Nucleobase-Functionalized 5-Aza-7-deazaguanine Ribo- A nd 2′-Deoxyribonucleosides: Glycosylation, Pd-Assisted Cross-Coupling, and Photophysical Properties

Article abstract of DOI:10.1021/acs.joc.9b01347

The special nucleobase recognition pattern of 5-aza-7-deazaguanine nucleosides makes them valuable for construction of homo purine DNA, silver-mediated base pairs, and expansion of the four letter genetic coding system. To widen the utility of 5-aza-7-deazaguanine nucleosides, side chains were introduced at position-7 of the nucleobase. As key compounds, 7-iodo nucleosides were synthesized. Nucleobase anion glycosylation of the iodo derivative of isobutyrylated 5-aza-7-deazaguanine with the bromo sugar of 2,3,5-tri-O-benzoyl-1-O-acetyl-d-ribofuranose gave the pure β-D anomeric N-9 glycosylation product (67%), whereas one-pot Vorbrüggen conditions gave only 42% of the iodinated nucleoside. The noniodinated nucleoside was formed in 84%. For the synthesis of 2′-deoxyribonucleosides, anion glycosylation performed with Hoffer's 2′-deoxyhalogenose yielded an anomeric mixture (α-D = 33% and β-D = 39%) of 2′-deoxyribonucleosides. Various side chain derivatives were prepared from nonprotected nucleosides by Pd-assisted Sonogashira or Suzuki-Miyaura cross-coupling. Among the functionalized ribonucleosides and anomeric 2′-deoxyribonucleosides, some of them showed strong fluorescence. Benzofuran and pyrene derivatives display high quantum yields in non-aqueous solvents and solvatochromism. Single-crystal X-ray analysis of 7-iodo-5-aza-7-deaza-2′-deoxyguanosine displayed intermolecular iodo-oxygen interactions in the crystal and channels filled with solvent molecules.

Full text of DOI:10.1021/acs.joc.9b01347

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Article
Nucleobase Functionalized 5-Aza-7-deazaguanine
Ribo- and 2'-Deoxyribonucleosides: Glycosylation, Pd-
Assisted Cross-Coupling and Photophysical Properties
Peter Leonard, Dasharath Kondhare, Xenia Jentgens, Constantin Daniliuc, and Frank Seela
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.9b01347 • Publication Date (Web): 04 Oct 2019
Downloaded from pubs.acs.org on October 7, 2019
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Nucleobase Functionalized 5-Aza-7-deazaguanine Ribo- and 2'-Deoxyribonucleosides:
Glycosylation, Pd-Assisted Cross-Coupling and Photophysical Properties
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a
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a
c
Peter Leonard , Dasharath Kondhare , Xenia Jentgens , Constantin Daniliuc and
Frank Seelaa,b*
aLaboratory of Bioorganic Chemistry and Chemical Biology, Center for Nanotechnology,
b
Heisenbergstrasse 11, 48149 Münster, Germany and Laboratorium für Organische und
Bioorganische Chemie, Institut für Chemie neuer Materialien, Universität Osnabrück,
c
Barbarastrasse 7, 49069 Osnabrück, Germany, Institut für Organische Chemie, Universität
Münster, Corrensstrasse 40, 48149 Münster, 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
Home page: www.seela.net
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ABSTRACT
The special nucleobase recognition pattern of 5-aza-7-deazaguanine nucleosides makes them
valuable for construction of homo purine DNA, silver-mediated base pairs and expansion of
the four letter genetic coding system. To widen the utility of 5-aza-7-deazaguanine
nucleosides, side chains were introduced at position-7 of the nucleobase. As key compounds
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-iodo nucleosides were synthesized. Nucleobase anion glycosylation of the iodo derivative
of isobutyrylated 5-aza-7-deazaguanine with the bromo sugar of 2,3,5-tri-O-benzoyl-1-O-
acetyl-D-ribofuranose gave the pure β-D anomeric N-9 glycosylation product (67%), whereas
one-pot Vorbrüggen conditions gave only 42% of the iodinated nucleoside. The non-iodinated
nucleoside was formed in 84%. For the synthesis of 2’-deoxyribonucleosides anion
glycosylation performed with Hoffer’s 2’-deoxyhalogenose yielded an anomeric mixture (α-D
=
33% and β-D = 39%) of 2’-deoxyribonucleosides. Various side chain derivatives were
prepared from non-protected nucleosides by Pd-assisted Sonogashira or Suzuki-Miyaura
cross-coupling. Among the functionalized ribonucleosides and anomeric 2'-
deoxyribonucleosides some of them showed strong fluorescence. Benzofuran and pyrene
derivatives display high quantum yields in non-aqueous solvents and solvatochromism. A
single-crystal X-ray analysis of 7-iodo-5-aza-7-deaza-2’-deoxyguanosine displayed
intermolecular iodo-oxygen interactions in the crystal and channels filled with solvent
molecules.
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TABLE OF CONTENTS
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O
I
Nucleobase Anion
Glycosylation
O
N
N
O
O
N
I
N
N
N
O
N
N
H
O
BzO
Br
OBz
N
N
BzO
H
H
BzO
BzO
OBz
O
N
N
N
N
NH2
O
HO
HO
R
R = H, OH
Suzuki-Miyaura coupling
Solvent dependent fluorescence
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INTRODUCTION
7
-Deazapurine nucleosides are common surrogates of purine nucleosides and serve as
versatile building blocks for many purposes such as DNA detection, sequencing and
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,2
imaging. Functionalization at the 7-position (purine numbering is used throughout the
manuscript) – a site which is not practical for purine nucleoside functionalization – provides
derivatives with side chains that have sufficient space in DNA as they protrude to the major
3
groove and are therefore well accommodated in the double helix. Side chains can stabilize
3a
3b
the DNA double helix , represent clickable residues , link fluorescent dyes or other reporter
3
c
groups to the nucleobases. Basic work on nucleosides with a pyrrolo[2,3-d]pyrimidine and
4
,5
pyrazolo[3,4-d]pyrimidine nucleosides skeleton was initiated by our laboratory and is
6
ongoing. Compared to this very little is known on nucleosides and oligonucleotides with
imidazo[1,2-a]-s-triazines (5-aza-7-deazapurines) as base having the nitrogen in the
bridgehead position. Only a few ribonucleosides, 2'-deoxyribonucleosides and
7
fluoronucleosides are existing . Side chain derivatives are totally unknown. Most of the
existing work has been reviewed.8
5
-Aza-7-deaza-2'-deoxyguanosine 1 and its α-D-anomer 3 form mismatches with dC. Stable
base pairs are generated when complementary nucleosides provide hydrogen atoms for the
9
formation of tridentated base pairs which are also formed when the base becomes
10
protonated. Homo purine DNA base pairs are developed by 1 with 2’-deoxyguanosine and
7
c
2
'-deoxyisoguanosine (motif I, Figure 1). Base pairs with “protonated” dC analogues are
9
used to expand the genetic alphabet by a six letter code. Also, programmable metal ion
11
mediated base pairs with dC exist in the presence of silver ions (motif III, Figure 1). The
importance of 5-aza-7-deazapurine α-D anomeric nucleosides has been demonstrated by the
formation of DNA with parallel strand orientation and of silver-mediated base pairs.10-12 For
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diagnostic and other purposes side chains are essential that carry reporter groups developing
fluorescence or allow further functionalization.
In this work, the functionalization of 5-aza-7-deazapurine ribonucleosides and 2’-
deoxyribonucleosides was investigated exactly at the same 7-position that has been used for
the functionalization of the widely used pyrrolo[2,3-d]pyrimidine and pyrazolo[3,4-
d]pyrimidine nucleosides (Figure 1).3,4,13 Until now, the only reported 7-functionalized
derivatives of the 5-aza-7-deazaguanine nucleosides are the 7-iodo derivative 4 and its α-D
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7f
anomer 5 prepared by our laboratory some time ago. As 7-iodo nucleosides represent key
compounds for the synthesis of side chain derivatives via cross-coupling reaction, the 7-iodo
ribonucleoside 6 was synthesized and the access of pure anomers of the 2’-
deoxyribonucleosides 4 and 5 was improved. A diversity of side chain derivatives of
anomeric 2'-deoxyribonucleosides and ribonucleosides were synthesized by Suzuki and
Sonogashira cross-coupling reactions performed on the iodo nucleosides 4-6 with a series of
alkynes and boronic acid derivatives (Figure 1). Among them, the benzofuran and pyrene side
chains were found to be particularly valuable as fluorescent reporter groups. Photophysical
properties of various derivatives e.g.7-9 were examined in different solvents and quantum
yields were determined. A single-crystal X-ray analysis of 7-iodo-5-aza-7-deaza-2'-
deoxyguanosine (4) was performed.
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O
O
O
N
4
(6)
5(5) 6(7)
N
O
3(1)N
HO
HN
HN
7
(8)
N
N 8(9)
N
N
H2N
HO
N 8a(4)
H2N
HO
N
H2N
HO
1(3)
HO
N
O
NH2
N
O
O
O
N
N
HO
1 R = H
R = OH
R
HO
HO
c7z8Gd
c7Gd
3
2
pyrrolo[2,3-d]pyrimidine
pyrazolo[3,4-d]pyrimidine
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imidazo[1,2-a][1,3,5]-triazine
Systematic numbering
Purine numbering)
(
H
N
H
H
O
N
H
O
N
H
N
N
O
O
N
N
H
N
N
N
N
N
N
N
N
N
N
O
N
1
H
dC
1
Ag
dC
N
1
N
O
N
H
iG
N
N
S
S
S
N
S
N
H
N
H
H
H
S
S
H
H
motif III
I
motif I
O
motif II
O
N
I
O
HO
N
N
N
N
N
N
N
NH2
H2N
HO
H2N
HO
N
HO
N
O
O
N
N
I
O
HO
OH
HO
5
6
4
O
HO
N
NH2
HO
N
O
N
O
N
O
N
N
N
N
N
N
O
N
H2N
H2N
HO
N
O
O
HO
HO
HO
OH
7
8
9
Figure 1. Various 7-deazaguanine 2’-deoxyribonucleosides and base pairs of 1 with iGd,
protonated dC and a silver-mediated construct. S corresponds to 2’-deoxyribose. iG
corresponds to 2'-deoxyisoguanosine. Fluorescent side chain derivatives 7-9 obtained from 7-
iodinated 5-aza-7-deazaguanine ribo- and 2'-deoxyribonucleosides and anomers 4-6 by cross-
coupling reactions.
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RESULTS AND DISCUSSION
Synthesis of the Anomeric 7-Iodo-5-aza-7-deazaguanine 2'-Deoxyribonucleosides 4 and 5
and the Ribonucleoside 6
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Previously, the syntheses of 7-iodo-2'-deoxyribonucleoside 4 and its α-D anomer 5 have been
7f
described. To this end, the isobutyrylated iodobase 10b was prepared by regioselective
iodination of isobutyrylated 5-aza-7-deazaguanine 10a with iodosuccinimide (→10b).
Following the literature procedure for the preparation of 10b, we were encountered with
difficulties to obtain the clean iodinated base. Single column chromatography was not
sufficient to remove the large amounts of succinimide formed during the reaction. To this end,
a modified procedure was developed. Nucleobase 10a was treated with N-iodosuccinimide as
7
f
described. After solvent removal, the remaining residue was suspended in water, filtered and
washed again. The residue was applied to column chromatography affording the pure base
1
0b (39%) (for details see the Exp. Section). Then, nucleobase base 10b was glycosylated as
described resulting in a mixture of anomers 12 and 13 (90% yield).7f,14 1H NMR spectra of the
prepurified glycosylation mixture revealed a β/α ratio of 3:2. Both anomers were separated by
7
f
crystallization and deprotected afterwards. Instead of crystallization of the α-anomer, the
mother liquor from crystallization of the β-anomer 12 was deprotected with NH /MeOH to
3
yield an inseparable mixture of nucleosides 4 and 5 (87% yield). For separation DMT residues
were introduced to protect the 5’-OH groups followed by column chromatography. This
resulted in the DMT derivative of the α-nucleoside 14 (40% yield) and its β-D counterpart 15
(12%). Compound 14 could be detritylated (trichloro acetic acid) to give 5 in 91% yield (for
details, see the Exp. Section and Scheme S1, Supporting Information).
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6
Scheme 1. Synthesis and Separation of Anomeric 7-Iodinated 5-Aza-7-deazaguanine 2’-
7
f
Deoxyribonucleosides and DMT Derivatives
Anomeric mixture of
12 and 13 (ratio 3:2)
O
O
O
I
TolO
TolO
0
1
2
3
4
5
6
7
8
9
0
1
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0
N
N
O
Cl
O
H
TolO
TolO
N
N
O
N
N
N
N
11
K2CO3, TDA-1
MeCN
H
O
N
N
O
+
TolO
90%
I
O
N
I
7f
(95%)
TolO
12
13
N
N
H
1. from mother liquor
N
N
1. from MeOH
1. 39% (31%)7f
7f
1. 33% (27%)7f
2. 95% (95%)
H
7f
crystallization by EtOAC/PE
crystallization
7f
7f
2. 92% (92%)
7f
7
f
2. NH /MeOH
10b7f
2. NH /MeOH
3
3
4
5
washing with NIS, CH Cl
2
2
water and FC
9% (35%)7f
3
O
O
I
DMTO
O
N
N
+
HO
N
DMT-Cl
N
O
NH2
N
N
N
O
N
NH2
4/5
N
N
N
O
N
N
H
DMTO
H
I
0a7b
1
HO
14
15
Next, the unknown 7-iodinated 5-aza-7-deazaguanine ribonucleoside 6 was prepared.
Attempts to iodinate the non-functionalized ribonucleoside 2 with N-iodosuccinimide failed.
Consequently, the ribonucleoside 6 was prepared by convergent synthesis utilizing the
isobutyrylated iodo base 10b and 1-O-acetyl-2,3,5-tri-O-benzoyl-ribofuranose 16. To this end,
two different glycosylation protocols were employed: the nucleobase anion glycosylation
(
method A) developed in our laboratory5a,b and the so-called one-pot glycosylation (method
1
5
B) previously used for 7-iodo-7-deaza-2'-deoxyguanosine . In method B the base 10b was
silylated using N,O-bis(trimethylsilyl) acetamide (BSA). Then, glycosylation was performed
with the protected ribose 16 using TMSOTf (Vorbrüggen conditions) in MeCN at 50°C for 16
h. After solvent evaporation and column chromatography compound 18 was isolated in 42%
yield. As various other unidentified products were formed the isolation of pure material was
time consuming. On the contrary, when the one-pot conditions were applied to the non-
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iodinated protected and unprotected nucleobases 10a and 19, the protected nucleosides 20a
and 20b were formed in 48% and 84% yield, respectively (Scheme 2).
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1
1
1
1
1
1
1
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2
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8
9
0
1
2
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8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
In order to increase ribonucleoside formation nucleobase anion glycosylation was performed.
The same glycosylation protocol was employed as described for the 2’-deoxyribonucleoside
synthesis reported above. To this end, the bromo sugar 17 was prepared in situ from the 1-
acetoxy compound 16 with a 30% solution of HBr in acetic acid overnight. This protocol does
16
not require HBr gas and works efficiently as it was demostrated earlier. Then, nucleobase
anion glycosylation was performed at rt using MeCN as solvent with potassium carbonate and
tris-[2-(2-methoxyethoxy)ethyl]amine (TDA-1) as catalyst. When the glycosylation was
performed with the 5-aza-7-deazapurine base 10a the reaction was sluggish and the yield of
2
0b was only 10% (Scheme S1, Supporting information). This is in line with reactions
1
7
performed on 7-deazapurines. In contrary, when the iodinated base 10b was employed the
reaction was successful and the protected glycosylation product 18 was isolated in 67% yield
(Scheme 2). Purification of the glycosylation products was easier than in case of the one-pot
glycosylation. Then, the protecting groups were removed with NH /MeOH at rt yielding the
3
iodinated ribonucleoside 6 in 86% yield. The results show that the nucleobase anion
glycosylation works only efficiently when the pyrrole or imidazole ring carries electron-
withdrawing substituents such as halogens, a phenomenon which was already observed in the
synthesis of pyrrolo[2,3-d] pyrimidine nucleosides.15,18 All new synthesized compounds were
1
13
characterized by H-, C NMR spectra as well as ESI-TOF mass spectra (see Experimental
1
13
13
section). The H- C correlated (HMBC and HSQC) NMR spectra were used to assign the C
NMR signals. For details see the Experimental section (for spectra, see the Supporting
Information).
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4
4
4
4
4
4
4
4
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5
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5
5
5
5
6
Scheme 2. Synthesis of 5-Aza-7-deazaguanine Ribonucleosidesa
i) Nucleobase Anion
Glycosylation
Method A
X
O
O
O
N
BzO
I
I
O
N
N
O
N
N
O
6
O
N
BzO
6 X = acetoxy
17 X = Br
OBz
I
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
N
iv)
N
N
N
H2N
HO
1
O
N
N
ii)
H
N
N
BzO
H
H
10b
BzO
OBz
HO
OH
iii) One-pot Vorbrüggen
Glycosylation
18
A: 67%
B: 42%
86%
Method B
OAc
O
BzO
BzO
OBz
16
O
O
O
N
N
N
O
N
N
O
i) One-pot Vorbrüggen
Glycosylation
N
iv)
N
N
RHN
N
2
H N
N
N
N
RHN
H
OAc
O
BzO
97a R = H
BzO
HO
1
1
7
b
0a R = i-Bu
BzO
OBz
BzO
OBz
HO
OH
16
Method B
2
85%
2
2
0a R = H, 48%
0b R = i-Bu, 84%
a
Reagents and Conditions: i) TDA-1, K CO , 4h, r.t.; ii) HBr in glacial acetic acid; iii) BSA,
2
3
TMSOTf, CH CN, 16 h, 50°C; iv) NH /MeOH r.t, 24 h.
3
3
X-ray Analysis of 4, Iodo-Oxygen Interactions and Channel Formation
Earlier, X-ray studies on iodo nucleosides showed that some compounds show strong iodo-
iodo interactions in the solid state. Also, an early report on 5-iodo-2’-deoxyuridine with iodo-
1
9
oxygen contacts exists. These observations prompted us to perform a single-crystal X-ray
analysis of compound 4 (Figure 2). As a result, we obtained deeper insight into conformation,
hydrogen bonding, nucleoside packing and the role of the iodo substituent of compound 4 in
the crystalline state.
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8
9
0
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5
6
7
8
9
0
Figure 2. A perspective view of 4 showing the atomic numbering scheme. H-atoms are
shown as small spheres of arbitrary size.
The three-dimensional structure of 4 is shown in Figure 2. For details of the X-ray
crystallographic analysis see the Supporting Information. According to the Flack parameter,
the anomeric centre shows R-configuration confirming the β-D anomer structure of 4. The
2
0
orientation of the nucleobase relative to the sugar residue is anti with χ (O4’-C1’-N9-C4) = -
139.9(6)°. The two principal puckering modes are C3’-endo (N) and C2’-endo (S) with
preferred values of P = 0 – 36° for C3’-endo and P = 144 – 190° for C2’-endo.21,22 The 2’-
3
deoxyribosyl residue of 4 shows a C3’-endo-C4’-exo ( T ) sugar pucker with P = 28.6° and τ
4
m
= 34.3°, referring to a N-type sugar conformation. This is different from the preferred sugar
7f
conformation of 4 observed in solution where the S conformation (62%) predominates. The
torsion angle γ (O5’-C5’-C4’-C3’) characterizes the orientation of the exocyclic 5’-hydroxy
group relative to the sugar ring. For 4, an antiperiplanar (trans) conformation is observed with
γ = -172.7(4)°.
Figure 3 displays the crystal packing mode and hydrogen bonds of compound 4. Hydrogen
bonding occurs solely between the nucleobase and the sugar moieties but not between two
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6
nucleobase moieties (Table S13, Supporting Information). The iodo substituent has a strong
contact to oxygen O3’ of the sugar residue (I7⋯O3’ = 2.794(4) Å, C7-I7⋯O3’ = 169.2(2)°)
while iodo⋯iodo interactions are not observed. The crystal structure of 4 is composed of
different layers and the molecules are ordered in a zig-zag-like arrangement. All
intermolecular contacts, hydrogen bonds and the I⋯O interaction, occur solely between
neighbouring layers.
0
1
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1
2
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5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
Interestingly, Figure 3c shows formation of channels within the crystal structure. The
channels are filled with solvent guest molecules, probably methanol molecules. However, due
to the lack of specific interactions between the solvent molecules and compound 4, the solvent
molecules cannot be accurately located and were removed by applying the software tool
PLATON/SQUEEZE.23
(
a)
(b)
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(c)
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0
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0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 3. (a), (b) Crystal packing of 4 showing the hydrogen bonds and the iodine and O3’
interaction between the different layers. (c) Space filling model of 4 showing channels within
the crystal structure (viewed from the a direction). Solvent molecules within the channels
were removed by applying the program PLATON/SQEEZE.23
Hirshfeld surface analysis and 2D fingerprint plots were used to visualize the intermolecular
2
4
25
interactions of compound 4 in the solid state. The Crystal Explorer 17 program was used
to carry out the Hirshfeld surface analysis mapped over a dnorm range of -0.5 to 1.5 Å, shape
index (-1.0 to 1.0 Å) and curvedness (-4.0 to 0.4 Å) as well as a 2D fingerprint plot analysis.
Figures 4a,b show the molecular Hirshfeld surfaces of 4 mapped over dnorm. For shape index
and curvedness surfaces see the Supporting Information (Figures S6a-d, Supporting
Information). The red areas indicate close contacts as these interactions are shorter than the
sum of van-der-Waals radii and show negative dnorm. The results of the Hirshfeld analyses are
consistent with the hydrogen bonding data (Table S13, Supporting Information). Most
important, also the strong interaction between iodine and oxygen O3’ is confirmed and
appears as two distinct spikes in the fingerprint plots with a proportion of 3.5% (Figure S6e,
Supporting Information).
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a)
0
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7
8
9
0
b)
Figure 4. Hirshfeld surface of compound 4 mapped with dnorm (-0.5 to 1.5 Å). (a) Front
view and (b) back view.
Synthesis of 7-Functionalized 5-Aza-7-Deazaguanine Nucleosides by Sonogashira and
Suzuki-Miyaura Cross-coupling
The 7-iodo-5-aza-7-deaza-2'-deoxyguanosine 4, its α-D anomer 5 as well as the iodo
ribonucleoside 6 were then the starting materials for the synthesis of a diversity of side chain
derivatives. To this end, alkynes were employed in Sonogashira cross-coupling or boronic
2
6
acids derivatives were used in Suzuki-Miyaura reactions. Among the various side chain
derivatives, particular attention was given to compounds that show fluorescence as
fluorescent 7-deazapurine nucleosides play a key role in the chemical manipulation of nucleic
27
acids. The α-anomeric coupling products were prepared as they are suitable for
incorporation in DNA with parallel chain orientation or to be used in silver-mediated hybrid
base pairs.10-12
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Three types of 7-functionalized nucleoside derivatives were synthesized: (i) β-D 2’-
deoxyribonucleosides carrying heterocyclic side chains and pyrene residues, (ii) α-D
nucleosides that can be incorporated in DNA with parallel chain orientation, (iii)
ribonucleosides as building blocks for RNA. Scheme 3 displays the various derivatives and
shows reaction details.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
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8
9
0
1
2
3
4
5
6
7
8
9
0
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3
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4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Scheme 3. Synthesis of 7-Functionalized 5-Aza-7-deazaguanine β-D- and α-D-2’-
Deoxyribonucleosides and β-D Ribonucleosidesa
O
N
O
N
I
Side chain
O
O
N
N
O
Suzuki-Miyaura (A) or
Sonogashira (B)
cross-coupling
N
N
O
HO
HO
Suzuki-Miyaura (A) or
Sonogashira (B)
cross-coupling
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
N
H N
N
H N
2
HO
N
O
NH2
2
N
HO
N
O
NH2
N
i) or ii)
N
N
i) or ii)
N
N
HO
HO
I
Side chain
HO
R
HO
R
8, 25-28
7, 9, 21-24
5
4
R = H
6 R = OH
O
O
O
O
O
N
O
O
N
N
N
N
O
N
N
O
N
N
N
N
O
N
N
H N
N
H N
N
H N
N
2
N
R
N
2
2
H N
N
H N
N
2
2
O
O
HO
HO
HO
HO
HO
HO
HO
HO
HO
23 R = H
9 R = OH
HO
21
7
22
24
Yield
76%
52%
54%
72%
65%
9 10 min
46%
6h
23
Reaction time
Reaction type
30 min
A
30 min
A
2h
B
10 min
A
A
B
O
O
O
O
O
HO
HO
HO
NH2
HO
HO
HO
N
HO
N
NH2 HO
N
NH2
HO
HO
N
N
N
N
O
NH2
N
O
NH2
N
N
N
N
N
N
N
N
N
N
N
N
O
O
O
O
O
25
26
27
28
8
Yield
Reaction time
Reaction type
65%
2h
B
51%
1h
A
9
4%
82%
30 min
A
31%
6h
30 min
A
B
O
N
I
O
N
HN
O
N
O
iii)6g
O
iii)
N
HN
H N
2
29
HN
52%
15 min
41%
0 min H N
HN
O
H N
2
N
1
2
N
HO
N
H N
N
2
O
O
HO
HO
O
HO
HO
29
HO
HO
3
0
31
HO
3
228
a
Reagents and Conditions: i) Suzuki-Miyaura cross-coupling (A): Na CO •10H O,
2
3
2
tetrakistriphenylphosphin Pd(0), CH CN/H O 1:1 and the corresponding boronic acid or
3
2
boronic acid pinacol ester at 105°C; ii) Sonogashira cross-coupling (B):
tetrakistriphenylphosphin Pd(0), CuI, N-ethyldiisopropylamine, DMF and the corresponding
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alkyne at r.t; iii) CsCO , Pd(OAc) , TPPTS, CH CN/H O 1:1 and the corresponding boronic
3
2
3
2
acid. For boronic acids and alkynes used in these reactions, see the Exp. Section.
For Suzuki-Miyaura couplings the iodo nucleosides 4-6 were suspended in CH CN/H O 1:1.
3
2
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Then, Pd(0) (0.1 eq.), sodium carbonate (8 eq.) and the corresponding boronic acid (4 eq.)
were added. In a few cases, the reaction proceeded in 10 min. For most compounds longer
reaction times were required (see Scheme 3). However, only traces of deiodination were
observed in all cases. Formation of the deiodinated nucleoside was confirmed by comparison
with an authentic sample. For comparative fluorescence studies the 7-substituted 7-
deazaguanine nucleosides 30-32 were prepared by Suzuki-Miyaura coupling. Compound 30
and 32 were already described in the literature6d,28, whereas compound 31 is new. For 30 and
3
1 different reaction conditions using a combination of TPPTS (triphenylphosphine-3,3’,3”-
trisulfonic acid trisodium salt hydrate) and Pd(OAc) were required. Sonogashira cross-
2
coupling reactions of the 5-aza-7-deazaguanine nucleosides 4 and 5 were performed in DMF
using tetrakistriphenylphosphin Pd(0) (0.1 eq.), CuI (0.2 eq.) and the corresponding alkyne (2-
4 eq.) at r.t. Here, the reaction time for complete consumption of starting material had to be
increased compared to Suzuki coupling leading to lower product yields as deiodination took
place (for details, see the Exp. Section).
Photophysical Properties of 7-Functionalized 5-Aza-7-deazaguanine Nucleosides
As canonical nucleosides of DNA and RNA are virtually non-fluorescent, natural nucleosides
2
9
were modified in various ways to generate fluorescence. A number of reviews appeared
3
0
reporting on this matter. We anticipated that 5-aza-7-deazaguanine nucleosides become
fluorescent when side chains are introduced that were already successfully employed in the
series of other 7-deazapurine nucleosides. Furthermore, microenvironmental factors such as
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4
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4
5
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5
5
5
5
6
polarity, viscosity, and base pairing influence the fluorescence of a particular nucleoside dye
conjugate. However, we learned from nucleobase functionalized pyrrolo[2,3-d]pyrimidine
7
7 8
(c G ) or pyrazolo[3,4-d]pyrimidine (c z G , Figure 1) derivatives that the nucleobase
d
d
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
structure has a significant impact on the fluorescence regarding emission maxima and
quantum yield.13,27c,28,29c,29f
To this end, compounds shown in Scheme 3 were inspected for their fluorescence behaviour.
It was found that the benzofuran derivatives 7 (β-D) and 26 (α-D) as well as pyrene
derivatives show significant fluorescence, whereas others (phenylacetylen, furan) are only
weakly fluorescent. Figure 5a displays UV and fluorescence excitation and emission spectra
of 7 and 26 determined in methanol. Both anomers show almost idendical UV and
fluorescence spectra with emission maxima at 352 nm when excited at 313 nm. Next,
photophysical properties of 7 were determined in solvents of different polarity and the
corresponding Stokes shift as well as quantum yields were determined (Table 1). The
emission spectra for β-D benzofuran nucleoside 7 in various solvents are displayed in Figure
5
b. It appears that fluorescence in aqueous solution is very low compared to other polar
solvents. As it was not clear if the phenomenon is due to the benzofuran residue or the 5-aza-
7-deazaguanine skeleton the related 7-deazaguanine benzofuran conjugate 30 was measured
in the same solvents (Figure 5c and Table 1). Fluorescence quantum yields of the 5-aza-7-
deazaguanine nucleoside 7 are almost 2-fold higher than those for the 7-deazaguanine
nucleoside 30. The order of solvent dependent fluorescence for 7 and 30 is very similar with
the highest quantum yields in DMSO/DMF with emission maxima which are red-shifted
compared to the other solvents. However, significant differences are observed among the two
classes of molecules for MeOH (Figure 5b,c). Also, the quantum yields are significantly
different (0.59 and 0.04, respectively). This indicates that the two molecules respond on
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solvent polarity changes in a different way. Furthermore, they do not show a linear change of
fluorescence when related to polarity.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(a)
(
b)
(
c)
O
N
O
N
R
R
O
N
N
O
HO
HN
H2N
HO
H2N
HO
N
HO
N
N
O
NH2
N
N
N
O
R
HO
HO
7
26
30
O
R =
Figure 5. (a) UV and fluorescence emission spectra of 7 (β-D) and 26 (α-D) measured in
methanol (50 µM for UV and 1 µM for fluorescence). Excitation wavelengths of nucleosides
7
(313 nm) and 26 (313 nm). Fluorescence emission spectra of nucleoside 7 (b) and 30 (c)
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7
8
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
were measured in various solvents with a nucleoside concentration of 1 µM (for details see
Table S1, Supporting Information).
Table 1. Photophysical Data of 7-Benzofuranosyl-5-aza-7-deaza-2’-deoxyguanosine (7) and
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
7
-Benzofuranosyl-7-deaza-2'-deoxyguanosine (30) Measured in Solvents of Different
Polaritya
Stokes
shift
λ
abs, max
λ
max, em
Solvent Excitation Emission
nm) (nm)
Φc
b
(Δν)
(
-1
(cm )
4000
3900
3500
3800
3700
3700
DMSO
DMF
MeOH
MeCN
318
316
313
312
364
0.69
0.68
0.59
0.47
0.26
0.03
360
352
354
354
352
7
Dioxane 314
Water
311
DMF
DMSO
MeCN
Dioxane 317
MeOH
Water
321
322
317
373
380
364
358
364
357
4300
4700
4100
3600
4100
3500
0.38
0.37
0.25
0.13
0.04
<0.01
3
0
317
317
O
R
O
N
R
N
N
O
HN
O
N
R =
H2N
HO
N
N
H2N
HO
O
HO
HO
7
30
a
b
The concentration of nucleosides 7 and 30 was 1 µM. The
Stokes shift was calculated from the equation ΔEphoton
=
c
hc(1/λabs
,
max – 1/ λmax, em). The fluorescence quantum yields (Φ)
2 4
were calculated using quinine sulfate in 0.1 M H SO (ΦSt = 0.54).
The conformation of the benzofuran moiety to the nucleobase forms a rotatable system, which
is sensitive to molecular crowding and viscosity. High viscous media restrict rotation and
3
1
enhance fluorescence. This phenomenon was already reported for 2'-deoxyuridine
nucleosides decorated with a benzofuran residue at position-5.31b-e Fluorescence was
significantly increased in glycerol or ethylenglycol compared to water. Now, this matter was
proven on the nucleoside benzofuran conjugate 7.
Figure 6 displays the fluorescence emission spectra of 7 in glycerol, water and mixtures
thereof. The nucleoside is low-emissive in water and also low-emissive in glycerol. However,
2
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in glycerol/water mixtures the fluorescence increases with increasing glycerol content.
Nevertheless, this is only valid for a glycerol content of about 50%. A higher proportion of
glycerol leads to a fluorescence decrease. So, nucleoside 7 behaves different compared to 2'-
deoxyuridine conjugates and shows the expected immobilization of the benzofuran moiety
accompanied by fluorescence increase only when the glycerol content of the water/glycerol
mixtures are below 50%. An explanation for this unusual behaviour cannot be given now,
however it is apparent from differences in the shape of the fluorescence spectra that other
factors account for this behaviour. In the ethylene glycol/water system the fluorescence
depends on the viscosity in the expected way. It increases with increasing solvent viscosity as
it was reported for pyrimidine benzofuran conjugates before (Figure 6b).31b-e
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Normally, the low fluorescence intensity of nucleoside 7 in water is a disadvantage for
application in bio-related systems. However, the phenomenon of fluorescence increase caused
by immobilization of the benzofuran moiety is applicable to detect binding to biopolymers
e.g. proteins or to study structural changes of oligonucleotide assemblies. As rotational
motion is reduced during polymer binding the fluorescence increase is a valuable tool to
detect interactions between molecules.29c,31c,31e,31f
2
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
a)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
b)
Figure 6. Fluorescence emission spectra of the 5-aza-7-deaza benzofuran conjugate 7 in (a)
ethylenglycol and (b) glycerol. Excitation wavelength of nucleoside 7 was 312 nm in glycerol
and 315 nm in ethylene glycol.
Next, the photophysical properties of the pyrene nucleoside conjugates were studied. Pyrene
3
2
is a polycylcic aromatic molecule representing the class of photoexcitable dyes. This
behaviour makes it useful for studies on nucleic acids and other biopolymers. The molecule
shows monomer and excimer fluorescence which is sensitive to the microenvionment.33
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Excimer fluorescence is a useful tool for the detection of environmental changes and
neighbouring effects in DNA duplexes.27c
The photophysical properties of 5-aza-7-deazaguanine pyrene conjugates 8, 9, 23, 24 and 28
were determined in various solvents of different polarity and compared to the pyrene
conjugates 31, 32 of the related 7-deaza-2’-deoxyguanosine (Table 2 and Table S3,
Supporting Information). The pyrene was either directly linked to position-7 of the nucleobase
or connected via an alkynyl linker. Figure 7 shows fluorescence spectra and solvent
dependencies.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(a)
(b)
(c)
(d)
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7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(
e)
(f)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
HO
HO
N
O
NH2
N
O
N
O
N
O
N
O
N
O
N
N
N
N
N
N
N
N
N
O
HN
H2N
HN
H2N
N
N
H2N
HO
N
H2N
HO
N
H2N
HO
N
O
9
O
O
O
HO
HO
HO
OH
HO
HO
HO
HO
32
24
28
31
23
Figure 7. (a) Fluorescence emission spectra of pyrene nucleosides determined in MeOH.
Fluorescence emission spectra of 9 (b), 23 (c), 24 (d), 31 (e) and 32 (f) measured in various
solvents with a nucleoside concentration of 1 µM. For compound 24 the concentration in
dioxane was 0.2 µM (for details see Table 2).
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Table 2. Photophysical Data of 7-Pyrenyl Nucleosides 9, 23, 24, 31 and 32 Measured in
Solvents of Different Polaritya
λ
abs, max
λ
max, em
Stokes
b
Φc
Solvent
Ex
nm]
Em
shift (Δν)
-1
[
[nm]
436
435
420
411
413
411
[cm ]
5800
5800
5300
4600
4900
4800
DMSO
DMF
348
347
343
346
343
343
0.68
0.59
0.59
0.56
0.52
0.45
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
MeCN
Dioxane
Water
9
MeOH
DMF
347
346
348
343
344
343
436
412
445
412
424
413
5900
4600
6300
4900
5500
4900
0.71
0.50
0.70
0.40
0.54
0.53
Dioxane
DMSO
MeOH
MeCN
Water
2
3
1
Dioxane
MeCN
MeOH
DMF
DMSO
Water
350
349
346
356
359
345
435
491
480
503
516
458
5600
8300
8100
8200
8500
7200
0.39
0.24
0.17
0.11
0.11
<0.01
3
DMF
397
392
400
396
391
391
462
435
471
429
425
426
3500
2500
3800
1700
2100
2300
0.57
0.57
0.56
0.52
0.46
0.24
MeCN
DMSO
Dioxane
MeOH
Water
24
32
d
Dioxane
MeCN
MeOH
DMF
DMSO
Water
389
389
389
389
389
389
448
526
504
545
559
535
3400
6700
5900
7400
7800
7000
0.60
0.08
0.06
0.03
0.03
<0.01
O
N
O
O
N
O
N
HN
N
O
HN
H2N
H2N
HO
N
N
R
H N
N
N
H N
N
N
N
2
2
N
O
O
O
HO
HO
HO
HO
HO
HO
2
31
4
HO
32
9
: R = OH
2
3: R = H
a
b
The concentration of nucleosides was 1 µM. The Stokes shift
was calculated from the equation ΔEphoton= hc(1/λabs
em). The fluorescence quantum yields (Φ) were calculated using
,
max – 1/ λmax,
c
d
quinine sulfate (1 µM) in 0.1 M H
with a concentration of 0.2 µM.
2
SO
4
(ΦSt = 0.54). Determined
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
According to Figure 7 and Table 2 pyrene nucleosides derived either from α- or β-D 5-aza-7-
deaza-2’-deoxyguanosine (23, 28) or the ribonucleoside 5-aza-7-deazaguanosine (9) show
almost identical fluorescence spectra. The emission maxima of the pyrene conjugate 23 are
significantly red-shifted (~20 nm) in polar aprotic solvents (DMF, DMSO) (Figure 7). Solvent
dependent measurements of the directly conjugated pyrene 5-aza-7-deazaguanine
ribonucleoside 23 gave high quantum yields in all cases (Φ = 0.4-0.7). The same is true valid
for nucleosides 9 and 28. Triple bond pyrene conjugates 8 and 24 show similar fluorescence
properties as the directly attached pyrene conjugate 23. Here, a red-shift of the excitation
wavelengths (~50 nm) leads to a reduced Stokes shift in all solvents. Furthermore, the
quantum yield in water is decreased (Figure 7 and Figure S1, Supporting Information). The
related 7-deaza-2’-deoxyguanosine pyrene conjugate 31 (directly connected) shows
fluorescence with much lower quantum yields and a stronger solvent dependency as observed
for the 5-aza-7-deazaguanine conjugate 23. The 7-deazaguanine pyrene nucleoside 32
connected via alkynyl linker is almost non-fluorescent. Only in dioxane the compound
becomes fluorescent (Figure 7f).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Main differences exist between fluorescence of the benzofuran 5-aza-7-deazguanine residues
7 and 26 and the pyrene conjugates 24 and 31. (i) The excitation and emission maxima of the
pyrene conjugates are significantly red-shifted. (ii) The directly connected pyrene conjugate
2
3 and 9 shows minor solvent dependencies whereas the benzofuran conjugates 7 and 26
show strong solvent dependency.
The different quenching effects observed for the fluorescence of the 5-aza-7-deazaguanine
and 7-deazaguanine pyrene conjugates most probably result from charge separation between
the pyrene residues and the nucleobases (intramolecular electron transfer or hole transfer). It
is reported that 7-deazapurine conjugates form a charge separated state with a nucleobase
radical cation and a radical anion of pyrene.27c,34 A similar behaviour is expected for 5-aza-7-
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deazapurine pyrene conjugates. The higher quantum yields of the 5-aza-7-deazapurine pyrene
conjugates might result from a higher oxidation potential of the 5-aza-7-deazaguanine to 7-
deazaguanine. None of the pyrene compounds shows excimer fluorescence.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
CONCLUSION
Nucleobase anion glycosylation of isobutyrylated 5-aza-7-deaza-7-iodoguanine (10b) with the
bromo sugar of 2,3,5-tri-O-benzoyl-1-O-acetyl-D-ribofuranose 16 gave the pure β-D
anomeric N-9 glycosylation product 18 in 67% yield. The corresponding one-pot
glycosylation performed with the silylated base under Vorbrüggen condition (TMSOtriflate)
resulted in lower yield and the glycosylation product was difficult to purify. When
glycosylation of the non-iodinated base 19 was performed using the Vorbrüggen protocol, the
7a
yield was the same (84%) as that reported previously under SnCl catalysis . All
4
glycosylation products – ribo- and anomeric 2'-deoxyribo compounds – were deprotected to
yield the free nucleosides 2-6. The crystal structure of 4 showed intermolecular contacts with
I⋯O interaction. Channels are formed in the crystal, that are filled with solvent guest
molecules.
With the help of iodo nucleosides as starting materials a diversity of side chain derivatives
were synthesized employing either Sonogashira or Suzuki-Miyaura cross-coupling. For the
first time, functionalized 5-aza-7-deazaguanine nucleosides were accessible which were
inspected for their photophysical properties. Among those, the benzofuran and pyrene
derivatives show strong fluorescence and high quantum yields. Fluorescence spectra
3
5
measured in various solvents show solvatochromism . Higher quantum yields were obtained
for 5-aza-7-deazaguanine nucleoside conjugates compared to those with a 7-deazaguanine
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
skeleton. The benzofuran conjugate 7 shows increase fluorescence in viscous media (ethylene
glycol, glycerol) compared to water.
Functionalized 5-aza-7-deaza nucleosides reported in this work can be utilized as starting
materials for chemical (phosphoramidites) or enzymatic synthesis (triphosphates) of modified
DNA or RNA. According to the special recognition pattern of the 5-aza-7-deazaguanine base
that lacks a hydrogen at position-1 base pairs with purines can be constructed7c,10 and
orthogonal base pairs with "protonated" dC analogues or silver ions can be formed to extent
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
9
the 4-letter code of the genetic system. The side chain derivatives with anomeric α-D
configuration represent building blocks for duplex DNA with parallel chain orientation.7c
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-
3
-1
-1
spectra were recorded on a UV-spectrophotometer: λmax (ε) in nm, ε in dm mol cm . NMR
1
spectra were measured at 599.74 MHz or 399.89 MHZ for H and 150.82 MHz or 100.56
1
3
1
13
MHz for C. H- C correlated (HMBC, HSQC) NMR spectra were used for the assignment
1
3
of the C signals (Tables S1-2, Supporting Information). The J values are given in Hz; δ
values in ppm relative to Me Si as internal standard. For NMR spectra recorded in DMSO-d ,
4
6
1
13
the chemical shift of the solvent peak was set to 2.50 ppm for H NMR and 39.50 ppm for C
NMR. ESI-TOF mass spectra of nucleosides were recorded on a Micro-TOF spectrometer.
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2
7f
2
-[(N -Isobutyryl)amino]-6-iodo-8H-imidazo[1,2-a]-s-triazin-4-one (10b). To the stirred
2
7b
suspension of 2-[(N -Isobutyryl)amino]-8H-imidazo[1,2-a]-s-triazin-4-one (10a; 5.4 g,
17.53 mmol) in anh. CH Cl (500 mL) was added N-iodosuccinimide (4.73 g, 21.03 mmol) in
2
2
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
one portion at r.t. Stirring was continued for 30 min and the solvent was evaporated. The
residue was stirred with water (25 mL) for 15 min. and then filtered. The residue was washed
with another 25 mL water. The dry residue was applied to FC (silica gel, CH Cl /MeOH
2
2
100:2). Evaporation of the main zone gave compound 10b (2.4 g, 39%) as yellowish powder.
3
−1
−1
TLC (silica gel, CH Cl /MeOH 20:1) R 0.2. λ (MeOH)/nm 237 (ε/dm mol cm 43300),
2
2
f
max
1
2
84 (10500). H NMR (600 MHz, DMSO-d )  0.98 (dd, J = 10.0, 6.9 Hz, 6H, CHCH ), 2.87
6
3
(p, J = 6.8 Hz, 1H, CHCH ), 4.70 (dd, J = 11.9, 5.9 Hz, 1H, H-5'), 4.77 (dd, J = 12.0, 4.2 Hz,
3
1
H, H-5’), 4.86 (td, J = 5.9, 4.2 Hz, 1H, H-4'), 6.20 (dd, J = 6.0, 4.0 Hz, 1H, H-2’), 6.28 (t, J
6.0 Hz, H-3’), 6.34 (d, J = 3.9 Hz, 1H, H-1’), 7.43-7.51 (m, 6H, arom. H), 7.63-7.67 (m,
=
3H, arom. H), 7.88-7.92 (m, 5H, H-7, arom. H), 7.95-7.98 (m, 2H, arom. H), 10.42 (s, 1H,
NH). Analytical data were identical to those obtained earlier.7f
Glycosylation of 10b with 2-Deoxy-3,5-di-O-toluoyl-α-
D
-erythro-pentofuranosyl Chloride
11). As described in the literature with 2-[(N -Isobutyryl)amino]-6-iodo-8H-imidazo[1,2-a]-
s-triazin-4-one 10b (800 mg, 2.30 mmol), K CO (1.01 g, 7.31 mmol), TDA-1 (0.1 mL, 0.29
7f
2
(
2
3
mmol), 2-deoxy-3,5-di-O-toluoyl-α-
D
-erythro-pentofuranosyl chloride (1.43 g, 3.68 mmol) in
CH CN (80 mL). After FC (silica gel, column, 12 x 4 cm, CH Cl /MeOH 100:1→95:5) an
3
2
2
1
anomeric mixture of 12 and 13 (1.44 g, 90%) was obtained as colorless foam. H NMR of the
anomeric mixture showed a 3:2 β to α ratio. Crystallization of the residue from MeOH
7
f
afforded the β-D anomer 12 (627 mg, 39%). Lit. : 31%. Crystallization from PE/EtOAC of
the solid obtained after evaporation of the mother liquor from the first crystallization afforded
2
9
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