Synthesis, hybridization characteristics, and fluorescence properties of oligonucleotides modified with nucleobase-functionalized locked nucleic acid adenosine and cytidine monomers

Article abstract of DOI:10.1021/jo500994c

Conformationally restricted nucleotides such as locked nucleic acid (LNA) are very popular as affinity-, specificity-, and stability-enhancing modifications in oligonucleotide chemistry to produce probes for nucleic acid targeting applications in molecular biology, biotechnology, and medicinal chemistry. Considerable efforts have been devoted in recent years to optimize the biophysical properties of LNA through additional modification of the sugar skeleton. We recently introduced C5-functionalization of LNA uridines as an alternative and synthetically more straightforward approach to improve the biophysical properties of LNA. In the present work, we set out to test the generality of this concept by studying the characteristics of oligonucleotides modified with four different C5-functionalized LNA cytidine and C8-functionalized LNA adenosine monomers. The results strongly suggest that C5-functionalization of LNA pyrimidines is indeed a viable approach for improving the binding affinity, target specificity, and/or enzymatic stability of LNA-modified ONs, whereas C8-functionalization of LNA adenosines is detrimental to binding affinity and specificity. These insights will impact the future design of conformationally restricted nucleotides for nucleic acid targeting applications.

Full text of DOI:10.1021/jo500994c

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Article
Synthesis, hybridization characteristics and fluorescence properties
of oligonucleotides modified with nucleobase-functionalized
LNA (Locked Nucleic Acid) adenosine and cytidine monomers
Mamta Kaura, Pawan Kumar, and Patrick J. Hrdlicka
J. Org. Chem., Just Accepted Manuscript • Publication Date (Web): 16 Jun 2014
Downloaded from http://pubs.acs.org on June 18, 2014
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Synthesis, hybridization characteristics and fluorescence properties of oligonucleotides
modified with nucleobase-functionalized LNA (Locked Nucleic Acid) adenosine and
cytidine monomers
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Mamta Kaura, Pawan Kumar and Patrick J. Hrdlicka*
Department of Chemistry, University of Idaho, Moscow, ID 83844-2343, USA
* Hrdlicka@uidaho.edu (+1 208-885-0108)
NH2
NH2
NH2
NH2
NH2
N
H
N
N
H2N
N
N
N
N
N
N
N
N
O
O
O
N
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
P
O
O
O
P
O
O
O P
O P
C5-functionalized LNA-C monomers
C8-functionalized LNA-A monomers
- extraordinary affinity/specificity
- enzymatic resistance
- decreased affinity/specificity
- anti vs syn conformations
ABSTRACT. Conformationally restricted nucleotides such as locked nucleic acids (LNAs) are
very popular as affinity-, specificity- and stability-enhancing modifications in oligonucleotide
chemistry to produce probes for nucleic acid targeting applications in molecular biology,
biotechnology and medicinal chemistry. Considerable efforts have been devoted in recent years
to optimize LNA’s biophysical properties through additional modification of the sugar skeleton.
We recently introduced C5-functionalization of LNA uridines as an alternative and synthetically
more straightforward approach to improve LNA’s biophysical properties. In the present report,
we set out to test the generality of this concept by studying the characteristics of oligonucleotides
modified with four different C5-functionalized LNA cytidine and C8-functionalized LNA
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adenosine monomers. The results strongly suggest that C5-functionalization of LNA pyrimidines
indeed is a viable approach for improving the binding affinity, target specificity and/or
enzymatic stability of LNA-modified ONs, whereas C8-functionalization of LNA adenosines is
detrimental to binding affinity and specificity. These insights will impact the future design of
conformationally restricted nucleotides for nucleic acid targeting applications.
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INTRODUCTION. Conformationally restricted nucleotides1,2 are widely used as affinity-,
specificity- and stability-enhancing modifications in oligonucleotides for nucleic acid targeting
applications in molecular biology, biotechnology and medicinal chemistry.3 Locked Nucleic
Acid (LNA, Figure 1),4 which was independently developed by Wengel5 and Imanishi,6 is one of
the most promising examples of this compound class. LNA-modified oligodeoxyribonucleotides
(ONs) form highly thermostable duplexes with complementary DNA/RNA; increases in thermal
denaturation temperatures (Tm's) of up to 10 °C per modification relative to unmodified duplexes
have been observed along with improved binding specificity. LNA has accordingly been used to
alter gene expression through binding to mRNA, pre-mRNA or miRNA, leading to the
development of LNA-based drug candidates against diseases of genetic origin.7 Given these
interesting properties, it is not surprising that a plethora of LNA analogues have been developed
over the past fifteen years, which aim to improve the biophysical properties of LNA.1,2,8 These
analogues have primarily focused on modification of the oxymethylene bridge spanning the
C2′/C4′-positions and/or introduction of minor-groove-oriented substituents on the bridge.
We began exploring nucleobase functionalization of LNA as an alternative and
synthetically more straightforward strategy to modulate the biophysical properties of LNA.9 For
example, we have shown that ONs modified with small C5-alkynyl-functionalized LNA uridine
(U) monomers display improved affinity, specificity and enzymatic stability relative to ONs
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modified with conventional LNA.10 Moreover, we have shown that ONs modified with C5-
pyrene-functionalized LNA-U monomers display more desirable photophysical properties than
ONs modified with the corresponding 2′-deoxyuridine monomers, presumably due to more well-
defined placement of the fluorophore in the major groove.10,11 Similar improvements have been
observed for ONs modified with diastereomeric C5-functionalized -L-LNA-U, which suggests
that C5-functionalization of uridines is a general and synthetically straightforward approach for
improvement of biophysical properties of conformationally restricted nucleotide building
blocks.11,12
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Intrigued by these results and keen to test the generality of this concept across LNA
nucleotides with other nucleobases, which would be important for therapeutic applications, we
set out to study a series of representative C5-functionalized LNA cytidine (C) and C8-
functionalized LNA adenosine (A) monomers (Figure 1). Unlike C5-functionalized pyrimidine
monomers, which predictably position the substituent toward the major groove of nucleic acid
duplexes,13,14 the binding modes of C8-functionalized purines are more complex.15 Bulky
substituents promote adoption of syn conformations about the glycosyl link (N1-C1′) to
minimize clashes between the C8-substituent and the sugar protons, whereas a more equal
distribution of syn and anti conformations is observed with medium sized substituents. The
conformational flexibility has been utilized to develop fluorophore-functionalized ON probes
with interesting photophysical properties.16 We hypothesized that the extreme pucker and
conformationally restricted nature of the LNA skeleton would influence the barrier between syn
and anti conformations of C8-functionalized LNA-A monomers, resulting in building blocks
with even stronger biases for a particular conformation. Here, we report the synthesis of four C5-
functionalized LNA-C and C8-functionalized LNA-A phosphoramidites, their incorporation into
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ONs and the characterization of the modified ONs by thermal denaturation, absorption, steady-
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6
state fluorescence and enzymatic stability experiments.
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8
NH2
9
NH2
NH2
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N
N
B
N
N
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O P
O
O P
O P
LNA
(B = 5MeC)
Monomer M
Monomer N
Monomer L
NH2
NH2
NH2
N
N
N
H
H
N
H2N
N
N
N
N
N
O
N
N
O
N
O
O
O
O
O
O
O
O
O
O P
O
O P O
O P
O
Monomer X
Monomer Y
Monomer Z
Figure 1. Structures of monomers discussed herein.
RESULTS AND DISCUSSION
Synthesis of C5-functionalized LNA-C phosphoramidites. Our synthetic strategy toward target
phosphoramidites 6a and 6b (Scheme 1) was prompted by: i) large-scale availability of starting
material 1, which is obtained from diacetone--D-allose in ~38% yield),10 ii) reports on
successful uracil-to-cytosine transformations for closely related LNA analogues,8k and iii) a
desire to introduce the C5-substitutent at the latest stage possible to reduce the total number of
synthetic steps. Thus, LNA C5-iodouridine 1 was first protected at the O3′-position as a tert-
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butyldimethylsilyl ether in the presence of TBDMS-Cl, N-methylimidazole and iodine17 to afford
nucleoside 2 in excellent yield. The uracil-to-cytosine conversion was realized using the
phosphoryl chloride/1,2,4-triazole/ammonia method18 to give nucleoside 3 in 72% yield.
Subsequent coupling of trimethylsilyl-protected acetylene or trifluroacetyl-protected
propargylamine to the C5-position of 3 at Sonogashira conditions19 proceeded smoothly to afford
nucleosides 4a and 4b in excellent yield. N4-Benzoylation of 6 was followed by O3′-desilylation
to furnish alcohols 5a and 5b in 70% and 41% yield, respectively. The low yield of 5b was in
part due to unexpected formation of LNA pyrrolocytosine 5c during the desilylation step (results
not shown). Attempts to change the order of reactions, i.e., conducting the Sonogashira coupling
after N4-bezoylation and O3′-desilylation, also led to LNA pyrrolocytosine formation (results
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not
shown).
O3′-phosphitylation
of
5a
and
5b
using
2-cyanoethyl-N,N-
diisopropylchlorophosphoramidite and Hünig’s base afforded targets 6a and 6b in moderate
yields.
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O
NH2
O
N
I
I
I
NH
NH
N
i) POCl3, 1,2,4-triazole,
CH3CN, Et3N, rt
O
N
O
N
O
DMTrO
DMTrO
DMTrO
O
ii) sat. NH3/MeOH, rt
O
O
TBDMS-Cl, I2, NMI
CH2Cl2, rt, 90%
72%, two steps
O
O
O
TBDMSO
TBDMSO
OH
1
2
3
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NHCOCF3
NH
R
NH2
R
NHBz
N
N
N
N
O
N
O
N
O
DMTrO
O
DMTrO
DMTrO
i) Bz2O, DMF, rt
ii) TBAF, THF, rt
R
, Pd(PPh3)4
O
O
CuI, DMF, Et3N, rt
O
O
O
TBDMSO
OH
OH
5c
4a
R = Me3Si- (81%)
5a R = H (70%)
5b
4b R = CF3CONHCH2- (85%)
R = CF3CONHCH2- (41%)
R
NHBz
N
N
O
DMTrO
(iPr)2N
PCl reagent
O
DIPEA, CH2Cl2, rt
O
O
P
O(CH2)2CN
6a
R = H (60%)
6b R = CF3CONHCH2- (62%)
Scheme 1. Synthesis of C5-functionalized LNA cytidine phosphoramidites 6a and 6b. NMI = N-
methylimidazole; PCl = 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite; DIPEA = N,N′-
diisopropylethylamine.
Synthesis of C8-functionalized LNA-A nucleosides. Known LNA adenosine diol 7, which is
obtained in ~25% yield from diacetone--D-allose,20 was identified as a convenient starting
material for the preparation of phosphoramidites 12 and 16 (Schemes 2 and 3). Treatment of 7
with molecular bromine in 1,4-dioxane and aqueous sodium acetate buffer (pH 4.5)21 afforded
nucleoside 8 in 85% yield. Subsequent O5′-dimethoxytritylation using standard conditions
provided 9 in 77% yield. This was followed by protection of the exocyclic amine of the adenine
moiety as a N,N-dimethyl formamidine group (90% yield).22 We found this approach to be
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higher yielding and more convenient than N6-benzoylation of nucleoside 9 via a transient
protection protocol23 (72% yield over three steps, see compound 10*, Scheme S1). A vinyl
moiety, which is converted to an aminoethyl group during standard oligonucleotide
deprotection,24 was introduced at the 8-position of 10 via a Stille coupling19 to give
functionalized nucleoside 11 in 75% yield. Subsequent O3′-phosphitylation using 2-cyanoethyl-
N,N-diisopropylchlorophosphoramidite afforded target phosphoramidite 12 in 64% yield.
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NH2
NH2
NH2
N
N
N
N
N
N
N
N
N
Br
Br
HO
HO
N
N
DMTrO
N
O
O
Br2, aq. NaOAc/AcOH (pH 4.5)
1,4-dioxane, rt, 85%
DMTrCl, DMAP
pyridine, rt, 77%
O
Me2NCH(OMe)2
DMF, 50 oC, 90%
O
O
O
OH
OH
OH
7
8
9
N
NMe2
N
N
NMe2
N
N
N
NMe2
N
N
N
N
N
N
N
Br
N
DMTrO
N
DMTrO
DMTrO
O
tetravinyl tin, Pd(PPh3)4
NMP, 70 oC, 75%
O
PCl reagent, DIPEA
CH2Cl2, rt, 64%
O
O
O
O
OH
OH
(iPr)2N
O
P
O(CH2)2CN
10
11
12
Scheme 2. Synthesis of C8-vinyl LNA adenosine phosphoramidite 12. DMTrCl = 4,4′-
dimethoxytrityl chloride; NMP = N-methyl pyrrolidone; PCl reagent = 2-cyanoethyl-N,N-
diisopropylchlorophosphoramidite; DIPEA = N,N′-diisopropylethylamine.
The reaction sequence had to be modified for the synthesis of C8-pyrene-functionalized LNA
adenosine phosphoramidite 16 (Scheme 3) as the Sonogashira reaction between C8-brominated
nucleoside 10 and 1-acetylenepyrene was sluggish and did not proceed to completion,
presumably due to steric interference from the O5′-DMTr group. Instead, unprotected C8-bromo
LNA adenosine 8 proved to be a viable substrate for Sonogashira coupling with 1-
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acetylenepyrene as nucleoside 13 was obtained in 61% yield. Subsequent O5′-
dimethoxytritylation (80%), N6-protection (90%) and O3′-phosphitylation (64%) finally
provided phosphoramidite 16.
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NH2
NH2
N
N
N
N
N
N
Py
HO
Py
DMTrO
N
N
O
PyC
CH, Pd(PPh3)4
DMTrCl, DMAP
O
8
CuI, DMF, Et3N, 50 oC, 61%
pyridine, 50 oC, 80%
O
O
N
OH
OH
13
14
N
NMe2
NMe2
N
N
N
N
N
Py
Py
N
DMTrO
N
DMTrO
N
O
PCl, DIPEA
CH2Cl2, rt, 71%
O
Me2NCH(OMe)2
DMF, 50 oC, 90%
O
O
OH
(iPr)2N
O
P
O(CH2)2CN
15
16
Scheme 3. Synthesis of C8-pyrene-functionalized LNA adenosine phosphoramidites 16.
DMTrCl 4,4′-dimethoxytrityl chloride; PCl reagent 2-cyanoethyl-N,N-
diisopropylchlorophosphoramidite; DIPEA = N,N′-diisopropylethylamine.
=
=
Conformational analysis of C8-functionalized LNA-A nucleosides. ROESY (rotating-frame
nuclear Overhauser effect correlation spectroscopy) spectra of selected nucleosides were
recorded to examine if C8-functionalization influences the syn-anti equilibrium about the
glycosyl link. Bulky C8-substituents are known to shift this equilibrium toward an increasing syn
preference in 2′-deoxyribonucleosides due to steric repulsion between the C8-substituent and the
sugar ring.15 However, these shifts are accompanied by changes in the sugar pucker, which likely
are more difficult with the conformationally restricted sugar skeleton of LNA.
ROESY spectra of C8-bromo or C8-vinyl substituted LNA-A nucleosides 10 and 11 offer
little evidence of significant syn populations (Figures S1 and S2). Most notably, there is an
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absence of cross peaks corresponding to through-space interactions between H2 and any of the
sugar protons. In fact, the only sign that nucleoside 11 adopts syn conformations is a through-
space coupling between H1′ and the penultimate proton of the vinyl group. In contrast, the
ROESY spectrum of nucleoside 13 displays numerous cross peaks consistent with syn
conformations (Figures S3 and S4), including through-space interactions between: i) H2 and
H2′/H3′/5′-OH, and ii) pyrene protons and H1′/H2′/H5′′/3′-OH.
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ON synthesis. Phosphoramidites 6a and 6b were used in machine-assisted solid-phase DNA
synthesis to incorporate monomers M and N into 9-mer mixed-sequence ONs, while
phosphoramidites 12 and 16 were used to incorporate monomers X and Y into 13-mer mixed-
sequence ONs. To examine if the LNA skeleton has any influence on the position of C8-
substituents, we also synthesized ONs modified with the DNA analogue of monomer Y (i.e.,
monomer Z, Figure 1). The following hand-coupling conditions were used (activator, coupling
time, coupling yield) for monomer M (4,5-dicyanoimidazole, 15 min, ~95%), monomer N
(pyridinium hydrochloride, 15 min, ~90%), monomers X/Y (5-(ethylthio)-1H-tetrazole, 20 min,
~95%) and monomer Z (5-[3,5-bis(trifluoromethyl)phenyl]-1H-tetrazole, 20 min, ~95%).
Suitable activators were identified through initial screening of common activators (results not
shown). The composition and purity of all modified ONs was ascertained by MALDI MS
analysis (Tables S1 and S2) and ion-pair reversed-phase HPLC, respectively.
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Thermal denaturation studies – experimental setup. Thermal denaturation temperatures of
duplexes between modified ONs and DNA/RNA targets were evaluated by UV-Vis thermal
denaturation experiments performed in a medium salt phosphate buffer ([Na+] = 110 mM). All
denaturation curves displayed monophasic sigmoidal transitions (Figures S5 and S6).
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Binding affinities/specificities of ONs modified with C5-functionalized LNA-C monomers. ONs
modified with C5-ethynyl or C5-aminopropynyl LNA-C monomers M and N display markedly
increased affinity toward complementary DNA and RNA targets relative to unmodified ONs
(Tm between +5.5 °C and +10.0 °C, Table 1); larger increases are observed with RNA targets.
The stabilizing effects of monomers M and N are additive as evidenced by the similar Tm per
modification values observed for triply modified B4 and singly modified B1-B3. Remarkably,
the duplex between triply modified N4 and complementary RNA exhibits an absolute Tm of 69
°C, which is 33 °C higher than the corresponding unmodified duplex. Interestingly, M- and N-
modified ONs display similar or slightly higher affinity toward DNA/RNA targets than
corresponding ONs modified with conventional LNA 5-methylcytidine (5MeC) monomer L,
which reinforces our recent observations with C5-functionalized LNA-U.10 Most likely, the
stabilizing properties of monomer N are the result of favorable stacking and electrostatic
interactions, in a similar manner as previously suggested for C5-aminopropynyl-modified DNA
monomers.14e,14h
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Table 1. Tm's of duplexes between L/M/N-modified ONs and complementary DNA or RNA.a
Tm (∆Tm/mod)/°C
3
4
5
6
7
8
9
DNA
RNA
3′-CGT AGA GTG
3′-CGU AGA GUG
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55
56
57
58
59
60
ON
Sequence
B =
L
M
N
L
M
N
40.0
40.5
42.5
46.0
45.0
46.0
5′-GBA TCT CAC
B1
(+5.0)
(+5.5)
(+7.5)
(+10.0)
(+9.0)
(+10.0)
41.0
40.5
43.0
44.0
45.0
44.5
5′-GCA TBT CAC
5′-GCA TCT BAC
5′-GBA TBT BAC
B2
B3
(+6.0)
(+5.5)
(+8.0)
(+8.0)
(+9.0)
(+8.5)
42.0
41.5
42.0
44.0
45.0
44.0
(+7.0)
(+6.5)
(+7.0)
(+8.0)
(+9.0)
(+8.0)
53.0
53.0
60.0
64.0
63.5
69.0
B4
(+6.0)
(+6.0)
(+8.3)
(+9.3)
(+9.2)
(+11.0)
a
Tm’s determined as maximum of the first derivative of denaturation curves (A260 vs T) recorded in Tm buffer ([Na+]
= 110 mM, [Cl-] = 100 mM, pH 7.0 (NaH2PO4/Na2HPO4)) using 1.0 µM of each strand. Tm’s are averages of at least
two measurements within 1.0 °C. “ΔTm/mod” = change in Tm per incorporation relative to unmodified reference
duplex. Tm's of unmodified DNA:DNA and DNA:RNA duplexes are 35 °C and 36 °C, respectively.
The binding specificities of singly or triply modified B2 and B4 were evaluated against
DNA/RNA targets with centrally mismatched nucleotides (Table 2). M- and N-modified ONs
discriminate mismatched targets very efficiently as evidenced by the large drops in Tm's of
mismatched duplexes. However, comparison with ONs modified with conventional LNA-C
monomers reveals that the C5-substituents of monomers M and N do not induce additional
improvements in binding specificity.
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Table 2. Discrimination of mismatched DNA/RNA targets by B2 and B4 ONs.a
3
4
5
6
7
8
DNA: 3′-CGT ABA GTG
ΔTm/°C
RNA: 3′-CGU ABA GUG
Tm/°C
G
Tm/°C
G
ΔTm/°C
C
9
B =
ON
D1
L2
Sequence
A
C
T
A
U
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15
16
17
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5′-GCA TCT CAC
5′-GCA TXT CAC
5′-GXA TXT XAC
5′-GCA TYT CAC
5′-GYA TYT YAC
5′-GCA TZT CAC
5′-GZA TZT ZAC
<-25.0
-25.0
-27.0
-24.5
-22.0
-24.5
-22.0
<-25.0
-28.0
-29.0
-25.0
-25.0
-26.5
-29.0
-21.5
-24.5
-25.0
-22.0
-21.0
-23.5
-24.0
-23.5
-25.0
-24.0
-21.0
-18.5
-19.0
-19.0
<-26.0
-26.0
-29.0
-28.0
-26.0
-28.0
-24.0
<-26.0
-26.0
-27.0
-26.0
-22.0
-23.0
-22.0
35.0
41.0
53.0
40.5
53.0
43.0
60.0
36.0
44.0
64.0
45.0
63.5
44.5
69.0
L4
M2
M4
N2
N4
a For experimental conditions, see Table 1. Tm = change in Tm relative to fully matched ON:DNA or ON:RNA
duplex (B=G).
Binding affinities/specificities of ONs modified with C8-functionalized LNA-A monomers. ONs
modified with C8-functionalized LNA-A monomers X or Y generally display lower affinity
toward complementary DNA than control ONs (Tm for X5-X8 and Y5-Y8 between -6.0 and
+0.5 °C, Table 3). Duplex destabilization is more pronounced when the monomers are flanked
by pyrimidines, most likely due to less efficient stacking with the smaller nucleobases (compare
Tm for B5/B7 with B6/B8, Table 3). Interestingly, only minor differences in Tm's are observed
between X- and Y-modified DNA duplexes despite the very different nature of the C8-
substituents. It is also noteworthy that DNA duplexes involving ONs modified with LNA-based
monomer Y or DNA-based monomer Z display similar Tm's, which indicates that the LNA
skeleton exerts little influence on the position of the destabilizing structural elements.
The binding specificities of X/Y/Z-modified ONs were determined by performing
thermal denaturation experiments using DNA targets with mismatched nucleotides opposite of
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the modified monomer (Table 3). As expected, unmodified duplexes with mismatched base pairs
display significantly lower Tm's than fully complementary duplexes. ONs modified with C8-
aminoethyl-functionalized LNA-A monomer X display less efficient discrimination of DNA
targets with centrally mismatched dA or dC nucleotides than unmodified ONs, but improved
discrimination of targets with mismatched dG nucleotides (compare Tm for mismatched DNA
duplexes involving D5-D8 and X5-X8, Table 3). The latter observation is interesting since the
dA moiety of mismatched dA:dG base pairs is known to have a preference for syn conformations
in certain sequence contexts.15d,25 We speculate that adoption of syn conformations is
energetically unfavorable for C8-aminoethyl LNA-A monomer X, leading to more destabilized
and thus better discriminated X:dG mismatches.
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Conversely, Y-modified ONs display poor binding specificity (compare Tm for
mismatched DNA duplexes involving Y5-Y8 and D5-D8, Table 3), which is indicative of a
preference for syn conformations and (partial) intercalation of the pyrene moiety, as intercalating
moieties are known to reduce binding specificity.26 Only very minor differences in binding
specificities are observed for Y- and Z-modified ONs.
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Table 3. Tm's of duplexes between centrally modified ONs and complementary or singly
5
6
mismatched DNA targets.a
7
8
9
Tm (ΔTm) / C
Mismatch ΔTm / C
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Sequence
B=
ON
D5
D6
D7
T
A
-11.0
-9.5
-8.0
-10.0
C
G
5′-GCGTT AAA TTGCG
5′-GCGTT CAC TTGCG
5′-GCGTT GAG TTGCG
48.5
55.0
55.5
48.5
-12.0
-13.5
-10.0
-14.0
-9.0
-4.5
-8.0
-5.0
D8 5′-GCGTT TAT TTGCG
5′-GCGTT AXA TTGCG
5′-GCGTT CXC TTGCG
5′-GCGTT GXG TTGCG
47.0 (-1.5)
50.0 (-5.0)
56.0 (+0.5)
44.0 (-4.5)
-5.0
-5.0
-6.0
-10.0
-11.0
-7.0
-11.0
-8.0
-12.0
-7.0
X5
X6
X7
-12.0
-8.0
X8 5′-GCGTT TXT TTGCG
5′-GCGTT AYA TTGCG
5′-GCGTT CYC TTGCG
5′-GCGTT GYG TTGCG
47.0 (-1.5)
49.0 (-6.0)
52.0 (-3.5)
44.5 (-4.0)
-1.0
-2.0
+0.5
-2.0
+1.0
-5.0
+1.0
-2.0
-1.0
-1.0
-3.5
+2.0
Y5
Y6
Y7
Y8 5′-GCGTT TYT TTGCG
5′-GCGTT AZA TTGCG
5′-GCGTT CZC TTGCG
5′-GCGTT GZG TTGCG
5′-GCGTT TZT TTGCG
46.5 (-2.0)
47.0 (-8.0)
53.5 (-2.0)
44.5 (-2.0)
-0.5
-1.0
-2.0
-2.5
+1.5
-5.0
±0.0
-1.5
-1.0
-0.5
-5.5
+0.5
Z5
Z6
Z7
Z8
a
For experimental conditions, see Table 1. “Mismatch ΔTm” = change in Tm relative to fully matched duplex (B =
T). DNA targets: 3′-CGCAA TBT AACGC (for B5), 3′-CGCAA GBG AACGC (for B6), 3′- CGCAA CBC
AACGC (for B7) and 3′- CGCAA ABA AACGC (for B8). For structures of monomers X-Z see Figure 1.
Photophysical characterization of ONs modified with C8-pyrene-functionalized LNA-A monomer
Y. To gain additional insight into the binding mode of the pyrene moiety of monomer Y,
absorption and steady state fluorescence emission spectra of Y-modified ONs were recorded in
the absence or presence of complementary or centrally mismatched DNA targets. Single-
stranded probes Y5-Y8 and the corresponding duplexes with complementary/mismatched DNA
targets have very similar UV-Vis absorption spectra including a well-defined absorption
maximum at ~420 nm as well as shoulders at ~385 nm and ~400 nm (Figure 2). The absence of
major hybridization-induced shifts in pyrene absorption maxima27 ( between -2 and +1 nm,
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Table S3) suggests that the pyrene moiety is in a similar microenvironment in the single-stranded
and double-stranded state. This is in agreement with the preference for syn conformations of
nucleoside 13, which would place the pyrene moiety of monomer Y in close contact with
flanking nucleobases regardless of the hybridization state. The most notable difference between
Y- and Z-modified ONs/duplexes is that the pyrene absorption maxima of single-stranded Z-
modified ONs are blue-shifted by 1-3 nm, which indicates weaker pyrene-nucleobase
interactions in the single-stranded state (Table S3 and Figure S7). Hence, subtle hybridization-
induced bathochromic shifts are observed for Z-modified ONs ( = -1 to +4 nm, Table S3).
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47
48
49
50
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53
54
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56
57
58
59
60
Figure 2. UV-Vis absorption spectra of single-stranded Y5-Y8 and the corresponding duplexes
with complementary (cDNA) or centrally mismatched DNA targets (MM – central mismatched
nucleotide is specified). Spectra were recorded in Tm buffer at T = 5 °C using each strand at 1.0
µM concentration.
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Steady-state fluorescence emission spectra (ex ~ 385 nm, T = 5 °C) of duplexes between Y5-Y8
and complementary or centrally mismatched DNA targets exhibit a broad emission maximum
centered at ~460 nm, which is indicative16a of strong electronic interactions between the pyrene
and adenine moieties (Figure 3). Up to two-fold increases in fluorescence intensity are observed
upon hybridization of Y5 or Y8 with DNA targets, whereas hybridization of Y6 or Y7 with DNA
results in subtle decreases in fluorescence intensity. The nature of the nucleotide opposite of the
modification does not appear to have a major influence on the fluorescence properties. The
different trends of Y5/Y8 and Y6/Y7, in which A/T and C/G base pairs flank monomer Y,
respectively, strongly suggest that monomer Y predominantly adopts syn conformations leading
to intercalation of pyrene and nucleobase-mediated quenching11,16b,28 of pyrene fluorescence.
Thus, in duplexes involving Y5 or Y8 the pyrene moiety is near weakly quenching A/T base
pairs, while it is near strongly quenching C/G base pairs in duplexes involving Y6/Y7.
Additional support for this hypothesis comes from the fact that the fluorescence intensity of
duplexes involving Y6 and Y7 is very low. Similar trends are seen for Z-modified duplexes,
again suggesting that the LNA skeleton only has a minor influence on the position of the C8-
fluorophore (Figure S8).
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Figure 3. Steady-state fluorescence emission spectra of single-stranded Y5-Y8 and the
corresponding duplexes with complementary (cDNA) or centrally mismatched DNA targets
(MM – central mismatched nucleotide is specified). Spectra were recorded in Tm buffer at T = 5
°C using each strand at 1.0 µM concentration; ex = 385 nm. Please note that different Y-axis
scales are used.
3′-Exonuclease stability of ONs modified with C5-functionalized LNA-C monomers. Prompted by
the interesting hybridization characteristics of M- and N-modified ONs, we set out to determine
the stability of singly modified M3 and N3 against the 3′-exonuclease snake venom
phosphordiesterase (SVPDE) by monitoring the change in absorbance at 260 nm of the ONs
(Figure 4). As expected, unmodified DNA strand D1 is rapidly cleaved, whereas conventional
LNA L3 exhibits moderate resistance against degradation by SVPDE (~70% cleavage within
~50 min). Gratifyingly, M3 and N3 are considerably more resistant toward degradation by
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SVPDE (~50% and ~30% cleavage within ~50 min, respectively), presumably since the C5-
substituents interferes with SVPDE activity. These results are in agreement with our
observations in the C5-alkynyl-functionalized LNA-U series,10 which suggests that conjugation
of alkynes to the C5-position of LNA pyrimidines is a general approach toward improving the
enzymatic stability of LNA-modified ONs.
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100
90
80
70
60
50
40
30
20
10
0
D1
L3
M3
N3
0
50
100
Time (min)
Figure 4. 3′-Exonuclease (SVPDE) degradation of singly modified B3 and reference strands.
Nuclease degradation studies were performed in magnesium buffer (50 mM Tris-HCl, 10 mM
Mg2+, pH 9.0) by using 3.3 M ONs and 0.03 U of SVPDE.
CONCLUSION. Taken together with the results from our recent reports,10 the current study
demonstrates that attachment of alkynes to the C5-position of LNA pyrimidines is a
straightforward approach for improving the binding affinity, target specificity and/or enzymatic
stability of LNA-modified ONs. This strategy extends to -L-LNA and presumably many other
conformationally restricted pyrimidines.12 In contrast, C8-functionalization of LNA adenosines
is detrimental to binding affinity and specificity, demonstrating that the beneficial effects of the
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LNA skeleton do not supersede the destabilizing effect of the C8-substituent. Similar effects
have been observed upon C5-functionalization of LNA uridines10 or N2-functionalization of 2′-
amino-LNA thymidines29 with large hydrophobic moieties.
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In summary, C5-functionalized LNA pyrimidines are interesting affinity-, specificity-
and stability-enhancing modifications that can be used by themselves or alongside other
chemically modified nucleotides for development of enabling tools for nucleic acid targeting
applications in molecular biology, biotechnology and medicinal chemistry.
EXPERIMENTAL SECTION
(1R,3R,4R,7S)-7-(tert-Butyldimethylsilyloxy)-1-(4,4′-dimethoxytrityloxymethyl)-3-(5-
iodouracil-1-yl)-2,5-dioxabicyclo[2.2.1]heptane (2). To a solution of nucleoside 110 (3.20 g,
4.67 mmol), I2 (3.50 g, 14.0 mmol) and N-methylimidazole (1.2 mL, 14.0 mmol) in anhydrous
CH2Cl2 (50 mL) was added TBDMS-Cl (1M in CH2Cl2, 5.6 mL, 5.60 mmol). The reaction
mixture was stirred at rt for 4h whereupon it was diluted with CH2Cl2 (100 mL) and sat. aq.
sodium thiosulfate (100 mL). The layers were separated and the organic layer was washed with
sat. aq. solution of sodium thiosulfate (2 × 200 mL). The combined aqueous phase was then
extracted with CH2Cl2 (100 mL). The combined organic layers were dried (Na2SO4) and
evaporated to dryness to afford a residue that was purified by column chromatography (0-40%
EtOAc in petroleum ether, v/v) to afford nucleoside 2 (3.30 g, 90%) as slight yellow solid
material. Rf = 0.5 (40% EtOAc in petroleum ether, v/v); ESI-HRMS m/z 821.1731 ([M+Na]+,
1
C37H43IN2O8Si·Na+, Calc. 821.1726; H NMR (DMSO-d6) δ 11.74 (s, 1H, ex, NH), 8.08 (s, 1H,
H6), 7.42-7.45 (m, 2H, Ar), 7.21-7.36 (m, 7H, Ar), 6.89 (d, 4H, J = 8.5 Hz, Ar), 5.48 (s, 1H,
H1′), 4.27 (s, 2H, H2′, H3′), 3.72-3.76 (m, 7H, 2×CH3O, H5′′), 3.65-3.68 (d, 1H, J = 8.0 Hz,
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H5′′), 3.33-3.36 (d, 1H, J = 11.0 Hz, H5′), 3.21-3.24 (d, 1H, J = 11.0 Hz, H5′), 0.71 (s, 9H,
Me3C), 0.04 (s, 3H, CH3Si), -0.03 (s, 3H, CH3Si); 13C NMR (DMSO-d6) δ 160.7, 158.11, 158.10,
149.8, 144.5, 143.0 (C6), 135.20, 135.17, 129.6 (Ar), 129.5 (Ar), 127.9 (Ar), 127.5 (Ar), 126.7
(Ar), 113.3 (Ar), 113.2 (Ar), 87.6, 87.2 (C1′), 85.6, 78.5 (C2′), 71.6 (C5′′), 70.4 (C3′), 69.0, 58.5
(C5′), 55.0 (CH3O), 25.3 (Me3C), 17.3, -4.9 (CH3Si), -5.4 (CH3Si).
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(1R,3R,4R,7S)-7-(tert-Butyldimethylsilyloxy)-1-(4,4′-dimethoxytrityloxymethyl)-3-(5-
iodocytosin-1-yl)-2,5-dioxabicyclo[2.2.1]heptane (3). To a cold stirred suspension of 1,2,4-
triazole (4.60 g, 66.3 mmol) in anhydrous acetonitrile (25 mL) was added freshly distilled POCl3
(1.5 mL, 15.6 mmol). The mixture was stirred at ~0 °C for 15 min, whereupon anhydrous
triethylamine (11.0 mL, 79.2 mmol) was added. After stirring for another 30 min at 0 °C, a
solution of nucleoside 2 (1.60 g, 2.00 mmol) in anhydrous acetonitrile (45 mL) was added. The
reaction mixture was stirred at rt for 3h at which point solvents were removed under reduced
pressure. The resulting residue was taken up in EtOAc (100 mL) and water (100 mL). The layers
were separated and the organic layer was washed with sat. aq. NaHCO3 (2 × 100 mL). The
combined aqueous layer was then extracted with EtOAc (100 mL). The combined organic phase
was dried (Na2SO4) and evaporated to dryness to afford a solid material that was dissolved in sat.
methanolic ammonia (150 mL). The reaction mixture was stirred at rt for ~12h at which point
solvent was evaporated off and the resulting residue was purified by column chromatography (0-
2% MeOH in CH2Cl2) to afford 3 (1.15 g 72%) as a slightly yellow solid material. Rf = 0.4 (2%
MeOH in CH2Cl2); ESI-HRMS m/z 820.1895 ([M+Na]+, C37H44IN3O7Si·Na+, Calc. 820.1885;
1H NMR (DMSO-d6) δ 8.06 (s, 1H, H6), 7.91 (br s, 1H, ex, NH), 7.43-7.46 (m, 2H, Ar), 7.21-
7.35 (m, 7H, Ar), 6.89-6.92 (m, 4H, Ar), 6.68 (br s, 1H, ex, NH), 5.47 (s, 1H, H1′), 4.26 (s, 1H,
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H2′), 4.20 (s, 1H, H3′), 3.72-3.75 (m, 7H, 2×CH3O, H5′′), 3.64-3.66 (d, 1H, J = 8.0 Hz, H5′′),
3.34-3.37 (d, 1H, J = 11.0 Hz, H5′), 3.18-3.21 (d, 1H, J = 11.0 Hz, H5′), 0.71 (s, 9H, Me3C),
7
8
9
13
0.00 (s, 3H, CH3Si), -0.06 (s, 3H, CH3Si); C NMR (DMSO-d6) δ 164.0, 158.1, 153.5, 145.4
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(C6), 144.5, 135.3, 135.2, 129.6 (Ar), 129.5 (Ar), 127.9 (Ar), 127.5 (Ar), 126.7 (Ar), 113.28
(Ar), 113.25 (Ar), 87.5 (C1′), 87.4, 85.6, 78.5 (C2′), 71.5 (C5′′), 70.3 (C3′), 58.6 (C5′), 56.7, 55.0
(CH3O), 25.3 (Me3C), 17.4, -5.0 (CH3Si), -5.3 (CH3Si).
(1R,3R,4R,7S)-7-(tert-Butyldimethylsilyloxy)-1-(4,4′-dimethoxytrityloxymethyl)-3-[5-
(trimethylsilylethynyl)cytosin-1-yl]-2,5-dioxabicyclo[2.2.1]heptane (4a). Nucleoside 3 (0.50
g, 0.63 mmol), Pd(PPh3)4 (72 mg, 0.06 mmol), CuI (24 mg, 0.13 mmol) and trimethylsilyl
acetylene (0.25 mL, 1.76 mmol) were added to anhydrous DMF (10 mL) and the reaction
chamber was degassed and placed under an argon atmosphere. To this was added anhydrous
Et3N (0.35 mL, 2.51 mmol) and the reaction mixture was stirred at rt for ~12 h at which point
solvent was evaporated off. The resulting residue was taken up in EtOAc (100 mL) and washed
with brine (2 × 50 mL) and sat. aq. NaHCO3 (50 mL). The combined aqueous layer was then
extracted with EtOAc (100 mL). The combined organic layer was dried (Na2SO4), evaporated to
dryness and the resulting residue purified by column chromatography (0-5% MeOH in CH2Cl2
(v/v) to afford nucleoside 4a (0.39 g, 81%) as a pale yellow solid material. Rf = 0.4 (5% MeOH
in CH2Cl2, v/v); ESI-HRMS m/z 790.3310 ([M+Na]+, C42H53N3O7Si2·Na+, Calc. 790.3314); 1H
NMR (DMSO-d6) δ 8.00 (s, 1H, H6), 7.86 (br s, 1H, ex, NH), 7.41-7.45 (m, 2H, Ar), 7.20-7.27
(m, 7H, Ar), 6.86-6.90 (m, 4H, Ar), 6.68 (br s, 1H, ex, NH), 5.43 (s, 1H, H1′), 4.28 (s, 1H, H2′),
4.21 (s, 1H, H3′), 3.75-3.78 (d, 1H, J = 7.5 Hz, H5′′), 3.72-3.73 (2s, 6H, CH3O), 3.69-3.71 (d,
1H, J = 7.5 Hz, H5′′), 3.30-3.36 (m, 2H, H5′), 0.73 (s, 9H, Me3C), -0.01 (s, 3H, CH3Si), -0.04 (s,
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9H, Me3Si), -0.07 (s, 3H, CH3Si); C NMR (DMSO-d6) δ 164.2, 158.10, 158.07, 152.9, 144.5,
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143.6 (C6), 135.3, 135.1, 129.7 (Ar), 129.4 (Ar), 127.8 (Ar), 127.5 (Ar), 126.6 (Ar), 113.18 (Ar),
113.15 (Ar), 99.7, 96.4, 89.7, 87.6 (C1′), 87.5, 85.5, 78.3 (C2′), 71.5 (C5′′), 70.4 (C3′), 58.6
(C5′), 55.0 (CH3O), 25.3 (Me3C), 17.4, -0.6 (Me3Si), -4.9 (CH3Si), -5.3 (CH3Si).
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(1R,3R,4R,7S)-7-(tert-Butyldimethylsilyloxy)-1-(4,4′-dimethoxytrityloxymethyl)-3-[5-(3-
trifluoroacetylaminopropyn-1-yl)cytosin-1-yl]-2,5-dioxabicyclo[2.2.1]heptane
(4b).
Nucleoside 3 (0.50 g, 0.63 mmol), Pd(PPh3)4 (72 mg, 0.06 mmol), CuI (24 mg, 0.13 mmol) and
2,2,2-trifluoro-N-(2-propynyl)acetamide30 (0.33 mL, 1.88 mmol) were added to anhydrous DMF
(10.0 mL) and the reaction chamber was degassed and placed under an argon atmosphere. To
this was added anhydrous Et3N (0.35 mL, 2.51 mmol) and the reaction mixture was stirred at rt
for ~12 h at which point solvent was evaporated off. The resulting residue was taken up in
EtOAc (100 mL) and washed with brine (2 × 50 mL) and sat. aq. NaHCO3 (50 mL). The
combined aqueous layer was back-extracted with EtOAc (100 mL) and the combined organic
layer was dried (Na2SO4) and evaporated to dryness. The resulting residue was purified by
column chromatography (0-7% MeOH in CH2Cl2 (v/v) to afford nucleoside 4b (0.44 g, 85%) as
a pale yellow solid material. Rf = 0.5 (7% MeOH in CH2Cl2, v/v); ESI-HRMS m/z 843.3040
([M+Na]+, C42H47F3N4O8Si·Na+, Calc. 843.3007); 1H NMR (DMSO-d6) δ 9.84 (t, 1H, ex, J = 4.7
Hz, NHCH2), 7.95 (br s, 2H, 1 ex, H2, NH2), 7.40-7.44 (m, 2H, Ar), 7.21-7.34 (m, 7H, Ar), 6.88-
6.92 (d, 4H, J = 8.5 Hz, Ar), 6.84 (br s, 1H, ex, NH2), 5.50 (s, 1H, H1′), 4.20 (s, 1H, H2′), 4.14-
4.19 (m, 2H, H3′, CH2NH), 4.06-4.12 (dd, 1H, J = 17.8 Hz, 4.7 Hz, CH2NH), 3.77-3.80 (d, 1H, J
= 8.0 Hz, H5′′), 3.74 (br s, 6H, CH3O), 3.69-3.72 (d, 1H, J = 8.0 Hz, H5′′), 3.40-3.44 (d, 1H, J =
11.0 Hz, H5′), 3.30-3.34 (d, 1H, J = 11.0 Hz, H5′), 0.70 (s, 9H, Me3C), -0.02 (s, 3H, CH3Si), -
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0.08 (s, 3H, CH3Si); 13C NMR (DMSO-d6) δ 164.6, 158.2, 158.1, 155.9 (q, J = 36.5 Hz, -
COCF3), 153.0, 144.7, 142.6 (C6), 135.2, 134.7, 129.8 (Ar), 129.5 (Ar), 127.8 (Ar), 127.4 (Ar),
126.6 (Ar), 115.7 (q, J = 286 Hz, CF3CO), 113.3 (Ar), 113.2 (Ar) , 89.7, 88.9, 87.5 (C1′), 87.3,
85.5, 78.6 (C2′), 75.6, 71.5 (C5′′), 70.3 (C3′), 58.6 (C5′), 55.0 (CH3O), 29.9 (CH2NH), 25.3
(Me3C), 17.4, -5.0 (CH3Si), -5.4 (CH3Si); 19F NMR (DMSO-d6) δ -74.8.
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(1R,3R,4R,7S)-3-[4-N-Benzoyl-5-ethynylcytosin-1-yl]-1-(4,4′-dimethoxytrityloxymethyl)-7-
hydroxy-2,5-dioxabicyclo[2.2.1]heptane (5a). To a solution of nucleoside 4a (0.73 g, 0.95
mmol) in anhydrous DMF (14.0 mL) was added benzoic anhydride (0.47 g, 2.10 mmol). The
reaction mixture was stirred at rt for 24 h, at which point it was concentrated to near dryness and
diluted with EtOAc (100 mL) and washed with brine (2×50 mL). The aqueous layer was back-
extracted with EtOAc (100 mL). The combined organic layer was dried (Na2SO4), evaporated to
dryness and the resulting residue (~ 0.73 g) dissolved in THF (20 mL). TBAF (1M in THF, 1.45
mL, 1.45 mmol) was added to this and the reaction mixture was stirred at rt for ~3.5 h at which
point EtOAc (100 mL) was added. The organic layer was washed with brine (2 × 50 mL) and
H2O (50 mL). The aqueous layer was back-extracted with EtOAc (100 mL). The combined
organic layer was dried (Na2SO4), evaporated to dryness and the resulting residue purified by
column chromatography (0-5% MeOH in CH2Cl2, v/v) to afford nucleoside 5a (0.46 g, 70% over
two steps) as a slightly yellow solid material. Rf = 0.3 (7% MeOH in CH2Cl2, v/v); ESI-HRMS
m/z 708.2294 ([M+Na]+, C40H35N3O8·Na+, Calc. 708.2316); 1H NMR31 (DMSO-d6) δ 12.78 (br s,
0.5H, ex, NHA), 10.81 (br s, 0.5H, ex, NHB), 7.9-8.3 (broad signal, 3H, Ar, H6), 7.60-7.65 (broad
signal, 1H, Ar), 7.50-7.55 (m, 2H, Ar), 7.42-7.46 (m, 2H, Ar), 7.30-7.36 (m, 6H, Ar), 7.22-7.26
(m, 1H, Ar), 6.91 (d, 4H, J = 9.0 Hz, Ar), 5.73 (br s, 1H, ex, 3′-OH), 5.54 (br s, 1H, H1′), 4.32 (s,
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1H, H2′), 4.08-4.10 (2s, 2H, H3′, HC≡C), 3.78-3.83 (m, 2H, H5′′), 3.75 (br s, 6H, CH3O), 3.48-
3.52 (1H, d, J = 11.0 Hz, H5′), 3.30-3.33 (1H, d, J = 11.0 Hz, H5′, partial overlap with H2O
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signal); C NMR (DMSO-d6) δ 158.1, 144.6, 135.3, 135.2, 132.7 (Ar), 129.7 (Ar), 129.6 (Ar),
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128.4 (Ar), 127.9 (Ar), 127.6 (Ar), 126.7 (Ar), 113.3 (Ar), 87.8 (C1′), 85.7, 78.6 (C2′), 75.6, 71.4
(C5′′), 69.3 (C3′), 58.9 (C5′), 55.0 (CH3O).
(1R,3R,4R,7S)-3-[4-N-Benzoyl-5-(3-trifluoroacetylaminopropyn-1-yl)cytosin-1-yl]-1-(4,4′-
dimethoxytrityloxymethyl)-7-hydroxy-2,5-dioxabicyclo[2.2.1]heptane
(5b).
Benzoic
anhydride (0.30 g, 1.34 mmol) was added to a solution of nucleoside 4b (0.50 g, 0.61 mmol) in
anhydrous DMF (10.0 mL) and the reaction mixture was stirred at rt for 24 h, at which point it
was evaporated to near dryness. The resulting residue was taken up in ethyl acetate (100 mL) and
washed with brine (2 × 50 mL). The aqueous layer was back-extracted with EtOAc (100 mL)
and the combined organic layers were dried (Na2SO4), evaporated to dryness and the resulting
residue (0.41 g) dissolved in THF (12 mL). To this was added TBAF (1M in THF, 0.17 mL, 0.57
mmol) and the reaction mixture was stirred at rt for ~3.5 h. At this point, EtOAc (100 mL) was
added and the organic layer was washed with brine (2×50 mL) and H2O (50 mL). The aqueous
layer was back-extracted with EtOAc (100 mL). The combined organic layer was dried
(Na2SO4), evaporated to dryness and the resulting residue purified by column chromatography
(0-40% EtOAc in petroleum ether, v/v) to afford nucleoside 5b (0.20 g, 41% yield) as a slightly
yellow solid material. Rf = 0.5 (40% EtOAc in petroleum ether, v/v); ESI-HRMS m/z 833.2415
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([M+Na]+, C43H37F3N4O9·Na+, Calc. 833.2405); H NMR (DMSO-d6) δ 12.86 (br s, 0.5H, ex,
NH), 10.46 (br s, 0.5H, ex, NH), 9.92 (br s, 1H, ex, NH), 7.9-8.2 (broad signal, 3H, Ar, H6),
7.59-7.64 (t, 1H, J = 7.0 Hz, Ar), 7.24-7.50 (m, 11H, Ar), 6.90-6.95 (m, 4H, Ar), 6.91-6.92 (d,
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2H, J = 2.5 Hz, Ar), 5.77 (br s, 1H, 3′-OH), 5.50 (s, 1H, H1′), 4.32 (s, 1H, H2′), 3.94-4.09 (m,
3H, H3′, CH2NH), 3.84 (br s, 2H, H5′′), 3.75 (s, 6H, CH3O), 3.59-3.62 (d, 1H, J = 11.0 Hz, H5′),
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3.29-3.32 (d, 1H, H5′, overlap with H2O signal); C NMR (DMSO-d6) δ 158.12, 158.08, 155.9
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(q, J = 35.6 Hz , COCF3), 144.7, 135.4, 134.9, 132.6 (Ar), 129.8 (Ar), 129.6 (Ar), 128.3 (Ar),
127.9 (Ar), 127.5 (Ar), 126.7 (Ar), 115.7 (q, J = 286 Hz, CF3CO), 113.3 (Ar), 113.2 (Ar), 87.8
(C1′),32 87.5, 85.6, 78.5 (C2′), 74.8, 71.4 (C5′′), 69.5 (C3′), 59.0 (C5′), 55.0 (CH3O), 29.4
(CH2NH); 19F NMR (DMSO-d6) δ -74.8.
(1R,3R,4R,7S)-3-[4-N-Benzoyl-5-(trimethylsilylethynyl)cytosin-1-yl]-7-[2-
cyanoethoxy(diisopropylamino)phosphinoxy]-1-(4,4′-dimethoxytrityloxymethyl)-2,5-
dioxabicyclo[2.2.1]heptane (6a). Nucleoside 5a (0.45 g, 0.65 mmol) was dried through co-
evaporation with anhydrous 1,2-dichloroethane (3×25 mL) and dissolved in anhydrous CH2Cl2
(12 mL). To this was added anhydrous N,N′-diisopropylethylamine (0.45 mL, 2.60 mmol) and 2-
cyanoethyl-N,N-diisopropylchlorophosphoramidite (0.32 mL, 1.42 mmol) and the reaction
mixture was stirred at rt for ~3.5 h. The reaction mixture was diluted with CH2Cl2 (50 mL),
washed with 5% aq. NaHCO3 (2×25 mL) and the combined aqueous layers back-extracted with
CH2Cl2 (50 mL). The combined organic layers were dried (Na2SO4), evaporated to dryness and
the resulting residue purified by column chromatography (0-4% MeOH in CH2Cl2, v/v) and
subsequent trituration from CH2Cl2 and petroleum ether to provide phosphoramidite 6a (0.34 g,
60%) as a yellow foam. Rf = 0.7 (3% MeOH in CH2Cl2, v/v); ESI-HRMS m/z 908.3429
([M+Na]+, C49H52N5O9P·Na+, Calc. 908.3395); 31P NMR (CDCl3) δ 149.9, 149.5.
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(1R,3R,4R,7S)-3-[4-N-Benzoyl-5-(3-trifluoroacetylaminopropyn-1-yl)cytosin-1-yl]-7-[2-
cyanoethoxy(diisopropylamino)phosphinoxy]-1-(4,4′-dimethoxytrityloxymethyl)-2,5-
dioxabicyclo[2.2.1]heptane (6b). Nucleoside 5b (0.25 g, 0.33 mmol) was dried through co-
evaporation with anhydrous 1,2-dichloroethane (2×10 mL) and dissolved in anhydrous CH2Cl2 (8
mL). To this was added anhydrous N,N′-diisopropylethylamine (0.23 mL, 1.33 mmol) and 2-
cyanoethyl-N,N-diisopropylchlorophosphoramidite (0.15 mL, 0.67 mmol) and the reaction
mixture was stirred at rt for ~3.5 h. The reaction mixture was diluted with CH2Cl2 (25 mL),
washed with 5% aq. NaHCO3 (2×10 mL) and the combined aqueous layers back-extracted with
CH2Cl2 (25 mL). The combined organic layers were dried (Na2SO4), evaporated to dryness and
the resulting residue purified by column chromatography (0-4% MeOH in CH2Cl2, v/v) and
subsequent trituration from CH2Cl2 and petroleum ether to provide phosphoramidite 6b (210 mg,
62%) as a yellow foam. Rf = 0.7 (4% MeOH in CH2Cl2, v/v); ESI-HRMS m/z 1033.3516
([M+Na]+, C52H54F3N6O10P·Na+, Calc. 1033.3483); 31P NMR (CDCl3) δ 150.0, 149.3.
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(1S,3R,4R,7S)-3-(8-Bromoadenin-9-yl)-7-hydroxy-1-hydroxymethyl-2,5-
dioxabicyclo[2.2.1]heptane (8). A solution of Br2 (0.28 mL, 5.07 mmol) in 1,4-dioxane (20 mL)
was added dropwise to a solution of known diol 78a (1.13 g, 4.05 mmol) in 1,4-dioxane (12 mL)
and 0.5 M aq. sodium acetate buffer (23.2 mL, pH 4.5). The reaction mixture was stirred at rt
overnight, at which point a sat. aq. solution of Na2S2O3 was added until the red color from
bromine disappeared. The solution was then carefully neutralized using 0.5 M aq. NaOH. The
mixture was concentrated to approximately half volume and allowed to stand at ~5 °C overnight.
The resulting precipitate was filtered off and washed with a cold solution of H2O and 1,4-
dioxane (1:1, v/v) to obtain C8-brominated LNA nucleoside 8 (1.23 g, 85%) as a slightly pale
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yellow solid material. Rf = 0.7 (10% MeOH in CH2Cl2, v/v ); ESI-HRMS m/z 379.9965
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([M+Na]+, C11H12BrN5O4·Na+, Calc. 379.9965); H NMR (DMSO-d6) δ 8.12 (s, 1H, H2), 7.43
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(br s, 2H, ex, NH2), 5.76 (s, 1H, H1′), 5.74 (d, 1H, ex, J = 4.3 Hz, 3′-OH), 4.95 (t, 1H, ex, J = 6.0
Hz, 5′-OH), 4.83 (s, 1H, H2′), 4.69 (d, 1H, J = 4.3 Hz H3′), 3.95-3.97 (d, 1H, J = 8.0 Hz, H5′′),
3.76-3.78 (d, 1H, J = 8.0 Hz, H5′′), 3.72 (d, 2H, J = 6.0 Hz, H5′); 13C NMR (DMSO-d6 ) δ 154.9,
152.4 (C2), 149.9, 126.4, 119.0, 88.5, 87.5 (C1′), 79.4 (C2′), 71.9 (C3′), 71.8 (C5′′), 57.1 (C5′).
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(1R,3R,4R,7S)-3-(8-Bromoadenin-9-yl)-1-(4,4′-dimethoxytrityloxymethyl)-7-hydroxy-2,5-
dioxabicyclo[2.2.1]heptane (9). Nucleoside 8 (0.50 g, 1.40 mmol), was co-evaporated with
anhydrous pyridine (10 mL) and redissolved in anhydrous pyridine (15 mL). DMAP (10 mg,
0.35 mmol) and 4,4′-dimethoxytrityl chloride (0.62 g, 1.82 mmol) were added to this solution
and the reaction mixture was stirred at rt for 6 h. Methanol (~1.25 mL) was added and the
solvents were evaporated off to furnish a crude material, which was partitioned between CH2Cl2
(~50 mL) and aq. NaHCO3 (25 mL). The phases were separated and the organic phase was
washed with aq. NaHCO3 (2  30 mL). The aqueous layer was back-extracted with CH2Cl2 (30
mL) and the combined organic layers were dried (NaSO4) and concentrated to dryness. The
resulting residue was coevaporated with toluene/abs. EtOH (2100 mL, 1:2 v/v) and purified by
silica gel column chromatography (0-5% MeOH in CHCl3, v/v) to provide nucleoside 9 (0.71 g,
77%) as a yellow solid material. Rf = 0.4 (5% MeOH in CH2Cl2, v/v ); MALDI-HRMS m/z
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682.1277 ([M+Na]+, C32H30BrN5O6·Na+, Calc. 682.1272); H NMR (DMSO-d6) δ 8.11 (s, 1H,
H2), 7.43 (bs, 2H, ex, NH2), 7.33-7.35 (d, 2H, Ar), 7.18-7.27 (m, 7H, Ar), 6.82-6.86 (m, 4H, Ar),
5.85 (s, 1H, H1′), 5.78 (d, 1H, ex, J = 5.0 Hz, 3′-OH), 5.05 (s, 1H, H2′), 4.64 (d, 1H, J = 5.0 Hz,
H3′), 3.97-4.02 (2d, 2H, J = 8.0 Hz, H5′′), 3.73 (s, 3H, CH3O), 3.72 (s, 3H, CH3O), 3.30-3.33 (d,
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1H, J = 11.0 Hz, H5′ - partial overlap with H2O), 3.21-3.24 (d, 1H, J = 11.0 Hz, H5′); 13C NMR
(DMSO-d6) δ 158.0, 154.9, 152.5 (C2), 149.9, 144.7, 135.4, 135.3, 129.7 (Ar), 129.6 (Ar), 127.7
(Ar), 127.6 (Ar), 126.7, 126.5 (Ar), 118.9, 113.1 (Ar), 87.1 (C1′), 86.6, 85.2, 78.8 (C2′), 72.7
(C3′), 72.0 (C5′′), 60.1 (C5′), 55.0 (CH3O).
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(1R,3R,4R,7S)-3-(8-Bromo-6-N-(dimethylamino)methylene-adenin-9-yl)-1-(4,4′-
dimethoxytrityloxymethyl)-7-hydroxy-2,5-dioxabicyclo[2.2.1]heptane
(10).
N,N-
dimethylformamide dimethyl acetal (0.17 mL, 1.20 mmol) was added to a solution of nucleoside
9 (0.32 g, 0.50 mmol) in anhydrous DMF (5.0 mL) and the reaction mixture was stirred for 5 h at
50 °C. All volatile components were removed and the resulting residue was taken up in ethyl
acetate (15 mL) and subsequently washed with brine (225 mL) and saturated aq. NaHCO3 (25
mL). The organic layer was dried (Na2SO4), evaporated to dryness and the resulting residue
purified by silica gel column chromatography (0-5% MeOH in CH2Cl2, v/v) to furnish
nucleoside 10 (0.31 g, 90%) as a pale yellow solid material. Rf = 0.5 (5% MeOH in CH2Cl2, v/v);
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MALDI-HRMS m/z 715.1848 ([M+H]+, C35H35BrN6O6·H+, Calc. 715.1874); H NMR (DMSO-
d6) δ 8.87 (s, 1H, CH(NMe2)), 8.37 (s, 1H, H2), 7.33-7.36 (m, 2H, Ar), 7.18-7.28 (m, 7H, Ar),
6.82-6.86 (m, 4H, Ar), 5.88 (s, 1H, H1′), 5.80 (d, 1H, ex, J = 5.0 Hz, 3′-OH), 5.08 (s, 1H, H2′),
4.65 (d, 1H, J = 5.0 Hz, H3′), 3.98-4.04 (2d, J = 7.5 Hz, H5′′), 3.72 (br s, 6H, CH3O), 3.32-3.34
(d, 1H, J = 11.0 Hz, H5′), 3.21-3.24 (d, 1H, J = 11.0 Hz, H5′), 3.20 (s, 3H, NCH3), 3.13 (s, 3H,
13
NCH3); C NMR (DMSO-d6) δ 158.00, 157.97, 157.7 (CH(NMe2)), 151.7 (C2), 144.6, 135.4,
135.3, 129.7 (Ar), 129.6 (Ar), 129.3, 127.7 (Ar), 127.63 (Ar), 126.58 (Ar), 125.4, 113.1 (Ar),
87.1 (C1′), 86.6, 85.2, 78.9 (C2′), 72.8 (C3′), 72.0 (C5′′), 60.2 (C5′), 55.0 (CH3O), 40.7 (NCH3),
34.6 (NCH3).
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Page 29 of 43
The Journal of Organic Chemistry
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(1R,3R,4R,7S)-1-(4,4′-Dimethoxytrityloxymethyl)-3-(6-N-benzoyl-8-bromo-adenin-9-yl)-7-
hydroxy-2,5-dioxabicyclo[2.2.1]heptane (10*). Nucleoside 9 (0.25 g, 0.38 mmol) was dried
through co-evaporation with pyridine (2×10 mL) and re-dissolved in anhydrous pyridine (5 mL).
To this was added trimethylchlorosilane (0.14 mL, 1.14 mmol) and the reaction mixture was
allowed to stir for 30 min at rt. At this point, BzCl (0.22 ml, 1.90 mmol) was added and the
reaction mixture was stirred at rt for 5 h. The reaction mixture was then cooled to 0 °C and water
(~1 mL) was added. After stirring for 15 min, aq. NH3 (29%, 5.0 mL) was added and the
suspension was stirred at rt for 30 min. The mixture was evaporated to near dryness and the
resulting taken up in CH2Cl2 (25 ml) and washed with 5% aq. NaHCO3 (2×10 mL). The organic
layer was evaporated to dryness and the resulting residue was purified using silica gel column
chromatography (0-5% MeOH in CH2Cl2, v/v) to obtain nucleoside 10* (210 mg, 72%) as a pale
brown solid material. Rf = 0.7 (5% MeOH in CH2Cl2, v/v ); ESI-HRMS: m/z 786.1534 ([M+Na]+,
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C39H34BrN5O7·Na+, calc. 786.1539); H NMR (DMSO-d6) δ 11.26 (s, 1H, ex, NH), 8.70 (s, 1H,
H2), 8.02-8.05 (d, 2H, J = 7.0 Hz, Ar), 7.63-7.67 (t, 1H, J = 7.0 Hz, Ar), 7.53-7.58 (d, 2H, J =
7.0 Hz, Ar), 7.34-7.38 (m, 2H, Ar), 7.18-7.30 (m, 7H, Ar), 6.84-6.88 (m, 4H, Ar), 5.95 (s, 1H,
H1′), 5.87 (d, 1H, ex, J = 5.0 Hz, 3′-OH), 5.16 (s, 1H, H2′), 4.62 (d, 1H, J = 5.0 Hz, H3′), 4.06-
4.08 (d, 1H, J = 8.0 Hz, H5′′), 4.02-4.05 (d, 1H, J = 8.0 Hz, H5′′), 3.72 (br s, 6H, CH3O), 3.38-
3.42 (d, 1H, J = 11.0 Hz, H5′), 3.22-3.25 (d, 1H, J = 11.0 Hz, H5′); 13C NMR (DMSO-d6) δ
165.6, 158.0, 152.4, 151.5 (C2), 149.2, 144.7, 135.5, 135.3, 133.1, 132.5 (Ar), 132.1, 129.7 (Ar),
129.6 (Ar), 128.5 (Ar), 128.4 (Ar), 127.8 (Ar), 127.6 (Ar), 126.6 (Ar), 125.4, 113.2 (Ar), 87.3
(C1′), 86.9, 85.2, 78.9 (C2′), 72.7 (C3′), 72.1 (C5′′), 60.3 (C5′), 55.0 (CH3O).
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