHz, CDCl
3
1
3 2
)
(
quartet), bs (broad signal) and m (multiplet). High Resolution Mass
spectra were performed on a Bruker maxis mass spectrometer by the
Fédération de Recherche” ICOA/CBM (FR2708) platform. Melting
(C‐2’), 81.4 (C‐3’), 70.1 (C‐5’), 27.2 (C(CH
3
)
2
), 25.3 (C(CH
3
)
2
). HRMS‐ESI
+
(
m/z) [M+H] calcd for C14
H
16ClN
6
O
6
399.0814, found 399.0812.
“
point was performed only after recrystallization in adequate solvent.
All reactions under microwave irradiation were performed using the
Microwave Biotage Initiator in 2–5 mL sealed tubes
5
(
0
’‐O‐(6‐Chloro‐1,2,4,5‐tetrazin‐3‐yl)‐uridine (4). Anhydrous ZnBr
250 mg, 1 mmol) was added to a solution of compound 3 (100 mg,
.25 mmol) in CH Cl (4 mL). Another batch of anhydrous ZnBr (250
2
2
2
2
mg) was introduced again after 5 h stirring at room temperature and
then the mixture was stirred overnight. The reaction was quenched
Jasco FP‐8200 Fluorescence Spectrometer (Jasco Corporation, Lisses,
France) equipped with a 150ꢀW xenon lamp was utilized for all
fluorescence measurements. The bandwidths for excitation and
emission spectra were adjusted at 5.0ꢀnm. Scan speed, data interval
and response were 200 nm/min, 0.5 nm and 0.5 sec, respectively.
with a saturated solution of EDTA.Na
was removed by extraction with CH
2
. The residual starting material
Cl and then the unprotected
2
2
uridine derivative was extracted from the aqueous layer with AcOEt
6
| Org. Biomol. Chem., 2018, 00, 1‐3
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DOI: 10.1039/C8OB01606B
Journal Name
ARTICLE
(
5 x 10 mL). The organic layer was dried over MgSO
4
, filtrated and RT, the mixture was diluted with AcOEt, washed with water (3 x 5
, filtrated and concentrated under
concentrated under reduced pressure. After a quick purification by mL), brine, dried over MgSO
4
flash column chromatography (AcOEt), s‐tetrazine 4 (58 mg, 64%) reduced pressure. The crude product was purified by flash column
1
was obtained as an orange‐pink oil. H NMR (400 MHz, DMSO‐d
6
) δ chromatography (hexanes/AcOEt 2:8) to obtain compound 7 (93.2
1
1
5
3
1.37 (d, J = 2.2 Hz, 1H, NH), 7.72 (d, J = 8.1 Hz, 1H, H‐6), 5.83 (d, J = mg, 36%) as an orange‐pink oil. H NMR (400 MHz, CDCl
.1 Hz, 1H, H‐1’), 5.62 (dd, J = 8.1, 2.2 Hz, 1H, H‐5), 4.82 (dd, J = 11.7, 1H, NH), 7.36 (d, J = 8.0 Hz, 1H, H‐6), 5.77 (d, J = 2.1 Hz, 1H, H‐1’),
.3 Hz, 1H, H‐5’a), 4.75 (dd, J = 11.7, 6.1 Hz, 1H, H‐5’b), 4.30 – 4.25 5.62 (dd, J = 8.0, 2.1 Hz, 1H, H‐5), 5.08 (dd, J = 6.5, 2.2 Hz, 1H, H‐2’),
3
) δ 8.71 (s,
(
m, 1H, H‐4’), 4.21 (dd, J = 5.2 Hz, 1H, H‐2’), 4.15 (dd, J = 5.2 Hz, 1H, 5.03 (dd, J = 6.2, 3.7 Hz, 1H, H‐3’), 4.84 (dd, J = 11.4, 5.5 Hz, 1H, H‐5’),
1
3
6
H‐3’). C NMR (101 MHz, DMSO‐d ) δ 166.3 (C‐Cl), 163.2 (C‐O), 163.0 4.79 (dd, J = 11.4, 3.5 Hz, 1H, H‐5’), 4.64 – 4.60 (m, 1H, H‐4’), 4.24 (s,
(
7
C
13
C‐4), 150.7 (C‐2), 141.0 (C‐6), 102.1 (C‐5), 88.9 (C‐1’), 80.9 (C‐4’), 3H, OCH
3
), 1.60 (s, 3H, CH
) δ 166.7 (C‐OCH ), 166.1 (C‐O), 162.9 (C‐4), 150.1 (C‐2), 142.3
(C‐6), 114.9 (C(CH ), 103.1 (C‐5), 94.8 (C‐1’), 85.2 (C‐4’), 84.6 (C‐2’),
1.1 (C‐3’), 69.2 (C‐5’), 57.0 (OCH ), 27.3 (C(CH ), 25.4 (C(CH ).
HRMS‐ESI (m/z) [M+H] calcd for C15 395.1310, found
95.1309.
3 3
), 1.38 (s, 3H, CH ). C NMR (101 MHz,
+
2.6 (C‐2’), 69.9 (C‐3’), 69.9 (C‐5’). HRMS‐ESI (m/z) [M+H] calcd for CDCl
359.0501, found 359.0501.
3
3
11
H
12ClN
6
O
6
3 2
)
8
3
3
)
2
3 2
)
5
’‐O‐(6‐Octanthio‐1,2,4,5‐tetrazin‐3‐yl)‐2’,3’‐O‐
+
19 6 7
H N O
isopropylideneuridine (5). A solution of 1‐octanethiol (0.04 mL, 0.25
mmol) and triethylamine (0.03 mL, 0.25 mmol) in acetonitrile (20 mL)
3
was added dropwise through a dropping funnel to a solution of s‐ 5’‐O‐(6‐Methoxy‐1,2,4,5‐tetrazin‐3‐yl)‐uridine
(8).
General
tetrazine 3 (50 mg, 0.13 mmol) in acetonitrile (20 mL). The mixture procedure used for tetrazine derivative 4 was followed with
was stirred at room temperature for 1 hour and the solvent was compound 7 (93.2 mg, 0.24 mmol) to give deprotected compound 8
1
evaporated.
CH Cl
NMR (400 MHz, CDCl
), 5.74 (d, J = 1.9 Hz, 1H, H‐1’), 5.73 (d, J = 8.1, 1.9 Hz, 1H, H‐5), 5.08 (m, 4H, H‐2’, H‐3’, H‐4’, OH), 4.21 (s, 3H, OCH
dd, J = 6.4, 1.9 Hz, 1H, H‐2’), 5.03 (dd, J = 6.4, 3.9 Hz, 1H, H‐3’), 4.88 acetone‐d ) δ 167.5 (C‐OCH ), 166.9 (C‐O), 164.5 (C‐4), 151.3 (C‐2),
4.78 (m, 2H, H‐5’), 4.64 – 4.58 (m, 1H, H‐4’), 3.26 (t, J = 7.4 Hz, 2H, 142.0 (C‐6), 102.6 (C‐5), 90.7 (C‐1’), 82.6 (C‐4’), 74.6 (C‐2’), 71.2 (C‐
Purification by flash column chromatography (80.3 mg, 97%) as a pink oil. H NMR (250 MHz, acetone‐d
6
) δ 10.30
1
(
2
2
/MeOH 96:4) gives compound 5 (60 mg, 94%) as a red oil. H (s, 1H, NH), 7.89 (d, J = 8.1 Hz, 1H, H‐6), 5.99 (d, J = 3.4 Hz, 1H, H‐1’),
3
) δ 9.30 (s, 1H, NH), 7.34 (d, J = 8.1 Hz, 1H, H‐ 5.75 (d, J = 8.1 Hz, 1H, H‐5), 4.92 – 4.80 (m, 3H, H‐5’, OH), 4.54 – 4.38
1
3
6
(
–
3
). C NMR (63 MHz,
6
3
+
CH
2
‐S), 1.77 (quint, J = 7.0 Hz, 2H), 1.58 (s, 3H, CH
), 1.37 (s, 3H, CH ), 1.35 – 1.21 (m, 8H, 4 x CH
t, J = 7.0 Hz, 3H, CH ) δ 172.6 (C‐S), 165.9
). 13C NMR (101 MHz, CDCl
C‐O), 163.3 (C‐4), 150.2 (C‐2), 142.4 (C‐6), 114.9 (C(CH ), 103.1 (C‐
), 95.0 (C‐1’), 85.3 (C‐4’), 84.6 (C‐2’), 81.2 (C‐3’), 69.0 (C‐5’), 31.9
3
), 1.46 (quint, J = 3’), 69.5 (C‐5’), 56.9 (CH
3
). HRMS‐ESI (m/z) [M+H] calcd for
), 0.87 355.0997, found 355.0999.
7.0 Hz, 2H, CH
2
3
2
12 15 6 7
C H N O
(
(
5
3
3
4
‐[N‐Methyl‐N‐(2‐O‐propargylethyl)]aminobenzaldehyde (10). To a
stirred suspension of NaH (60%, 3.91 g, 0.098 mol) in distilled THF (50
mL) cooled in an ice bath and under argon, were dripped over 20 min
3 2
)
(
(
[
CH
CH
2
‐S), 31.1 (CH
), 25.4 (C(CH
+
2
), 29.3 (CH
2
), 29.2 (CH
), 22.7 (C(CH ), 14.2 (CH
S 509.2177, found 509.2175.
2
), 28.9 (CH
2 2
), 28.8 (CH ), 27.2
a
solution of commercially available 4‐[N‐(2‐hydroxyethyl)‐N‐
2
3
)
2
3
)
2
3
). HRMS‐ESI (m/z)
methyl]aminobenzaldehyde (10.29 g, 0.057 mol) in distilled THF (90
mL) followed by a solution of propargyl bromide (80% in toluene, 9.2
mL, 0.083 mol) over 5 minutes. After 15 h, the excess of sodium
M+H] calcd for C22
H
33
N
6
O
6
5
’‐O‐(6‐Phenylthio‐1,2,4,5‐tetrazin‐3‐yl)‐2’,3’‐O‐
isopropylideneuridine (6). A solution of thiophenol (0.03 mL, 0.25 hydride was destroyed by addition of crushed ice in the mild and
mmol) and triethylamine (0.03 mL, 0.25 mmol) in acetonitrile (20 mL) organic solvent was evaporated under reduced pressure. The residue
was added dropwise through a dropping funnel to a solution of s‐ was then partitioned in a mixture CH
tetrazine 3 (50 mg, 0.13 mmol) in acetonitrile (20 mL). The mixture aqueous layer was extracted with CH
was stirred at room temperature for 2 h and the solvent was were combined, washed with brine, dried over MgSO
evaporated under reduced pressure. Purification by flash column under reduced pressure and purified by column chromatography
2
Cl
2
/H
(2 x 50 mL). Organic layers
, evaporated
2
O (150/150 mL) and the
2
Cl
2
4
chromatography (CH
2
Cl
2
/MeOH 96:4) gives compound 6 (24 mg, (hexanes/EtOAc from 9:1 to 6:4) to give 62% of the desired
1
1
4
–
1
5
3
1%) as a red oil. H NMR (400 MHz, CDCl
3
) δ 9.03 (s, 1H, NH), 7.66 compound 10 (7.72 g, 0.036 mol) as a brown oil. H NMR (400 MHz,
7.64 (m, 2H, CH(Ar)), 7.49 – 7.45 (m, 3H, CH(Ar)), 7.29 (d, J = 8.0 Hz, CDCl
H, H‐6), 5.72(d, J = 1.9 Hz, 1H, H‐1’), 5.70 (d, J = 8.0, 1.9 Hz, 1H, H‐ = 9.2 Hz, 2H, 2 x CHAr), 4.15 (d, J = 2.3 Hz, 2H, O‐CH
), 5.05 (dd, J = 6.4, 1.9 Hz, 1H, H‐2’), 5.00 (dd, J = 6.4, 4.0 Hz, 1H, H‐ 5.3 Hz, 2H, O‐CH ), 3.66 (t, J = 5.3 Hz, 2H, N‐CH ), 3.10 (s, 3H, N‐CH
’), 4.79 (d, J = 4.6 Hz, 2H, H‐5’), 4.58 (dd, J = 4.6 Hz, 1H, H‐4’), 1.58 2.43 (t, J = 2.3 Hz, 1H, H‐C≡). 13C NMR (100 MHz, CDCl
) δ 190.4 (CHO),
) δ 173.1 (C‐ 153.6, 132.2 (CHAr), 125.5, 111.1 (CHAr), 79.4 (Cq C≡C), 74.9 (CHC≡C),
3
) δ 9.73 (s, 1H, HCHO), 7.73 (d, J = 9.2 Hz, 2H, 2 x CHAr), 6.73 (d, J
‐C≡), 3.73 (t, J =
),
2
2
2
3
3
13
(s, 3H, CH
3
), 1.36 (s, 3H, CH
3
). C NMR (101 MHz, CDCl
3
S), 166.0 (C‐O), 163.1 (C‐4), 150.1 (C‐2), 142.4 (C‐6), 135.6 (CHAr), 67.3 (O‐CH
2
), 58.6 (O‐CH
2
), 52.0 (N‐CH
2 3
), 39.4 (N‐CH ). HRMS‐ESI m/z
+
1
9
30.5 (CHAr), 130.0 (CHAr), 126.5 (CAr‐S), 114.9 (C(CH
5.0 (C‐1’), 85.2 (C‐4’), 84.5 (C‐2’), 81.2 (C‐3’), 69.1 (C‐5’), 27.3
3
)
2
), 103.0 (C‐5), [M+H] calcd for C13
H
16NO
2
218.1177, obsd 218.1165.
+
(E)‐2‐(2‐[4‐(N‐Methyl‐N‐(2‐O‐propargylethyl)amino)styryl]‐6‐(tert‐
butyl)‐4H‐pyran‐4‐ylidene)malononitrile (11). To a stirred solution
of aldehyde 10 (5.95 g, 0.027 mol) and 4‐(dicyanomethylene)‐2‐(tert‐
(
C(CH
3
)
2
), 25.4 (C(CH
3
)
2
). HRMS‐ESI (m/z) [M+H] calcd for
20 21 6 6
C H N O S 473.1238, found 473.1236.
5
’‐O‐(6‐Methoxy‐1,2,4,5‐tetrazin‐3‐yl)‐2’,3’‐O‐
mixture of 2’,3’‐O‐ mL) under argon, was added piperidine (2.9 mL, 0.029 mol). The
isopropylideneuridine (189 mg, 0.67 mmol) and 3‐chloro‐6‐methoxy‐ mixture was heated at 85 °C overnight. After completion of the
s‐tetrazine (195 mg, 1.33 mmol) in anhydrous CH Cl (3 mL) was reaction, the mild was cooled to room temperature and the
introduced 2,4,6‐collidine (0.36 mL, 2.66 mmol). After 72 h stirring at precipitate was filtered and washed with cold CH CN (10 mL)
3
butyl)‐6‐methyl‐4H‐pyran (5.40 g, 0.025 mol) in distilled CH CN (60
isopropylideneuridine (7). To
a
2
2
3
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ARTICLE
Journal Name
followed by cyclohexane (200 mL). The filtrate was evaporated under 2H, CH‐(C‐C=O)), 7.77 – 7.69 (m, 2H, CH‐(CH‐C‐C=O)), 7.18 (d, J = 9.5
reduced pressure and the residue was then triturated (sonication) Hz, 1H, CH‐(C(NH ))), 7.15 – 7.05 (m, 2H, NH ), 7.03 (d, J = 9.5 Hz, 1H,
with a minimum of cold CH CN then filtered. Combination of the two CH‐(C(NH))), 3.61 (q, J = 7.0 Hz, 2H, CH
2
2
3
2
N), 2.63 (td, J = 7.0, 2.6 Hz,
1
3
solids afforded 84% of the title compound 11 (10.6 g, 0.026 mol) as 2H, CH
an orange/red solid. Mp 156°C. H NMR (400 MHz, CDCl
2
), 2.12 (t, J = 2.6 Hz, 1H, H‐C≡). C NMR (101 MHz, CDCl
3
) δ
1
3
) δ 7.43 (d, 184.0 (C=O‐(C‐C(NH
2
))), 183.2 (C=O‐(C‐C(NH))), 146.5, 144.5, 134.8,
J = 9.2 Hz, 2H, 2 x CHAr), 7.34 (d, J = 16.0 Hz, 1H, CH=), 6.74 (d, J = 8.7 134.2, 132.7 (CH‐(CH‐C‐C=O)), 132.4 (CH‐(CH‐C‐C=O)), 128.7 (CH‐
Hz, 2H, 2 x CHAr), 6.59 (d, J = 2.3 Hz, 1H, CHpyran), 6.51 (d, J = 2.3 Hz, (C(NH ))), 126.4 (CH‐(C‐C=O)), 122.4 (CH‐(C(NH))), 111.1 (C‐N), 110.2
2
1
H, CHpyran), 6.50 (d, J = 16.9 Hz, 1H, CH=), 4.16 (d, J = 2.7 Hz, 2H, O‐ (C‐N), 81.2 (C≡), 70.7(C≡), 41.9 (CH
2
N), 20.0 (CH
2
). HRMS‐ESI (m/z)
+
CH
2
), 3.74 (t, J = 5.7 Hz, 2H, O‐CH
), 2.43 (t, J = 2.5 Hz, 1H, H‐C≡), 1.37 (s, 9H, 3 x CH
C NMR (100 MHz, CDCl
29.9 (CHAr), 122.8, 115.9, 115.8, 113.2 (CH=), 112.3 (CHAr), 105.7
), 58.7,
), 36.8 (Cq, tBu), 28.3 (CH3 tBu). HRMS‐
Na 436.2001, obsd 436.1992.
2
), 3.64 (t, J = 5.7 Hz, 2H, N‐CH
2
), [M+H] calcd for C18
H
15
N
2
O
2
291.1128, found 291.1131.
3
.09 (s, 3H, N‐CH
3
3
).
1
3
2‐[2‐tert‐Butyl‐6‐[(E)‐2‐[4‐[2‐[[1‐[[4‐(2,4‐dioxopyrimidin‐1‐yl)‐2,2‐
dimethyl‐3a,4,6,6a‐tetrahydrofuro[3,4‐d][1,3]dioxol‐6‐
yl]methyl]triazol‐4‐yl]methoxy]ethyl‐methyl‐
amino]phenyl]vinyl]pyran‐4‐ylidene]propanedinitrile (18). 5’‐
azido‐5’‐deoxy‐2’,3’‐O‐isopropylideneuridine (150 mg, 0.49 mmol),
Tag 11 (201 mg, 0.49 mmol), sodium ascorbate (73 mg, 0.29 mmol)
3
) δ 172.0, 160.3, 156.9, 150.7, 138.4 (CH=),
1
(
CHpyran), 102.5 (CHpyran), 79.5, 77.4, 74.9 (HC≡), 67.4 (O‐CH
2
5
ESI m/z [M+Na] calcd for C26
7.9, 52.3 (N‐CH
2
), 39.4 (N‐CH
3
+
H
27
N
3
O
2
5
‐(Diethylamino)‐2‐nitrosophenol chlorhydrate (13). 1M HCl and copper sulfate pentahydrate (30 mg, 0.15 mmol) were
solution in ethanol was prepared by adding dropwise AcCl (5.7 mL, suspended in a 1:1 mixture of water and ethanol (2.5 mL each) in a
.10 mol) in 100 mL of EtOH at 0 °C and the solution stirred for 15 closed microwave reaction vessel. After microwave‐irradiation for 3
minutes. Diethylaminophenol 12 (5.00 g, 30.3 mmol) was added and min at 80 °C, the mixture was concentrated under reduced pressure.
then amyl nitrite (4.0 mL, 30 mmol, 1 eq) was added dropwise at 0 °C The crude residue was extracted with CH Cl , washed with brine,
and the solution stirred for 2.5 h. Et O was added (ca. 500 mL) and dried with MgSO , filtrated and concentrated under reduced
the precipitate was filtered, washed with Et O and vacuum pressure. Purification by flash column chromatography
0
2
2
2
4
2
dried. Nitrosophenol was obtained as a brown powder (5.32 g, 23.1 (hexanes/AcOEt 3:7 followed by CH
2
Cl
2
/MeOH 96:4) afforded
1
1
mmol, 76%). H NMR (400 MHz, CD
3
OD) ꢏ 7.70 (d, J = 10.4 Hz, 1H, H‐ compound 18 (154 mg, 99%) as a red oil. H NMR (400 MHz, CDCl
3
) δ
3
3
), 7.20 (dd, J = 10.4, 2.4 Hz, 1H, H‐4), 6.40 (d, J = 2.4 Hz, 1H, H‐6), 9.36 (s, 1H, NH), 7.49 (s, 1H, H‐triazole), 7.41 (d, J = 8.9 Hz, 2H), 7.32
.95 (q, J = 7.2 Hz, 2H, N‐CH ), 3.87 (q, J = 7.2 Hz, 2H, N‐CH ), 1.39 (t, (d, J = 15.9 Hz, 1H, H‐alkene), 7.03 (d, J = 8.0 Hz, 1H, H‐6), 6.68 (d, J =
). C NMR (100 MHz, CD OD): ꢏ 164.0, 145.8, 8.9 Hz, 2H), 6.59 (d, J = 2.1 Hz, 1H, H‐pyran), 6.51 (d, J = 2.1 Hz, 1H,
), 15.5 (CH ), 14.0 (CH ). H‐pyran), 6.49 (d, J = 15.9 Hz, 1H, H‐alkene), 5.72 (dd, J = 8.0, 1.7 Hz,
2
2
1
3
J = 7.2 Hz, 6H, 2 x CH
1
3
3
24.8 (C‐3), 121.0 (C‐4), 98.9 (C‐6), 49.8 (CH
2
3
3
1
2
H, H‐5), 5.51 (d, J = 1.7 Hz, 1H, H‐1’), 5.06 (dd, J = 6.5, 1.7 Hz, 1H, H‐
’), 4.95 (dd, J = 6.5, 4.3 Hz, 1H, H‐3’), 4.71 (dd, J = 14.0, 4.1 Hz, 1H,
9
‐(Diethylamino)‐2‐(prop‐2‐yn‐1‐yloxy)‐5H‐benzo[a]phenoxazin‐5‐
one (15). To a solution of phenol 13 (1.52 g, 6.59 mmol) in DMF (50
mL) was added 1,6‐dihydroxynaphtalene (1.06 g, 6.62 mmol). The
mixture was refluxed for 4 h. Compound 14 can be isolated by
preparative chromatography. To the resulting solution was added
H‐5’), 4.65 (dd, J = 14.0, 7.1 Hz, 1H, H‐5’), 4.63 (s, 2H, C‐CH
2
‐O), 4.49
‐O), 3.60
‐N ), 1.53 (s, 3H, C(CH ),
). 13C NMR (101 MHz, CDCl
(
ddd, J = 7.1, 4.3, 4.1 Hz, 1H, H‐4’), 3.73 (t, J = 5.9 Hz, 2H, CH
t, J = 5.9 Hz, 2H, CH ‐N), 3.04 (s, 3H, CH
.37 (s, 9H, CH (tBu)), 1.34 (s, 3H, C(CH
δ 172.0 (C‐pyran), 163.2 (C‐4), 160.4 (C‐pyran), 156.9, 150.9, 150.1
C‐2), 145.0 (C‐triazole), 143.4 (C‐6), 138.5 (CH‐alkene), 129.9 (CHAr),
23.9 (CH‐triazole), 122.5, 115.9, 115.0 (s, C(CH ), 113.1 (CH‐
alkene), 112.1 (2C, CHAr), 105.6 (CH‐pyran), 103.0 (C‐5), 102.5 (CH‐
pyran), 96.4 (C‐1’), 86.4 (C‐4’), 84.4 (C‐2’), 81.9 (C‐3’), 67.9 (CH ‐O),
‐N), 36.7
2
(
1
2
3
3 2
)
3
)
3
3
)
2
2 3
K CO (3.51 g, 25.4 mmol, 4 eq), then 3‐bromoprop‐1‐yne (80%, 2.8
mL, 25 mmol, 4 eq) was added dropwise and stirred 1 h at 115 °C.
After concentration under reduced pressure, the crude was purified
by column chromatography (hexanes/EtOAc 2:1) to obtain alkyne 15
(
1
3 2
)
(
708 mg, 30% for two steps) as a dark green powder. Mp 184°C.
2
1
H NMR (400 MHz, CDCl
.6 Hz, 1H, H‐1), 7.59 (d, J = 9.1 Hz, 1H, H‐11), 7.24 (dd, J = 8.7, 2.7
Hz, 1H, H‐3), 6.65 (dd, J = 9.1, 2.7 Hz, 1H, H‐10), 6.44 (d, J = 2.7 Hz,
3
) ꢏ 8.24 (d, J = 8.7 Hz, 1H, H‐4), 8.13 (d, J =
6
4.7 (C‐CH
C(tBu)), 28.3 (CH
43 8 7
calcd for C38H N O 723.3249, found 723.3246.
2
‐O), 57.8 (CN), 52.1 (CH
). HRMS‐ESI (m/z) [M+H]+
), 27.21 (CH ), 25.38 (CH
3
2 3
‐N), 51.9 (C‐5’), 39.2 (CH
2
(
3
3
1
7
6
1
1
H, H‐8), 6.30 (s, 1H, H‐6), 4.89 (d, J = 2.4 Hz, 2H, CH
.1 Hz, 4H, 2 x N‐CH
), 2.59 (t, J = 2.4 Hz, 1H, H‐C≡), 1.26 (t, J = 7.1 Hz, 2‐[2‐tert‐Butyl‐6‐[(E)‐2‐[4‐[2‐[[1‐[[5‐(2,4‐dioxopyrimidin‐1‐yl)‐3,4‐
H, 2 x CH ) ꢏ 183.3 (C=O), 160.2, 152.3, dihydroxy‐tetrahydrofuran‐2‐yl]methyl]triazol‐4‐
). 13C NMR (100 MHz, CDCl
2
‐C≡), 3.46 (q, J =
2
3
3
51.0, 147.0, 139.8, 134.1, 131.3 (C‐11), 128.0 (C‐4), 126.4, 124.9, yl]methoxy]ethyl‐methyl‐amino]phenyl]vinyl]pyran‐4‐
18.5 (C‐3), 109.7 (C‐10), 107.2 (C‐1), 105.4 (C‐6), 96.3 (C‐8), 78.1 (‐ ylidene]propanedinitrile (19). To a solution of compound 18 (154
C≡), 76.1 (H‐C≡), 56.2 (CH
2
‐C≡), 45.2 (CH
2 3
), 12.7 (CH ). HRMS‐ESI m/z mg, 0.21 mmol) in water (0.12 mL) was added TFA (1.08 mL). The
+
[M+H] calcd for C23
H
21
N
2
O
3
373.1547, found 373.1545.
mixture was stirred at room temperature until completion (10 min).
After evaporation under reduced pressure, the crude product was
1‐Amino‐4‐(but‐3‐ynylamino)anthracene‐9,10‐dione (Tag 16). To a
extracted with AcOEt, washed with NaHCO
MgSO , filtrated and concentrated. The product was purified by flash
column chromatography (CH Cl /MeOH 96:4 to 90:10) followed by
CH Cl /MeOH/ammoniac (90:9:1) to give compound 19 (146 mg,
9%) as a red solid. H NMR (400 MHz, DMSO‐d
.00 (s, 1H, H‐triazole), 7.56 – 7.51 (m, 3H, H‐6, H‐Ar), 7.45 (d, J = 15.9
3
, brine, dried with
solution of 1,4‐diaminoanthraquinone (500 mg, 2.10 mmol) and
CO (1.2 g, 8.40 mmol) in ACN (8 mL) was added 4‐bromobutyne
0.85 mL, 9.06 mmol) in ACN (2 mL). The solution was stirred at 65 °C
for 6 days. After concentration under reduced pressure, the residue
was purified by flash column chromatography (CH Cl then
CH Cl /MeOH 99:1) to give compound 16 as a blue solid (62 mg,
4
K
2
3
2
2
(
2
2
1
9
8
6
) δ 11.37 (s, 1H, NH),
2
2
2
2
Hz, 1H, H‐alkene), 7.03 (d, J = 15.9 Hz, 1H, H‐alkene), 6.76 – 6.73 (m,
1
1
3
0%). H NMR (400 MHz, CDCl ) δ 10.74 (s, 1H, NH), 8.43 – 8.31 (m,
8
| Org. Biomol. Chem., 2018, 00, 1‐3
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DOI: 10.1039/C8OB01606B
Journal Name
ARTICLE
3
H, H‐Ar, H‐pyran), 6.41 (d, J = 2.0 Hz, 1H, H‐pyran), 5.74 (d, J = 5.6 4.10 (ddd, J = 5.5 Hz, 1H, H‐2’), 4.01 (ddd, J = 5.5 Hz, 1H, H‐3’), 3.49
13
Hz, 1H, OH), 5.63 (dd, J = 8.1, 1.4 Hz, 1H, H‐5), 5.51 (d, J = 5.6 Hz, 1H, (q, J = 6.8 Hz, 4H, CH
2
‐N), 1.16 (t, J = 7.0 Hz, 6H, CH
3
). C NMR (101
)),
‐O), 4.16 – 4.10 151.73 (O‐C‐(CHC=O)), 150.8 (C‐2), 150.6 (C‐Ar), 146.4 (C‐Ar), 142.33
m, 1H, H‐4’), 4.08 (dd, J = 10.5, 5.6 Hz, 1H, H‐2’), 3.97 (dd, J = 10.5, (C‐triazole), 141.1 (C‐6), 138.1 (C=N), 133.5 (CAr), 130.9 (CHAr), 127.2
H‐1’), 5.39 (d, J = 5.3 Hz, 1H, OH), 4.71 (dd, J = 14.4, 4.4 Hz, 1H, H‐5’), MHz, DMSO‐d
.62 (dd, J = 14.4, 7.7 Hz, 1H, H‐5’), 4.53 (s, 2H, C‐CH
(
6 2
) δ 181.3 (C=O), 162.9 (C‐4), 160.6 (C‐Ar‐(OCH
4
2
5
.1 Hz, 1H, H‐3’), 3.65 – 3.57 (m, 4H, CH
2
‐N, CH
2
‐O), 2.98 (s, 3H, CH
3
‐
(CHAr), 125.5 (CH‐triazole), 125.2 (CAr), 123.9 (CAr), 117.9 (CHAr), 110.1
13
N), 1.35 (s, 9H, CH
3
(tBu)). C NMR (101 MHz, DMSO‐d
6
) δ 172.3 (C‐ (CHAr), 106.8 (CHAr), 104.1 (CH‐C=O), 102.1 (C‐5), 95.9 (CHAr), 88.8 (C‐
pyran), 162.9 (C‐4), 160.9 (C‐pyran), 156.5, 150.6 (C‐2, (C‐Ar)‐N), 1’), 81.7 (C‐4’), 72.1 (C‐2’), 70.6 (C‐3’), 61.5 (CH
2
‐O), 51.3 (C‐5’), 44.4
+
1
1
1
4
5
43.9 (s, C‐triazole), 141.1 (s, C‐6), 138.6 (s, CH‐alkene), 130.1 (CAr), (CH
2
‐N), 125 (2
x
CH
3
). HRMS‐ESI (m/z) [M+H] calcd for
24.6 (CH‐triazole), 122.1, 115.8, 112.9 (CH‐alkene), 111.7 (CAr),
04.9 (CH‐pyran), 102.1 (C‐1’), 101.5 (CH‐pyran), 88.7 (C‐5), 81.7 (C‐
C H N O 642.2307, found 642.2303.
32 32 7 8
1‐[6‐[[4‐[2‐[(4‐Amino‐9,10‐dioxo‐1‐anthryl)amino]ethyl]triazol‐1‐
’), 72.0 (C‐2’), 70.5 (C‐3’), 67.1 (CH
2
‐O), 63.5 (C‐CH
‐N), 36.3 (C(tBu)), 27.5 (CH
683.2936, found
2
‐O), 54.9 (CN),
yl]methyl]‐2,2‐dimethyl‐3a,4,6,6a‐tetrahydrofuro[3,4‐
d][1,3]dioxol‐4‐yl]pyrimidine‐2,4‐dione (22). Following the general
procedure
1.2 (C‐5’), 51.0 (CH
2
‐N), 39.0 (CH
+
3
3
).
HRMS‐ESI (m/z) [M+H] calcd for C34
6
H
39
N
8
O
7
used
for
18,
5’‐azido‐5’‐deoxy‐2’,3’‐O‐
83.2932.
isopropylideneuridine (97 mg, 0.31 mmol), Tag 16 (91 mg, 0.31
1‐[6‐[[4‐[[9‐(Diethylamino)‐5‐oxo‐benzo[a]phenoxazin‐2‐
mmol), sodium ascorbate (47 mg, 0.19 mmol) and copper sulfate
yl]oxymethyl]triazol‐1‐yl]methyl]‐2,2‐dimethyl‐3a,4,6,6a‐
2
pentahydrate (19 mg, 0.09 mmol) were reacted in H O/EtOH (1:1) (4
tetrahydrofuro[3,4‐d][1,3]dioxol‐4‐yl]pyrimidine‐2,4‐dione (20). mL) to yield compound 22 as a blue solid (125 mg, 67%) after
Following the procedure used for 18 from 5’‐azido‐5’‐deoxy‐2’,3’‐O‐ purification by flash column chromatography (hexanes/AcOEt 1:9
1
isopropylideneuridine (83 mg, 0.27 mmol), Tag 15 (100 mg, 0.27 then CH
2
Cl
2
/MeOH 96:4). H NMR (400 MHz, DMSO‐d
6
) δ 11.46 (d, J
mmol), sodium ascorbate (40 mg, 0.16 mmol) and copper sulfate = 1.9 Hz, 1H, NHuracil), 10.83 (t, J = 5.7 Hz, 1H, NHAQ), 8.53 – 8.25 (m,
pentahydrate (15 mg, 0.08 mmol) in H O/EtOH (1:1) (4 mL), 2H, NH ), 8.25 – 8.18 (m, 2H, CH‐(C‐C=O)), 8.00 (s, 1H, H‐triazole),
compound 20 was obtained as a purple solid (143 mg, 78%) after 7.81 – 7.74 (m, 2H, CH‐(CH‐C‐C=O)), 7.64 (d, J = 8.0 Hz, 1H, H‐6), 7.42
2
2
purification by flash column chromatography (hexanes/AcOEt 3:7 (d, J = 9.6 Hz, 1H, CH‐(C(NH))), 7.30 (d, J = 9.6 Hz, 1H, CH‐(C(NH
2
))),
1
then CH
2
Cl
2
/MeOH 95:5). H NMR (400 MHz, DMSO‐d
6
) δ 11.49 (s, 5.77 (d, J = 1.8 Hz, 1H, H‐1’), 5.62 (dd, J = 8.0, 1.9 Hz, 1H, H‐5), 5.10
1
=
H, NH), 8.33 (s, 1H, H‐triazole), 8.07 – 8.00 (m, 2H, H‐Ar), 7.69 (d, J (dd, J = 6.5, 1.8 Hz, 1H, H‐2’), 4.87 (dd, J = 6.5, 4.5 Hz, 1H, H‐3’), 4.73
7.6 Hz, 1H, H‐6), 7.56 (d, J = 8.8 Hz, 1H, H‐Ar), 7.32 (d, J = 8.4 Hz, 1H, (dd, J = 14.2, 4.5 Hz, 1H, H‐5’), 4.62 (dd, J = 14.2, 7.7 Hz, 1H, H‐5’),
H‐Ar), 6.78 (d, J = 8.5 Hz, 1H, H‐Ar), 6.59 (s, 1H, H‐Ar), 6.15 (s, 1H, CH‐ 4.36 (dt, J = 7.7, 4.5 Hz, 1H, H‐4’), 3.72 (q, J = 6.9 Hz, 2H, CH
C=O), 5.80 (s, 1H, H‐1’), 5.65 (d, J = 7.6 Hz, 1H, H‐5), 5.34 (s, 2H, CH (t, J = 6.9 Hz, 2H, CH ), 1.45 (s, 3H, CH ), 1.26 (s, 3H, CH
). 13C NMR
O), 5.18 – 5.11 (m, 1H, H‐2’), 4.95 – 4.88 (m, 1H, H‐3’), 4.80 (dd, J = (63 MHz, DMSO‐d ) δ 181.3 (C=O‐(C‐C(NH ))), 180.5 (C=O‐(C‐C(NH))),
4.1, 5.0 Hz, 1H, H‐5’), 4.68 (dd, J = 14.1, 7.6 Hz, 1H, H‐5’), 4.50 – 4.36 163.3 (C‐4), 150.3 (C‐2), 146.4, 146.1, 144.12 (s, C‐triazole), 143.4 (C‐
2
N), 3.01
2
‐
2
3
3
6
2
1
(
m, 1H, H‐4’), 3.55 – 3.40 (m, 4H, CH
2
‐N), 1.48 (s, 3H, C(CH
3
)
2
), 1.29 6), 134.1, 133.7, 132.3 (CH‐(CH‐C‐C=O)), 132.2 (CH‐(CH‐C‐C=O)),
). C NMR (101 MHz, 129.6 (CH‐(C(NH ))), 125.7 (2 x C, CH‐(C‐C=O)), 123.7 (CH‐(C(NH))),
)), 152.2 (O‐ 123.2 (CH‐triazole), 113.4 (C(CH ), 108.1 (C‐N), 107.9 (C‐N), 101.8
C‐(CHC=O)), 151.3 (C‐Ar), 150.8 (C‐2), 146.9 (O‐C‐(CH‐Ar)), 144.0 (C‐ (C‐5), 93.2 (C‐1’), 85.3 (C‐4’), 83.6 (C‐2’), 81.3 (C‐3’), 51.2 (C‐5’), 41.8
), 142.9 (C‐triazole), 138.6 (C=N), 133.9 (C‐Ar), 131.4 (CH‐Ar), 127.7 (CH N), 26.8 (CH ), 25.7 (CH ), 25.1 (CH
). HRMS‐ESI (m/z) [M+H]+
CHAr), 125.7 (CAr), 125.6 (s, CH‐triazole), 124.4 (CAr), 118.3 (CHAr), calcd for C30 600.2201, found 600.2200.
13
(s, 3H, C(CH
3
)
2
), 1.16 (t, J = 6.3 Hz, 6H, CH
3
2
DMSO‐d
6
) δ 181.8 (C=O), 163.8 (C‐4), 161.0 (C‐Ar‐(OCH
2
3 2
)
6
(
2
3
2
3
30 7 7
H N O
1
5
13.9 (C(CH
3 2
) ), 110.5 (CHAr), 107.3 (CHAr), 104.5 (CH‐C=O), 102.4 (C‐
1
‐[5‐[[4‐[2‐[(4‐Amino‐9,10‐dioxo‐1‐anthryl)amino]ethyl]triazol‐1‐
), 96.4 (CHAr), 93.8 (C‐1’), 85.8 (C‐4’), 84.1 (C‐2’), 81.9 (C‐3’), 61.9
yl]methyl]‐3,4‐dihydroxy‐tetrahydrofuran‐2‐yl]pyrimidine‐2,4‐
dione (23). General procedure used for 19 was followed with 22 (250
mg, 0.42 mmol) and TFA (2.2 mL) in H O (0.2 mL), to afford
2
compound 23 (199 mg, 85%) as a blue solid after purification by flash
(
CH
2
‐O), 51.8 (C‐5’), 44.9 (CH
2
‐N), 27.3 (CH
3
), 25.5 (CH
3
), 12.9 (2 x
+
CH
6
3
). HRMS‐ESI (m/z) [M+H] calcd for C35
H
36
N O
7 8
682.2620, found
82.2618.
1
1
‐[5‐[[4‐[[9‐(Diethylamino)‐5‐oxo‐benzo[a]phenoxazin‐2‐
column chromatography (CH
(400 MHz, DMSO‐d
‐yl]pyrimidine‐2,4‐dione (21). The same procedure used for 19 was 2H, NH ), 8.22 (dd, J = 5.8, 3.2 Hz, 2H, CH‐(C‐C=O)), 7.97 (s, 1H, H‐
followed with 20 (25 mg, 0.04 mmol) and TFA (0.19 mL) in H O (0.02 triazole), 7.84 – 7.71 (m, 2H, CH‐(CH‐C‐C=O)), 7.45 (d, J = 8.0 Hz, 1H,
mL), to afford compound 21 (17 mg, 74%) as a purple solid after H‐6), 7.42 (d, J = 9.5 Hz, 1H, CH‐(C(NH))), 7.30 (d, J = 9.5 Hz, 1H, CH‐
2
Cl
2
/MeOH from 95:5 to 90:10). H NMR
yl]oxymethyl]triazol‐1‐yl]methyl]‐3,4‐dihydroxy‐tetrahydrofuran‐
2
6
) δ 10.84 (t, J = 5.7 Hz, 1H, NHAQ), 8.68 – 8.25 (brs,
2
2
purification by flash column chromatography (CH
H NMR (400 MHz, DMSO‐d ) δ 11.37 (s, 1H, NH), 8.29 (s, 1H, H‐ 4.70 (dd, J = 4.8, 14.0 Hz, 1H, H‐5’), 4.61 (dd, J = 7.4, 14.0 Hz, 1H, H‐
6
2
Cl
2
/MeOH 90:10). (C(NH
2
))), 5.75 (d, J = 5.2 Hz, 1H, H‐1’), 5.55 (d, J = 8.0 Hz, 1H, H‐5),
1
triazole), 8.05 (d, J = 2.5 Hz, 1H, H‐Ar), 8.04 (d, J = 8.6 Hz, 1H, H‐Ar), 5’), 4.20 – 4.09 (m, 1H, H‐4’), 4.05 (t, J = 5.2 Hz, 1H, H‐2’), 3.96 (t, J =
7
=
=
5
=
.59 (d, J = 9.1 Hz, 1H, H‐Ar), 7.55 (d, J = 8.1 Hz, 1H, H‐6), 7.33 (dd, J 5.2 Hz, 1H, H‐3’), 3.73 (q, J = 6.7 Hz, 2H, CH
2
N), 3.02 (t, J = 7.1 Hz, 2H,
1
3
8.6, 2.5 Hz, 1H, H‐Ar), 6.79 (dd, J = 9.1, 2.5 Hz, 1H, H‐Ar), 6.61 (d, J CH
2
). C NMR (101 MHz, DMSO‐d
6
) δ 181.3 (C=O‐(C‐C(NH ))), 180.5
2
2.5 Hz, 1H, H‐Ar), 6.17 (s, 1H, CH‐C=O), 5.77 (d, J = 5.5 Hz, 1H, H‐1’), (C=O‐(C‐C(NH))), 164.4 (C‐4), 151.5 (C‐2), 146.3 (Cquat), 146.1 (Cquat),
.64 (d, J = 8.1 Hz, 1H, H‐5), 5.53 (d, J = 5.5 Hz, 1H, OH‐2’), 5.41 (d, J 144.0 (C‐triazole), 140.7 (C‐6), 134.1, 133.7, 132.3 (CH‐(CH‐C‐C=O)),
5.5 Hz, 1H, OH‐3’), 5.35 (s, 2H, CH ‐O), 4.78 (dd, J = 14.4, 4.2 Hz, 1H, 132.2 (CH‐(CH‐C‐C=O)), 129.7 (CH‐(C(NH ))), 125.8 (CH‐(C‐C=O)),
2 2
H‐5’), 4.70 (dd, J = 14.4, 7.6 Hz, 1H, H‐5’), 4.24 – 4.15 (m, 1H, H‐4’), 125.7 (CH‐(C‐C=O)), 123.7 (CH‐(C(NH))), 123.4 (CH‐triazole), 108.1 (C‐
This journal is © The Royal Society of Chemistry 20xx
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DOI: 10.1039/C8OB01606B
ARTICLE
Journal Name
N), 107.9 (C‐N), 102.1 (C‐5), 89.1 (C‐1’), 81.6 (C‐4’), 72.3 (C‐2’), 70.6 2‐[2‐[(E)‐2‐[4‐[2‐[[1‐[[4‐(6‐Aminopurin‐9‐yl)‐2,2‐dimethyl‐
+
(
C‐3’), 51.2(C‐5’), 41.9 (CH
2
N), 25.8 (CH
2
). HRMS‐ESI (m/z) [M+H]
3a,4,6,6a‐tetrahydrofuro[3,4‐d][1,3]dioxol‐6‐yl]methyl]triazol‐4‐
yl]methoxy]ethyl‐methyl‐amino]phenyl]vinyl]‐6‐tert‐butyl‐pyran‐
calcd for C27
H
N
26 7
O
7
560.1888, found 560.1884.
4
‐ylidene]propanedinitrile (29) Following general procedure used
for 18, 5’‐azido‐5’‐deoxy‐2’,3’‐O‐isopropylideneadenosine (140 mg,
.42 mmol) was reacted with Tag 11 (174 mg, 0.0.42 mmol), sodium
ascorbate (63 mg, 0.25 mmol) and copper sulfate pentahydrate (25
mg, 0.13 mmol) in H O/EtOH (1:1) (4 mL) to afford compound 29 as
an orange oil (256 mg, 81%) after purification by flash column
5
‐Bromopentyl
2‐(N‐ethyl‐N‐{4’‐[(2’’‐chloro‐4’’‐nitrophenyl)‐
phazo]phenyl}amino)ethyl ether (Tag 24) was prepared following
0
2
3
the procedure reported by Isaad et al. from commercially available
‐[4‐(2‐chloro‐4‐nitrophenylazo)‐N‐ethylphenylamino]ethanol
Disperse Red 13) in 59% yield.
2
(
2
chromatography (AcOEt/CH
2
Cl
2
/MeOH 3:1:0.1 then CH
) δ 8.33 (s, 1H, H‐2), 7.78 (s, 1H, H‐8),
2 2
Cl /MeOH
1
‐[6‐[[5‐[2‐[4‐[(E)‐(2‐Chloro‐4‐nitro‐phenyl)azo]‐N‐ethyl‐
1
96:4). H NMR (400 MHz, CDCl
3
anilino]ethoxy]pentylamino]methyl]‐2,2‐dimethyl‐3a,4,6,6a‐
tetrahydrofuro[3,4‐d][1,3]dioxol‐4‐yl]pyrimidine‐2,4‐dione (26). To
a solution of 5’‐amino‐5’‐deoxy‐2’,3’‐O‐isopropylideneuridine (240
mg, 0.85 mmol) and K CO (88 mg, 0.64 mmol) in DMF (50 mL) was
2 3
added red Tag 24 (205 mg, 0.42 mmol). The resulting mixture was
stirred at room temperature for 16 h. The mixture was extracted with
4
AcOEt, washed with water (5 x 150 mL), brine, dried with MgSO ,
filtrated and concentrated under reduced pressure. The crude
product was purified by flash column chromatography
7.37 (d, J = 8.8 Hz, 2H, CHAr), 7.31 (d, J = 15.8 Hz, 1H, H‐alkene), 7.23
(
s, 1H, H‐triazole), 6.65 (d, J = 8.8 Hz, 2H, CHAr), 6.58 (d, J = 1.7 Hz, 1H,
H‐pyran), 6.50 (d, J = 1.7 Hz, 1H, H‐pyran)), 6.47 (d, J = 15.8 Hz, 1H,
H‐alkene), 6.05 (d, J = 1.8 Hz, 1H, H‐1’), 5.89 (s, 2H, NH ), 5.40 (dd, J
6.3, 1.8 Hz, 1H, H‐2’), 5.19 (dd, J = 6.3, 3.6 Hz, 1H, H‐3’), 4.77 (dd, J
14.3, 4.2 Hz, 1H, H‐5’), 4.70 (dd, J = 14.3, 7.4 Hz, 1H, H‐5’), 4.63 –
.56 (m, 1H, H‐4’), 4.55 (s, 2H, C‐CH O), 3.67 (t, J = 5.6 Hz, 2H, CH O),
.56 (t, J = 5.6 Hz, 2H, CH N), 2.99 (s, 3H, CH N), 1.59 (s, 3H, C(CH ),
.36 (s, 12H, C(CH , CH ) δ 171.9
(tBu)). 13C NMR (101 MHz, CDCl
2
=
=
4
3
1
2
2
2
3
3 2
)
3
)
2
3
3
(
(
CH
2
Cl
2
/MeOH from 99:1 to 97:3) to give compound 26 as a red oil
1
(C‐pyran), 160.3 (C‐pyran), 156.9, 155.8, 153.3 (C‐2), 150.9, 149.1,
127 mg, 43%). H NMR (400 MHz, CDCl
3
) δ 8.37 (d, J = 2.4 Hz, 1H, H‐
1
1
44.9 (C‐triazole), 140.2 (C‐8), 138.4 (CH‐alkene), 129.8 (2 x C, CHAr),
23.7, 122.4, 120.5, 115.9, 115.8, 115.08, 112.9 (CH‐alkene), 112.0
Ar), 8.13 (dd, J = 8.9, 2.4 Hz, 1H, H‐Ar), 7.92 (d, J = 8.7 Hz, 2H, H‐Ar),
7
8
.76 (d, J = 8.9 Hz, 1H, H‐Ar), 7.32 (d, J = 8.0 Hz, 1H, H‐6), 6.76 (d, J =
.7 Hz, 2H, H‐Ar), 5.74 (d, J = 8.0 Hz, 1H, H‐5), 5.69 (d, J = 2.5 Hz, 1H,
(2C, CHAr), 105.5 (CH‐pyran), 102.4 (CH‐pyran), 90.7 (C‐1’), 85.5 (C‐
4
5
’), 84.0 (C‐2’), 82.0 (C‐3’), 67.8 (CH
2.0 (CH N), 51.7 (C‐5’), 39.1 (CH
), 25.4 (C(CH ). HRMS‐ESI (m/z) [M+H] calcd for
, 746.3521 found 746.3519.
2
O), 64.6 (C‐CH
2
O), 57.8 (2C, CN),
H‐1’), 4.91 (dd, J = 6.6, 2.5 Hz, 1H, H‐2’), 4.75 (dd, J = 6.6, 4.5 Hz, 1H,
H‐3’), 4.09 (d, J = 4.5 Hz, 1H, H‐4’), 3.92 – 3.83 (m, 2H, H‐5’), 3.62 (s,
2
3
N), 36.7, 28.2 (CH
3
+
(tBu)), 27.2
(C(CH
3
)
2
3 2
)
4H, N‐CH
2H, CH O), 1.67 – 1.58 (m, 4H, CH
– 1.36 (m, 2H, CH ), 1.34 (s, 3H, C(CH
1.24 (t, J = 7.0 Hz, 3H, CH
153.7 (C‐N=N), 152.5 (C‐N), 151.0 (C‐2), 147.4 (C‐NO
2
‐CH
2
‐O), 3.55 (q, J = 7.0 Hz, 2H, CH
N, CH ), 1.56 (s, 3H, C(CH
), 1.27 – 1.20 (m, 2H, CH
). C NMR (101 MHz, CDCl ) δ 162.9 (C‐4),
), 144.6 (C‐
2
N), 3.44 (t, J = 6.4 Hz,
), 1.44
),
C
39 44 11 5
H N O
2
2
2
3 2
)
2
3
)
2
2
2‐[2‐[(E)‐2‐[4‐[2‐[[1‐[[5‐(6‐Aminopurin‐9‐yl)‐3,4‐dihydroxy‐
1
3
3
3
tetrahydrofuran‐2‐yl]methyl]triazol‐4‐yl]methoxy]ethyl‐methyl‐
amino]phenyl]vinyl]‐6‐tert‐butyl‐pyran‐4‐ylidene]propanedinitrile
2
N=N), 140.1 (C‐6), 134.2 (C‐Cl), 127.5 (CHAr), 126.4 (CHAr), 123.0
CHAr), 118.4 (CHAr), 115.1 (C(CH ), 111.9 (2 x C, CHAr), 102.6 (C‐5),
4.7 (C‐1’), 87.6 (C‐4’), 84.9 (C‐2’), 81.7 (C‐3’), 71.7 (CH O), 68.6
OCH ‐(CH N)), 50.8 (NCH ‐(CH O)), 46.5 (CH N), 41.5 (C‐5’), 30.1,
), 25.8 (CH ), 23.9, 12.6 (CH ). HRMS‐ESI
700.2856, found 700.2856.
(
30). Following general procedure used for 19, compound 29 (240
mg, 0.32 mmol) was reacted with TFA (1.6 mL) in H O (0.2 mL) to
afford compound 30 as a dark red solid (186 mg, 82%) after
(
9
(
2
3 2
)
2
2
2
2
2
2
2
purification by flash column chromatography (CH
2
Cl
2
/MeOH 95:5 to
9.7 (CH
m/z) [M+H] calcd for C33
2
NH), 27.6, 27.6 (CH
3
3
3
1
8
8
7
0:20) as eluent. H NMR (400 MHz, DMSO‐d
6
) δ 8.26 (s, 1H, H‐2),
+
(
H
43ClN
7
O
8
.16 (s, 1H, H‐8), 7.84 (s, 1H, H‐triazole), 7.53 (d, J = 8.5 Hz, 2H, HAr),
.43 (d, J = 15.9 Hz, 1H, H‐alkene), 7.32 (s, 2H, NH ), 7.00 (d, J = 15.9
2
1‐[5‐[[5‐[2‐[4‐[(E)‐(2‐Chloro‐4‐nitro‐phenyl)azo]‐N‐ethyl‐
Hz, 1H, H‐alkene), 6.74 (d, J = 2.1 Hz, 1H, H‐pyran), 6.69 (d, J = 8.5 Hz,
anilino]ethoxy]pentylamino]methyl]‐3,4‐dihydroxy‐
tetrahydrofuran‐2‐yl]pyrimidine‐2,4‐dione (27). The procedure
used for 19 was followed with 26 (160 mg, 0.23 mmol) and TFA (1.2
2
1
–
4
2
d
1
H, HAr), 6.40 (d, J = 2.1 Hz, 1H, H‐pyran), 5.91 (d, J = 5.4 Hz, 1H, H‐
’), 5.62 (d, J = 5.5 Hz, 1H, OH‐2’), 5.50 (d, J = 4.4 Hz, 1H, OH‐3’), 4.81
4.72 (m, 2H, H‐5’), 4.69 (d, J = 5.5 Hz, 1H, H‐2’), 4.46 (s, 2H, C‐CH
.28 – 4.24 (m, 2H, H‐3’, H‐4’), 3.56 (t, J = 3.6 Hz, 4H, O‐CH CH2‐N),
N), 1.34 (s, 9H, CH3, (tBu)). C NMR (101 MHz, DMSO‐
) δ 172.2 (C‐pyran), 160.9 (C‐pyran), 156.4, 156.1, 152.6 (C‐2),
50.6, 149.2, 143.8, 139.9 (C‐8), 138.5 (CH‐alkene), 130.0 (2 x C,
2
O),
2
mL) in H O (0.1 mL), to afford compound 27 (105 mg, 70%) as a black
2
solid after purification by flash column chromatography
1
3
.91 (s, 3H, CH
3
1
(
CH
2
Cl
2
/MeOH from 95:5 to 80:20). H NMR (400 MHz, DMSO‐d
6
) δ
6
8
.42 (s, 1H, H‐Ar), 8.24 (d, J = 8.5 Hz, 1H, H‐Ar), 7.93 (d, J = 7.9 Hz, 1H,
H‐6), 7.85 (d, J = 8.8 Hz, 2H, H‐Ar), 7.78 (d, J = 8.5 Hz, 1H, H‐Ar), 6.91
d, J = 8.8 Hz, 2H, H‐Ar), 5.86 – 5.67 (m, 2H, H‐1’, H‐5), 4.11 – 4.05 (m,
CHAr), 124.4, 122.0, 119.2, 115.8, 115.7, 112.8 (CH‐alkene), 111.7 (2
x C, CHAr), 104.9 (CH‐pyran), 101.5 (CH‐pyran), 87.8 (C‐1’), 82.4 (C‐4’),
(
1
3
2
H, H‐2’), 3.97 – 3.90 (m, 1H, H‐3’), 3.85 – 3.70 (m, 3H, H‐4’, CH
.70 – 3.50 (m, 6H, N‐CH ‐CH ‐O, CH N), 3.41 (s, 2H, CH O), 2.86 –
.73 (m, 2H, H‐5’), 1.50 (s, 4H, CH ), 1.34 – 1.23 (m, 2H, CH ), 1.17 (t,
). 13C NMR (101 MHz, DMSO‐d
) δ 161.8 (C‐4), 152.4
C‐N=N), 152.2 (C‐N), 150.7 (C‐2), 146.6 (C‐NO ), 143.2 (C‐N=N),
2
NH),
7
2.5 (C‐2’), 70.9 (C‐3’), 67.0 (CH
CH N), 36.3 (CH N), 27.52 (CH
for C36 , 706.3208 found 706.3201.
2
O), 63.5 (C‐CH
(tBu)). HRMS‐ESI (m/z) [M+H] calcd
2
O), 51.3 (C‐5’), 50.9
2
2
2
2
+
(
2
3
3
2
2
40 11 5
H N O
J = 6.7 Hz, 3H, CH
(
3
6
2
1
1
2
39.7 (C‐6), 132.2 (C‐Cl), 126.7 (2 x C, CAr), 125.7 (CHAr), 123.4 (CHAr),
18.0 (CHAr), 111.8 (CHAr), 101.0 (C‐5), 89.1 (C‐1’), 84.8 (C‐4’), 73.1 (C‐
Conflicts of interest
There are no conflicts to declare.
’), 70.2 (C‐3’, CH
2
O), 67.8 (OCH
NH), 28.7, 26.8, 23.0, 12.0 (CH
660.2543, found 660.2542.
2
‐(CH
2
N)), 49.7 (CH
2
N), 45.4 (NCH
2
‐
(
CH
2
O)), 42.9 (C‐5’), 40.6 (CH
2
3
). HRMS‐
+
Acknowledgements
ESI (m/z) [M+H] calcd for C30
H
7 8
39ClN O
1
0 | Org. Biomol. Chem., 2018, 00, 1‐3
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Or gP al en ai cs e& d Bo i on mo to al edc j uu lsat r mC ha re gmi ni ss try
View Article Online
DOI: 10.1039/C8OB01606B
Journal Name
ARTICLE
This work was supported by institutional support from the
16. Y.‐H. Gong, P. Audebert, G. Clavier, F. Miomandre, J. Tang, S.
University of Orleans and from ANR‐10‐TECS‐0004.
Badré, R. Méallet‐Renault, E. Naidus, New J. Chem., 2008, 32,
1235‐1242.
1
7. G. Wang, K. Sakthivel, V. Rajappan, T. W. Bruice, K. Tucker, P.
Fagan, J. Brooks, T. Hurd, J. M. Leeds, P. D. Cook, Nucleosides
Nucleotides Nucleic Acids, 2004, 23, 317‐337.
Notes and references
1.
a) L.P. Jordheim, D. Durantel, F. Zoulim, C. Dumontet, Nat. Rev.
Drug Discov., 2013, 12, 447–464. b) L. A. Agrofoglio, S. R.
Challand in “Acyclic, Carbocyclic and L‐Nucleosides”; Kluwer
Academic Publishers: Dordrecht, 1998. c) E. De Clercq, A. Holý,
Nat. Rev. Drug Discov., 2005, 4, 928–940.
1
1
2
8. A. Procopio, M. Gaspari, M. Nardi, M. Oliverio, A. Tagarelli, G.
Sindona, Tetrahedron Lett., 2007, 48, 8623‐8627.
9. Z. Novák, B. Bostai, M. Csékei, K. Lőrincz, A. Kotschy,
Heterocycles, 2003, 60, 2653‐2668.
0. a) R. Lucas, R. Zerrouki, R. Granet, P. Krausz, Y. Champavier,
Tetrahedron, 2008, 64, 5467‐5471; b) X. Chi, P. Pahari, K.
Nonaka, S. G. Van Lanen, J. Am. Chem. Soc., 2011, 133,
2.
a) R. W. Sinkeldam, N. J. Greco, Y. Tor, Chem Rev., 2010, 110,
2
579–2619; b) M. S. Noe, Y. Xie, Y. Tor, in “Methods for
Studying Nucleic Acid/Drug Interactions”; CRC Press, 2011, pp.
59–182; c) U. Asseline, Curr. Org. Chem., 2006, 10, 491–518;
d) D. M. Jameson, J. F. Eccleston, Methods Enzymol., 1997, 178,
63‐390; e) A. A. Tanpure, M. G. Pawar, S. G. Srivatsan, Isr. J.
14452–14459.
1
2
1. a) N. Yotapan, C. Charoenpakdee, P. Wathanathavorn, B.
Ditmangklo, H.‐A. Wagenknecht, T. Vilaivan, Belstein J. Org.
Chem., 2014, 10, 2166‐2174; b) M. Börgardts, K. Verlinken, M.
Neidhardt, T. Wöhrle, A. Herbst, S. Laschat, C. Janiak, T. J. J.
Müller, RSC Adv., 2016, 6, 6209‐6222; c) M. S. J. Briggs, I.
Bruce, J. N. Miller, C. J. Moody, A. C. Simmonds, E. Swann, J.
Chem. Soc, Perkin Trans. 1, 1997, 1051‐1058.
3
Chem., 2013, 53, 366–378.
3
.
.
T. Hiratsuka, K. Uchida, Biochim. Biophys. Acta, 1973, 320, 365‐
6
47.
4
a) T. Hiratsuka, Eur. J. Biochem., 2003, 270, 3479‐3485; b) C. M.
McKeen, L. J. Brown, J. T. Nicol, J. M. Mellor, T. Brown, Org.
Biomol. Chem., 2003, 1, 2267–2275. c) H. Asanuma, T. Yoshida,
T. Ito, M. Komiyama, Tetrahedron Lett. 1999, 40, 7995–7998.
a) A. R. Katritzky, L. Khelashvili, J. Kovacs, K. Shanab, Chem. Biol.
Drug. Des.,2009, 73, 396–402; b) M. Adamczyk, S. R. Akireddy,
P. G. Mattingly, R. E. Reddy, Tetrahedron, 2003, 59, 5749–5761.
A. Krstulja, S. Lettieri, A.J. Hall, V. Roy, P. Favetta, L. A.
2
2. P. M. Chaudhary, S. R. Chavan, F. Shirazi, M. Razdan, P.
Nimkar, S. P. Maybhate, A. P. Likhite, R. Gonnade, B. G.
Hazara, M. V. Deshpande, S. R. Deshpande, Bioorg. Med.
Chem., 2009, 17, 2433‐2440.
5.
2
2
2
3. J. Isaad, M. Rolla, R. Bianchini, Eur. J. Org. Chem., 2009, 2748‐
2
764.
4. L. Li, X. Shen, Q.‐H. Xu, S. Q. Yao, Angew. Chem., Int. Ed., 2013,
2, 424‐428.
5. (a) S. Vertuani, A. Baldisserotto, K. Varani, P. A. Borea, B. De
Marcos Maria Cruz, L. Ferraro, S. Manfredini, A. Dalpiaz, Eur. J.
Med. Chem., 2012, 54, 202‐209. (b) L. R. Comstock, S. R.
Rajski, Tetrahedron, 2002, 58, 6019‐6026.
6
.
.
Agrofoglio, Macromol. Biosci. 2017, 17, 1700250.
C. Dejous, H. Hallil, V. Raimbault, J.L. Lachaud, B. Plano, R.
Delépée, P. Favetta, L. A. Agrofoglio, D. Rebière, Sensors,
5
7
2
016, 16, 915; b) L. A. Agrofoglio, A. Krstulja, C. De Schutter,
P. Favetta, R. Delépée, V. Roy, C. Dejous, H. Hallil, J.‐L.
Lachaud, N. Lebal, N. Omar Aouled, V. Raimbault, D. Rebière,
S. Dulong, F. Levi, C. Junot, F. Théodoro, A. Pruvost, R. Vidal,
IRBM 2014, 35, 66–71; c) N. Lebal, D. Rebière, R. Delepée, L.
A. Agrofoglio, B. Plano, H. Hallil, A. Krstulja, C. Dejous, Micro
Amp Nano Lett. 2013, 8, 563–566.
8
.
.
A. Krstulja, S. Lettieri, A. J. Hall, R. Delépée, P. Favetta, L. A.
Agrofoglio, Anal. Bioanal. Chem., 2014, 406, 6275‐6284.
a) K. M. Koczula, A. Gallotta, Essays Biochem., 2016, 60, 111‐
9
1
2
20; b) G. Kim, J. Lim, C. Mo, J. Biosys. Eng., 2015, 40, 277‐
83.
10. N. S. Freeman, C. E. Moore, L. M. Wilhelmsson, Y. Tor, J. Org.
Chem., 2016, 81, 4530‐4539.
1
1
1. G. Clavier, P. Audebert, Chem. Rev., 2010, 110, 3299‐3314.
2. a) Y.‐H. Gong, F. Miomandre, R. Méallet‐Renaud, S. Badré, L.
Galmiche, J. Tang, P. Audebert, G. Clavier, Eur. J. Org. Chem.,
2
009, 6121‐6128; b) J. Malinge, C. Allain, L. Galmiche, F.
Miomandre, P. Audebert, Chem. Mater., 2011, 23, 4599‐4605.
3. A. M. Hossion, R. Islam, R. K. Kandahary, T. Nagamatsu,
Synthesis, 2009, 16, 2689‐2696.
1
1
1
4. J. P. Ferris, V. R. Rao, T. A. Newton, J. Org. Chem., 1979, 44,
4
378‐4381.
5. C. Ribes, E. Falomir, J. Murga, Tetrahedron, 2006, 62, 1239‐
244.
1
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Org. Biomol. Chem., 2018, 00, 1‐3 | 11
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Organic & Biomolecular Chemistry
Page 12 of 12
View Article Online
DOI: 10.1039/C8OB01606B
A novel set of visibly coloured dye-labeled 5’-nucleosides including 1,2,4,5-tetrazine (orange),
dicyanomethylene-4H-pyran (yellow), benzophenoxazinone (pink), 9,10-anthraquinone (blue) and
azobenzene (red) chromophores, were prepared, and their absorption and fluorescence spectra
recorded. Such nucleodyes have great potential for future competitive lateral flow test MIP-based
strips.
N
N
NC
CN
N
N
N
N
H N
2
O
O
O
N
H
N
O
N
O
NH
N
N
N
MeO
O
O
O
N
Et2N
N
HO OH
N
H
N
N
N
O
O
(
5
O
O
N
N
O2N
N
Cl