Alkynylated and Dendronized 5-Aza-7-deazaguanine Nucleosides: Cross-Coupling with Tripropargylamine and Linear Alkynes, Click Functionalization, and Fluorescence of Pyrene Adducts ?

Article abstract of DOI:10.1021/acs.joc.0c00926

The change of the recognition face of 5-aza-7-deazaguanine bridgehead nucleosides with respect to purine nucleosides permits the construction of new purine-purine or purine-pyrimidine base pairs in DNA and RNA. Clickable derivatives of 5-aza-7-deazaguanine were synthesized by introducing ethynyl, 1,7-octadiynyl, and tripropargylamino side chains in the 7-position of the 5-aza-7-deazapurine moiety by Sonogashira cross-coupling. Click reactions were performed with 1-azidomethylpyrene by the copper-catalyzed azide-alkyne cycloaddition. The copper(I)-catalyzed click reaction on the tripropargylamino nucleoside was significantly faster and higher yielding than that for nucleosides carrying linear alkynyl chains. Also, this reaction could be performed with copper(II) as the catalyst. An autocatalyzed cycle was suggested in which the click product acts as a catalyst. Pyrene click adducts of linear alkynylated nucleosides showed pyrene monomer emission, while tripropargylamino adducts showed monomer and excimer fluorescence. The fluorescence intensities of the 5-aza-7-deazaguanine nucleosides were higher than those of their 7-deazaguanine counterparts. The reported clickable nucleosides can be utilized to functionalize or to cross-link monomeric nucleosides or DNA for diagnostic or imaging purposes and other applications in nucleic acid chemistry and biotechnology.

Full text of DOI:10.1021/acs.joc.0c00926

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Article
Alkynylated and Dendronized 5-Aza-7-deazaguanine Nucleosides:
Cross-Coupling with Tripropargylamine and Linear Alkynes,
Click Functionalization and Fluorescence of Pyrene Adducts
Dasharath Kondhare, Aigui Zhang, Peter Leonard, and Frank Seela
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.0c00926 • Publication Date (Web): 23 Jul 2020
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Alkynylated and Dendronized 5-Aza-7-deazaguanine Nucleosides: Cross-Coupling with
Tripropargylamine and Linear Alkynes, Click Functionalization and Fluorescence of
Pyrene Adducts†
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Dasharath Kondharea, Aigui Zhanga, Peter Leonarda and Frank Seelaa,b*
aLaboratory of Bioorganic Chemistry and Chemical Biology, Center for Nanotechnology,
Heisenbergstrasse 11, 48149 Münster, Germany and bLaboratorium für Organische und
Bioorganische Chemie, Institut für Chemie neuer Materialien, Universität Osnabrück,
Barbarastrasse 7, 49069 Osnabrück, Germany
Prof. Dr. Frank Seela
Phone: +49 (0)251-53406-500; Mobile: +49 (0)173-7250-297
Fax: +49 (0)251-53406-587
E-mail: Frank.Seela@uni-osnabrueck.de
Homepage: www.seela.net
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TABLE OF CONTENTS
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Clickable side chains
O
N
X
(CH2)4
Ia
: X =
Ib
: X =
N
N
O
I-IIIc
: X = tripropargylamine
N
H2N
HO
Click adducts
N
N
Py
N
IaPy
:
3
HO
R
N
N
N
N
Py
Py
N
N
I
II
III
: R = H
IcPy
:
: R = OH
N
: R = araF
Fluorescence of pyrene click adducts (Py)
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ABSTRACT
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The change of the recognition face of 5-aza-7-deazaguanine bridgehead nucleosides with
respect to purine nucleosides permits the construction of new purine-purine or purine-
pyrimidine base pairs in DNA and RNA. Clickable derivatives of 5-aza-7-deazaguanine were
synthesized by introducing ethynyl, 1,7-octadiynyl and tripropargylamino side chains in the 7-
position of the 5-aza-7-deazapurine moiety by Sonogashira cross-coupling. Click reactions
were performed with 1-azidomethylpyrene by the copper-catalyzed azide-alkyne
cycloaddition. The copper(I) catalyzed click reaction on the tripropargylamino nucleoside was
significantly faster and higher yielding than that for nucleosides carrying linear alkynyl
chains. Also, this reaction could be performed with copper(II) as catalyst. An autocatalyzed
cycle was suggested in which the click product acts as a catalyst. Pyrene click adducts of
linear alkynylated nucleosides showed pyrene monomer emission while tripropargylamino
adducts showed monomer and excimer fluorescence. The fluorescence intensities of the 5-
aza-7-deazaguanine nucleosides were higher than those of their 7-deazaguanine counterparts.
The reported clickable nucleosides can be utilized to functionalize or to cross-link monomeric
nucleosides or DNA for diagnostic or imaging purposes and other applications in nucleic acid
chemistry and biotechnology.
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INTRODUCTION
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Bridgehead 5-aza-7-deazaguanine nucleosides contain a heterocyclic skeleton that differs
from those of 7-deazapurine or 8-aza-7-deazapurine nucleosides (purine numbering is used
throughout the results and discussion section). Due to the shift of nitrogen-7 to the bridgehead
position-5, nitrogen-1 becomes the proton acceptor site and is no longer a donor site as
existing in related guanine nucleosides. This change does not only affect the physical
properties of this class of nucleosides but also changes base pairing.1 As a consequence, 5-
aza-7-deazaguanine forms base pairs with guanine and isoguanine.1b It produces stable
“purine” DNA with parallel and antiparallel chain orientation (Figure 1).1b Base pairing with
protonated dC has been reported1a and special dC analogs were used to expand the genetic
alphabet by a six letter code.1c Also, programmable metal ion mediated base pairs with dC
exist in the presence of silver ions.2 Recently, 7-functionalized derivatives were synthesized
in the series of ribo- and 2’-deoxyribonucleosides.3 Earlier work on non-functionalized 5-aza-
7-deazaguanine nucleosides has been reviewed.4 Current work is cited in the manuscript.
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H
H
H N
O
N
N
O
H N
N
N
N
N
dG
R
N
z5Gd
N
N
z5Gd N
iGd
N
O
H
H
N
O
N
R
R
R
N
N
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N
H
N
H
H
H
motif II: ps orientation
motif I: aps orientation
Figure 1. Base pair motifs for “purine” DNA with antiparallel (aps) and parallel (ps) chain
orientation.1b z5Gd corresponds to 5-aza-7-deaza-2'-deoxyguanosine, iGd corresponds to 2'-
deoxyisoguanosine and R corresponds to 2'-deoxy-D-ribofuranosyl.
Recently, fluorescent residues have been connected directly to the base moiety of 5-aza-7-
deazaguanine nucleosides.3 Nonetheless, with respect to oligonucleotide synthesis this
requires conversion of each particular nucleoside conjugate to an individual phosphoramidite
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building block which can be later employed in solid-phase synthesis. A more universal
approach represents the use of click chemistry.5 The method allows functionalization with a
diversity of azido compounds and is therefore versatile for many applications.6
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Now, clickable derivatives of 5-aza-7-deazaguanine were synthesized by introducing alkynyl
side chains in the 7-position of the 5-aza-7-deazapurine moiety by Sonogashira cross-
coupling. This position has steric freedom in canonical B-DNA and it is anticipated that this is
also valid for "purine" nucleic acids.
In more detail, the synthesis of 5-aza-7-deazaguanine nucleosides with linear alkynyl chains
or dendritic triprogargylamino residues is described in this work and various sugar residues
were introduced at the nucleoside glycosylation site-9 (nucleosides 1-3; Figure 2). This makes
the molecules suitable for click reactions, expands the functionalities on the sugar moiety and
provides 5-aza-7-deazapurine compounds with new properties as nucleosides or components
of oligonucleotides. With the alkynylated and dendronized nucleosides 2a-c and 3a-d in hand
click libraries can be produced. To demonstrate the utility of the compounds a pyrene azide
was clicked to the nucleosides and steady-state fluorescence was investigated. During this
work, it became apparent that tripropargylamine click cycloaddition can be induced by
copper(II) ions in the absence of ascorbic acid as reducing agent. By serendipity, we also
found that TBTA {tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine} acts as a catalyst in the
Sonogashira cross-coupling reaction.
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O
N
R
N
N
N
O
O
O
I
N
H2N
HO
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5
N
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7
3
1
N
2
N
N
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N
N
H2N
N
9
H2N
N
8a
8
1
S
S
HO
3a-d
1a-c
Purine numbering
2a-c
Systematic numbering
S
corresponds to
(CH2)4
SiMe3
3a:
3b:
3c:
3d:
R=
R=
R=
R=
O
O
O
HO
HO
HO
F
Si(i-Pr)3
HO
HO
OH
HO
c
a
b
Figure 2. Structures of sugar-modified 5-aza-7-deazaguanine nucleosides used in this study.
RESULTS AND DISCUSSION
Synthesis of 7-Iodo-5-aza-7-deazaguanine Bridgehead Nucleosides. For the synthesis of
the tripropargylamine and alkynyl nucleosides 2a-c and 3a-d the 7-iodo compounds 1a-c
were the precursors. The synthesis of the 7-iodonucleosides 1a and 1b has already been
reported employing Vorbrüggen conditions or nucleobase anion glycosylation3,7, whereas 1c
is unknown. Earlier, the unsubstituted 5-aza-7-deazaguanine 2’-arabinofluoro nucleoside was
synthesized.8a As fluoro nucleosides play an important role in nucleoside and oligonucleotide
chemistry8b, the 7-iodo derivative 1c was prepared by nucleobase anion glycosylation of a
suitably protected nucleobase and the corresponding sugar derivative (Scheme 1).
In more detail, the fluoroarabino sugar 6 was prepared in situ from 5.9 Then, nucleobase anion
glycosylation was carried out in MeCN with the protected nucleobase 4 and excess of 6. DBU
(1,8-diazabicyclo[5.4.0]undec-7-ene) was used as base to generate the nucleobase anion. The
reaction gave a mixture of the protected anomers 7/8 in 73% overall yield isolated after flash
chromatography. The ratio of anomers 7/8 (1:1, β to α) was determined from the intensity of
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anomeric 1H NMR signals (Figure S10, Supporting Information). This mixture gave only one
spot on TLC and was inseparable by column chromatography. However, the pure protected β-
anomer 7 was obtained by crystallization from methanol in 30% yield. Deprotection of 7 in 7
N NH3/MeOH afforded the β-D anomer 1c in 87% yield. As the α-D anomer 8 could not be
isolated from the mother liquor as clean material, another separation method was chosen. The
mother liquor containing mainly the α-anomer was deprotected in 7 N NH3/MeOH to yield an
inseparable mixture of nucleosides 9 and 1c in 85%. Then, the mixture of the 5-aza-7-
deazaguanine 2’-deoxyribonucleoside α/β-anomers 9/1c could be separated as 5’-O-DMT
derivatives. Protection with DMT chloride in pyridine followed by flash chromatography
afforded the pure α-nucleoside 10 (faster migrating) in 60% and the β-anomer 11 in 10% yield
(slower migrating). Deprotection with trichloroacetic acid furnished the pure anomers 9 (α-D,
88%) and 1c (β-D, 84%).
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All compounds were characterized by mass spectra and high resolution 1H, 13C{1H} and 2D
NMR spectra (Figures S10-89, Supporting Information). 13C{1H}NMR chemical shifts are
shown in Table S1, Supporting Information). The anomeric configuration of the fluoro
nucleoside 1c was assigned by long range couplings from H-7 and C-7 to C2’-F (Figure S21,
Supporting Information) which indicate β-D configuration.10 Also, the signals for H-1’ (0.1
ppm), H-2’ (0.24 ppm) and H-4’ (0.4 ppm) as well as the carbon signals for C-1’ (5 ppm), C-
2’ (3 ppm), C-3’ (~1 ppm) and C-4’ (3 ppm) are shifted when the anomeric configuration is
altered from β-D to α-D. Furthermore, the coupling constants for 2J(C1’-C2’-F) and (C2’-
C2’-F) are different by 17 to 20 Hz. These observations are in line with reports on anomers of
non-iodinated 2’-fluoro nucleosides.8a
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Scheme 1. Synthesis and Separation of Anomeric 7-Iodo-5-aza-7-deaza-2’-deoxyfluoro
arabinofuranosyl Nucleosidesa
7
8
9
O
N
O
N
I
I
O
N
I
O
I
OBz
N
N
O
N
N
O
O
F
N
N
ii)
iii)
iv)
N
N
O
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BzO
i)
N
N
N
N
N
NH2
73%
87%
30%
N
H
H
N
N
+
H
BzO
O
F
O
F
H
O
F
BzO
BzO
HO
4
5
6
= OBz
= Br
BzO
BzO
HO
1c
7/8
7
+ 7/8 i
n mother liquor
O
F
O
HO
DMTO
F
HO
HO
N
N
NH2
N
NH2
N
vi)
N
N
N
N
O
F
88%
HO
I
I
O
O
HO
9
N
NH2
N
I
10
(60%)
v)
iv)
7/8
mother liquor
N
N
85%
O
N
I
O
N
N
vi)
9/1c
1c
N
NH2
84%
O
DMTO
F
11 (10%)
HO
aReagents and conditions: (i) HBr in glacial acetic acid, 16 h, rt; (ii) DBU, MeCN, 16 h, rt;
(iii) Crystallization from MeOH; (iv) 7N NH3/MeOH; (v) DMT-Cl, pyridine, 3.5 h, rt; (vi)
3% TCA in CH2Cl2, 16 h, rt.
Conversion of 7-Iodo Nucleosides to Alkynyl and Tripropargylamine Nucleosides. Next,
alkynyl side chains (linear and dendronized) were introduced to nucleosides 1a-c. For this,
Sonogashira cross-coupling reactions were performed.11 Compounds 1a-c were treated with
excess of alkyne in DMF in the presence of Pd(0) and copper(I) (Scheme 2, Table 1) (for
details, see the Experimental Section). The reaction performed on 1a with 1,7-octadiyne was
slow and significant deiodination took place. To access the product, the reaction was
monitored on TLC and was stopped before completion. Long reaction time resulted in
decomposition. Due to deiodination and decomposition, the yield was only between 12-24%.
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For the synthesis of the ethynyl nucleoside 3d cross-coupling reactions were performed with
TMS- and TIPS-acetylene. Compounds 3b (TMS) and 3c (TIPS) were obtained in moderate
yields after a reaction time of 24 h (39% for 3b and 36% for 3c) but significant deiodination
took place also in these cases. To solve this problem, Sonogashira reaction was performed on
the nucleobase 4. Unfortunately, deiodination occurred and an alkynylated product could not
be isolated. The reaction time of Sonogashira-cross coupling reactions performed on
nucleosides could be shortened with the help of TBTA {tris[(1-benzyl-1H-1,2,3-triazol-4-
yl)methyl]amine} but led to lower or marginally higher yields (for details, see Table S2,
Supporting Information). We anticipate that copper(I)ions are stabilized by TBTA as reported
for click reactions.12 Next, the silyl protecting groups were removed from 3b and 3c with 1 M
TBAF/THF to afford nucleoside 3d in 69% and 70% yield, respectively.
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Scheme 2. Synthesis of 5-Aza-7-deazaguanine Ribo-, 2’-Deoxyribo, and 2’-
Fluoroarabino Nucleosides with Tripropargyl Side Chains and 2’-Deoxyribonucleosides
with Alkynyl Side Chainsa
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N
O
N
O
N
I
N
N
O
N
N
O
N
N
H2N
HO
H2N
HO
i)
2a
: 63%
2b
: 52%
R2
R1
R2
R1
2c
: 59%
HO
2a-c
R1, R2 = H; R1 = OH; R2 = H; R1 = H; R2 = F
HO
1a-c
a:
b:
c:
O
N
O
N
R
I
N
N
O
N
N
N
N
H2N
HO
H2N
HO
ii) or iii)
O
HO
HO
1a
3a-d
ii) 23%, iii) 12%
ii) 39%, iii) 33%
ii) 36%, iii) 43%
(CH2)4
3a:
3b:
3c:
R=
R=
R=
SiMe3
Si(i-Pr)3
iv)
3b
3c
69% from
70% from
3d:
R=
aReagents and conditions: (i) Alkyne (10 eq.), Pd(PPh3)4, Et3N, CuI, DMF, rt; (ii) alkyne,
Pd(PPh3)4, Et3N, CuI, DMF, rt; (iii) alkyne, Pd(PPh3)4, Et3N, CuI, DMF, rt with TBTA (Table
S2, Supporting Information). (iv) 1 M TBAF in THF.
Contrary to the linear alkynes, Sonogashira cross-coupling of 1a with tripropargylamine was
efficient and the dendronized nucleoside 2a was obtained in 63% yield in 6 h. No
deiodination was observed. With these results in hand, Sonogashira cross-coupling reactions
with tripropargylamine were performed on nucleosides 1b and 1c yielding the ribonucleoside
2b in 52% yield and the 2’-fluorinated nucleoside 2c in 59%. The modification of the sugar
residue causes a significant change on the hydrophobic character of the nucleosides. As
demonstrated by the chromatographic mobilities on a reversed phase HPLC column (Figure
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S6, Supporting Information) the fluoro nucleoside is the most lipophilic compound and the
ribonucleoside the most hydrophilic.
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Incorporation of the side chains was confirmed by strong shifts of the nucleobase carbon-7 in
the 13C{1H} NMR spectra and the appearance of new signals for the side chains (Table S1,
Supporting Information). From previous studies on 5-tripropargylamine-2’-deoxyuridine it is
known that the introduction of side chains has an impact on the pKa value of the nucleobase.
As pK values are important for the recognition in base pairing they were determined from UV
spectral changes for compounds 1c, 2a-2c and 3d. They were found to be as follows 2a-2c
(pKa = 3.4), tripropargylamino 3d (pKa = 3.5), 7-iodinated 1a7 and 1c (pKa = 3.6) and non-
iodinated 5-aza-7-deaza-2’-deoxyguanosine (pKa = 3.8)13 (Figures S1-5, Supporting
Information). The data reflect protonation of the nucleobases. As the pK values are all in the
same range influence of the side chain on base protonation is small.
Click Reactions Performed On Linear and Dendronized Nucleosides with
Pyrenemethylazide under Copper(I) and Copper(II) Catalysis. The Cu(I)-catalyzed
Huisgen-Sharpless-Meldal cycloaddition5 – “click chemistry” has become a key reaction to
functionalize nucleosides and nucleic acids. The reaction has been performed in aqueous
solution or mixtures of organic solvents and water.6 To prove the applicability of the click
reaction to nucleosides 2a, 3a and 3d, the click functionalization was studied using 1-
azidomethyl pyrene (12) as second component. First, the reactions were performed under
standard conditions with CuSO4•5H2O and sodium ascorbate as reducing agent in a THF/t-
BuOH/H2O (3:1:1) mixture at rt. The copper(I)-catalyzed reaction with the linear ethynyl and
1,7-octadiynyl derivatives 3a and 3d was complete after 16 h affording the pyrene adducts 14
and 15 (63% and 49% yield, respectively) (Scheme 3). In case of the tripropargylamine
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nucleoside 2a both terminal alkyne groups underwent click functionalization leading to the
double click adduct 13 in 68% yield within 3 h.
7
8
9
Scheme 3. Synthesis of Pyrene Click Conjugatesa
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13
14
15
16
17
18
19
20
21
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23
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N
N
N
N
N3
O
N
N
i) 68%, 3h
N
N
N
O
ii) 67%, 24h
N
N
N
H2N
HO
O
N
+
N
N
12
N
H2N
HO
HO
O
2a
HO
13
N
N
N
N
N
O
N
O
N
O
N
O
N
N
N
N
O
N
N
O
N
N
O
N
N
O
N
N
N
N
H2N
HO
H2N
HO
H2N
HO
H2N
HO
i)
63%, 16h
i)
49%, 16h
12
+
12
+
HO
HO
HO
HO
3d
14
3a
15
aReagents and conditions: (i) CuSO4•5H2O, sodium ascorbate, t-BuOH/THF/H2O (3:1:1); (ii)
CuSO4•5H2O, t-BuOH/THF/H2O (3:1:1).
Usually, copper(I) is used as catalyst to promote the azide alkyne click reaction and to control
regioselectivity.5,6 However, the reaction can be also performed with copper(II) when the
copper ions are chelated.14 Earlier, we reported on a stepwise click reaction employing a
pyridine bis-azide.14e This click reaction was performed with copper(II) as catalyst which
chelates to the azido group next to a pyrimidine nitrogen. Only this azido group formed a
click product with an alkyne. Apparently, in an initial phase the chelated copper(II) is reduced
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to copper(I), thereby forming the triazole click product. Only a mono click product was
formed as the reaction is fast on this site compared to the site of the non-chelating azido
group. Later, the second azido group could be functionalized by a second click reaction under
classical copper(I) catalysis making the protocol to a stepwise process in which different
alkynes can be used in each step.14 In this stepwise process, the chelation of copper(II) is
crucial for the reduction of copper(II) to copper(I). This shows that chelated copper(II) ions
can be used to perform click reactions. The use of copper(II) in click reactions without using
ascorbic acid has also been reported for other cases in which copper(II) ions were chelated by
ligands.14 As only copper(I) is able to catalyze the click reaction the reduction of copper(II) is
always required.
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Whitesides et al. described an autocatalytic cycle for the copper azide alkyne cycloaddition
(CuAAC) reaction performed on tripropargylamine and 2-azidoethanol.15 The reaction used
copper(II) instead of copper(I). The reaction is promoted by (i) coordinating copper(II) and its
reduction to copper(I) and (ii) by enhancing the catalytic reactivity of chelated copper(I) in
the cycloaddition step. After an initiation phase in which copper(II) is reduced to copper(I), an
autocatalytic cycle starts in which click products catalyze the click reaction. An autocatalytic
cycle for a regular click reaction was already suggested by Fokin16a, Devaraj for self-
reproducing catalyst driving repeated phospholipid synthesis16b and Binder for multivalent
poly(acrylate)s and poly(isobutylene)s.16c Single-crystal X-ray diffraction of the Cu(I)
complex of TBTA revealed an unusual dinuclear dication with one triazole unit bridging two
metal centers. The structure of the complex of TBTA with Cu(II) in the crystalline state is
trigonal bipyramidal and can be reduced to the active 'click' catalyst by sodium ascorbate,
copper metal, or other reducing agents.17
As the side chain of the tripropargylamine nucleoside 2a shows structural similarities to the
free tripropargylamine we anticipated a similar reaction sequence can occur when compound
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2a is clicked to pyrene azide. Accordingly, the click reaction of 2a with pyrene azide was
carried out in the presence of Cu(II) in absence of ascorbic acid as reducing agent in a THF/t-
BuOH/water (3:1:1) mixture at rt and were monitored by TLC. In the beginning, a very slow
formation of click products took place compared to the click functionalization with Cu(I)
reported above. After 8 hours the formation of traces of the double and mono click product
along with a major amount of starting material was observed on TLC. In the next 12 h,
prominent formation of the double click adduct 13 took place together with the mono click
product and starting material. After additional 4 hours, the double click reaction was complete
(total reaction time 24 h) and the conjugate 13 was isolated in 67% yield (Scheme 3).
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13
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R
R
N
N
N
N
N
Cu(II) Cu(I)
N
N
(I)
R
O
N
Cu
N
Glaser coupling
or solvent oxidation
N
N
N
-
RCH2 N3
N
N
N
O
N
O
N
-
RCH2
N3, THF/t-BuOH/H2O
N
S
THF/t-BuOH/H2O
H2N
N
N
N
N
2a
+
N
N
H2N
H2N
S
S
13
Autocatalytic function
as copper(I) complex
R
R
N3
N
N
N
N
N
N
-
RCH2 N3
=
(II)
(I)
Cu(II) Cu(I)
Chelate complex
THF/t-BuOH/H2O
Cu
Cu
N
N
O
O
12
N
N
N
N
N
S: 2'-Deoxy--D-ribofuranosyl
N
H2N
N
H2N
N
S
S
Figure 3. Schematic view on the reduction of copper(II) and the autocatalytic function of
compound 13 copper(I) complex. Copper (I) and copper (II) complexes can display different
structures as reported.17
We anticipate that in an initial phase copper(II) is reduced to copper(I) by oxidation of two
alkynes (Glaser coupling) or solvent molecules as it has been reported by Whitesides.15 The
so-formed copper(I) catalyzes the click reaction to a monofunctional click product which was
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not isolated but detected on TLC. Copper(II) can be reduced to copper(I) as free ion or
chelated by the click adduct. The copper(II) complex might be more easily reduced as the
non-chelated copper(II) ions. The copper(I) click product complex can catalyze the next click
reaction. As soon as the first click products were formed an autocatalytic cycle starts. The
autocatalytic cycle forms more and more of the active Cu(I) complex which accelerates the
reaction in an autocatalytic way. So, the copper(I) click product catalyst is generated during
product formation. Indeed, addition of the double click product 13 in an early stage of the
reaction accelerates the reaction. Autocatalysis plays a major role in the processes of life.
Here, it is illustrated by a copper catalyzed click reaction performed on a dendronized
nucleoside. This process should not take place in the case of the 1,7-octadiyne nucleoside 3a
or the ethynyl nucleoside 3d. In fact, click reactions performed on 3a and 3d using copper(II)
without reducing agent are very sluggish.
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The identity of click products was confirmed by ESI-TOF and 1H, 13C{1H} and 2D NMR
spectra. A signal for the triazole hydrogen H-C(5) (δH = 8.49-8.59 ppm) appeared, whereas
the signals for the acetylenic protons for 2a (δH 3.23 ppm), 3a (δH 2.77 ppm) and 3d (δH 4.58
ppm) disappeared. In the 13C{1H} NMR spectra the absence of the two terminal C C carbon
signals and the appearance of two new double bond carbon signals for the 1,2,3-triazole
moiety are observed (Table S1, Supporting Information).
Fluorescence of Pyrene Click Conjugates. Having the pyrene click conjugates 13-15 in
hand their fluorescence properties were determined with respect to linker structure, solvent
changes and related 7-deazapurine nucleosides. First, the fluorescence of the pyrene click
conjugates 13-15 with ethynyl, octadiynyl and tripropargyl side chains was measured in
methanol (Figure 4). In all three cases the excitation wavelength was 341 nm. The
fluorescence monomer emission of the pyrene residues was the lowest for the tripropargyl
compound 13 and the highest for the octadiynyl nucleoside 15. Furthermore, conjugate 15
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with the octadiynyl linker shows high fluorescence in water (Figure 5a), whereas for 13 and
14 the fluorescence in water is low (Figures 5c,e). Apparently, a sufficient linker length is
important for the solvation with water molecules or an intramolecular interaction between the
nucleobase and the dye is controlled by the linker. The tripropargylamino nucleoside carrying
two pyrene residues shows excimer fluorescence (Figure 5e).
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Excimer fluorescence occurs when one pyrene molecule in the excited state forms a contact
dimer with a second molecule in the ground state.18 In the bis-click adduct of the
tripropargylamine nucleoside 13 the two pyrene residues are linked to the nucleobase by short
flexible chains. Thus, in solution the local concentration of the chromophores is greatly
enhanced compared to monochromophoric systems and excimer emission can be detected at
low concentration. According to observations on non-nucleoside systems, the extent of
excimer emission is limited by the probability of the molecule to reach a conformation
suitable for excimer emission. Conformational changes of the connecting chains have to be
faster than the excimer forming step.18b Furthermore, it has been discussed that the pyrene
residues have to be apart when light is absorbed and the excitation is localized in only one of
the two pyrene residues forming the excimer.18b This is actual the case for the bis-click adduct
13 and thus the nucleoside is attractive to be used as environmentally sensitive reporter.
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Figure 4. Fluorescence emission spectra of the 5-aza-7-deazaguanine pyrene click conjugates
13-15 measured in MeOH (10 µM) and for 15 in water (5 µM). Excitation wavelength was
341 nm for 13-15 in MeOH and 344 nm for 15 in water.
Next, the fluorescence dependency of the click conjugates was evaluated in a series of
different solvents. According to Figure 5, the fluorescence among the 5-aza-7-deazaguanine
conjugates showed significant differences. Strong fluorescence was observed for the
octadiynyl conjugate 15 in water, whereas the conjugates 13 and 14 show only weak
fluorescence. For all three compounds the fluorescence is high in dioxane, but the order of
fluorescence intensity changes in DMF, DMSO, MeOH and MeCN. Also, Stoke’s shifts were
measured and quantum yields were determined (Table 1).
For the tripropargylamino conjugate 13, the intensity ratio of monomer to excimer emission is
the highest in aprotic polar dioxane (IM/IE: ~2:1), whereas the ratio is almost 1:1 in polar
protic solvents like MeOH and water and is altered in unipolar aprotic MeCN (IM/IE:1:2)
The highest monomer emission is observed in dioxane and the highest excimer emission is
found in DMSO. In water the system shows low monomer and excimer emission. Very little
changes regarding wavelength are observed among the conjugates in the various solvents.
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a)
a
b)
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d)
c)
e)
f)
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
O
N
O
N
O
N
O
N
O
N
O
N
N
N
O
HN
H2N
HN
H2N
N
N
O
HN
N
N
O
N
N
N
N
N
N
H2N
HO
H2N
HO
H2N
HO
H2N
HO
O
O
O
HO
HO
HO
HO
HO
HO
HO
HO
13
18
15
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Figure 5. (a) Fluorescence emission spectra of 5-aza-7-deazaguanine conjugates 15 (a), 14 (c)
and 13 (e) as well as 7-deazaguanine conjugates 16 (b), 17 (d) and 18 (f) measured in various
solvents with a nucleoside concentration of 10 µM. For 15 in water the nucleoside
concentration was 5 µM.
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Table 1. Photophysical Data of Click Conjugates 13-18 Measured in Solvents of Different
Polaritya
λabs, max
Ex [nm]
342
344
343
340
341
344
λmax, em
Em [nm]
395
396
396
395
395
396
Stokes shift
(Δν)b cm-1]
3900
d
Solvent
Φc
IM/IE
Dioxane
DMSO
DMF
MeCN
MeOH
Water
0.123
0.123
0.101
0.060
0.043
0.337
3800
3900
4100
4000
15
16
14
17
3800
Dioxane
DMSO
DMF
MeCN
MeOH
Water
343
344
343
341
340
341
395
396
396
395
395
395
3800
3800
3900
4000
4100
4000
0.026
0.011
0.014
0.008
0.009
0.006
Dioxane
DMSO
DMF
MeCN
MeOH
Water
343
344
343
341
341
341
395
396
395
395
395
395
3800
3800
3800
4000
4000
4000
0.124
0.014
0.011
0.028
0.052
0.174
Dioxane
DMSO
DMF
MeCN
MeOH
Water
342
344
343
341
341
341
395
396
396
394
394
395
3900
3800
3900
3900
3900
4000
0.021
0.017
0.012
0.007
0.007
0.013
Monomer
Excimer
Dioxane
DMSO
DMF
MeCN
MeOH
Water
343
344
343
341
341
347
395
397
396
395
396
396
471
477
477
474
472
478
7900
8100
8200
8200
8100
7900
0.175
0.105
0.111
0.058
0.080
0.045
3:1
1.2:1
1:1
1:2
1:1.3
1:1
13
18
Dioxane
DMSO
DMF
MeCN
MeOH
342
343
344
340
340
395
396
396
395
395
478
477
477
472
471
8300
8200
8100
8200
8200
0.069
0.052
0.030
0.034
0.037
2.2:1
1.3:1
1.8:1
1:2
1:2
Water
341
395
478
8400
0.018
1:1
a The concentration of nucleosides was 10 µM. For 15 in water the concentration was 5 µM. b The
Stokes shift was calculated from the equation ΔEphoton= hc(1/λabs max
– 1/ λmax, em). c The fluorescence
,
quantum yields (Φ) were calculated using quinine sulfate (1 µM) in 0.1 M H2SO4 (ΦSt = 0.54). d IM:
monomer intensity at the monomer emission maximum. IE: excimer intensity at the excimer emission
maximum.
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Then, the solvent dependent fluorescence of the 5-aza-7-deazaguanine nucleoside conjugates
were compared with 7-deazaguanine conjugates 16-1819 having exactly the same structure but
with nitrogen in position-7 and not in position-5 (Figure 5). It is apparent, that in all solvents
the steady state fluorescence of the 5-aza-7-deazaguanine conjugates is significantly higher
than the fluorescence of the 7-deazaguanine compounds (Figure 5). Also, the quantum yields
shown in Table 1 differ significantly in both series with always higher quantum yields for the
5-aza-7-deazaguanine nucleosides.
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Fluorescence quenching can be induced by fluorescence resonance energy transfer (FRET) or
charge separation (intramolecular electron transfer or hole transfer) between the dye and the
nucleobase.20 The low oxidation potential of 7-deazaguanine nucleosides was made
responsible for its strong fluorescence quenching caused by a charge transfer between base
and the dye. Apparently, pyrene cannot be oxidized by the 5-aza-7-deazaguanine base
resulting in higher fluorescence of its click products.
CONCLUSION
The synthesis of 5-aza-7-deazaguanine nucleosides with linear alkynyl chains or dendritic
triprogargylamino residues was performed. Various sugar residues were introduced at the
nucleoside glycosylation site-9. From 5-aza-7-deazaguanine nucleosides with ribo, 2’-
deoxyribo and fluoroarabino sugar residues clickable derivatives were synthesized for the
copper(I) promoted Huisgen-Sharpless-Meldal cycloaddition. Ethynyl, 1,7-octadiynyl and
tripropargylamino side chains were introduced in the 7-position of the nucleobase by
Sonogashira cross-coupling employing 5-aza-7-deaza-7-iodoguanine nucleosides and
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corresponding alkynes. TBTA shortened the reaction times of Sonogashira cross-coupling
reactions but led to lower or marginally higher yields.
7
8
9
Click reactions with tripropargylamino nucleosides and 1-azidomethylpyrene were
significantly faster and higher yielding with respect to nucleosides carrying linear alkynyl side
chains. Furthermore, the reaction could be performed with copper(II) as catalyst, whereas
copper(I) is used for regular click reactions.
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An autocatalyzed cycle is suggested in which copper(I) is chelated by the click product and
the next click reaction is catalyzed by this copper(I) click complex. We observed that addition
of click products at an early stage of the reaction accelerated product formation. This fulfills
the criteria of autocatalysis.
Pyrene click adducts of 5-aza-7-deazaguanine nucleosides with linear and dendronized side
chains show significantly higher fluorescence than previously reported 7-deazaguanine
conjugates and similar intensities as 7-deaza-8-azaguanine compounds.20d Due to the absence
of a hydrogen atom at nitrogen-1 the recognition face of 5-aza-7-deazaguanine differs from
that of the other guanine nucleosides. Consequently, other base pairs are formed that can now
be decorated with fluorescent tags.1b Furthermore, the clickable nucleosides reported in this
work can be utilized to prepare libraries of antiviral active 5-aza-7-deazaguanine
nucleosides21 or being applicable for diagnostic or imaging purposes in DNA biotechnology.
EXPERIMENTAL SECTION
General Methods and Materials. All chemicals and solvents were of laboratory grade as
obtained from commercial suppliers and were used without further purification. Thin-layer
chromatography (TLC) was performed on TLC aluminium sheets covered with silica gel 60
F254 (0.2 mm). Flash column chromatography (FC): silica gel 60 (40-60 μM) at 0.4 bar. UV-
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spectra were recorded on a UV-spectrophotometer: λmax (ε) in nm, ε in dm3 mol-1 cm-1.
13C{1H} NMR spectra were measured at 599.74 MHz or 399.89 MHz for 1H and 150.82
MHz, 100.56 MHz or 75.47 MHz for 13C. 1H-13C correlated (HMBC, HSQC) NMR spectra
were used for the assignment of the 13C signals (Table S1, Supporting Information). The J
values are given in Hz; δ values in ppm relative to Me4Si as internal standard. For NMR
spectra recorded in DMSO-d6, the chemical shift of the solvent peak was set to 2.50 ppm for
1H NMR and 39.50 ppm for 13C NMR. ESI-TOF mass spectra of nucleosides were recorded
on a Micro-TOF spectrometer.
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Fluorescence Studies. Fluorescence measurements were performed with a F-2500
fluorescence spectrophotometer equipped with a cooling device or a F-7000 fluorescence
spectrophotometer (Hitachi, Tokyo, Japan). Fluorescence spectra of nucleoside ‘click’
conjugates were measured in different solvents. For solubility reasons, all click conjugates (2-
4 mg) were dissolved in 0.5 mL of DMSO and then diluted with 9.5 mL of methanol. This
solution was used as a stock solution (400 µmol). All measurements were performed with
identical concentrations of 10 µM. Quantum yields Φ were determined by using quinine
sulfate in 0.1 M sulfuric acid as a standard with a known Φ of 0.54.22
The quantum yield Φ (x) of the unknown compound can be calculated by the following
equation:
Φ (x) = Φ (ST)(AST/AX)(FX/FST)(η2X/η2ST)
where Φ (ST) is the quantum yield of the standard, A is the absorbance at the excitation
wavelength, F is the integrated area of the emission curve, the subscripts X and ST refer to
unknown and standard and η is the refractive index of solvent.
3,5-Di-O-benzoyl-2-deoxy-2-fluoro-α-D-arabinofuranosyl Bromide (6).9 Compound 6 was
prepared according to a literature protocol.9 To a solution of 1,3,5-tri-O-benzoyl-2-deoxy-2-
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fluoro-α-D-arabinofuranose 5 (1.5 g, 3.21 mmol) in CH2Cl2 (15 mL) was added a 30% soln.
of HBr in acetic acid (1.5 mL). The mixture was stirred at rt for 16 h and evaporated to
dryness. The remaining syrup was dissolved in CH2Cl2 (20 mL), washed with sat. aq.
NaHCO3 soln. (10 mL), dried over Na2SO4, filtrated and concentrated to syrup. The syrup of
compound 6 was used for the next step without any further purification.
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2-[(N2-Isobutyryl)amino]-8-(3,5-di-O-benzoyl-2-deoxy-2-fluoro-β-D-arabinofuranosyl)-
6-iodo-imidazo[1,2-a]-s-triazin-4(8H)-one (7) and 2-[(N2-Isobutyryl)amino]-8-(3,5-di-O-
benzoyl-2-deoxy-2-fluoro-β-D-arabinofuranosyl)-6-iodo-imidazo[1,2-a]-s-triazin-4(8H)-
one (8). The halogenose 6 was dissolved in CH3CN (8 mL) and introduced into a suspension
of compound 4 (500 mg, 1.44 mmol) and DBU (263 mg, 244 µL, 1.73 mmol) in MeCN (10
mL). Then, the reaction mixture was stirred at ambient temperature for 16 h and evaporated to
dryness. After FC (silica gel, column 12 x 4 cm, CH2Cl2/MeOH 100:1→95:5) an anomeric
mixture of 7 and 8 (723 mg, 73%) was obtained as colorless foam. 1H NMR of the anomeric
mixture showed a 1:1 β to α ratio.
2-[(N2-Isobutyryl)amino]-8-(3,5-di-O-benzoyl-2-deoxy-2-fluoro-β-D-arabinofuranosyl)-
6-iodo-imidazo[1,2-a]-s-triazin-4(8H)-one (7). The anomeric mixture of 7/8 (723 mg, 73%)
was dissolved in MeOH (15 mL) under gentle warming for 3 min. After cooling to rt, the β-D
anomer 7 precipitated as white powder. The precipitate was filtered-off and washed with
MeOH (5 mL) affording the compound 7 (299 mg, 30%) as colorless solid. TLC (silica gel,
CH2Cl2/MeOH, 95:5) Rf 0.4. λmax (MeOH)/nm 283 (ε/dm3 mol−1 cm−1 11 700). 1H NMR
(DMSO-d6, 600 MHz): δ 1.04 (dd, J = 6.9, 1.6 Hz, 6H, (CH3)2CH), 2.89 (hept, J = 6.8 Hz,
1H, CH(CH3)2), 4.65 – 4.80 (m, 3H, H-4', H-5'), 5.72 (ddd, J = 50.5, 4.3, 2.7 Hz, 1H, H-2'),
5.90 (ddd, J = 18.7, 4.6, 2.6 Hz, 1H, H-3'), 6.45 (dd, J = 16.8, 4.2 Hz, 1H, H-1'), 7.50 – 7.54
(m, 2H, arom. H), 7.56 – 7.61 (m, 2H, arom. H), 7.65 – 7.69 (m, 2H, arom. H), 7.71 – 7.75
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(m, 1H, H-8), 7.97 – 8.01 (m, 2H, arom. H), 8.05 – 8.08 (m, 2H, arom. H), 10.42 (s, 1H, NH).
13C{1H}NMR (DMSO-d6,151 MHz): δ 19.0, 19.0, 34.6, 59.3, 63.7, 75.9, 76.1, 78.4, 78.5,
81.9, 82.1, 92.3, 93.5, 123.9, 123.9, 128.6, 128.8, 128.8, 129.2, 129.6, 133.5, 134.0, 150.1,
150.3, 160.0, 164.7, 165.4, 175.7. HRMS (ESI-TOF) m/z: [M + Na+] calcd for
C28H25FIN5O7Na, 712.0675; found, 712.0672.
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2-Amino-8-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)-6-iodo-8H-imidazo[1,2-a]-s-triazin-
4-one (1c). From 7: Compound 7 (100 mg, 0.15 mmol) was suspended in 7N NH3/MeOH (20
mL) and the mixture was stirred at rt overnight. The solvent was evaporated, and the residue
was purified by FC (silica gel, column 10 x 2 cm, CH2Cl2/MeOH 100:1→85:15). From the
main zone compound 1c (54 mg, 87%) was obtained as colorless solid. TLC (silica gel,
CH2Cl2/MeOH, 90:10) Rf 0.4. λmax (MeOH)/nm 266 (ε/dm3 mol−1 cm−1 13 500), 260 (12600).
1H NMR (DMSO-d6, 600 MHz): δ 3.56 – 3.70 (m, 2H, H-5'), 3.80 (q, J = 4.7 Hz, 1H, H-4'),
4.33 (dt, J = 18.5, 4.7 Hz, 1H, H-3'), 5.08 – 5.21 (m, 2H, H-2', HO-5'), 5.96 (d, 1H, HO-3'),
6.14 (dd, J = 13.4, 4.5 Hz, 1H, H-1'), 7.01 and 7.05 (2s, 2H, NH2), 7.49 (d, J = 1.8 Hz, 1H, H-
8). 13C{1H} NMR (DMSO-d6, 151 MHz): δ 57.6, 60.0, 71.8, 71.9, 81.1, 81.2, 83.5, 83.6, 94.5,
95.7, 121.9, 121.9, 150.0, 150.4, 150.4, 164.2. HRMS (ESI-TOF) m/z: [M + Na+] calcd for
C10H11FIN5O4Na, 433.9732; found, 433.9720.
From 11: Compound 11 (100 mg, 0.14 mmol) was dissolved in CH2Cl2 (5 mL) and treated
with trichloroacetic acid (1 mL of a 3% soln in CH2Cl2). The mixture was stirred for 16 h at
rt. Then, the reaction mixture was neutralized with triethylamine (50 µL) and the solvent was
evaporated. The remaining residue was applied to FC (silica gel, column 10 x 3 cm, CH2Cl2/
MeOH, 100:0→85:15). From the main zone, compound 1c was obtained as a colorless solid
(49 mg, 84%). Analytical data were identical to those described above.
Separation of the Anomeric Glycosylation Mixture of 7/8 by 4,4’-Dimethoxytritylation.
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In a separate glycosylation experiment, the mother liquor obtained from crystallization of the
β-D anomer 7 was evaporated to a colorless foam (424 mg, 0.62 mmol). Then, 7N
NH3/MeOH (100 mL) was added and the reaction mixture was stirred overnight at rt. The
solvent was evaporated and the remaining residue was applied to FC (silica gel, column 12 x
3 cm, CH2Cl2/MeOH 100:1→85:15). Evaporation of the main zone gave an anomeric mixture
of 9 and 1c as an off-white solid (216 mg, 85%). Then, 140 mg (0.34 mmol) of this mixture
were dissolved in pyridine (5 mL), 4,4’-dimethoxytritylchloride (231 mg, 0.68 mmol) was
added in three equal portions and the reaction mixture was stirred for 3.5 h at ambient
temperature. Then, the solvent was evaporated and the remaining syrup was dissolved in
CH2Cl2 (20 mL) washed with sat. aq. NaHCO3 soln. (10 mL), dried over Na2SO4, filtrated
and applied to FC (silica gel, column 10 x 2 cm, CH2Cl2/MeOH, 100:0→97:3).
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2-Amino-8-[2-deoxy-2-fluoro-5-O-(4,4′-dimethoxytriphenylmethyl)-α-D-
arabinofuranosyl]-6-iodo-8H-imidazo[1,2-a]-s-triazin-4-one (10). From the faster
migrating zone, compound 10 was obtained as colorless foam (145 mg, 60%). TLC (silica gel,
CH2Cl2/MeOH, 95:5) Rf 0.4. λmax (MeOH)/nm 270 (ε/dm3 mol−1 cm−1 15 600). 1H NMR
(DMSO-d6, 600 MHz): δ 3.09 – 3.15 (m, 2H, H-5'), 3.73 and 3.74 (2s, 6H, OCH3) ; 4.34 (d, J
= 19.5 Hz, 1H, H-4'), 4.47 (q, J = 5.0 Hz, 1H, H-3'), 5.39 (dt, J = 50.9, 2.7 Hz, 1H, H-2’), 6.03
(d, J = 3.8 Hz, 1H, 3’-OH), 6.07 (dd, J = 15.9, 2.4 Hz, 1H, H-1'), 6.78-6.83 (m, 4H, arom. H),
7.00 and 7.03 (2s, 2H, NH2), 7.16-7.29 (m, 5H, arom. H), 7.32 (t, J = 7.7 Hz, 2H, arom. H),
7.38-7.44 (m, 2H, arom. H), 7.52 (s, 1H, H-8). 13C{1H} NMR (DMSO-d6, 151 MHz): δ 54.9,
55.0, 57.7, 62.8, 73.7, 73.9, 85.3, 85.3, 85.4, 86.3, 86.6, 98.1, 99.9, 112.6, 113.2, 121.6, 126.7,
127.6, 127.8, 129.6, 129.6, 135.3, 135.4, 144.7, 150.0, 150.5, 158.1, 164.1. HRMS (ESI-TOF)
m/z: [M + Na+] calcd for C31H29FIN5O6Na, 736.1039; found, 736.1032.
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2-Amino-8-[2-deoxy-2-fluoro-5-O-(4,4′-dimethoxytriphenylmethyl)-β-D-
arabinofuranosyl]-6-iodo-8H-imidazo[1,2-a]-s-triazin-4-one (11). From the slower
migrating zone the β-D- anomer 11 was obtained (24 mg, 10%) as colorless foam. TLC (silica
gel, CH2Cl2/MeOH, 95:5) Rf 0.3. λmax (MeOH)/nm 267 (ε/dm3 mol−1 cm−1 16 000). 1H NMR
(DMSO-d6, 600 MHz): δ 3.16 – 3.30 (2m, 2H, H-5'), 3.74 (s, 6H, OCH3), 4.00 (d, 1H, H-4’),
4.33 (dq, J = 18.4, 4.6 Hz, 1H, H-3'), 5.13 (dt, J = 52.2, 3.9 Hz, 1H, H-2'), 6.00 (d, J = 4.7 Hz,
1H, OH-3'), 6.19 (dd, J = 15.2, 4.3 Hz, 1H, H-1’), 6.85 – 6.96 (m, 4H, arom. H), 7.03 and
7.07 (2s, 2H, NH2), 7.16 (d, J = 2.0 Hz, 1H, H-8), 7.20 – 7.36 (m, 5H, arom. H), 7.32 (dd, J =
8.5, 7.1 Hz, 2H, arom. H), 7.37 – 7.46 (m, 2H, arom. H), 7.52 (s, 1H, H-8). 13C{1H} NMR
(DMSO-d6, 151 MHz) : δ 55.0, 55.1, 57.8, 63.1, 72.8, 73.0, 81.0, 81.2, 82.0, 82.0, 85.5, 94.0,
95.9, 113.2, 121.5, 126.7, 127.6, 127.8, 129.6, 129.6, 135.3, 135.4, 144.7, 149.9, 150.6, 158.1,
164.2. HRMS (ESI-TOF) m/z: [M + Na+] calcd for C31H29FIN5O6Na, 736.1039; found,
736.1028.
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2-Amino-8-(2-deoxy-2-fluoro-α-D-arabinofuranosyl)-6-iodo-8H-imidazo[1,2-a]-s-triazin-
4-one (9). Compound 10 (100 mg, 0.14 mmol) was dissolved in CH2Cl2 (5 mL) and treated
with trichloroacetic acid (1 mL of a 3% soln. in CH2Cl2) for 16 h at rt. The reaction mixture
was neutralized with triethylamine (100 µL) and the solvent was evaporated. The remaining
residue was applied to FC (silica gel, column 10 x 3 cm, CH2Cl2/ MeOH, 100:0→85:15).
From the main zone, compound 9 was obtained as a colorless solid (51 mg, 88%). TLC (silica
gel, CH2Cl2/MeOH, 9:1) Rf 0.3. λmax (MeOH)/nm 266 (ε/dm3 mol−1 cm−1 13 200), 260
(12200). 1H NMR (DMSO-d6, 600 MHz): δ 3.45 – 3.56 (m, 2H, H-5'), 4.21 (q, J = 5.0 Hz,
1H, H-4'), 4.28 – 4.36 (m, 1H, H-3'), 5.01 (t, J = 5.7 Hz, 1H, OH-5'), 5.39 (dt, J = 51.2, 3.1
Hz, 1H, H-2'), 5.96 – 5.99 (m, 1H, OH-3'), 6.03 (dd, J = 15.9, 2.8 Hz, 1H, H-1'), 7.01 and
7.03 (2s, 2H, NH2), 7.51 (s, 1H, H-8). 13C{1H} NMR (DMSO-d6, 151 MHz): δ 57.8, 60.5,
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73.1, 73.2, 86.0, 86.2, 86.7, 86.7, 98.6, 99.8, 121.5, 150.0, 150.6, 164.1. HRMS (ESI-TOF)
m/z: [M + Na+] calcd for C10H11FIN5O4Na, 433.9732; found, 433.9726.
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General Procedure for Sonogashira Cross-Coupling of Nucleosides 1a-c with
Tripropargylamine without TBTA. To a suspension of the particular nucleoside (0.25
mmol, 1 eq.) in DMF (2 mL) in an oven dried round bottom flask Pd(PPh3)4 (30 mg, 0.025
mmol, 0.1 eq.), Et3N (69 µL, 0.50 mmol, 2 eq.), CuI (10 mg, 0.050 mmol, 0.2 eq.), and
tripropargyl amine (360 µL, 2.50 mmol, 10 eq.) were added. The reaction mixtures were
stirred at rt for 6 h. Then, the solvent was evaporated and the remaining residues were purified
by FC. From the main zones, compounds 2a-c were obtained.
2-Amino-8-(2-deoxy-β-D-erythro-pentofuranosyl)-6-{3-[di(prop-2-yn-1-yl)amino]prop-1-
yn-1-yl}-8H-imidazo[1,2-a]-s-triazin-4-one (2a). As described above with compound 1a.
Purification by FC (silica gel, column 15 x 2 cm, CH2Cl2/MeOH, 87:13) and evaporation of
the main zone gave compound 2a (62 mg 63%) as pale yellow solid. TLC (silica gel, CH2Cl2/
MeOH, 9:1) Rf 0.4. λmax (MeOH)/nm 273 (ε/dm3 mol−1 cm−1 16 200). 1H NMR (DMSO-d6,
600 MHz): δ 2.17 (ddd, J = 13.2, 6.1, 3.4 Hz, 1H, H-2'α), 2.36 (ddd, J = 13.1, 7.3, 5.8 Hz, 1H,
H-2'β), 3.23 (t, J = 2.4 Hz, 2H, 2 x C≡CH), 3.47 (d, J = 2.5 Hz, 4H, 2 x CH2), 3.48 – 3.57 (m,
2H, H-5'), 3.63 (s, 2H, CH2), 3.79 (td, J = 4.3, 2.8 Hz, 1H, H-4'), 4.30 (dq, J = 6.7, 3.5 Hz,
1H, H-3'), 4.98 (t, J = 5.4 Hz, 1H, OH-5'), 5.29 (d, J = 3.9 Hz, 1H, OH-3'), 6.14 (dd, J = 7.2,
6.1 Hz, 1H, H-1'), 7.00 and 7.02 (2s, 2H, NH2), 7.78 (s, 1H, H-8). 13C{1H} NMR (DMSO-d6,
151 MHz): δ 39.1, 41.0, 41.2, 42.0, 61.3, 70.2, 72.9, 76.0, 79.1, 82.9, 87.7, 90.9, 104.9, 119.5,
149.7, 150.0, 165.0. HRMS (ESI-TOF) m/z: [M + Na+] calcd for C19H20N6O4Na, 419.1438;
found, 419.1428.
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Sonogashira Cross-Coupling of Compound 1a with Tripropargylamine and TBTA: To a
suspension of compound 1a in DMF (2 mL) in an oven dried round bottom flask Pd(PPh3)4
(30 mg, 0.025 mmol, 0.1 eq.), Et3N (69 µL, 0.50 mmol, 2 eq.), CuI (10 mg, 0.050 mmol, 0.2
eq.), TBTA {tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amin)}(26.5 mg, 0.2 eq.) and
tripropargylamine (360 μL, 2.50 mmol, 10 eq.) were added. The reaction time was 2.5 h until
most of the staring material was consumed. The solvent was evaporated, and the remaining
residue was applied to FC (silica gel, column 15 × 2 cm, CH2Cl2/MeOH, 87:13). From the
main zone, compound 2a was obtained as pale yellow solid (49 mg, 49%). Analytical data
were identical to those described above.
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2-Amino-8-(β-D-ribofuranosyl)-6-{3-[di(prop-2-yn-1-yl)amino]prop-1-yn-1-yl}-8H-
imidazo[1,2-a]-s-triazin-4-one (2b). As described above without TBTA and compound 1b.
After purification by FC (silica gel, column 15 x 2 cm, CH2Cl2/MeOH, 87:13) compound 2b
(54 mg 52%) was obtained as pale yellow solid. TLC (silica gel, CH2Cl2/ MeOH, 85:15) Rf
0.4. λmax (MeOH)/nm 273 (ε/dm3 mol−1 cm−1 15 900). 1 H NMR (DMSO-d6, 400 MHz): δ 3.23
(t, J = 2.4 Hz, 2H, 2 x C≡CH), 3.47 (d, J = 2.5 Hz, 4H, 2 x CH2), 3.53 (ddd, J = 12.0, 5.4, 3.8
Hz, 1H, H-5’), 3.60 (dd, J = 5.3, 3.9 Hz, 1H, 2H-5'), 3.64 (s, 2H, CH2), 3.87 (q, J = 3.8 Hz,
1H, H-4'), 4.06 (td, J = 4.8, 3.6 Hz, 1H, H-3'), 4.24 (q, J = 5.3 Hz, 1H, H-2'), 5.07 (t, J = 5.4
Hz, 1H, OH-5'), 5.15 (d, J = 4.7 Hz, 1H, OH-3'), 5.46 (d, J = 5.5 Hz, 1H, OH-2'), 5.76 (d, J =
5.5 Hz, 1H, H-1'), 7.02 and 7.04 (2s, 2H, NH2), 7.82 (s, 1H, H-8). 13C{1H} NMR (DMSO-d6,
101 MHz): δ 41.1, 42.0, 60.9, 70.0, 72.8, 73.6, 75.9, 79.0, 85.3, 86.4, 91.0, 105.0, 119.5,
149.7, 150.5, 165.0. HRMS (ESI-TOF) m/z: [M + Na+] calcd for C19H20N6O5Na, 435.1387;
found, 435.1384.
2-Amino-8-(2-deoxy-2-fluoro-α-D-arabinofuranosyl)-6-{3-[di(prop-2-yn-1-
yl)amino]prop-1-yn-1-yl}-8H-imidazo[1,2-a]-s-triazin-4-one (2c). As described above
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without TBTA and compound 1c. After purification by FC (silica gel, column 15 x 2 cm,
CH2Cl2/MeOH, 87:13) compound 2b (61 mg 59%) was obtained as pale yellow solid. TLC
(silica gel, CH2Cl2/ MeOH, 85:15) Rf 0.4. λmax (MeOH)/nm 272 (ε/dm3 mol−1 cm−1 15 200).
1H NMR (DMSO-d6, 600 MHz): δ 3.23 (t, J = 2.4 Hz, 2H, 2 x C≡CH), 3.47 (d, J = 2.5 Hz,
4H, 2 x CH2), 3.58 – 3.70 (m, 4H, CH2 and 2 x H-5'), 3.82 (q, J = 4.8 Hz, 1H, H-3'), 4.34
(ddd, J = 18.1, 5.3, 4.1 Hz, 1H, H-4'), 5.16 (dt, J = 52.4, 4.3 Hz, 2H, H-2', OH-5'), 6.01 (m,
1H, OH-3'), 6.16 (dd, J = 13.0, 4.5 Hz, 1H, H-1'), 7.06 and 7.11 (2s, 2H, NH2), 7.72 (s, 1H,
H-8). 13C{1H} NMR (DMSO-d6, 151 MHz): δ 41.2, 42.0, 60.0, 71.7, 71.9, 72.6, 76.0, 79.1,
81.1, 81.2, 83.7, 83.7, 91.1, 94.3, 95.6, 95.7, 104.9, 120.4, 149.5, 150.0, 159.0, 162.0, 162.7,
164.9. HRMS (ESI-TOF) m/z: [M + Na+] calcd for C19H19FN6O4Na, 437.1344; found,
437.1344.
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General Procedure for Sonogashira Cross-Coupling of Nucleoside 1a with Linear
Alkynes without TBTA (Method A). To a suspension of the particular nucleoside (0.25
mmol, 1 eq.) in DMF (2 mL) in an oven dried round bottom flask Pd(PPh3)4 (30 mg, 0.025
mmol, 0.1 eq.), Et3N (69 µL, 0.50 mmol, 2 eq.), CuI (10 mg, 0.050 mmol, 0.2 eq.), and the
linear alkyne (for eq., see the particular compound) were added. The reaction mixture was
stirred at rt (for reaction time, see the particular compound). Then, the solvent was evaporated
and the remaining residue was and purified by FC. From the main zone, compounds 3a-c were
obtained.
General Procedure for Sonogashira Cross-Coupling of Nucleosides 1a with Linear
Alkynes with TBTA (Method B). To a suspension of the particular nucleoside (0.25 mmol, 1
eq.) in DMF (2 mL) in an oven dried round bottom flask Pd(PPh3)4 (30 mg, 0.025 mmol, 0.1
eq.), Et3N (67 µL, 0.50 mmol, 2 eq.), CuI (10 mg, 0.050 mmol, 0.2 eq.), TBTA {tris[(1-
benzyl-1H-1,2,3-triazol-4-yl)methyl]amin)}(26.5 mg, 0.2 eq.) and the linear alkyne (for eq.,
29
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