Parallel stranded DNA stabilized with internal sugar cross-links: Synthesis and click ligation of oligonucleotides containing 2′-propargylated isoguanosine

Article abstract of DOI:10.1021/jo4012706

Internal sugar cross-links were introduced for the first time into oligonucleotides with parallel chain orientation by click ligation. For this, the 2′- or 3′-position of the isoguanosine ribose moiety was functionalized with clickable propargyl residues, and the synthesis of propargylated cytosine building blocks was significantly improved. Phosphoramidites were prepared and employed in solid-phase synthesis. A series of oligo-2′-deoxyribonucleotides with parallel (ps) and antiparallel (aps) strand orientation were constructed containing isoguanine-cytosine, isoguanine-isocytosine, and adenine-thymine base pairs. Complementary oligonucleotides with propargylated sugar residues were ligated in a stepwise manner with a chelating bis-azide under copper catalysis. Cross-links were introduced within a base pair or in positions separated by two base pairs. From T_m stability studies it is evident that cross-linking stabilizes DNA with parallel strand orientation strongly (ΔT_m from +16 to +18.5 C) with a similar increase as for aps DNA.

Full text of DOI:10.1021/jo4012706

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Article
Parallel Stranded DNA Stabilized with Internal
Sugar Cross-links: Synthesis and Click Ligation of
Oligonucleotides Containing 2’-Propargylated Isoguanosine
Suresh S Pujari, and Frank Seela
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/jo4012706 • Publication Date (Web): 05 Aug 2013
Downloaded from http://pubs.acs.org on August 7, 2013
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Parallel Stranded DNA Stabilized with Internal Sugar Crossꢀlinks: Synthesis and Click
Ligation of Oligonucleotides Containing 2’ꢀPropargylated Isoguanosine
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Suresh S. Pujari and Frank Seela*
Laboratory of Bioorganic Chemistry and Chemical Biology, Center for Nanotechnology,
Heisenbergstraße 11, 48149 Münster, Germany and Laboratorium 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; Fax: +49 (0) 251 53406 857
Eꢀmail: Frank.Seela@uniꢀosnabrueck.de
Homepage: www.seela.net
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ABSTRACT
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Internal sugar crossꢀlinks were introduced for the first time into oligonucleotides with parallel
chain orientation by click ligation. For this, the 2’ꢀ or 3’ꢀposition of the isoguanosine ribose
moiety was functionalized with clickable propargyl residues, and the synthesis of
propargylated cytosine building blocks was significantly improved. Phosphoramidites were
prepared and employed in solidꢀphase synthesis. A series of oligoꢀ2’ꢀdeoxyribonucleotides
with parallel (ps) and antiparallel (aps) strand orientation was constructed containing
isoguanineꢀcytosine, isoguanineꢀisocytosine and adenineꢀthymine base pairs. Complementary
oligonucleotides with propargylated sugar residues were ligated in a stepwise manner with a
chelating bisꢀazide under copper catalysis. Crossꢀlinks were introduced within a base pair or
in positions separated by two base pairs. From Tm stability studies it is evident that crossꢀ
linking stabilizes DNA with parallel strand orientation strongly (∆Tm from +16 to +18.5°C)
with a similar increase as for aps DNA.
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INTRODUCTION
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The polymorphic nature of DNA1 offers numerous opportunities to construct DNA based
nanostructures which find widespread applications from material science to DNA diagnostics
and medicine.2 As canonical duplex DNA is formed by WatsonꢀCrick base pairing, it exhibits
chains with antiparallel strand (aps) orientation. A new family of parallel stranded (ps) duplex
DNA was realized by changing the WatsonꢀCrick motif to the reverse mode and/or by
replacing dG by 2’ꢀdeoxyisoguanosine (isoGd) and dC by 2’ꢀdeoxyꢀ5ꢀmethylisocytidine
(isoCd) (Figure 1).3 Such kind of parallel hybridization offers new opportunities for designing
new oligonucleotide hybridization probes, antisense constructs or nanodevices.4 Advantages
of the ps hybridization over naturally occurring aps hybrids include, e.g., the higher resistance
of these molecules against nucleolytic enzymes or the application of the alternative
hybridization system for diagnostic or nanoscopic purposes.5 Despite having such favourable
properties, dAꢀdT rich ps DNA is less stable than corresponding aps DNA.6 However,
stability of ps DNA was increased by the introduction of isoGdꢀdC or isoCdꢀdG base pairs.3
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Figure 1. Schematic presentation of duplex DNA with (a) antiparallel strand orientation
incorporating WatsonꢀCrick dAꢀdT or isoCdꢀisoGd base pairs and (b) parallel chain
orientation with isoGdꢀdC, isoCdꢀdG or dAꢀdT Donohue base pairs.
Different approaches for crossꢀlinking of DNA have been reported in literature.7 Recently, we
developed and reported on the Cu(I) catalyzed azideꢀalkyne8 “bisꢀclick” reaction as an
effective tool for crossꢀlinking nucleosides or oligonucleotides and to construct
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macromolecular assemblies.9 As an extension, to investigate its viability as well as to
diversify the relevance of the developed bisꢀclick protocol, the bisꢀclick reaction was
performed with sugar modified DNA as well.10 Further, a “stepwise click” protocol was
employed using a large excess of bisꢀazide (alkyne : bisꢀazide, 1:15). To circumvent the use
of excess azide, we recently reported on a chemoselective click reaction, where equimolar
amounts of bisꢀazide can be used to construct monofunctionalized oligonucleotides; though
this protocol is limited to only chelating bisꢀazides.11For illustration see figure 2 (A to C).
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Crossꢀlinks obtained by click chemistry have a favourable stabilizing effect on aps duplexes.9ꢀ
12 As DNA with parallel strand orientation shows another helix structure than aps DNA, it
was of utmost interest to perform crossꢀlinking on ps DNA and to study the impact on ps
duplex stability. Herein, we studied this phenomenon on 2’ꢀOꢀpropargylated DNA. In this
regard, the new building blocks 1 and 2 were synthesized, while the synthesis of known
building blocks 3 and 413 was significantly improved (Figure 2). These compounds were
further employed in oligonucleotide solidꢀphase synthesis, and the oligonucleotides were used
for the construction of ps and aps DNA. Different bifunctional azides (514 and 615; Figure 2)
and crossꢀlinking protocols were verified on antiparallel stranded duplexes. The most
promising protocol – crossꢀlinking with chelating bisꢀazide 6 – was used to implement crossꢀ
links in duplexes with parallel chain orientation. The stability of the sugar crossꢀlinked and
noncrossꢀlinked ps duplexes was compared, and the influence of crossꢀlinks at various
positions was studied.
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Figure 2. Upper part: sugar modified nucleobases and bifunctional azides used for crossꢀ
linking. Lower part: schematic illustration (A to C) of the stepwise click reaction using bisꢀ
azide 6.
RESULTS AND DISCUSSION
1. Synthesis of Monomers. Current methods describing the synthesis of propargylated
nucleosides are often laborious and low yielding.13, 16 Herein, we present an efficient synthetic
protocol for the propargylation of the earlier described cytidine nucleosides 3 and 4, and
applied this method to the preparation of the newly synthesized isoguanosine derivatives 1
and 2. The propargylation of cytidine was performed in DMF using NaH and TBAI for 72 h
at 55°C giving a mixture of the 2’ꢀO and 3’ꢀOꢀpropargylated regioisomers 3 and 4 in 64%
overall yield.16b From the 1H NMR spectrum of the crude reaction mixture an isomeric ratio
for the 2’ꢀO (3) to 3’ꢀOꢀisomer (4) of 4.5:1 was detected (based on the integration of the
C≡CH signal; Figure S6, supporting information). Thus, formation of the 2’ꢀOꢀisomer (3) is
favored. As the separation of these two regioisomers was difficult on that stage, the isomeric
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mixture was treated successively with DMTꢀchloride and benzoyl chloride,17 yielding the
protected compounds 8 and 9 (54% combined yield) which could be easily separated (Scheme
1). Compound 8 was deprotected with 28% aq. NH3 at 60°C overnight and successively
treated with acetic acid to give the propargylated nucleoside 3 in 92% yield. Phosphitylation
of 8 and 9 gave the building blocks 10 and 11 in 76% and 74% yield, respectively (Scheme
1).
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Scheme 1. Synthesis of 2’ꢀ and 3’ꢀOꢀPropargyl Derivatives of Cytidinea
NHBz
N
O
N
(iv), (v)
92%
DMTO
3
O
44%
NH2
N
NH2
N
NH2
N
N
N
N
OH O
8
O
(ii), (iii)
54%
O
O
(i)
HO
HO
HO
+
NHBz
N
64%
O
O
O
N
OH OH
O
OH
4 minor
(inseparable mixture)
OH
O
O
7
3 major
DMTO
10%
O
O
OH
NHBz
N
NHBz
9
N
N
O
N
(vi)
O
(vi)
74%
8
9
DMTO
DMTO
76%
O
O
O
P
O
O
O
P
NC(H2C)2O
N(i-Pr)2
NC(H2C)2O
N(i-Pr)2
10
11
aReagents and conditions: (i) NaH, TBAI, DMF, propargyl bromide, 55°C, 72 h; (ii) DMTꢀ
Cl, DMAP, pyridine, 3 h, rt; (iii) BzCl, TMSCl, pyridine, rt, 3 h; (iv) 28% aq. NH3, 60°C,
overnight; (v) AcOH, 1 h, rt; (vi) NC(CH2)2OP(Cl)N(iꢀPr)2, DIPEA, DCM, 1 h, rt.
As described in literature, isoguanosine derivatives can be obtained from 2ꢀaminoadenosine
(12) by selective deamination of the 2ꢀamino group.18 Therefore, compound 12 was chosen as
precursor for the synthesis of the propargylated isoguanosine derivatives 1 and 2. Treatment
of 12 with NaH and TBAI in DMF at 55°C resulted in the formation of the regioisomers 13
and 14 in moderate overall yield (43%) due to the incomplete conversion of the starting
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material, which was recovered in 25% (Scheme 2). Different from the propargylated cytidine
derivatives, the 2’ꢀO and 3’ꢀOꢀpropargylated compounds 13 and 14 could be easily separated.
From the 1H NMR spectrum of the crude reaction mixture an isomeric ratio of 3.2:1 was
determined for 13 : 14 (integration of the C≡CH signal; Figure S23, supporting information).
Moreover, a 6ꢀ10 ppm downfield shift is observed in the 13C NMR spectrum upon sugar
propargylation at the particular carbon. In general, we can use this observation for
determining the sugar propargylation sites.
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Scheme 2. Synthesis of 2’ꢀ and 3’ꢀOꢀPropargyl Isoguanosine Derivatives 1 and 2a
NH2
NH2
NH2
N
N
N
N
N
N
N
N
N
H2N
HO
N
O
H2N
HO
N
O
(i)
H2N
HO
N
O
+
43%
OH O
O
OH
OH OH
12
13 (31.7%)
14 (11.3%)
(ii) 58%
(ii) 68%
NH2
N
NH2
N
HN
HN
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N
O
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O
N
HO
HO
O
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O
OH
OH
O
1
2
aReagents and conditions: (i) NaH, TBAI, DMF, propargyl bromide, 55°C, 48 h, 25%
recovery of 12; (ii) NaNO2, AcOH, 50°C, 20 min.
Next, compounds 13 and 14 were subjected to deamination with NaNO2 in acetic acid (50°C,
20 min) to give nucleosides 1 and 2 in 58% and 68% yield, respectively.18 After protection of
the amino group of 1 and 2 using N,Nꢀdimethylacetamide dimethylacetal in MeOH at room
temperature for 4 h, compounds 15 (79%) and 16 (89%) were obtained (Scheme 3). The 2ꢀ
hydroxy group of compounds 15 and 16 was protected with N,Nꢀdiphenylcarbamoyl chloride
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in pyridine for 40 min at room temperature to afford compounds 17 (82%) and 18 (68%).
Protection of the 5’ꢀOH groups by DMT chloride under standard reaction conditions gave 19
and 20 (70% and 74%).19 Subsequently, phosphitylation furnished the phosphoramidite
building block 21 in good yield (84%) and the 3’ꢀOꢀpropargyl derivative 22 in moderate yield
(37%) (Scheme 3).
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Scheme 3. Synthesis of 2’ꢀ and 3’ꢀOꢀPropargylated Isoguanosine Building Blocksa
CH3
CH3
CH3
CH3
(H3C)2N
C
N
(H3C)2N
C N
(H3C)2N
C
N
(H3C)2N C N
N
N
N
N
N
N
N
N
N
N
HN
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1
or
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(iii)
DPCO
DMTO
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DMTO
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O
N
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HO
N
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O
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OR1 OR2
OR1 OR2
OR1 OR2
O
O
15 or 16
17 or 18
19 or 20
P
NC(H2C)2O
CH3
N(i-Pr)2
21
(H3C)2N
C
N
15, 17, 19: R1 = H, R2
=
N
N
N
(iv)
DPCO
DMTO
N
20
16, 18, 20: R1
=
R2 = H
O
O
O
P
NC(H2C)2O
22
N(i-Pr)2
aReagents and conditions: (i) N,Nꢀdimethylacetamide dimethylacetal, MeOH, 4 h, rt; 79% for
15; 89% for 16. (ii) N,Nꢀdiphenylcarbamoyl chloride, pyridine, 30 min, rt; 82% for 17; 68%
18. (iii) DMTꢀCl, pyridine, 90 min, rt; 70% for 19; 74% for 20. (iv) NC(CH2)2OP(Cl)N(iꢀPr)2,
DIPEA, DCM, 2 h, rt; 84% for 21; 37% for 22.
All synthesized compounds were characterized by 1H NMR and 13C NMR spectra as well as
elemental analysis. DEPTꢀ135 and 1Hꢀ13C gatedꢀdecoupled NMR spectra were used to assign
the 13C NMR signals. For details see experimental section (Tables 3, 4 and for spectra see
supporting information).
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2. Synthesis and Crossꢀlinking of Oligonucleotides.
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2.1. Oligonucleotide Synthesis. As in this study we wanted to focus on oligonucleotides with
the native 3’ꢀ5’ꢀphosphodiester linkage, we solely subjected 2’ꢀOꢀpropargylated
phosphoramidite building blocks 10 and 21 to solidꢀphase oligonucleotide synthesis. Thus, a
series of 12ꢀmer and 25ꢀmer oligonucleotides containing the propargylated nucleoside
residues 1 and 3 at various positions were designed which are able to form duplexes with
either parallel or antiparallel chain orientation. The oligonucleotides were prepared on solidꢀ
phase using the standard protocol of phosphoramidite chemistry. The coupling yields of the
modified building blocks were always higher than 98%. The synthesized oligonucleotides
were characterized by MALDIꢀTOF mass spectra and HPLC (Tables 1 and 5).
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2.2. Crossꢀlinking of 2’ꢀOꢀPropargylated Oligonucleotides to Homodimers with
Nonchelating and Chelating BisꢀAzides. Two procedures for the “click” crossꢀlinking of
propargylated oligonucleotides were studied. The first method utilizes symmetrical
nonchelating bisꢀazide 5,14 the second makes use of the unsymmetrical bisꢀazide 615e
containing a chelating and a nonchelating azido group. Both procedures were performed in a
stepwise manner (“stepwise click”) with monofunctionalized azido compounds as
intermediates.
At first, the reaction was performed with ODNꢀ27 and bisꢀazide 5. For the synthesis of
monofunctionalized ODNꢀ33 bearing one azido group, a large excess of azide 5 (ODNꢀ27 to
azide 5, 1:15) was used (Scheme 4 and experimental part).9 In a similar way,
monofunctionalized ODNs 5’ꢀd(TA1* GTC AAT ACT)ꢀ3’ (32), 5’ꢀd(TAG GTC AAT
A3*T)ꢀ3’ (34), and 5’ꢀd(AGT ATT GA3* CTA)ꢀ3’ (35) were synthesized (for detailed
formulas see Figure S1, supporting information). A second click reaction was carried out next
with the monofunctionalized ODNꢀ33 and propargylated ODNꢀ27 affording the crossꢀlinked
homodimer ODNꢀ37. For comparison, a number of propargylated oligonucleotides (25, 27,
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29, 31) were crossꢀlinked by the bisꢀclick procedure with equimolar amounts of the
oligonucleotide and bisꢀazide 5 (for details see experimental part). By this method, a number
of interstrand crossꢀlinked homodimeric oligonucleotides (ODNs 36ꢀ39) were constructed
(Scheme 4 and Table 1). These ODNs are identical to the crossꢀlinked oligonucleotide
adducts obtained by the stepwise click procedure.
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Scheme 4. Stepwise Click Reaction of Propargylated DNA with Symmetrical
Nonchelating BisꢀAzide 5
Next, the chemoselective stepwise click functionalization using chelating azide 6 was
investigated on propargylated oligonucleotides. As the procedure consists of a stepwise
protocol, formation of homodimers is suppressed. The chelating effect of the azide leads to
the exclusive formation of monofunctionalized click adducts still carrying an azido
functionality without using an excess of azide (oligonucleotide to azide ratio, 1:1).11
To prove the efficacy of the chemoselective click protocol, the reaction was initially studied
on the propargylated nucleoside 3 using the bisꢀazide 6. The first click was carried out in the
presence of Cu(II)acetate in MeOH/CH2Cl2 (1:1) at room temperature without reducing agent,
giving the monofunctionalized nucleoside 23 in 60% yield. Formation of a bifunctionalized
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click product was not observed. In the second step, compound 23 was used as azide precursor
and was reacted with a second molecule of 3 in the presence of Cu(OAc)2ꢁ H2O and sodium
ascorbate as a reducing agent affording the crossꢀlinked compound 24 in 77% yield (Scheme
5). Conclusively, we were able to show that the chemoselective click reaction can be carried
out in good yield for sugar modified nucleosides. Consequently, the chemoselective stepwise
click reaction was utilized to crossꢀlink propargylated oligonucleotides containing nucleosides
1 or 3 (Table 1).
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47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 5. ChelateꢀAssisted Chemoselective Click Reaction on Sugar Modified
Nucleoside 3a
aReagents and conditions: (i) Cu(OAc)2ꢁ H2O, MeOH/CH2Cl2 (1:1, v/v), 8 h, rt; (ii)
Cu(OAc)2ꢁ H2O, sodium ascorbate, 3, MeOH/CH2Cl2 (1:1, v/v), overnight, rt.
Then, the stepwise chemoselective click reaction was applied to propargylated ODNꢀ25,
which was reacted with bisꢀazide 6 in the presence of TBTAꢀCu(OAc)2•H2O complex with
NaHCO3 as catalyst in H2O/DMSO/tꢀBuOH (4:3:1) at room temperature (Scheme 6). After
completion of the reaction (8 h), the monofunctionalized ODNꢀ40 still carrying one azido
group was obtained in good yield. Owing to this promising result, other monofunctionalized
oligonucleotides, namely 5’ꢀd(TAG GT23 AAT ACT)ꢀ3’ (41), 5’ꢀd(TAG GTC AAT A23T)ꢀ
3’ (42) and 5’ꢀd(AGT ATT GA23 CTA)ꢀ3’ (43), were synthesized in a similar way.
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Next, monofunctionalized ODNꢀ40 still bearing a nonchelating azido group was used as a
substrate for the second click reaction. For that, ODNꢀ25 was treated with a CuSO4ꢀTBTA
complex. Then, the monofunctionalized ODNꢀ40 was added, and the reaction proceeded in
the presence of TCEP and benzoic acid in H2O/DMSO/tꢀBuOH at room temperature for 1 h
(Scheme 6) to afford click adduct ODNꢀ44. Accordingly, crossꢀlinked ODNs 45ꢀ47 were
synthesized in good yields (Table 1). The purity and molecular masses of the
monofunctionlized and crossꢀlinked oligonucleotides were approved by RPꢀ18 HPLC and
MALDIꢀTOF mass spectrometry (Tables 1 and 5).
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11
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13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
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30
31
32
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36
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39
40
41
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44
45
46
47
48
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50
51
52
53
54
55
56
57
58
59
60
Encouraged by the promising results of the stepwise chelateꢀassisted click reaction for
oligonucleotides forming aps duplexes, we consequently used this protocol for the first time
for the crossꢀlinking of 2’ꢀOꢀpropargylated oligonucleotides forming ps duplexes (see next
section).
Scheme 6. Stepwise Click Reaction of Sugar Modified DNA with Unsymmetrical
Chelating BisꢀAzide 6
NH2
N
NH2
N
N
N
N
N
HN
HN
N
N
N
N3
N
Cu(OAc)
2-TBTA
O
O
+
6
H2O/DMSO/t-BuOH
O
O
O
O
ODN-25 : azide 6 = 1:1
O
5'-d(T-A)
5'-d(T-A)
O
(G-T-C-A-A-T-A-C-T)-3'
O
(G-T-C-A-A-T-A-C-T)-3'
O
ODN-40
ODN-25
O
(A-T)-5'
3'-d(T-C-A-T-A-A-C-T-G)
O
NH2
N
N
O
HN
O
ODN-25
CuSO
4 -TBTA, TCEP
N
Linker
O
O
N
ODN-40
N
N
benzoic acid,
H2O/DMSO/t-BuOH
O
NH
NH2
O
O
5'-d(T-A)
(G-T-C-A-A-T-A-C-T)-3'
O
N
N
N
Linker =
ODN-44
N
N
N
N
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Earlier studies on sugar crossꢀlinked homodimers hybridized with complementary
oligonucleotides thereby forming ligated duplexes showed that crossꢀlinking at the center of
the duplex is slightly destabilizing, while for crossꢀlinking at the termini of the helices
stabilization was observed.10 A similar trend is also found for the aps homodimeric duplexes
presented in Table 1 with ꢂTm ranging from ꢀ6°C to +2°C compared to the noncrossꢀlinked
duplexes.
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19
20
21
22
23
24
25
26
27
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39
40
41
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Table 1. Tm Values and MALDIꢀTOF Masses of Sugar Propargylated Duplexes and CrossꢀLinked Duplexes (Homodimers)a
6
7
8
9
N3
N3
N3
N
N3
O
M.W. calcd b Tm
M.W. found [°C]
M.W. calcd
M.W. found
Tm
[°C]
M.W. calcd
M.W. found
Tm
[°C]
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18
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21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
Duplex
Homoꢀcrossꢀlinked ODNs
Duplex
Homoꢀcrossꢀlinked ODNs
Duplex
5’ꢀd(TA1 GTC AAT ACT)
5’ꢀd(TA1 GTC AAT ACT)
7586.1
7584.4
5’ꢀd(TA1 GTC AAT ACT) (25)
3’ꢀd(ATiC CAG TTA TGA) (26)
3698.4
53
7553.1
7552.5
│
(36)
49
41
52
44
│
(44)
47
41
51
48
26•36•26
26•44•26
3697.5
5’ꢀd(TA1 GTC AAT ACT)
5’ꢀd(TA1 GTC AAT ACT)
5’ꢀd(TAG GT3 AAT ACT)
5’ꢀd(TAG GT3 AAT ACT)
5’ꢀd(TAG GT3 AAT ACT) (27)
3’ꢀd(ATC CAG TTATGA) (28)
3698.4
46
7553.1
7553.6
7586.1
7585.1
│
(37)
│
(45)
28•37•28
29•38•29
30•39•30
28•45•28
29•46•29
30•47•30
3697.3
5’ꢀd(TAG GT3 AAT ACT)
5’ꢀd(TAG GT3 AAT ACT)
5’ꢀd(TAG GTC AAT A3T)
5’ꢀd(TAG GTCAA3 ACT)
5’ꢀd(TAG GTCAAT A3T) (29)
3’ꢀd(ATC CAG TTA TGA) (28)
3698.4
50
7553.1
7554.4
7586.1
7585.4
│(38)
│
(46)
3697.4
5’ꢀd(TAG GTC AAT A3T)
5’ꢀd(TAG GTCAA3 ACT)
3’ꢀd(ATC 3AG TTA TGA)
3’ꢀd(ATC 3AG TTA TGA)
5’ꢀd(TAG GTCAAT ACT) (30)
3’ꢀd(ATC 3AG TTA TGA) (31)
3698.4
49
7553.1
7553.7
7586.1
7585.2
│
(39)
│
(47)
3697.1
3’ꢀd(ATC 3AG TTA TGA)
3’ꢀd(ATC 3AG TTA TGA)
a Measured at 260 nm in a 1 M NaCl solution containing 100 mM MgCl2 and 60 mM Naꢀcacodylate (pH 7.0) with 2.5 ꢃM singleꢀstrand concentration of each strand in noncrossꢀ
linked duplexes and 5 ꢄM of crossꢀlinked duplexes. The Tm values were determined from the melting curves using the software MELTWIN, version 3.0. b The molecular masses
refer to the propargylated oligonucleotides. ODNs 36ꢀ39 were prepared by both stepwise and bisꢀclick reaction using nonꢀchelating azide 5. ODNs 44ꢀ47 were prepared by
stepwise click reactions using chelating bisazide 6.
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2.3. Crossꢀlinking of Propargylated Oligonucleotides to Heterodimers with Parallel
Chain Orientation.
3
4
5
6
7
8
9
General Aspects. Parallel stranded duplex DNA was proposed by Pattabiraman already in
198620 and represents a unique DNA structure with both sugarꢀphosphate chains pointing to
the same direction.21 A series of manuscripts were published by our laboratory and by others
reporting on the chemical synthesis and physical properties of ps DNA.22ꢀ23 These studies
demonstrated that the structural features and spectroscopic properties, enzymatic recognition
as well as drugꢀbinding properties of ps DNA are different from native DNA with antiparallel
chains.4d,24 However, up to now it is unknown whether the concept of crossꢀlinking via sugar
modified oligonucleotides using click chemistry can be realized for parallel stranded duplexes
in a similar way as observed for aps DNA despite their structural differences (grooves of
almost identical size).
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42
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44
45
46
47
48
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50
51
52
53
54
55
56
57
58
59
60
2.4. Synthesis of Heterodimeric Oligonucleotides with Parallel and Antiparallel Chain
Orientation. For the construction of crossꢀlinked ps duplexes, oligonucleotides containing
dAꢀdT sequence elements and 2’ꢀOꢀpropargyl modifications were used (1, 3, A*; for the
structure of A* see Table 2), which are able to form duplexes with either parallel or
antiparallel chain orientation (Table 2).
The stepwise chelateꢀassisted click reaction protocol was now applied to ODNꢀ50 (Table 2)
containing 2’ꢀOꢀpropargylated isoguanosine (1) as described in section 2.2. In the chelateꢀ
assisted first click reaction, ODNꢀ50 was reacted with bisꢀazide 6 in the presence of TBTAꢀ
Cu(OAc)2•H2O complex at room temperature. After completion of the reaction (8 h), the
monofunctionalized ODNꢀ55 was obtained in good yield. Accordingly, the
monofunctionalized ODNs 3’ꢀd(TT1az TTT TTT TAT TAA AAT TTA TAA A)ꢀ5’ (ODNꢀ
56), 5’ꢀd(AA23 AAA AAA ATA ATT TTA AAT ATT T)ꢀ3’ (ODNꢀ57), 5’ꢀd(AazAA AAA
AAA ATA ATT TTA AAT ATT T)ꢀ3’ (ODNꢀ58) and 5’ꢀd(AazAC AAA AAA ATA ATT
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TTA AAT ATT T)ꢀ3’ (ODNꢀ59) were synthesized (for the structure of Aaz see Table 2). For
the second click reaction, monofunctionalized ODNꢀ55 still carrying the nonchelating azido
group was reacted with complementary ODNꢀ60 (CuSO4ꢀTBTA complex, TCEP, benzoic
acid, H2O/DMSO/tꢀBuOH) to give the crossꢀlinked ps duplex ODNꢀ61 (Table 2). It should be
noted that by this protocol, we were able to construct a sugar crossꢀlink within the parallel
base pair of 2’ꢀOꢀpropargylated isoguanosine (1) and cytidine (3) as a component of a duplex
with parallel strand orientation. Similarily, other heterodimeric DNA constructs with ps strand
orientation and internal sugar crossꢀlinks were synthesized (ODNꢀ52 and ODNꢀ54). However,
contrary to ODNꢀ61, the 2’ꢀOꢀpropargyl modification of ODNꢀ52 and ODNꢀ54 was not part
of a base pair. Instead, the crossꢀlink was constructed between sugar residues separated by
one dAꢀdT base pair (Table 2). For comparison, the reactions described above were also
performed on ODNs with aps orientation. All monofunctionalized and crossꢀlinked
oligonucleotides were characterized by MALDIꢀTOF mass spectrometry (Table 5), ionꢀ
exchange HPLC or gel electrophoresis (Figure 4) confirming the integrity of the DNA
adducts.
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23
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25
26
27
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35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The results clearly indicate that the chemoselective ligation protocol using bisꢀazide 6 can be
used to construct heterodimeric DNA adducts with internal sugar crossꢀlinks at proximal
(base pairs) and distant positions. We anticipate that the high local concentration of the
duplex facilitates the reaction sites situated near the flexible end of the duplex to come
together, leading to efficient crossꢀlinking. This protocol can be applied as efficiently to
duplexes with ps orientation as well as to duplexes with aps orientation, despite any
differences in the helical structure of ps and aps DNA.
3. Characterization of Crossꢀlinked Oligonucleotide Adducts. Ionꢀexchange HPLC and
denaturing gel electrophoresis (PAGE) were used to analyze monofunctionalized click
adducts as well as heterodimeric DNA constructs with parallel strand orientation. Figure 3a
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shows the ionꢀexchange chromatography profile of the monofunctionalized 25ꢀmer ODNꢀ55
with a retention time of 17.6 min. On the contrary, the heterodimeric ps adduct ODNꢀ61
obtained after crossꢀlinking of monofunctionalized ODNꢀ55 and propargylated ODNꢀ60
shows a retention time of 20.6 min, confirming the formation of the interstrand crossꢀlink
within a base pair (mobility depends on the number of negative charges – phospodiester
anions – of the oligonucleotides). Similar to ODNꢀ61, a retention time of 20.0 min was
observed for ps adduct ODNꢀ54 with a crossꢀlink of sugar residues at a distant position
(Figure 3b).
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25
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44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(a)
(b)
Figure 3. Ionꢀexchange HPLC elution profiles of (a) monofunctionalized ODNꢀ55 and
interstrand crossꢀlinked ODNꢀ61; (b) Crossꢀlinked ODNꢀ54. Ionꢀexchange chromatography
was performed on a 4 x 250 mm DNA Pac PAꢀ100 column, using the following buffer
system: (A) 25 mM TrisꢀHCl, 10% MeCN, pH 7.0; (B) 25 mM TrisꢀHCl, 1.0 M NaCl, and
10% MeCN, pH 7.0. Elution gradient: 0ꢀ30 min 20ꢀ80% B in A with a flow rate of 0.75 mL
minꢀ1.
Sugar crossꢀlinking of ps oligonucleotide duplexes was also confirmed by denaturing
PAGE.10 As shown in Figure 4a, the crossꢀlinked adducts ODNꢀ61 (lane 3) and ODNꢀ54 (lane
4) with parallel strand orientation migrate much slower than the 2’ꢀOꢀpropargylated starting
material (lane 1, ODNꢀ50 and lane 2, ODNꢀ60) and the corresponding monofunctionalized
oligonucleotides still bearing one azido group (lane 5: ODNꢀ55, lane 6: ODNꢀ57, lane 7:
ODNꢀ58).
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The crossꢀlinked adducts ODNꢀ66 (lane 3) and ODNꢀ65 (lane 4) with antiparallel strand
orientation (Figure 4b) show a comparable mobility as the corresponding parallel stranded
crossꢀlinked adduct ODNꢀ52 (lane 5) which proves the successful sugar crossꢀlinking of ps
duplexes.
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54
55
56
57
58
59
60
1 2 3 4 5 6
(a)
7
1 2 3 4 5 6 7
(b)
Figure 4. Denaturing PAGE analysis of monofunctionalized oligonucleotides and crossꢀ
linked click adducts on a 17% polyacrylamide gel. (a) Lane 1: ODNꢀ50; 2: ODNꢀ60; 3: crossꢀ
linked ps ODNꢀ61; 4: crossꢀlinked ps ODNꢀ54; 5: monofunctionalized ODNꢀ55; 6:
monofunctionalized ODNꢀ57; 7: monofunctionalized ODNꢀ58. (b) Lane 1:
monofunctionalized ODNꢀ56; 2: monofunctionalized ODNꢀ59; 3: crossꢀlinked aps ODNꢀ66;
4: crossꢀlinked aps ODNꢀ65; 5: crossꢀlinked ps ODNꢀ52: 6: ODNꢀ50; 7: ODNꢀ51.
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4. Stability of Crossꢀlinked Duplexes. The structure of an aps WatsonꢀCrick helix is
significantly different from that of ps DNA. Theoretical studies have shown that the groove
size of a dAꢀdTꢀrich WatsonꢀCrick (aps) helix is 11 Å for the minor and 20 Å for the major
groove, and 13 and 16 Å for the minor and major grooves, respectively, for the ps DNA.25
The helix sense of ps DNA is right handed, the DNA has about 10.7 bp per turn and the axial
rise per base pair is 3.4 Å leading to the height of one helix turn of 36.4 Å, compared to aps
DNA with 10 bp per turn and a height of one helix turn of 34 Å. These findings were also
supported by NMR experiments.26 Moreover, it was shown that dAꢀdTꢀrich ps DNA is less
stable than its aps counterpart.25 Consequently, different protocols were developed to stabilize
ps DNA by chemical modification. Our laboratory has introduced tridentate base pairs of
dGꢁisoCd and dCꢁisoGd which stabilize ps DNA (Figure 5). 7ꢀHalogenated 7ꢀdeazaꢀ2’ꢀ
deoxyisoguanosines, 7ꢀsubstituted 8ꢀazaꢀ7ꢀdeazaꢀ2’ꢀdeoxyisoguanosines as well as the Gꢀ
clamp have been used for this purpose.3d, 27Additionally, ps DNA was stabilized by
incorporation of modified purine nucleosides in the reverse WatsonꢀCrick base pair (Donohue
pair) as it was reported for dAꢀdT base pairs in aps DNA (Figure 5).28 A third possibility,
which is the topic of this manuscript, is the crossꢀlinking of ps DNA. However, it stays to
proof if ligation at “identical” sequence positions in aps and ps DNA can stabilize duplexes in
a similar way. Due to the structural differences of ps and aps duplexes, structural
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modifications can induce more steric stress on ps DNA than on aps DNA. Such a negative
phenomenon can obscure the favourable stability increase induced by crossꢀlinking.
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H
H
S
N
N
H
O
N
O
N
N
H
O
N
O
N
N
N
N
N
H
H
N
N
N
S
S
S
reverse Watson - Crick/
Donohue base pair
dA - dT
Watson - Crick base pair
dA - dT
9
H
H
S
S
10
11
12
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36
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40
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60
N
N
O
H
H
O
N
N
O
N
N
H
N
N
O
N
N
N
N
N
N
H
N
N
S
S
H
H
H
H
isoGd - dC
dG - isoCd
H
N
H
O
N
N
N
O
H
N
N
N
N
S
H
S
H
isoGd - isoCd
S: corresponds to 2'-deoxyribofuranosyl
Figure 5. Base pair motifs of ps and aps DNA; arrows indicate chain orientation.
Table 2 summarizes the Tm data obtained form crossꢀlinked and noncrossꢀlinked
oligonucleotides synthesized in this study and compare the effect of crossꢀlinks in ps and aps
DNA. The influence of the different helical geometries (groove size) on stability changes
caused by crossꢀlinking is demonstrated. The crossꢀlinks were indroduced between
heterodimeric complementary oligonucleotides at the termini of duplexes (i) in a fully
matching sequence with the crossꢀlink in a two base pair distance (52); (ii) in a mismatched
sequence with the crossꢀlink in a two base pair distance (54) and (iii) in a fully matching
sequence with the crossꢀlink within a base pair (61).
At first, the influence of the clickable sugar modification on duplex stability is compared
(Table 2) and leads to the following conclusions: (i) as reported before,6 the 25ꢀmer ps
duplexes are less stable than the corresponding aps duplexes, which results from the
thermodynamically less stable dAꢀdT Donohue base pairs compared to the WatsonꢀCrick
pairs; (ii) the introduction of propargylated residues (1, 3, A*) leads to a slight destabilization
of the ps duplexes compared to the unmodified parent duplex 48 • 49, independent of the
position of the propargylated residue or of a matching or mismatching situation and (iii) the
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additional functionalization of the side chains by bisꢀazide 6 generating an azidoꢀmodified
side chain stabilizes ps and aps duplexes slightly more than the shorter propargyl chain.
Different to the noncrossꢀlinked duplexes, crossꢀlinked DNA generated with bisꢀazide 6 and
propargylated sugar residues leads to a strong stabilization of ps DNA. In addition, a
destabilized mismatched ps duplex is as efficiently stabilized by crossꢀlinking as a fully
matching duplex. From Table 2, it is apparent that the crossꢀlinks stabilize both ps and aps
duplexes significantly. The increase in Tm is similar in both cases, showing that the crossꢀlinks
are well accepted in ps and aps duplexes.
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Table 2. Tm Values of Sugar Modified and Sugar Crossꢀlinked ps and aps Duplexesa
b
c
b
d
Tm
∆Tm
∆Tm
Tm
∆Tm
∆Tm
ps Duplexes
aps Duplexes
[°C] [°C]
[°C]
[°C]
[°C]
[°C]
9
5’ꢀd(TTT TTT TTT TAT TAA AAT TTA TAA A)ꢀ3’ (48)
5’ꢀd(AAA AAA AAA ATA ATT TTA AAT ATT T)ꢀ3’ (49)
5’ꢀd(TTT TTT TTT TAT TAA AAT TTA TAA A)ꢀ3’ (48)
3’ꢀd(AAA AAA AAA ATA ATT TTA AAT ATT T)ꢀ5’ (62)
42.0
40.0
ꢀꢀ
56.0
59.0
ꢀꢀ
ꢀꢀ
ꢀꢀ
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
5’ꢀd(T T 1 TTT TTT TAT TAA AAT TTA TAA A)ꢀ3’ (50)
5’ꢀd(A*AC AAA AAA ATA ATT TTA AAT ATT T)ꢀ3’ (51)
3’ꢀd(TT 1 TTT TTT TAT TAA AAT TTA TAA A)ꢀ5’ (63)
5’ꢀd(A*AiC AAA AAA ATA ATT TTA AAT ATT T)ꢀ3’ (64)
ꢀ2.0
+14.0
ꢀ6.5
+3.0
+14.0
ꢀ4.0
+11.0
+15.0
+16.0
5’ꢀd(TT1 TTT TTT TAT TAA AAT TTA TAA A)ꢀ3’
3’ꢀd(TT 1 TTT TTT TAT TAA AAT TTA TAA A)ꢀ5’
56.0
35.5
54.0
70.0
52.0
67.0
┌┘
(52)
┌─┘
(65)
5’ꢀd(A*AC AAA AAA ATA ATT TTA AAT ATT T)ꢀ3’
5’ꢀd(A*AiC AAA AAA ATA ATT TTA AAT ATT T)ꢀ3’
5’ꢀd(T T 1 TTT TTT TAT TAA AAT TTA TAA A)ꢀ3’ (50)
5’ꢀd(A*AA AAA AAA ATA ATT TTA AAT ATT T)ꢀ3’ (53)
3’ꢀd(T T1 TTT TTT TAT TAA AAT TTA TAA A)ꢀ5’ (63)
5’ꢀd(A*AA AAA AAA ATA ATT TTA AAT ATT T)ꢀ3’ (53)
+18.5
ꢀ
5’ꢀd(TT1 TTT TTT TAT TAA AAT TTA TAA A)ꢀ3’
3’ꢀd(TT1 TTT TTT TAT TAA AAT TTA TAA A)ꢀ5’
+12.0
+11.0
┌┘
(54)
┌─┘
(66)
5’ꢀd(A*AA AAA AAA ATA ATT TTA AAT ATT T)ꢀ3’
5’ꢀd(TT1az TTT TTT TAT TAA AAT TTA TAA A)ꢀ3’ (55)
5’ꢀd(AA3 AAA AAA ATA ATT TTA AAT ATT T)ꢀ3’ (60)
5’ꢀd(A*AA AAA AAA ATA ATT TTA AAT ATT T)ꢀ3’
5’ꢀd(T T1 TTT TTT TAT TAA AAT TTA TAA A)ꢀ3’ (50)
3’ꢀd(AAiC AAA AAA ATA ATT TTA AAT ATT T)ꢀ5’ (67)
5’ꢀd(TT1az TTT TTT TAT TAA AAT TTA TAA A)ꢀ3’ (55)
3’ꢀd(AAiC AAA AAA ATA ATT TTA AAT ATT T)ꢀ5’ (67)
38.0
37.0
ꢀ4.0
ꢀ5.0
54.5
55.0
ꢀ
ꢀ1.5
ꢀ1.0
5’ꢀd(TT1 TTT TTT TAT TAA AAT TTA TAA A)ꢀ3’ (50)
5’ꢀd(AA3 AAA AAA ATA ATT TTA AAT ATT T)ꢀ3’ (60)
+0.5
+18.5
5’ꢀd(TT1 TTT TTT TAT TAA AAT TTA TAA A)ꢀ3’
ꢀꢀ
55.5
+13.5
ꢀꢀ
ꢀꢀ
ꢀꢀ
│
(61)
5’ꢀd(AA3 AAA AAA ATA ATT TTA AAT ATT T)ꢀ3’
aMeasured at 260 nm in 0.1 M NaCl, 10 mM MgCl2, 10 mM Naꢀcacodylate (pH 7.0) with 5 ꢃM + 5 ꢃM singleꢀstrand concentration for noncrossꢀlinked oligonucleotides and 1
ꢄM for crossꢀlinked oligonucleotides. b ꢂTm was calculated as Tm crossꢀlinked duplex − Tm propargylated duplex. c ꢂTm was calculated as Tm modified duplex − Tm unmodified
duplex using 48•49 as comparison. d ꢂTm was calculated as Tm modified duplex − Tm unmodified duplex using 48•62 as comparison. Monofunctionalized and crossꢀlinked ODNs
were prepared by stepwise click reactions.
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Conclusion and Outlook
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9
In this work, we demonstrated that complementary oligonucleotides with propargylated sugar
residues and parallel strand orientation can be ligated efficiently in a stepwise manner with
chelating bisꢀazides as it was observed for antiparallel duplexes. For this, a series of
propargylated homodimeric and heterodimeric duplexes were prepared. Corresponding
phosphoramidites of propargylated isoGd were prepared, and the synthesis of propargylated
dC was improved. From the above findings, it is apparent that the crossꢀlinks stabilize both ps
and aps duplexes significantly. The increase in Tm is similar in both cases, clearly indicating
that the crossꢀlinks are well accommodated in ps DNA leading to the strong stabilization. This
was rather unexpected as the different helix geometries of ps and aps DNA can induce more
steric stress on ps DNA than on aps DNA. The application of the method expands the
repertoire of construction tools for larger assemblies and nanodevices based on DNA
including walkers, tweezers and nanomachines.29
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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. Thinꢀlayer
chromatography (TLC) was performed on TLC aluminum sheets covered with silica gel 60
F254 (0.2 mm). Flash column chromatography (FC): silica gel 60 (40ꢀ60 ꢃM) at 0.4 bar. UV
spectra were recorded on a spectrophotometer; λmax in nm, ε in dm3 molꢀ1 cmꢀ1. NMR spectra
were measured at 300.15 MHz for 1H and 75.48 MHz for 13C and 121.52 MHz for 31P. The J
values are given in Hz; δ values in ppm relative to Me4Si as internal standard. For NMR
spectra recorded in DMSOꢀd6, the chemical shift of the solvent peak was set to 2.50 ppm for
1H NMR and 39.50 ppm for 13C NMR. The 13C NMR signals were assigned on the basis of
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DEPTꢀ135 and 1Hꢀ13C gatedꢀdecoupled NMR spectra (for coupling constants see Tables S1
and S2, supporting information). Reversedꢀphase HPLC was carried out on a 250 x 4 mm RPꢀ
18 LiChrospher 100 column with a HPLC pump connected with a variable wavelength
monitor, a controller and an integrator. Gradients used for HPLC chromatography: A =
MeCN; B = 0.1 M (Et3NH)OAc (pH 7.0)/MeCN, 95:5. Conditions: (I) 3 min 15% A in B, 12
min 15ꢀ50% A in B, and 5 min 50ꢀ10% A in B, flow rate 0.7 mL minꢀ1; (II) 0ꢀ25 min 0ꢀ20%
A in B, flow rate 0.7 mL minꢀ1. Melting curves were measured with a UVꢀVIS
spectrophotometer equipped with a thermoelectrical controller with a heating rate of 1°C/min.
The Tm values were determined from the melting curves using the software MELTWIN,
version 3.0. (J. A. Mc Dowell, 1996). ESIꢀTOF mass spectra of the nucleosides were
measured with a MicroꢀTOF spectrometer. Molecular masses of oligonucleotides were
determined by MALDIꢀTOF mass spectrometry in the linear positive mode with 3ꢀ
hydroxypicolinic acid (3ꢀHPA) as a matrix (Tables 1 and 5).
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Table 3. 13C NMR Table for Cytidine Derivativesa
Pyridynyl
linker
C(2)b C(4)b C(5)b C(6)b
CH2 C(1’) C(2’) C(3’) C(4’) C(5’) C=O OMe
Triazole
ꢀꢀ
C≡C
7c 155.3 165.4
93.9
94.1
96.2
141.3
141.3
144.5
ꢀꢀ
ꢀꢀ
89.0
73.8
80.4
69.3
68.0
83.9
84.3
60.5
60.2
ꢀꢀ
ꢀꢀ
ꢀꢀ
ꢀꢀ
ꢀꢀ
80.1
77.3
154.6 165.2
154.2 163.2
154.3 163.1
154.9 165.6
155.0 165.6
56.8 87.1
ꢀꢀ
ꢀꢀ
ꢀꢀ
3
8
ꢀꢀ
ꢀꢀ
80.9
80.2
57.2 88.5d 82.1
67.8 86.0d 61.7 167.4 55.0
79.9
77.7
ꢀꢀ
96.0
93.8
93.9
144.5
141.1
141.0
56.7 91.3
71.9
81.2
81.2
80.0
67.9
67.9
86.1
84.0
84.0
61.1 167.2 56.1
9
155.1, 149.2
137.4, 130.9
122.1
155.1, 149.1
131.2, 122.2
122.2
50.6
87.3
54.1
144.2
124.8
ꢀꢀ
ꢀꢀ
60.0
60.0
ꢀꢀ
ꢀꢀ
ꢀꢀ
ꢀꢀ
23
24
50.0
87.3
54.0
144.4, 144.2
124.8, 124.2
a Measured in DMSOꢀd6 at 298 K. b Pyrimidine numbering. c Reference 30a. d Tentative.
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Table 4. 13C NMR Table for Purineꢀ2,6ꢀdiamine and Isoguanine Nucleosidesa
C(2)b,c C(4)b,c C(5)b C(6)b,c C(8)b
C(1’) C(2’) C(3’) C(4’) C(5’) CH=N NMe2 =CMe C = O
C≡C
CH2
ꢀꢀ
12e
13
14
156.3
156.2
156.3
151.4 113.6 160.1 136.3
151.4 113.4 160.1 135.8
151.4 113.6 160.0 136.1
ꢀꢀ
87.1
73.2
79.8
72.7
73.0
79.5
72.5
79.7
72.2
79.8
77.3
79.8
72.0
70.7
68.9
77.2
70.9
68.8
77.3
68.9
77.3
68.7
61.3
69.0
76.7
85.5 61.8
84.8 61.6
83.3 61.6
85.6 61.7
85.4 61.4
83.9 61.7
85.5 61.6
83.9 61.8
84.9 61.2
83.6 61.3
84.8 63.6
85.7 63.1
ꢀꢀ
ꢀꢀ
ꢀꢀ
ꢀꢀ
ꢀꢀ
ꢀꢀ
ꢀꢀ
ꢀꢀ
ꢀꢀ
ꢀꢀ
ꢀꢀ
ꢀꢀ
ꢀꢀ
ꢀꢀ
ꢀꢀ
ꢀꢀ
ꢀꢀ
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
79.7
77.6
56.9 86.2
57.2 86.9
80.5
77.6
ꢀꢀ
ꢀꢀ
isoGf 158.3
ꢀꢀd
ꢀꢀd
ꢀꢀd
109.8 155.9 138.4
109.2 155.3 137.9
109.6 155.6 138.4
ꢀꢀ
ꢀꢀ
87.8
ꢀꢀ
ꢀꢀ
79.1
77.3
ꢀꢀd
56.6 86.6
57.2 87.7
56.8 86.7
57.2 87.6
56.8 86.4
57.2 87.3
57.2 85.7
57.0 87.1
ꢀꢀ
ꢀꢀ
1
80.4
77.7
ꢀꢀd
ꢀꢀ
ꢀꢀ
2
79.1
77.5
ꢀꢀd
38.0
ꢀꢀd
38.1
156.2
156.2
160.6
160.8
160.8
160.8
153.6 113.1 156.9 140.2
153.7 113.4 156.9 140.5
151.4 123.7 155.3 141.7
151.5 124.0 155.3 141.9
151.5 123.8 158.0 141.9
151.5 123.9 158.0 141.9
163.9
164.0
162.3
162.4
162.5
162.4
18.8
18.8
17.2
17.3
17.3
17.2
15
16
17
18
19
20
80.4
77.7
ꢀꢀ
151.7
77.5
79.5
37.6
38.2
151.8
151.9
151.9
80.3
77.3
37.7
38.3
79.6
77.7
37.7
38.4
80.1
77.5
37.7
38.3
a Measured in DMSOꢀd6 at 298 K. b Purine numbering. c Tentative. d Not detected. e Reference 30b. f Reference 30c.
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Oneꢀpot Synthesis of 4ꢀ(Benzoylamino)ꢀ1ꢀ[5ꢀOꢀ(4,4’ꢀdimethoxytrityl)ꢀ2ꢀOꢀ(propargyl)ꢀβꢀ
Dꢀribofuranosyl]ꢀ2Hꢀpyrimidinꢀ2ꢀone (8) and 4ꢀ(Benzoylamino)ꢀ1ꢀ[5ꢀOꢀ(4,4’ꢀ
dimethoxytrityl)ꢀ3ꢀOꢀ(propargyl)ꢀβꢀDꢀribofuranosyl]ꢀ2Hꢀpyrimidinꢀ2ꢀone (9). Cytidine 7
(1.0 g, 4.11 mmol) was dissolved in hot anhydrous DMF (25 mL) under an argon atmosphere.
The solution was cooled to 5°C, and NaH (0.13 g, 3.3 mmol, 60% dispersion in mineral oil)
was subsequently added, followed by the addition of tetrabutylammoniumiodide (TBAI) (0.68
g, 1.85 mmol) and propargyl bromide (0.49 g, 4.11 mmol). The reaction mixture was allowed
to stir for 3 days at 55°C. After completion of the reaction (TLC monitoring), evaporation of
the solvent under reduced pressure gave a suspension, which was absorbed on silica gel. FC
(silica gel, CH2Cl2/MeOH, 9:1) gave a chromatographically inseparable mixture of 2’ꢀOꢀ
propargylcytidine (3) and 3’ꢀOꢀpropargylcytidine (4) as colourless solid (0.74 g, 64%). TLC
(silica gel, CH2Cl2/MeOH, 9:1) Rf 0.6.
3
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This solid was repeatedly coꢀevaporated with pyridine (3 x 10 mL) and suspended in pyridine
(20 mL). The suspension was treated with dimethoxytrityl chloride (1.73 g, 5.12 mmol),
triethylamine (0.52 g, 5.12 mmol) and 4ꢀ(dimethylamino)pyridine (0.13 g, 1.07 mmol) and
stirred at room temperature for 4.5 h. After consumption of the starting material (TLC
monitoring, CH2Cl2/acetone, 8:2), trimethylsilyl chloride (1.39 g, 12.81 mmol) was added and
stirring was continued at room temperature for 30 min. Then, benzoyl chloride (1.80 g, 12.81
mmol) was added, and stirring was continued for another 3 h. The reaction mixture was
cooled in an iceꢀbath and 5 mL of water was added. After 5 min, 10 mL of 28ꢀ30% aq.
ammonia was added and stirring was continued at r.t. for another 30 min. The reaction
mixture was evaporated to dryness. The remaining residue was dissolved in dichloromethane
(100 mL) and extracted with 5% aq. NaHCO3 (50 mL). The aqueous layer was washed with
dichloromethane (2 x 100 mL). The organic layer was collected, dried over Na2SO4 and
concentrated under reduced pressure. The residue was coꢀevaporated with toluene (3 x 10
mL) and subjected to FC (silica gel, column 15 x 3 cm, CH2Cl2/acetone, 80:20). From the fast
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migrating zone compound 8 (0.77 g, 44%) was isolated as yellowish foam, and from the
slower migrating zone compound 9 (0.18 g, 10%) was isolated as a colourless foam.
6
7
8
9
4ꢀ(Benzoylamino)ꢀ1ꢀ[5ꢀOꢀ(4,4’ꢀdimethoxytrityl)ꢀ2ꢀOꢀ(propargyl)ꢀβꢀDꢀribofuranosyl]ꢀ2Hꢀ
pyrimidinꢀ2ꢀone (8). TLC (silica gel, CH2Cl2/acetone, 80:20) Rf 0.4. λmax (MeOH)/nm 260
(ε/dm3 molꢀ1 cmꢀ1 23 500), 235 (32 600), 304 (11 600). 1H NMR (DMSOꢀd6, 300 MHz): δ
3.49 (t, J = 2.4 Hz, 1H, C≡CH), 3.76 (s, 6H, 2x OCH3), 4.05ꢀ4.08 (m, 2H, C3’ꢀH, C4’ꢀH),
4.33ꢀ4.40 (m, 1H, C2’ꢀH), 4.49 (d, J = 2.1 Hz, 2H, CH2), 5.34 (d, J = 7.2 Hz, 1H, C3’ꢀOH),
5.89 (d, J = 1.5 Hz, 1H, C1’ꢀH), 6.88ꢀ6.94 (m, 4H, ArꢀH), 7.14 (d, J =7.5 Hz, 1H, C5ꢀH),
7.24ꢀ7.43 (m, 9H, ArꢀH), 7.49ꢀ7.54 (m, 2H, ArꢀH ), 7.60ꢀ7.65 (m, 1H, ArꢀH ), 7.98ꢀ8.01 (m,
2H, ArꢀH), 8.38 (d, J = 7.5 Hz, 1H, C6ꢀH), 11.33 (br s, 1H, NH). Anal. calcd. for C40H37N3O8
(687.7): C 69.86, H 5.42, N 6.11. Found: C 70.00, H 5.49, N 6.10.
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4ꢀ(Benzoylamino)ꢀ1ꢀ[5ꢀOꢀ(4,4’ꢀdimethoxytrityl)ꢀ3ꢀOꢀ(propargyl)ꢀβꢀDꢀribofuranosyl]ꢀ2Hꢀ
pyrimidinꢀ2ꢀone (9). TLC (silica gel, CH2Cl2/acetone, 80:20) Rf 0.1. λmax (MeOH)/nm 260
(ε/dm3 molꢀ1 cmꢀ1 24 300), 236 (32 400), 304 (11 300). 1H NMR (DMSOꢀd6, 300 MHz): δ
3.52 (t, J = 2.4 Hz, 1H, C≡CH), 3.76 (s, 6H, 2x OCH3), 4.17ꢀ4.19 (m, 2H, C5’ꢀH, C4’ꢀH),
4.24 (d, J = 2.4 Hz, 1H, C3’ꢀH), 4.32ꢀ4.41 (m, 3H, CH2, C2’ꢀH), 5.77ꢀ5.79 (m, 2H, C2’ꢀOH,
C1’ꢀH), 6.92ꢀ6.65 (m, 4H, ArꢀH), 7.14 (d, J = 7.2 Hz, 1H, C5ꢀH), 7.24ꢀ7.44 (m, 9H, ArꢀH),
7.49ꢀ7.54 (m, 2H, ArꢀH ), 7.60ꢀ7.65 (m, 1H, ArꢀH ), 7.99ꢀ8.02 (m, 2H, ArꢀH), 8.46 (d, J = 7.2
Hz, 1H, C6ꢀH), 11.29 (s, 1H, NH). Anal. calcd. for C40H37N3O8 (687.7): C 69.86, H 5.42, N
6.11. Found: C 70.00, H 5.60, N 6.01.
4ꢀ(Benzoylamino)ꢀ1ꢀ[5ꢀOꢀ(4,4’ꢀdimethoxytrityl)ꢀ2ꢀOꢀ(propargyl)ꢀβꢀDꢀribofuranosyl]ꢀ2Hꢀ
pyrimidinꢀ2ꢀone 3ꢀOꢀ(2ꢀCyanoethyl)ꢀN,Nꢀdiisopropylphosphoramidite (10). Compound 8
(0.58 g, 0.84 mmol) was dissolved in dry CH2Cl2 (15 mL) and subsequently (iꢀPr)2NEt (0.457
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g, 594 ꢄL, 3.54 mmol) and 2ꢀcyanoethyl diisopropylphosphoramidochloridite (0.398 g, 375
ꢄL, 1.68 mmol) were added, and the solution was stirred for 1 h at rt. After completion of the
reaction (TLC monitoring), the reaction mixture was diluted with CH2Cl2 (30 mL) and was
poured into 5% aq. NaHCO3 (50 mL), extracted with CH2Cl2 (3 × 20 mL). The combined
organic layers were dried over Na2SO4 and the solvent was evaporated. The residual foam
was applied to FC (silica gel, CH2Cl2/acetone, 90:10). Evaporation of the main zone afforded
10 (0.57 g, 76%) as a light yellow foam. TLC (silica gel, CH2Cl2/acetone, 80:20) Rf 0.8. 31P
NMR (CDCl3, 121.5 MHz): 150.33, 150.43.
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4ꢀ(Benzoylamino)ꢀ1ꢀ[5ꢀOꢀ(4,4’ꢀdimethoxytrityl)ꢀ3ꢀOꢀ(propargyl)ꢀβꢀDꢀribofuranosyl]ꢀ2Hꢀ
pyrimidinꢀ2ꢀone 2ꢀOꢀ(2ꢀCyanoethyl)ꢀN,Nꢀdiisopropylphosphoramidite (11). As described
for 10. Compound 9 (0.2 g, 0.29 mmol) in dry CH2Cl2 (10 mL), (iꢀPr)2NEt (0.157 g, 204 ꢄL,
1.22 mmol) and 2ꢀcyanoethyl diisopropylphosphoramidochloridite (0.137 g, 129 ꢄL, 0.58
mmol). FC (silica gel, CH2Cl2/acetone, 90:10). Evaporation of the main zone afforded 11
(0.192 g, 74%) as a colourless foam. TLC (silica gel, CH2Cl2/acetone, 80:20) Rf 0.7. 31P NMR
(CDCl3, 121.5 MHz): 149.7.
4ꢀAminoꢀ1ꢀ[(2ꢀOꢀpropargyl)ꢀβꢀDꢀribofuranosyl]ꢀ2Hꢀpyrimidinꢀ2ꢀone (3). Compound 8
(0.4 g, 0.58 mmol) was dissolved in 28% aq. ammonia (50 mL) and stirred at 60°C overnight
in an autoclave. The solvent was evapoated, 85% aq. AcOH (10 mL) was added, and the
solution was stirred at rt for 1 h. After completion of the reaction, the solvent was evaporated,
and the residue was coꢀevaporated with ethanol and subjected to FC (silica gel,
CH2Cl2/MeOH, 85:15) to give 3 (0.15 g, 92%) as a colourless solid. TLC (silica gel,
CH2Cl2/MeOH, 80:20) Rf 0.3. λmax (MeOH)/nm 272 (ε/dm3 molꢀ1 cmꢀ1 9 800). 1H NMR
(DMSOꢀd6, 300 MHz): δ 3.42 (t, J = 2.4 Hz, 1H, C≡CH), 3.53ꢀ3.57 (m, 1H, C5’ꢀH), 3.65ꢀ
3.69 (m, 1H, C5’ꢀH), 3.82ꢀ3.83 (m, 1H, C4’ꢀH), 3.94 (t, J = 2.4 Hz, 1H, C3’ꢀH), 4.04ꢀ4.11
29
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