C5-alkynyl-functionalized α-L-LNA: Synthesis, thermal denaturation experiments and enzymatic stability

Article abstract of DOI:10.1021/jo5006153

Major efforts are currently being devoted to improving the binding affinity, target specificity, and enzymatic stability of oligonucleotides used for nucleic acid targeting applications in molecular biology, biotechnology, and medicinal chemistry. One of

Full text of DOI:10.1021/jo5006153

Article
pubs.acs.org/joc
C5-Alkynyl-Functionalized α‑L‑LNA: Synthesis, Thermal Denaturation
Experiments and Enzymatic Stability
Pawan Kumar,†,‡ Bharat Baral,† Brooke A. Anderson,† Dale C. Guenther,† Michael E. Østergaard,†
Pawan K. Sharma,‡ and Patrick J. Hrdlicka*,†
†Department of Chemistry, University of Idaho, Moscow, Idaho 83844-2343, United States
‡Department of Chemistry, Kurukshetra University, Kurukshetra 136119, India
S
* Supporting Information
ABSTRACT: Major efforts are currently being devoted to improving the binding affinity, target specificity, and enzymatic
stability of oligonucleotides used for nucleic acid targeting applications in molecular biology, biotechnology, and medicinal
chemistry. One of the most popular strategies toward this end has been to introduce additional modifications to the sugar ring of
affinity-inducing conformationally restricted nucleotide building blocks such as locked nucleic acid (LNA). In the preceding
article in this issue, we introduced a different strategy toward this end, i.e., C5-functionalization of LNA uridines. In the present
article, we extend this strategy to α-L-LNA: i.e., one of the most interesting diastereomers of LNA. α-L-LNA uridine monomers
that are conjugated to small C5-alkynyl substituents induce significant improvements in target affinity, binding specificity, and
enzymatic stability relative to conventional α-L-LNA. The results from the back-to-back articles therefore suggest that C5-
functionalization of pyrimidines is a general and synthetically straightforward approach to modulate biophysical properties of
oligonucleotides modified with LNA or other conformationally restricted monomers.
properties of α-L-LNA through modification or expansion of
the oxymethylene bridge spanning the C2′ and C4′ positions of
α-L-LNA and/or introduction of small branching substituents
onto the conformationally restricted furanose skeleton.10a,11
Our continued interest in LNA chemistry and C5-function-
alized pyrimidine DNA building blocks4c,12 prompted us to
pursue the synthesis of C5-alkynyl-functionalized LNA uridine
(U) monomers.13 We hypothesized that C5 substituents could
be used to modulate the characteristics of LNA pyrimidines.
Our preliminary results on a small set of C5-alkynyl-
functionalized LNA-U were promising and supported this
hypothesis.13 Thus, oligodeoxyribonucleotides (ONs) modified
with LNA-U monomers that are conjugated to small alkynes
display increased target affinity and binding specificity along
with moderately improved protection against 3′-exonucleases.
ONs modified with large C5-functionalized LNA-U monomers
display very high enzymatic stability, albeit at the expense of
INTRODUCTION
■
Significant efforts have been devoted to the development of
conformationally restricted nucleotides.1−3 Oligonucleotides
that are modified with such building blocks often display high
affinity toward nucleic acid targets and are accordingly used for
a variety of applications in molecular biology, biotechnology,
and medicinal chemistry.4 Locked nucleic acid (LNA),5−7
which is also known as bridged nucleic acid (BNA),8 is one of
the most promising members of this compound class, as it
displays some of the highest affinities toward complementary
DNA/RNA targets reported to date (increases in duplex
thermal denaturation temperatures, Tm’s, of up to +10 °C per
modification have been observed). One of the diastereoisomers
of LNA, i.e., α-L-LNA (α-L-ribo configuration; Figure 1)
displays similar hybridization characteristics9 and lower
hepatotoxicity10a and has accordingly been studied as a
potential modification for antisense, antigene, and decoy
oligonucleotides.10 The interesting properties of α-L-LNA
have spurred the development of many analogues, all of
which have focused on further improving the biophysical
Received: March 15, 2014
© XXXX American Chemical Society
A
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Figure 1. Structures of nucleotide monomers studied herein.
a
Scheme 1. Synthesis of Key Intermediate 9
a
Abbreviations: BSA, N,O-bis(trimethylsilyl)acetamide; U, uracil-1-yl; CAN, ceric ammonium nitrate; DMTr, 4,4′-dimethoxytrityl.
target affinity. Motivated by these results, we set out to (i)
study a greater number of C5-functionalized LNA-U
monomers14 and (ii) explore if this strategy for modulation
of biophysical properties can be applied to other conforma-
tionally restricted nucleotides.
Here, we present the synthesis of six different C5-alkynyl-
functionalized α-L-LNA-U phosphoramidites, their incorpo-
ration into ONs, and the characterization of these modified
ONs via thermal denaturation, enzymatic stability, and steady-
state fluorescence emission experiments. These monomers
were selected to ensure a representation of C5 substituents with
different sizes and polarities (Figure 1) and to facilitate direct
comparison with corresponding C5-alkynyl-functionalized
LNA-U.14
RESULTS AND DISCUSSION
■
Synthesis of α-L-LNA Key Intermediate 9. Our synthetic
route to C5-functionalized α-L-LNA-U phosphoramidites
11S−Y is inspired by the optimized routes to LNA15 and α-
L-LNA,9b as well as our recent synthesis of C5-alkynyl-
functionalized LNA nucleotides.13,14 Thus, fully protected
glycosyl donor 1, which is obtained from diacetone-α-D-glucose
in six steps and ∼30% overall yield,9b,16 was used as a starting
material (Scheme 1). One-pot glycosylation of 1 with
persilylated uracil under Vorbruggen conditions afforded
̈
nucleoside 2 in 85% yield via anchimeric assistance. Treatment
of 2 with hydrogen chloride in methanol afforded O2′-
deacetylated nucleoside 3 in 97% yield. We found these
conditions to be preferable to the use of cold dilute methanolic
ammonia,9b which results in the formation of small amounts of
xylo-LNA byproducts. Subsequent O2′-mesylation of 3
B
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a
Scheme 2. Synthesis of C5-Alkynyl-Functionalized α-L-LNA-U Phosphoramidites 11S−Y
a
Abbreviation: PCl-reagent, 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite.
Table 1. Thermal Denaturation Data for C5-Alkynyl-Functionalized α-L-LNA and Reference Strands against Complementary
a
DNA/RNA
ΔTm/mod (°C)
ON
duplex
B =
L
S
V
W
X
Y
Z
B1
5′-GTG ABA TGC
3′-CAC TAT ACG
+4.0
+7.5
+7.0
+9.5
+1.0
−2.5
−1.0
D2
D1
B2
5′-GTG ATA TGC
3′-CAC BAT ACG
+2.0
+8.5
+4.0
+9.5
+4.0
+9.0
+5.5
+3.5
+8.5
+4.0
+11.0
+5.0
+9.0
+6.0
+4.0
+7.0
+4.0
+8.5
+5.0
+8.5
+6.0
+5.5
+9.5
−0.5
+0.5
nd
−10.0
−2.0
nd
−10.5
0.0
D1
B3
5′-GTG ATA TGC
3′-CAC TAB ACG
D1
B4
5′-GTG ATA TGC
3′-CAC BAB ACG
+6.5
+0.5
+1.5
−8.5
+2.5
+2.0
B1
R2
5′-GTG ABA TGC
3′-CAC UAU ACG
+12.5
+7.0
+3.5
+2.5
+2.5
nd
+1.0
+0.5
−0.5
nd
R1
B2
5′-GUG AUA UGC
3′-CAC BAT ACG
R1
B3
5′-GUG AUA UGC
3′-CAC TAB ACG
+12.0
+7.5
R1
B4
5′-GUG AUA UGC
3′-CAC BAB ACG
a
ΔTm = change in Tm values relative to unmodified reference duplexes D1:D2 (Tm ≡ 29.5 °C), D1:R2 (Tm ≡ 27.0 °C), and D2:R1 (Tm ≡ 27.0 °C).
Tm values are determined as the first-derivative maximum of denaturation curves (A260 vs T) recorded in medium salt phosphate buffer ([Na+] = 110
mM, [Cl−] = 100 mM, pH 7.0 (NaH2PO4/Na2HPO4)), using 1.0 μM of each strand. Tm values are averages of at least two measurements within 1.0
°C. See Figure 1 for structures of monomers. nd = not determined. Data for L1−L4 were previously reported in ref 11b.
provided activated nucleoside 4 in 98% yield. Treatment of 4
with aqueous sodium hydroxide resulted in a cascade
reaction,17 i.e., formation of an O2,O2′-anhydronucleoside,
hydrolysis of the anhydronucleoside, and ring formation via
intramolecular nucleophilic displacement, to afford α-L-LNA
nucleoside 5 in quantitative yield. Subsequent protecting group
manipulations entailing nucleophilic substitution of the O5′-
mesylate of 5 (98%), O5′-debenzoylation of 6 (95%), and O3′-
debenzylation of 7 (85%), using catalytic transfer hydro-
genation conditions known to minimize undesired uracil C5−
C6 reduction (formic acid and Pd(OH)2/C),18 proceeded
smoothly to afford diol 8. Next, a reaction sequence entailing
O3′,O5′-diacetylation, C5-iodination, O3′,O5′-deacylation, and
O5′-dimethoxytritylation converted diol 8 into key intermedi-
ate 9 in high yield without purification of intermediates. Direct
C5-iodination of 8 and subsequent O5′-dimethoxytritylation to
directly afford key intermediate 9 in only two steps is also
possible but less attractive, due to lower overall yield and more
complicated purification (see Scheme S1, Supporting Informa-
tion). Hence, key intermediate 9 is obtained in ∼45% overall
yield and four chromatographic purification steps from glycosyl
donor 1 (Scheme 1).
Synthesis of C5-Alkynyl-Functionalized α-L-LNA
Phosphoramidites. Sonogashira reactions19 between key
C
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Table 2. Discrimination of Mismatched DNA/RNA Targets by Singly Modified C5-Alkynyl-Functionalized α-L-LNA and
a
Reference ONs
DNA: 3′-CAC TBT ACG
ΔTm
RNA: 3′-CAC UBU ACG
ΔTm
Tm
Tm
ON
sequence
A
C
G
T
A
C
G
U
D1
L1
S1
5′-GTG ATA TGC
5′-GTG ALA TGC
5′-GTG ASA TGC
5′-GTG AVA TGC
5′-GTG AWA TGC
5′-GTG AXA TGC
5′-GTG AYA TGC
5′-GTG AZA TGC
29.5
33.5
37.0
36.5
39.0
30.5
27.0
28.5
−16.5
−23.5
−24.0
−23.5
−25.0
−18.0
<−17.0
−13.5
−8.0
−13.5
−17.0
−15.5
−17.0
−15.5
<−17.0
−13.5
−15.5
−17.5
−23.0
−22.0
−22.5
−18.5
<−17.0
−8.5
27.0
36.5
38.0
35.5
39.5
30.5
28.0
28.5
<−17.0
−22.5
−27.0
−21.0
−23.5
−15.0
<−18.0
<−18.5
−4.5
−8.0
−11.0
−8.5
−11.0
−7.0
−11.0
−13.0
<−17.0
−22.5
−25.0
−21.0
−24.5
−20.0
<−18.0
<−18.5
V1
W1
X1
Y1
Z1
a
For experimental conditions and sequences see Table 1. ΔTm = change in Tm value relative to fully matched ON:DNA or ON:RNA duplex (B = A).
Data for L1 previously reported in reference 11b.
a
Table 3. Discrimination of Mismatched DNA/RNA Targets by Doubly Modified C5-Alkynyl-Functionalized α-L-LNA
DNA: 5′-GTG ABA TGC
ΔTm
RNA: 5′-GUG ABA UGC
ΔTm
Tm
Tm
ON
sequence
T
A
C
G
U
A
C
G
D2
L4
S4
3′-CAC TAT ACG
3′-CAC LAL ACG
3′-CAC SAS ACG
3′-CAC VAV ACG
3′-CAC WAW ACG
3′-CAC ZAZ ACG
29.5
37.0
37.0
37.5
42.0
30.5
<−19.5
−23.0
−18.0
−23.0
−21.5
<−20.5
−16.5
−18.0
−16.5
−15.5
−21.5
−14.5
−7.5
−17.0
−12.5
−12.0
−14.5
<−20.5
27.0
38.0
39.0
38.5
41.5
30.5
−16.0
−17.5
−15.0
−14.0
−14.5
−15.5
−16.0
−19.0
−17.0
−15.5
−16.5
<−20.5
−11.0
−14.0
−13.5
−14.0
−12.5
−16.0
V4
W4
Z4
a
For experimental conditions and sequences see Table 1. ΔTm = change in Tm value relative to fully matched duplexes (B = T).
intermediate 9 and different terminal alkynes20 provided C5-
alkynyl-functionalized LNA uridines 10 in moderate to
excellent yield (Scheme 2). Desilylation of 10S′ using TBAF
afforded 10S in 78% yield. Finally, O3′-phosphitylation of
nucleosides 10 using 2-cyanoethyl-N,N′-diisopropylchlorophos-
phoramidite provided target phosphoramidites 11S−11Y in
52−84% yield.
Structural Verification of α-L-LNA Nucleosides. As
expected,9b the 1H NMR signals of H1′, H2′, and H3′ of the α-
L-LNA nucleosides appear as singlets or narrow doublets (J < 2
Hz),21 since the torsion angles defined by H1′−C1′−C2′_-H2′
and H2′−C2′−C3′−H3′ are restricted to +gauche and −gauche
conformations, respectively. Moreover, the ROESY spectrum of
α-L-LNA diol 8 exhibits through-space couplings between (i)
H6 and H5″, (ii) H1′, H2′ and H3′, and (iii) H5′ and H3′,
whereas no through-space coupling between H2′ and H6 is
observed (Figure S1, Supporting Information). These observa-
tions are fully consistent with the proposed stereochemical
configuration.
Incorporation of C5-Alkynyl-Functionalized α-L-LNA
Monomers into ONs. Novel phosphoramidites 11S−Y (and
the known phosphoramidite of monomer Z22) were used to
incorporate monomers S−Z into 9-mer mixed-sequence ONs
via machine-assisted DNA synthesis. Standard procedures were
applied except for the use of extended hand-coupling times
during incorporation of C5-alkynyl-functionalized α-L-LNA
monomers (generally 15 min with 4,5-dicyanoimidazole as an
activatorsee the Experimental Section). The composition
and purity of all modified ONs was verified by MALDI-MS
(Table S1, Supporting Information) and ion-pair reversed-
phase HPLC, respectively. Unmodified reference DNA and
RNA strands are denoted D1/D2 and R1/R2, respectively,
while ONs containing a single incorporation of a modified
nucleotide in the 5′-GTG ABA TGC context are denoted S1,
V1, W1, and so on. Similar conventions are used for ONs in the
B2−B4 series.
Thermal Denaturation Studies. The thermostabilities of
duplexes between singly or doubly modified 9-mer ONs and
DNA/RNA complements were determined by thermal
denaturation experiments conducted in medium salt phosphate
buffer ([Na+] = 110 mM). Thermal denaturation temperatures
of modified duplexes are discussed relative to unmodified
reference duplexes unless otherwise mentioned (Table 1).
As previously noted,11b ONs modified with conventional α-
L-LNA-T monomer L form very thermostable duplexes,
especially with RNA targets, although considerable sequence
variation is observed (ΔTm = +2.5 to +9.5 °C, Table 1). ONs
modified with α-L-LNA uridines that are conjugated to small
alkynes at the C5 position generally result in the formation of
even more thermostable duplexes (compare ΔTm values for S/
V/W-modified ONs with L-modified ONs, Table 1). The effect
is most pronounced with W-modified ONs, which result in
additional duplex stabilization on the order of 1.0−5.5 °C
relative to ONs modified with conventional α-L-LNA-T. We
initially attributed these effects to improved base stacking
(larger aromatic surface area due to extended conjugation) and
reduced electrostatic repulsion (partially positively charged
aminopropynyl shielding negatively charged strands).23 How-
ever, analysis of thermodynamic parameters for the formed
duplexes indicates that the structural underpinnings accounting
for these results are more complex (vide infra). In contrast, α-
L-LNA-U monomers that are conjugated to large hydrophobic
entities reduce duplex thermostability relative to conventional
α-L-LNA, presumably due to unfavorable steric interactions
D
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a
Table 4. Thermodynamic Parameters for Formation of Duplexes Modified with Select C5-Functionalized α-L-LNA Monomers
+complementary DNA
+complementary RNA
ΔG298 [ΔΔG298
]
ΔH [ΔΔH]
−T298ΔS [Δ(T298ΔS)]
ΔG298 [ΔΔG298
]
ΔH [ΔΔH]
−T298ΔS [Δ(T298ΔS)]
ON
sequence
(kJ/mol)
(kJ/mol)
(kJ/mol)
(kJ/mol)
(kJ/mol)
(kJ/mol)
D1
D2
5′-GTG ATA TGC
3′-CAC TAT ACG
−42
−42
−314
−314
271
271
−36
−39
−278
−293
241
254
L1
L2
L3
5′-GTG ALA TGC
3′-CAC LAT ACG
3′-CAC TAL ACG
−49 [−7]
−45 [−3]
−48 [−6]
−349 [−35]
−311 [+3]
−302 [+12]
300 [+29]
266 [−5]
254 [−17]
−48 [−12]
−44 [−5]
−46 [−7]
−308 [−30]
−306 [−13]
−295 [−2]
260 [+19]
262 [+8]
249 [−5]
S1
S2
S3
5′-GTG ASA TGC
3′-CAC SAT ACG
3′-CAC TAS ACG
−49 [−7]
−45 [−3]
−48 [−6]
−333 [−19]
−276 [+38]
−325 [−11]
284 [+13]
231 [−40]
275 [+4]
−47 [−11]
−45 [−6]
−48 [−9]
−290 [−12]
−299 [−6]
−318 [−25]
243 [+2]
254 [ 0]
270 [+16]
V1
V2
V3
5′-GTG AVA TGC
3′-CAC VAT ACG
3′-CAC TAV ACG
−51 [−9]
−49 [−7]
−51 [−9]
−406 [−92]
−391 [−77]
−343 [−29]
354 [+83]
342 [+71]
292 [+21]
−50 [−14]
−46 [−7]
−49 [−10]
−302 [−24]
−340 [−47]
−289 [+4]
252 [+11]
293 [+39]
240 [−14]
W1
W2
W3
5′-GTG AWA TGC
3′-CAC WAT ACG
3′-CAC TAW ACG
−49 [−7]
−46 [−4]
−49 [−7]
−317 [−3]
−312 [+2]
−271 [+43]
267 [−4]
266 [−5]
222 [−49]
−50 [−14]
−46 [−7]
−49 [−10]
−302 [−24]
−340 [−47]
−289 [+4]
252 [+11]
293 [+39]
240 [−14]
X1
X2
X3
5′-GTG AXA TGC
3′-CAC XAT ACG
3′-CAC TAX ACG
−44 [−2]
−41 [−3]
−43 [−1]
−304 [+10]
−294 [+20]
−287 [+27]
260 [−11]
253 [−18]
244 [−27]
−44 [−8]
−41 [−2]
−42 [−3]
−319 [−41]
−245 [+48]
−283 [+10]
275 [+34]
204 [−50]
241 [−13]
Y1
Y2
Y3
5′-GTG AYA TGC
3′-CAC YAT ACG
3′-CAC TAY ACG
−39 [+3]
−36 [+6]
−39 [+3]
−313 [+1]
−305 [+9]
−324 [−10]
274 [+3]
269 [−2]
285 [−14]
−40 [−4]
−40 [−1]
−39 [ 0]
−313 [−35]
−325 [−32]
−310 [−17]
273 [+32]
285 [+31]
271 [+17]
a
Parameters were determined from thermal denaturation curves, which were recorded as described in Table 1. ΔΔG298, ΔΔH, and Δ(T298ΔS) are
calculated relative to reference duplexes D1:D2, D1:R2, and D2:R1.
and/or disruption of the hydration sphere in the major groove
(note ΔTm values for X/Y/Z-modified ONs; Table 1).
Nevertheless, many of the X/Y/Z-modified duplexes display
thermostabilities similar to those of the unmodified reference
duplexes.
should be designed in a manner that places likely single-
nucleotide polymorphism (SNP) sites directly opposite the
modified nucleotide for optimal thermal discrimination of
singly mismatched targets.
Thermodynamic Parameters for Duplex Formation.
Thermodynamic parameters for formation of duplexes modified
with C5-functionalized α-L-LNA-U monomers were derived
from thermal denaturation curves via curve fitting.24 In
agreement with the Tm data, duplexes modified with conven-
tional α-L-LNA thymidines are 3−12 kJ/mol more stable than
unmodified reference duplexes (see ΔΔG298 values for L1−L3,
Table 4). Duplexes that are modified with α-L-LNA-U
monomers conjugated to small alkynes display duplex stabilities
comparable to or slightly higher than those of duplexes
modified with conventional α-L-LNA-U monomers, while X/Y-
modified duplexes are less stable (compare ΔΔG298 values for
S/V/W series and X/Y series vs L series, Table 4).
The underlying structural underpinnings accounting for the
additional stabilization of S/V/W-modified duplexes are not as
clear as those for the corresponding C5-alkynyl-functionalized
LNA, which are stabilized by more favorable enthalpy, a
phenomenon that we ascribed to improved base stacking.14 For
example, the additional stabilization of V-modified DNA
duplexes is enthalpic in nature, whereas the situation is more
ambiguous with V-modified DNA:RNA duplexes (compare
ΔΔH and Δ(T298ΔS) values for V vs L series, Table 4). In
contrast, S/W-modified duplexes are generally stabilized by
more favorable entropy (compare ΔΔH and Δ(T298ΔS) values
for S/W series vs L series, Table 4). The lower stability of DNA
The binding specificities of ONs with a single central
modification (B1 series) were evaluated against centrally
mismatched DNA/RNA targets (Table 2). As previously
reported,11b ONs modified with conventional α-L-LNA
monomer L display significantly improved binding specificity
relative to the unmodified reference strand, as evidenced by the
greater drops in Tm values of mismatched duplexes (compare
ΔTm values for L1 and D1, Table 2). Interestingly, the high-
affinity ONs S1/V1/W1 display similar or slightly improved
binding specificity in comparison to conventional α-L-LNA L1
(compare ΔTm values for S1/V1/W1 and L1, Table 2),
whereas improvements are less pronounced for ONs modified
with hydrophobic C5-functionalized α-L-LNA-U monomers
(compare ΔTm’s for X1/Y1/Z1 and L1, Table 2).
The binding specificities of ONs with two next-nearest
neighbor modifications (B4 series) were determined using
DNA/RNA targets with a mismatched nucleotide opposite the
central 2′-deoxyriboadenosine (Table 3). α-L-LNA-T modified
L4 displays improved binding specificity relative to the
unmodified reference D2. Unlike the observations in the B1
series, ONs modified with C5-alkynyl-functionalized monomers
do not result in additional improvements in mismatch
discrimination (compare ΔTm values for the B4 series, Table
3). This suggests that C5-alkynyl-functionalized α-L-LNA
E
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duplexes modified with hydrophobic monomers X and Y is due
to unfavorable enthalpic contributions, whereas Y-modified
duplexes with RNA are destabilized by unfavorable entropic
contributions. Additional studies are clearly needed to fully
delineate the underlying reasons that govern these observations.
However, the mechanisms through which the C5-alkynyl
substituents exert their influence on duplex thermostability
appear to be different between LNA-U and α-L-LNA-U
nucleotides. This is not necessarily surprising, since incorpo-
ration of LNA and α-L-LNA nucleosides is known to have
different effects on global duplex geometries, i.e., LNA
nucleotides tune duplexes toward more RNA-like geometries,25
whereas α-L-LNA nucleotides leave duplexes globally un-
perturbed.26
that the C5 substituents interfere with SVPDE’s mode of
action. Expectedly, these trends are more pronounced with
doubly modified ONs (B4 series). Thus, considerable amounts
of conventional L4 and C5-ethynyl-functionalized α-L-LNA S4
remain after 2 h (<70% and <40% cleavage, respectively). It is
noteworthy that C5-aminopropynyl-functionalized α-L-LNA
W4, following a brief period of degradation of the 1−3
nucleotides closest to the 3′-end, is inert to further degradation.
Fluorescence Properties of Z-Modified ONs. Steady-
state fluorescence emission spectra of Z-modified ONs and the
corresponding duplexes with complementary or mismatched
DNA targets were recorded to evaluate the diagnostic potential
of these probes. Hybridization of singly Z-modified ONs with
complementary DNA/RNA generally results in significantly
increased fluorescence emission and the formation of duplexes
with two broad emission maxima at ∼388 and ∼401 nm
(Figures 3 and Figures S2 and S3 (Supporting Information)).
3′-Exonuclease Stability of C5-Alkynyl-Functionalized
α-L-LNA. Encouraged by the promising hybridization proper-
ties of S- and W-modified ONs, we set out to study their
stability in the presence of snake venom phosphodiesterase
(SVPDE), a 3′-exonuclease (Figure 2). As expected,
Figure 3. Steady-state fluorescence emission spectra of single-stranded
Z1 and corresponding duplexes with complementary or mismatched
DNA/RNA strands (mismatched nucleotide opposite to modification
in parentheses). Conditions: λex 344 nm, T = 5 °C, each
oligonucleotide used in 1 μM concentration. Note that different axis
scales are used.
Figure 2. 3′-Exonuclease degradation of singly (top, 3′-CAC BAT
ACG) and doubly modified (bottom, 3′-CAC BAB ACG) C5-
functionalized α-L-LNA and reference strands. Nuclease degradation
studies were conducted in magnesium buffer (50 mM Tris-HCl, 10
mM Mg2+, pH 9.0) using [ON] = 3.3 μM and 0.03 U of snake venom
phosphodiesterase.
In contrast, hybridization of doubly modified Z4 with DNA/
RNA complements results in decreased monomer emission
along with increased excimer emission (λem ∼510 nm, Figures
S4 and S5 (Supporting Information)), which is consistent with
the formation of pyrene−pyrene dimers in the major
groove.12c,27
Interestingly, the fluorescence intensity of Z1 is sensitive to
the nature of the nucleotide opposite to the modification;
hybridization with matched DNA/RNA targets results in the
formation of highly fluorescent duplexes, whereas incubation
with centrally mismatched targets results in much lower
unmodified D2 is degraded rapidly (>95% degradation after
15 min), while conventional α-L-LNA L1 displays moderate
resistance against SVPDE-mediated degradation (∼95%
degradation after 2 h). Interestingly, C5-ethynyl- and C5-
aminopropynyl-functionalized α-L-LNA S1 and W1 confer
additional protection against SVPDE (<80% and <60%
degradation after 2 h, respectively), which strongly suggests
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FAB-HRMS m/z 563.0998 ([M + H]+, C21H25N2O12S2·H+, calcd
563.1005); 1H NMR (DMSO-d6) δ 11.40 (d, 1H, ex, J = 2.0 Hz, NH),
7.76 (d, 1H, J = 8.0 Hz, H6), 7.33−7.39 (m, 5H, Ph), 6.09 (d, 1H, J =
6.0 Hz, H1′), 5.69 (dd, 1H, J = 8.0 Hz, 2.0 Hz, H5), 5.54 (t, 1H, J =
6.0 Hz, H2′), 4.68−4.71 (d, 1H, J = 12.0 Hz, CH2Ph), 4.65−4.68 (d,
1H, J = 12.0 Hz, CH2Ph), 4.58−4.62 (d, 1H, J = 11.0 Hz, H5′), 4.39−
4.47 (m, 4H, H3′, H5′, 2 × H5″), 3.27 (s, 3H, CH3SO2), 3.20 (s, 3H,
CH3SO2), 2.02 (s, 3H, CH3); 13C NMR (DMSO-d6) δ 169.6, 162.7,
150.4, 140.2 (C6), 137.1, 128.3 (Ar), 127.91 (Ar), 127.86 (Ar), 102.7
(C5), 84.6 (C1′), 81.8, 80.7 (C3′), 77.3 (C2′), 72.5 (CH2Ph), 68.3
(C5″), 67.9 (C5′), 36.7 (CH3SO2), 36.6 (CH3SO2), 20.3 (CH3).
1-[3-O-Benzyl-5-O-(methanesulfonyl)-4-C-(methanesulfony-
loxymethyl)-α-L-threo-pentofuranosyl]uracil (3). Method A. A 1
M solution of HCl in MeOH (50 mL) was added to a solution of
nucleoside 2 (2.81 g, 5.00 mmol) in MeOH (30 mL). and after the
reaction mixture was stirred at room temperature for 24 h, the solvent
was evaporated off. The resulting residue was dissolved in CH2Cl2
(100 mL), and the organic phase was washed with saturated aqueous
NaHCO3 (2 × 100 mL). The aqueous phase was then back-extracted
with CH2Cl2 (2 × 50 mL). The combined organic phase was dried
(Na2SO4) and evaporated to dryness to afford analytically pure
nucleoside 3 (2.52 g, 97%) as a white solid material: Rf = 0.4 (4%
MeOH in CH2Cl2, v/v); FAB-HRMS m/z 521.0900 ([M + H]+,
fluorescence intensities (Figure 3). Presumably this is due to
different positioning of the pyrene moiety in matched vs
mismatched duplexes in a similar manner as proposed for the
corresponding DNA analogue of monomer Z.28 According to
this hypothesis, the pyrene moiety is directed into the
nonquenching environment of the major groove in matched
duplexes, whereas it is intercalating in mismatched duplexes,
leading to nucleobase-mediated quenching29 of pyrene
fluorescence. In contrast, hybridization of doubly modified Z4
with centrally mismatched DNA/RNA targets results in a less
intense excimer signal but more pronounced monomer
emission (Figures S4 and S5 (Supporting Information)). This
suggests that the presence of mismatches in the vicinity of two
Z monomers positioned as next-nearest neighbors perturbs
pyrene−pyrene stacking in a manner similar to that observed
with other pyrene array forming probes.12a,30
We have recently studied the fluorescent properties of longer
Z-modified probes in detail.22 In comparison to ONs modified
with the analogous DNA monomer,28 Z-modified probes (i)
display slightly larger increases in fluorescence intensity upon
hybridization with complementary DNA, (ii) result in
formation of more brightly fluorescent duplexes, and (iii)
discriminate single-nucleotide polymorphisms more efficiently
in AT-rich sequence contexts. In summary, Z-modified ONs are
interesting probes for the discrimination of single-nucleotide
polymorphisms for applications in nucleic acid diagnostics.
1
C19H24N2O11S2·H+, calcd 521.0894); H NMR (DMSO-d6) δ 11.42
(d, 1H, ex, J = 2.0 Hz, NH), 7.76 (d, 1H, J = 8.0 Hz, H6), 7.29−7.40
(m, 5H, Ph), 6.12 (d, 1H, ex, J = 5.0 Hz, 2′-OH), 5.92 (d, 1H, J = 7.5
Hz, H1′), 5.68 (dd, 1H, J = 8.0 Hz, 2.0 Hz, H5), 4.74−4.77 (d, 1H, J =
12.0 Hz, CH2Ph), 4.67−4.69 (d, 1H, J = 12.0 Hz, CH2Ph), 4.33−4.52
(m, 5H, H2′, 2 × H5′, 2 × H5″), 4.19−4.21 (d, 1H, J = 6.5 Hz, H3′),
3.23 (s, 3H, CH3SO2), 3.17 (s, 3H, CH3SO2); 13C NMR (DMSO-d6)
δ 162.8, 150.8, 140.4 (C6), 137.5, 128.2 (Ar), 127.7 (Ar), 127.6 (Ar),
102.5 (C5), 86.0 (C1′), 82.8 (C3′), 80.9, 75.7 (C2′), 72.3 (CH2Ph),
68.9 (C5″), 68.2 (C5′), 36.7 (CH3SO2), 36.6 (CH3SO2).
CONCLUSION
■
Attachment of small alkynyl entities (ethynyl, hydroxypropynyl,
aminopropynyl) to the C5 position of α-L-LNA uridines
significantly increases the target affinity, binding specificity, and
enzymatic stability of oligonucleotides modified with these
building blocks in comparison to conventional α-L-LNA
uridines. On the other hand, attachment of larger alkynyl
groups (derivatives of stearic acid, cholesterol, and pyrene)
counteracts the stabilization provided by the extended
conjugation. Suitably designed C5-functionalized α-L-LNA
uridines are therefore interesting oligonucleotide modifications
for nucleic acid targeting applications in molecular biology,
biotechnology, and medicinal chemistry. The results from the
back-to-back articles strongly suggest that C5 functionalization
of pyrimidines is a general and synthetically convenient
approach for improving the pharmacodynamic properties of
oligonucleotides modified with LNA or other conformationally
restricted monomers.
Method B. To a solution of nucleoside 2 (1.50 g, 2.66 mmol) in
MeOH (50 mL) was added saturated methanolic ammonia (50 mL).
The solution was stirred for 2 h at room temperature, whereupon
solvents were evaporated off. The resulting residue was purified by
silica gel column chromatography (0−3% MeOH in CH2Cl2, v/v) to
afford nucleoside 3 (1.11 g, 80%) as a white solid material with
physical data as reported above.
1-[3-O-Benzyl-2,5-O-bis(methanesulfonyl)-4-C-(methanesul-
fonyloxymethyl)-α-L-threo-pentofuranosyl]uracil (4). Nucleo-
side 3 (10.0 g, 19.2 mmol) was coevaporated with anhydrous pyridine
(2 × 75 mL) and redissolved in anhydrous pyridine (120 mL). To this
was added methanesulfonyl chloride (MsCl, 1.8 mL, 23.3 mmol), and
the reaction mixture was stirred for 4 h at room temperature,
whereupon it was poured into saturated aqueous NaHCO3 (200 mL)
and extracted with CH2Cl2 (2 × 200 mL). The organic phase was
dried (Na2SO4) and concentrated to dryness to afford analytically pure
nucleoside 4 (11.3 g, 98%) as a slightly brown solid material: Rf = 0.4
(2% MeOH in CH2Cl2, v/v); FAB-HRMS m/z 599.0675 ([M + H]+,
1
C20H26N2O13S3·H+, calcd 599.0670); H NMR (DMSO-d6) δ 11.48
EXPERIMENTAL SECTION
(d, 1H, ex, J = 2.0 Hz, NH), 7.77 (d, 1H, J = 8.0 Hz, H6), 7.32−7.43
(m, 5H, Ph), 6.25 (d, 1H, J = 7.5 Hz, H1′), 5.70 (dd, 1H, J = 8.0 Hz,
2.0 Hz, H5), 5.54 (t, 1H, J = 7.0 Hz, H2′), 4.72 (s, 2H, CH2Ph), 4.58−
4.64 (2d, 2H, J = 11.0 Hz, J = 7.0 Hz, H5′, H3), 4.41−4.49 (m, 3H,
H5′, 2 × H5″), 3.28 (s, 3H, CH3), 3.25 (s, 3H, CH3), 3.18 (s, 3H,
CH3); 13C NMR (DMSO-d6) δ 162.7, 150.5, 140.3 (C6), 136.8, 128.3
(Ar), 128.1 (Ar), 128.0 (Ar), 102.8 (C5), 83.6 (C1′), 81.1 (C2′), 81.0,
80.7 (C3′), 73.0 (CH2Ph), 68.5 (C5″), 67.7 (C5′), 37.8 (CH3SO2)
36.7 (CH3SO2), 36.6 (CH3SO2).
(1S,3R,4S,7R)-7-Benzyloxy-1-methanesulfonyloxymethyl-3-
(uracil-1-yl)-2,5-dioxabicyclo[2.2.1]heptane (5). To a solution of
nucleoside 4 (9.87 g, 16.5 mmol) in 1,4-dioxane/H2O (60 mL, 1/1, v/
v) was added aqueous NaOH (2 M, 50 mL, 0.10 mol). After it was
stirred at room temperature for 4 h, the reaction mixture was
neutralized by 10% aqueous AcOH and diluted with EtOAc (300 mL)
and the phases were separated. The organic phase was washed with
saturated aqueous NaHCO3 (100 mL) and H2O (100 mL), dried
(Na2SO4), and evaporated to dryness to afford nucleoside 5 (7.00 g,
■
1-[2-O-Acetyl-3-O-benzyl-5-O-(methanesulfonyl)-4-C-(meth-
anesulfonyloxymethyl)-α-L-threo-pentofuranosyl]uracil (2).
Glycosyl donor 1 (6.10 g, 12.0 mmol) and uracil (2.70 g, 24.0
mmol) were coevaporated with anhydrous CH3CN (100 mL) and
resuspended in anhydrous CH3CN (150 mL). To this was added N,O-
bis(trimethylsilyl)acetamide (BSA; 10.4 mL, 41.9 mmol), and the
solution was refluxed until homogeneous. After the mixture was cooled
to room temperature, trimethylsilyl triflate (TMSOTf, 5.5 mL, 29.9
mmol) was added and the reaction mixture was refluxed for 28 h,
whereupon it was concentrated to near dryness. The resulting residue
was taken up in EtOAc (200 mL), and the organic phase was washed
with saturated aqueous NaHCO3 (2 × 100 mL) and brine (100 mL).
The aqueous phase was back-extracted with EtOAc (100 mL), and the
combined organic layers were dried (Na2SO4) and evaporated to
dryness. The resulting residue was purified by silica gel column
chromatography (0−2% MeOH in CH2Cl2, v/v) to afford nucleoside
2 (4.80 g, 85%) as a white foam: Rf = 0.5 (2% MeOH in CH2Cl2, v/v);
G
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quantitative) as a slightly brown solid material, which was used in the
next step without further purification: Rf = 0.5 (80% EtOAc in
petroleum ether, v/v); FAB-HRMS m/z 425.1018 ([M + H]+,
C18H20N2O8S·H+, calcd 425.1013); 1H NMR (DMSO-d6) δ 11.39 (br
s, 1H, ex, NH), 7.80 (d, 1H, J = 8.0 Hz, H6), 7.35−7.41 (m, 5H, Ph),
5.97 (s, 1H, H1′), 5.63 (d, 1H, J = 8.0 Hz, 1.2 Hz, H5), 4.55−4.73 (m,
5H, 2 × CH2Ph, H2′, 2 × H5′), 4.50 (s, 1H, H3′), 4.04−4.08 (d, 1H, J
= 8.5 Hz, H5″), 3.99−4.03 (d, 1H, J = 8.5 Hz, H5″), 3.23 (s, 3H,
CH3); 13C NMR (DMSO-d6) δ 163.0, 150.2, 140.3 (C6), 137.5, 128.3
(Ar), 127.7 (Ar), 127.5 (Ar), 100.6 (C5), 86.7, 86.6 (C1′), 79.2 (C3′),
76.4 (C2′), 71.9 (C5″), 71.2 (CH2Ph), 65.4 (C5′) 36.9 (CH3SO2). A
trace impurity of 1,4-dioxane was identified at 66.3 ppm in the 13C
NMR spectrum.31
(1S,3R,4S,7R)-1-Benzoyloxymethyl-7-benzyloxy-3-(uracil-1-
yl)-2,5-dioxabicyclo[2.2.1]heptane (6). To a solution of nucleoside
5 (7.00 g, 16.5 mmol) in anhydrous DMF (300 mL) was added
NaOBz (7.00 g, 48.6 mmol), and the reaction mixture was stirred at 90
°C for 20 h, whereupon it was cooled to room temperature and
poured into ice-cold water (500 mL). The solution was extracted with
EtOAc (2 × 300 mL) and the organic phase washed with H2O (2 ×
150 mL) and brine (100 mL). The organic phase was dried (Na2SO4)
and concentrated to dryness to afford analytically pure nucleoside 6
(7.24 g, 98%) as a slightly brown solid material: Rf = 0.6 (80% EtOAc
in petroleum ether, v/v); FAB-HRMS m/z 451.1505 ([M + H]+,
C24H22N2O7·H+, calcd 451.1500); 1H NMR (DMSO-d6) δ 11.38 (br s,
1H, ex, NH), 7.98 (dd, 2H, J = 8.5 Hz, 1.0 Hz, Ar), 7.82 (d, 1H, J = 8.0
Hz, H6), 7.66−7.71 (t, 1H, J = 7.5 Hz, Ar), 7.51−7.55 (m, 2H, Ar),
7.25−7.38 (m, 5H, Ar), 6.00 (s, 1H, H1′), 5.62 (dd, 1H, J = 8.0 Hz,
8.0 Hz, 1.5 Hz, H5), 4.66−4.76 (m, 3H, 2 × CH2Ph, H5′), 4.62 (s,
1H, H2′), 4.59 (s, 1H, H3′), 4.54−4.58 (d, 1H, J = 12.5 Hz, H5′),
4.14−4.17 (d, 1H, J = 8.5 Hz, H5″), 4.07−4.10 (d, 1H, J = 8.5 Hz,
H5″); 13C NMR (DMSO-d6) δ 165.2, 163.1, 150.3, 140.5 (C6), 137.6,
133.5 (Ar), 129.4 (Ar), 129.1, 128.7 (Ar), 128.2 (Ar), 127.6 (Ar),
127.5 (Ar), 100.5 (C5), 87.0, 86.6 (C1′), 79.3 (C3′), 76.3 (C2′), 72.1
(C5″), 71.1 (CH2Ph), 59.8 (C5′).
residue was purified by silica gel column chromatography (0−16%
MeOH in CH2Cl2, v/v) to afford nucleoside 8 (0.94 g, 85%) as a white
solid material: Rf = 0.4 (15% MeOH in CH2Cl2, v/v); FAB-HRMS m/
1
z 257.0760 ([M + H]+, C10H12N2O6·H+, calcd 257.0768); H NMR
(DMSO-d6) δ 11.34 (s, 1H, ex, NH), 7.80 (d, 1H, J = 8.0 Hz, H6),
5.87 (s, 1H, H1′), 5.85 (d, ex, J = 4.0 Hz, 3′-OH), 5.63 (d, 1H, J = 8.0
Hz, H5), 4.93 (t, ex, J = 5.5 Hz, 5′-OH), 4.27 (d, 1H, J = 4.0 Hz, H3′),
4.21 (s, 1H, H2′), 3.88−3.94 (2d, 2H, J = 8.5 Hz, H5″), 3.73 (d, 2H, J
= 5.5 Hz, 2 × H5′); 13C NMR (DMSO-d6) δ 163.2, 150.3, 140.4 (C6),
100.2 (C5), 90.9, 86.5 (C1′), 78.7 (C2′), 72.5 (C3′), 71.9 (C5″), 57.4
(C5′).
(1S,3R,4S,7R)-1-(4,4′-Dimethoxytrityloxymethyl)-7-hydroxy-
3-(5-iodoracil-1-yl)-2,5-dioxabicyclo[2.2.1]heptane (9). Ac2O
(0.21 mL, 2.20 mmol) was added to a solution of nucleoside 8
(0.25 g, 1.00 mmol) in anhydrous pyridine (10 mL) and the reaction
mixture was stirred at 60 °C for 14 h. After it was cooled to room
temperature, the reaction mixture was diluted with saturated aqueous
NaHCO3 (30 mL) and CH2Cl2 (30 mL) and the phases were
separated. The organic phase was washed with saturated aqueous
NaHCO3 (20 mL) and the combined aqueous phase back-extracted
with CH2Cl2 (2 × 20 mL). The combined organic layers were dried
(Na2SO4), evaporated to dryness, and coevaporated with toluene/
absolute EtOH (2 × 30 mL, 1/2, v/v). The resulting residue,
tentatively assigned as the O3′,O5′-diacetylated nucleoside, was used
in the next step without further purification (Rf = 0.5 (2% MeOH in
CH2Cl2, v/v); FAB-MS m/z 341 ([M + H]+)).
To a solution of the crude O3′,O5′-diacetylated α-L-LNA uridine in
glacial AcOH (10 mL) were added iodine (160 mg, 0.62 mmol) and
ceric ammonium nitrate (CAN, 235 mg, 0.50 mmol), and the reaction
mixture was stirred at 80 °C for 50 min. After it was cooled to room
temperature, the reaction mixture was evaporated to dryness and taken
up in CH2Cl2 (50 mL). The organic phase was washed with saturated
aqueous NaHCO3 (2 × 20 mL) and H2O (20 mL). The combined
aqueous phase was back-extracted with CH2Cl2 (2 × 20 mL). The
combined organic layers were dried (Na2SO4) and evaporated to
dryness. The resulting residue, tentatively assigned as the C5-iodo-
O3′,O5′-diacetylated nucleoside, was used in the next step without
further purification (Rf = 0.5 (3% MeOH in CH2Cl2, v/v); FAB-
HRMS m/z 466.9966 ([M + H]+, C14H15IN2O8·H+, calcd 466.9946)).
The crude C5-iodo-O3′,O5′-diacetylated nucleoside was dissolved
in saturated methanolic ammonia (30 mL) and stirred in a sealed flask
at room temperature for 12 h. The reaction mixture was evaporated to
dryness, affording a residue that was tentatively assigned as the C5-
iodo α-L-LNA diol and used in the next step without further
purification (Rf = 0.4 (15% MeOH in CH2Cl2, v/v); FAB-HRMS m/z
382.9735 ([M + H]+, C10H11IN2O6·H+, calcd 382.9740)).
(1R,3R,4S,7R)-7-Benzyloxy-1-hydroxymethyl-3-(uracil-1-yl)-
2,5-dioxabicyclo[2.2.1]heptane (7). Method A. To a solution of
nucleoside 6 (5.00 g, 11.1 mmol) dissolved in MeOH (100 mL) was
added saturated methanolic ammonia (100 mL). The reaction mixture
was stirred at room temperature for 14 h in a sealed flask. The solvent
was then evaporated and the resulting residue purified by silica gel
column chromatography (0−7% MeOH in CH2Cl2,v/v) to afford
nucleoside 7 (3.66 g, 95%) as a white solid material: Rf = 0.4 (7%
MeOH in CH2Cl2, v/v); FAB-HRMS m/z 347.1230 ([M + H]+,
1
C17H18N2O6·H+, calcd 347.1243); H NMR (DMSO-d6) δ 11.36 (s,
1H, ex, NH), 7.80 (d, 1H, J = 8.0 Hz, H6), 7.30−7.40 (m, 5H, Ph),
5.88 (s, 1H, H1′), 5.62 (d, 1H, J = 8.0 Hz, H5), 5.05 (t, 1H, ex, J = 5.5
Hz, 5′-OH), 4.63−4.71 (2d, 2H, J = 12.0 Hz, CH2Ph), 4.52 (s, 1H,
H2′), 4.33 (s, 1H, H3′), 3.92−3.98 (2d, 2H, J = 8.5 Hz, H5″), 3.73−
3.78 (m, 2H, 2 × H5′); 13C NMR (DMSO-d6) δ 163.1, 150.3, 140.4
(C6), 137.9, 128.3 (Ar), 127.6 (Ar), 127.4 (Ar), 100.3 (C5), 90.1
(C4′), 86.5 (C1′), 79.3 (C3′), 76.3 (C2′), 72.4 (C5″), 71.1 (CH2Ph),
57.1 (C5′).
The crude C5-iodo α-L-LNA diol was dried through coevaporation
with anhydrous pyridine (10 mL) and redissolved in anhydrous
pyridine (10 mL). To this was added 4,4′-dimethoxytrityl chloride
(DMTrCl, 0.40 g, 1.20 mmol) and the reaction mixture was stirred at
room temperature for 16 h, whereupon it was diluted with saturated
aqueous NaHCO3 (20 mL) and CH2Cl2 (25 mL). The phases were
separated, and the organic phase was washed with saturated aqueous
NaHCO3 (20 mL). The aqueous phase was back-extracted with
CH2Cl2 (2 × 20 mL). The combined organic layers were dried
(Na2SO4), evaporated to near dryness, and coevaporated with
toluene/absolute EtOH (2 × 30 mL, 1/2, v/v). The resulting residue
was purified by silica gel column chromatography (0−4.5% MeOH in
CH2Cl2, v/v) to afford nucleoside 9 (0.48 g, 70%, over four steps) as a
slightly yellow solid material: Rf = 0.5 (5% MeOH in CH2Cl2, v/v);
Method B. To a solution of nucleoside 6 (1.50 g, 3.33 mmol) in
THF/H2O (25 mL, 1/1, v/v) was added aqueous NaOH (2 M, 10.0
mL, 20.0 mmol), and the reaction mixture was stirred at room
temperature for 4 h, whereupon it was carefully neutralized with 10%
aqueous AcOH at 0 °C and diluted with EtOAc (50 mL). The organic
phase was washed with saturated aqueous NaHCO3 (50 mL) and the
combined aqueous phase extracted with EtOAc (2 × 50 mL). The
combined organic layers were dried (Na2SO4) and concentrated to
dryness to afford nucleoside 7 (1.05 g, 91%) as a slightly brown solid.
(1R,3R,4S,7R)-7-Hydroxy-1-hydroxymethyl-3-(uracil-1-yl)-
2,5-dioxabicyclo[2.2.1]heptane (8). To a solution of nucleoside 7
(1.50 g, 4.32 mmol) in THF/MeOH (100 mL, 9/1, v/v) were added
Pd(OH)2/C (20 wt %, 0.60 g) and 88% aqueous formic acid (2.3 mL,
61.1 mmol) from a freshly opened bottle. The reaction mixture was
refluxed for 24 h, whereupon it was cooled to room temperature. The
catalyst was filtered off and washed with excess MeOH, and the
combined filtrates were concentrated to dryness. The resulting crude
+
FAB-HRMS m/z 684.0980 ([M]+, C31H29IN2O8 , calcd 684.0969); 1H
NMR (DMSO-d6) δ 11.78 (s, 1H, ex, NH), 8.16 (s, 1H, H6), 7.23−
7.43 (m, 9H, Ar), 6.92 (d, 4H, J = 8.5 Hz, Ar), 5.92 (d, 1H, ex, J = 4.5
Hz, 3′-OH), 5.89 (s, 1H, H1′), 4.35 (d, 1H, J = 4.5 Hz, H3′), 4.27 (s,
1H, H2′), 3.98−4.02 (d, 1H, J = 8.5 Hz, H5″), 3.92−3.96 (d, 1H, J =
8.5 Hz, H5″), 3.75 (s, 6H, 2 × CH3O), 3.34−3.37 (d, 1H, J = 10.5 Hz,
H5′), 3.28−3.31 (d, 1H, J = 10.5 Hz, H5′ - partial overlap with H2O);
13C NMR (DMSO-d6) δ 160.6, 158.134, 158.126, 149.9, 144.7, 144.3
(C6), 135.2, 135.1, 129.64, 129.62 (Ar), 127.9 (Ar), 127.5 (Ar), 126.7
H
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(Ar), 113.3 (Ar), 89.3, 87.2 (C1′), 85.3, 78.8 (C2′), 72.9 (C3′), 72.3
(C5″), 67.7, 60.0 (C5′), 55.0 (CH3O).
1H, J = 7.5 Hz, 1.2 Hz, Ar), 7.50−7.54 (m, 2H, Ar), 7.17−7.44 (m,
9H, Ar), 6.90 (d, 4H, J = 9.0 Hz, Ar), 5.94 (br s, 2H, 1 ex, H1′, 3′-
OH), 5.20 (s, 2H, CH2OBz), 4.42 (d, 1H, J = 4.5 Hz, H3′), 4.28 (s,
1H, H2′), 4.01−4.04 (d, 1H, J = 8.0 Hz, H5″), 3.90−3.94 (d, 1H, J =
8.0 Hz, H5″), 3.73 (s, 6H, 2 × OCH3), 3.29 (s, 2H, H5′); 13C NMR
(DMSO-d6) δ 165.0, 161.4, 158.1, 149.2, 144.7, 144.0 (C6), 135.2,
135.1, 133.6 (Ar), 129.68 (Ar), 129.66 (Ar), 129.2 (Ar), 129.0, 128.8
(Ar), 127.8 (Ar), 127.5 (Ar), 126.6 (Ar), 113.2 (Ar), 96.1, 89.4, 87.3
(C1′), 86.6, 85.3, 79.2, 78.6 (C2′), 72.7 (C3′), 72.2 (C5″), 59.7 (C5′),
55.0 (CH3O), 53.2 (CH2OBz).
(1S,3R,4S,7R)-1-(4,4′-Dimethoxytrityloxymethyl)-7-hydroxy-3-[5-
(3-trifluoroacetylaminopropyn-1-yl)uracil-1-yl]-2,5-dioxabicyclo-
[2.2.1]heptane (10W). Nucleoside 9 (0.50 g, 0.73 mmol), Pd(PPh3)4
(90 mg, 0.07 mmol), CuI (30 mg, 0.14 mmol), 2,2,2-trifluoro-N-
(prop-2-ynyl)acetamide33 (180 mg, 1.46 mmol), and Et3N (0.4 mL,
2.84 mmol) in anhydrous DMF (10 mL) were reacted as described in
the representative Sonogashira protocol and stirred at room
temperature for 12 h. After workup and purification, nucleoside
10W (0.43 g, 84%) was obtained as a slightly brown solid material: Rf
= 0.5 (5% MeOH in CH2Cl2, v/v); MALDI-HRMS m/z 730.1976
([M + Na]+, C36H32F3N3O9·Na+, calcd 730.1983); 1H NMR (DMSO-
d6) δ 11.76 (s, 1H, ex, NH(U)), 10.04 (t, ex, 1H, J = 5.5 Hz, NHCH2),
7.94 (s, 1H, H6), 7.21−7.43 (m, 9H, Ar), 6.91 (d, 4H, J = 9.0 Hz, Ar),
5.94−5.96 (m, 2H, 1ex, H1′, 3′-OH), 4.44 (d, 1H, J = 4.0 Hz, H3′),
4.25−4.29 (m, 3H, H2′, CH2NH), 3.98−4.00 (d, 1H, J = 8.5 Hz,
H5″), 3.92−3.95 (d, 1H, J = 8.5 Hz, H5″), 3.74 (s, 6H, 2 × OCH3),
3.28−3.32 (m, 2H, H5′, overlap with H2O); 13C NMR (DMSO-d6) δ
161.5, 158.1, 156.0 (q, J = 36.3 Hz, COCF3) 149.2, 144.7, 143.4 (C6),
135.2, 135.1, 129.72 (Ar), 129.69 (Ar), 127.8 (Ar), 127.6 (Ar), 126.7
(Ar), 115.7 (q, J = 287 Hz, CF3), 113.2 (Ar), 96.5, 89.4, 87.3, 87.1
(C1′), 85.3, 78.6 (C2′), 75.4, 72.7 (C3′), 72.3 (C5″), 59.7 (C5′), 55.0
(CH3O), 29.5 (CH2NH).
Representative Protocol for Sonogashira Coupling Reac-
tions (10S′−Z). The key intermediate 9, Pd(PPh3)4, CuI, and alkyne
were added to anhydrous DMF (quantities and volumes specified
below), and the reaction chamber was degassed and placed under an
argon atmosphere. To this was added Et3N, and the reaction mixture
was stirred in the dark at room temperature (unless otherwise
mentioned) for 6−12 h, whereupon the solvents were evaporated. The
resulting residue was dissolved in EtOAc (100 mL), and the organic
phase was washed with brine (2 × 50 mL) and saturated aqueous
NaHCO3 (50 mL). The combined aqueous phase was back-extracted
with EtOAc (100 mL). The combined organic phase was dried
(Na2SO4) and evaporated to dryness and the resulting residue purified
by silica gel column chromatography (0−5% MeOH in CH2Cl2, v/v)
to afford the desired product.
(1S,3R,4S,7R)-1-(4,4′-Dimethoxytrityloxymethyl)-7-hydroxy-3-[5-
(trimethylsilylethynyl)uracil-1-yl]-2,5-dioxabicyclo[2.2.1]heptane
(10S′). Nucleoside 9 (0.68 g, 1.00 mmol), Pd(PPh3)4 (120 mg, 0.10
mmol), CuI (40 mg, 0.20 mmol), trimethylsilylacetylene (0.42 mL,
3.00 mmol), and Et3N (0.60 mL, 4.27 mmol) in anhydrous DMF (10
mL) were reacted as described in the representative Sonogashira
protocol, and the mixture was stirred at room temperature for 12 h.
After work-up and purification, nucleoside 10S′ (0.55 g, 84%) was
obtained as a slightly brown solid material: Rf = 0.5 (5% MeOH in
CH2Cl2, v/v); FAB-HRMS m/z 655.2462 ([M + H]+, C36H38N2O8Si·
1
H+, calcd 655.2476); H NMR (DMSO-d6) δ 11.75 (s, 1H, ex, NH),
7.93 (s, 1H, H6), 7.21−7.43 (m, 9H, Ar), 6.90 (d, 4H, J = 8.5 Hz, Ar),
5.94 (d, 1H, ex, J = 4.5 Hz, 3′-OH), 5.92 (s, 1H, H1′), 4.38 (d, 1H, J =
4.5 Hz, H3′), 4.26 (s, 1H, H2′), 3.99−4.02 (d, 1H, J = 8.5 Hz, H5″),
3.93−3.96 (d, 1H, J = 8.5 Hz, H5″), 3.74 (s, 6H, 2 × CH3O), 3.34 (s,
2H, H5′), 0.21 (s, 9H, Me3Si); 13C NMR (DMSO-d6) δ 161.3, 158.1,
149.1, 144.6, 143.7 (C6), 135.2, 135.1, 129.7 (Ar), 129.6 (Ar), 127.8
(Ar), 127.6 (Ar), 126.7 (Ar), 113.2 (Ar), 97.8, 97.1, 96.9, 89.4, 87.3
(C1′), 85.3, 78.6 (C2′), 72.7 (C3′), 72.3 (C5″), 60.0 (C5′), 55.0
(CH3O), −0.12 (Me3Si).
(1S,3R,4S,7R)-1-(4,4′-Dimethoxytrityloxymethyl)-7-hydroxy-3-[5-
(3-octadecanoylaminopropyn-1-yl)uracil-1-yl]-2,5-dioxabicyclo-
[2.2.1]heptane (10X). Nucleoside 9 (0.34 g, 0.50 mmol), Pd(PPh3)4
(60 mg, 0.05 mmol), CuI (20 mg, 0.10 mmol), N-(prop-2-
ynyl)stearamide,14 (0.28 g, 1.00 mmol) and Et3N (0.30 mL, 2.13
mmol) in anhydrous DMF (10 mL) were reacted as described in the
representative Sonogashira protocol, and the mixture was stirred at 40
°C for 6 h. After workup and purification, nucleoside 10X (0.24 g,
55%) was obtained as a brown solid material: Rf = 0.5 (5% MeOH in
CH2Cl2, v/v); ESI-HRMS m/z 900.4813 ([M + Na]+, C52H67N3O9·
(1S,3R,4S,7R)-1-(4,4′-Dimethoxytrityloxymethyl)-3-(5-ethynylura-
cil-1-yl)-7-hydroxy-2,5-dioxabicyclo[2.2.1]heptane (10S). TBAF in
THF (1M, 1.2 mL, 1.2 mmol) was added to a solution of nucleoside
10S′ (0.53 g, 0.81 mmol) in THF (20 mL) and the reaction mixture
was stirred at room temperature for 2 h. EtOAc (50 mL) was added,
and the solution was washed with brine (2 × 30 mL) and H2O (30
mL). The combined aqueous phase was back-extracted with EtOAc
(30 mL). The combined organic layer was dried (Na2SO4) and
concentrated to dryness and the resulting residue purified by silica
column chromatography (0−5% MeOH in CH2Cl2, v/v) to afford
nucleoside 10S (0.37 g, 78%) as a light brown solid material: Rf = 0.5
(5% MeOH in CH2Cl2, v/v); ESI-HRMS m/z 605.1918 ([M + Na]+,
1
Na+, calcd 900.4770); H NMR (CDCl3) δ 7.89 (s, 1H, H6), 7.24−
7.46 (m, 9H, Ar), 6.87 (d, 4H, J = 9.0 Hz, Ar), 5.97 (s, 1H, H1′), 5.88
(t, ex, 1H, J = 5.0 Hz, NHCH2), 4.55 (s, 1H, H2′), 4.48 (s, 1H, H3′),
4.27−4.29 (m, 2H, CH2NH), 4.10−4.13 (d, 1H, J = 9.0 Hz, H5″),
3.97−4.01 (d, 1H, J = 9.0 Hz, H5″), 3.81 (s, 6H, 2 × CH3O), 3.50−
3.57 (2d, 2H, J = 11.0 Hz, H5′), 2.16−2.19 (m, 2H, CH2CONH),
1.60−1.64 (m, 2H, CH2CH2CONH), 1.20−1.25 (m, 28H, 14 × CH2),
0.89 (t, 3H, J = 7.0 Hz, CH3CH2); 13C NMR (CDCl3) δ 172.8, 161.6,
158.77, 158.76, 149.0, 144.3, 142.7 (C6), 135.23, 135.21, 130.04 (Ar),
130.03, (Ar), 128.04 (Ar), 127.98 (Ar), 127.1 (Ar), 113.4 (Ar), 98.5,
89.8, 89.6, 88.0 (C1′), 86.7, 78.9 (C2′), 74.5, 74.3 (C3′), 72.8 (C5″),
59.5 (C5′), 55.3 (CH3O), 36.5 (CH2CONH), 31.9 (CH2), 30.1
(CH2NH), 29.69 (CH2), 29.68 (CH2), 29.66 (CH2), 29.65 (CH2),
29.63 (CH2), 29.5 (CH2), 29.353 (CH2), 29.345 (CH2), 29.33 (CH2),
25.5 (CH2CH2CONH), 22.7 (CH2), 14.1 (CH3).
1
C33H30N2O8·Na+, calcd 605.1894); H NMR (DMSO-d6) δ 11.74 (s,
1H, ex, NH), 8.03 (s, 1H, H6), 7.23−7.43 (m, 9H, Ar), 6.90 (d, 4H, J
= 8.5 Hz, Ar), 5.90−5.96 (m, 2H, 1ex, H1′, 3′-OH), 4.40 (s, 1H, H3′),
4.28 (s, 1H, H2′), 4.18 (s, 1H, HCC), 4.01−4.04 (d, 1H, J = 8.5 Hz,
H5″), 3.91−3.94 (d, 1H, J = 8.5 Hz, H5″), 3.75 (s, 6H, 2 × CH3O),
3.32 (br s, 2H, H5′); 13C NMR (DMSO-d6) δ 161.6, 158.1, 149.2,
144.7, 143.7 (C6), 135.2, 135.1, 129.7 (Ar), 128.9 (Ar), 127.8 (Ar),
127.6 (Ar), 126.7 (Ar), 113.2 (Ar), 96.4, 89.4, 87.3 (C1′), 85.3, 83.4
(HCC), 78.7 (C2′), 76.3, 72.8 (C3′), 72.2 (C5″), 59.8 (C5′), 55.0
(CH3O).
(1S,3R,4S,7R)-3-[5-(3-Benzoyloxypropyn-1-yl)uracil-1-yl]-1-(4,4′-
dimethoxytrityloxymethyl)-7-hydroxy-2,5-dioxabicyclo[2.2.1]-
heptane (10V). Nucleoside 9 (0.50 g, 0.73 mmol), Pd(PPh3)4 (90 mg,
0.07 mmol), CuI (30 mg, 0.14 mmol), prop-2-ynyl benzoate32 (180
mg, 1.12 mmol), and Et3N (0.40 mL, 2.84 mmol) in anhydrous DMF
(10 mL) were reacted as described in the representative Sonogashira
protocol, and the mixture was stirred at room temperature for 12 h.
After workup and purification, nucleoside 10V (0.40 g, 76%) was
obtained as a slightly brown solid material: Rf = 0.5 (5% MeOH in
CH2Cl2, v/v); ESI-HRMS m/z 739.2234 ([M + Na]+, C41H36N2O10·
Na+, calcd 739.2262); 1H NMR (DMSO-d6) δ 11.79 (s, 1H, ex, NH),
8.03 (s, 1H, H6), 7.98 (dd, 2H, J = 8.3 Hz, 1.2 Hz, Ar), 7.65−7.69 (td,
(1S,3R,4S,7R)-3-[5-(3-Cholesterylcarbonylaminopropyn-1-yl)-
uracil-1-yl]-1-(4,4′-dimethoxytrityloxymethyl)-7-hydroxy-2,5-
dioxabicyclo[2.2.1]heptane (10Y). Nucleoside 9 (0.34 g, 0.50 mmol),
Pd(PPh3)4 (60 mg, 0.05 mmol), CuI (20 mg, 0.10 mmol),
cholesterylprop-2-ynyl-carbamate amine34 (0.47 g, 1.00 mmol), and
Et3N (0.30 mL, 2.13 mmol) in anhydrous DMF (8 mL) were reacted
as described in the representative Sonogashira protocol and the
reaction mixture was stirred at room temperature for 12 h. After
workup and purification, nucleoside 10Y (0.39 g, 76%) was obtained
as a slightly yellow solid material: Rf = 0.5 (5% MeOH in CH2Cl2, v/
v); ESI-HRMS m/z 1046.5533 ([M + Na]+, C62H77N3O10·Na+, calcd
1
1046.5501); H NMR (CDCl3) δ 8.92 (bs, 1H, ex, NH(U)), 7.89 (s,
1H, H6), 7.23−7.46 (m, 9H, Ar), 6.87 (d, 4H, J = 9.0 Hz, Ar), 5.98 (s,
I
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1H, H1′), 5.36 (d, 1H, J = 5.0 Hz, HCC-Chol), 5.12 (t, ex, 1H, J =
5.0 Hz, NHCH2), 4.47−4.56 (m, 3H, H2′, HC-O-Chol, H3′), 4.20 (d,
2H, J = 5.0 Hz, CH2NH), 4.11−4.15 (d, 1H, J = 9.0 Hz, H5″), 3.98−
4.02 (d, 1H, J = 9.0 Hz, H5″), 3.81 (s, 6H, 2 × CH3O), 3.50−3.58 (2d,
2H, J = 11.0 Hz, H5′), 0.87−2.36 (m, 40H, Chol), 0.69 (s, 3H, CH3-
Chol); 13C NMR (CDCl3) δ 161.6, 158.74, 158.73, 149.0, 144.3, 142.7
(C6), 139.8, 135.3, 132.1 (Ar), 132.0 (Ar), 130.04 (Ar), 130.03 (Ar),
128.5 (Ar), 128.4 (Ar), 128.02 (Ar), 128.00 (Ar), 127.1 (Ar), 122.5
(CHC-chol), 113.4 (Ar), 98.6, 89.9, 88.0 (C1′), 86.6, 78.9 (C2′),
74.9 (CH-O-chol), 74.4, 74.3 (C3′),35 72.8 (C5″), 59.5 (C5′), 56.7
(CH-chol), 56.2 (CH-chol), 55.2 (CH3O), 50.0 (CH-chol), 42.3, 39.8
(CH2-chol), 39.5 (CH2-chol), 38.5 (CH2−chol), 37.0 (CH2−chol),
36.6, 36.2 (CH2−chol), 35.8 (CH-chol), 31.9 (CH-chol), 31.8
(CH2NH and CH2-chol overlap), 28.2 (CH2-chol), 28.1 (CH2-chol),
28.0 (CH-chol), 24.3 (CH2-chol), 23.8 (CH2-chol), 22.8 (CH3-chol),
22.5 (CH3-chol), 21.0 (CH2-chol), 19.3 (CH3-chol), 18.7 (CH3-chol),
11.9 (CH3-chol).
Representative Protocol for Phosphitylation. Unless other-
wise mentioned, the following protocol was used. Alcohol 10 was dried
through coevaporation with anhydrous 1,2-dichloroethane (2 × 10
mL) and dissolved in anhydrous CH2Cl2. To this solution was added
anhydrous EtN(iPr)2 (DIPEA) and 2-cyanoethyl-N,N-diisopropyl-
chlorophosphoramidite (PCl-reagent), and the reaction mixture was
stirred at room temperature until analytical TLC showed full
conversion of the starting material (2−3 h) (quantities and volumes
specified below). The reaction mixture was diluted with CH2Cl2 (25
mL) and washed with 5% aqueous NaHCO3 (2 × 10 mL) and the
combined aqueous phase back-extracted with CH2Cl2 (2 × 10 mL).
The combined organic layers were dried (Na2SO4) and evaporated to
dryness, and the resulting residue was purified by silica gel column
chromatography (0−3% MeOH in CH2Cl2, v/v) and subsequently
triturated from CH2Cl2 and petroleum ether to provide phosphor-
amidite 11.
(1S,3R,4S,7R)-7-[2-Cyanoethoxy(diisopropylamino)-
phosphinoxy]-1-(4,4′-dimethoxytrityloxymethyl)-3-(5-ethynyluracil-
1-yl)-2,5-dioxabicyclo[2.2.1]heptane (11S). Nucleoside 10S (0.35 g
0.60 mmol), DIPEA (0.50 mL, 2.88 mmol), and PCl-reagent (0.20
mL, 0.87 mmol) in anhydrous CH2Cl2 (10 mL) were reacted, worked
up, and purified as described in the representative phosphitylation
protocol to provide 11S (0.39 g, 83%) as a white foam: Rf = 0.5 (2%
MeOH in CH2Cl2, v/v); ESI-HRMS m/z 805.2943 ([M + Na]+,
C42H47N4O9P·Na+, calcd 805.2958); 31P NMR (CDCl3) δ 150.2,
149.9.
(1S,3R,4S,7R)-3-[5-(3-Benzoyloxypropyn-1-yl)uracil-1-yl]-7-[2-
cyanoethoxy(diisopropylamino)phosphinoxy]-1-(4,4′-dimethoxytri-
tyloxymethyl)-2,5-dioxabicyclo[2.2.1]heptane (11V). Nucleoside
10V (0.35 g, 0.49 mmol) was coevaporated with anhydrous 1,2-
dichloroethane (2 × 7 mL) and redissolved in anhydrous CH2Cl2 (7
mL). To this solution were added DIPEA (425 μL, 2.44 mmol) and N-
methylimidazole (31 μL, 0.39 mmol), followed by dropwise addition
of the PCl-reagent (220 μL, 0.98 mmol). The reaction mixture was
stirred at room temperature for 3 h, whereupon it was evaporated to
near dryness. The resulting residue was purified by silica gel column
chromatography (0−3% MeOH in CH2Cl2) and subsequently
triturated from CH2Cl2 and petroleum ether to provide 11V (0.28
g, 62%) as a white foam: Rf = 0.5 (3% MeOH in CH2Cl2); ESI-HRMS
m/z 939.3332 ([M + Na]+, C50H53N4O11P·Na+, calcd 939.3341); 31P
NMR (CDCl3) δ 150.1, 149.8.
(11X). Nucleoside 10X (170 mg, 0.19 mmol), DIPEA (145 μL, 0.83
mmol), and PCl-reagent (61 μL, 0.27 mmol) in anhydrous CH2Cl2 (2
mL) were mixed and reacted as described in the representative
phosphitylation protocol. After it was stirred at room temperature for
3 h, the mixture was diluted with EtOAc (20 mL) and washed with
H2O (2 × 30 mL). The organic phase was dried (Na2SO4) and
evaporated to dryness, and the resulting residue was purified by silica
gel column chromatography (0−70% EtOAc in petroleum ether, v/v)
and subsequently triturated from CH2Cl2 and petroleum ether to
provide 11X (107 mg, 52%) as a white foam: Rf = 0.4 (5% MeOH in
CH2Cl2, v/v); ESI-HRMS m/z 1100.5859 ([M
+
Na]+,
C61H84N5O10P·Na+, calcd 1100.5848); 31P NMR (CDCl3) δ 150.2,
149.9.
(1S,3R,4S,7R)-3-[5-(3-Cholesterylcarbonylaminopropyn-1-yl)-
uracil-1-yl]-7-[2-cyanoethoxy(diisopropylamino)phosphinoxy]-1-
(4,4′-dimethoxytrityloxymethyl)-2,5-dioxabicyclo[2.2.1]heptane
(11Y). Nucleoside 10Y (150 mg, 0.15 mmol), DIPEA (105 μL, 0.59
mmol), and PCl-reagent (50 μL, 0.21 mmol) in anhydrous CH2Cl2
(1.5 mL) were mixed and reacted as described in the representative
phosphitylation protocol. After it was stirred at room temperature for
3 h, the reaction mixture was diluted with EtOAc (20 mL) and washed
with H2O (2 × 30 mL). The organic layer was dried (Na2SO4) and
evaporated to dryness, the resulting residue was purified by silica gel
column chromatography (0−70% EtOAc in petroleum ether, v/v);
subsequent trituration from CH2Cl2 and petroleum ether provided
11Y (102 mg, 57%) as a white foam: Rf = 0.4 (5% MeOH in CH2Cl2,
v/v); ESI-HRMS m/z 1246.6519 ([M + Na]+, C71H94N5O11P·Na+,
calcd 1246.6579); 31P NMR (CDCl3) δ 150.2, 149.9.
Alternative Route to (1S,3R,4S,7R)-1-(4,4′-Dimethoxytrity-
loxymethyl)-7-hydroxy-3-(5-iodoracil-1-yl)-2,5-dioxabicyclo-
[2.2.1]heptane (9). Nucleoside 1236 (200 mg, 0.52 mmol) was
coevaporated with anhydrous pyridine (10 mL) and redissolved in
anhydrous pyridine (10 mL). To this was added 4,4′-dimethoxytrityl
chloride (DMTrCl, 230 mg, 0.68 mmol), and the reaction mixture was
stirred at room temperature for 16 h. At this point, saturated aqueous
NaHCO3 (20 mL) and CH2Cl2 (25 mL) were added and the phases
were separated. The organic phase was washed with saturated aqueous
NaHCO3 (20 mL). The combined aqueous phase was back-extracted
with CH2Cl2 (2 × 20 mL). The combined organic layer was dried
(Na2SO4), concentrated to near dryness, and coevaporated with
toluene/absolute EtOH (2 × 30 mL, 1/2, v/v). The resulting crude
product was purified by silica gel column chromatography (0−4.5%
MeOH in CH2Cl2, v/v) to afford nucleoside 9 (250 mg, 70%) as a
light yellow solid material.
(1R,3R,4S,7R)-7-Hydroxy-1-hydroxymethyl-3-(5-iodoracil-1-
yl)-2,5-dioxabicyclo[2.2.1]heptane (12). To a solution of nucleo-
side 8 (200 mg, 0.78 mmol) in glacial AcOH (10 mL) were added
iodine (119 mg, 0.47 mmol) and ceric ammonium nitrate (213 mg,
0.39 mmol), and the reaction mixture was stirred at 80 °C for 50 min.
After it was cooled to room temperature, the mixture was evaporated
to dryness and the resulting residue purified by silica gel column
chromatography (0−16% MeOH/CH2Cl2, v/v) to afford 1236 (240
mg, 80%) as a white solid material: Rf = 0.4 (15% MeOH in CH2Cl2,
v/v); FAB-HRMS m/z 382.9735 ([M + H]+, C10H11IN2O6·H+, calcd
1
382.9740); H NMR (DMSO-d6) δ 11.76 (s, 1H, ex, NH), 8.09 (s,
1H, H6), 5.88 (d, 1H, ex, J = 4.5 Hz, 3′-OH), 5.84 (s, 1H, H1′), 4.97
(t, 1H, ex, J = 5.4 Hz, 5′-OH), 4.25 (d, 1H, J = 4.5 Hz, H3′), 4.23 (s,
1H, H2′), 3.93−3.96 (d, 1H, J = 8.5 Hz, H5″), 3.78−3.81 (d, 1H, J =
8.5 Hz, H5″), 3.70−3.77 (m, 2H, 2 × H5′); 13C NMR (DMSO-d6) δ
160.5, 149.9, 144.2 (C6), 91.2, 87.1 (C1′), 78.7 (C2′), 72.4 (C3′),
72.0 (C5″), 67.7, 57.5 (C5′).
Synthesis of Oligodeoxyribonucleotides and Biophysical
Characterization Studies. Unmodified DNA and RNA strands were
obtained from commercial suppliers and used without further
purification. L1−4 were prepared and characterized with respect to
identity (MALDI-MS) and purity (>80%, ion-pair reverse-phase
HPLC) in a previous study.11b ONs modified with C5-alkynyl-
functionalized α-L-LNA monomers were synthesized, purified,
structurally characterized, and utilized in biophysical experiments
essentially as described for the corresponding C5-alkynyl-function-
(1S,3R,4S,7R)-7-[2-Cyanoethoxy(diisopropylamino)-
phosphinoxy]-1-(4,4′-dimethoxytrityloxymethyl)-3-[5-(3-trifluoroa-
cetylaminopropyn-1-yl)uracil-1-yl]-2,5-dioxabicyclo[2.2.1]heptane
(11W). Nucleoside 10W (0.25 g 0.35 mmol), DIPEA (0.30 mL, 1.7
mmol), and PCl-reagent (0.10 mL, 0.45 mmol) in anhydrous CH2Cl2
(10 mL) were reacted, worked up, and purified as described in the
representative phosphitylation protocol to provide 11W (0.27 g, 84%)
as a white foam: Rf = 0.5 (2% MeOH in CH2Cl2, v/v); ESI-HRMS m/
z 930.3068 ([M + Na]+, C45H49F3N5O10P·Na+, calcd 930.3080); 31P
NMR (CDCl3) δ 150.2, 149.9.
(1S,3R,4S,7R)-7-[2-Cyanoethoxy(diisopropylamino)-
phosphinoxy]-1-(4,4′-dimethoxytrityloxymethyl)-3-[5-(3-octadeca-
noylaminopropyn-1-yl)uracil-1-yl]-2,5-dioxabicyclo[2.2.1]heptane
J
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alized LNA in the preceding article.14 The following hand-coupling
conditions (coupling time; activator; phosphoramidite solvent) were
used for incorporation of monomers L-Z into ONs: monomers S/V/
W/X (15 min; 0.25 M 4,5-dicyanoimidazole in CH3CN; CH3CN),
monomer Y (15 min; 0.25 M 5-[3,5-bis(trifluoromethyl)phenyl]-1H-
tetrazole37 in CH3CN; CH3CN), and monomer Z (30 min; 0.25 M
4,5-dicyanoimidazole in CH3CN; CH2Cl2).
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ASSOCIATED CONTENT
* Supporting Information
■
S
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Text and figures giving general experimental details, NMR
spectra for all new compounds, ROESY spectra for compound
8, MS data for all new modified ONs, and additional
fluorescence data. This material is available free of charge via
the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail for P.J.H.: hrdlicka@uidaho.edu.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We appreciate financial support from Idaho NSF EPSCoR, the
BANTech Center at the University of Idaho, and Award
Number GM088697 from the National Institute of General
Medical Sciences, National Institutes of Health. A scholarship
from the College of Graduate Studies (M.E.Ø.) is appreciated.
We thank Dr. Alexander Blumenfeld (Department of
Chemistry, University of Idaho), Dr. Gary Knerr (Department
of Chemistry, University of Idaho), and Dr. Lee Deobald (EBI
Murdock Mass Spectrometry Center, University of Idaho) for
NMR and mass spectrometric analyses. We thank Mr. Daniel J.
Raible (University of Idaho) for the synthesis of alkyne labels.
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