Functionalization of 2′-amino-LNA with additional nucleobases

Article abstract of DOI:10.1039/b901028a

Thymin-1-yl-acetic acid and adenin-9-yl-acetic acid have been coupled to the N2′-atom of a 2′-amino-LNA thymine nucleoside, and these "double-headed" LNA monomers have been incorporated into oligodeoxyribonucleotides via their corresponding phosphoramidit

Full text of DOI:10.1039/b901028a

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www.rsc.org/obc | Organic & Biomolecular Chemistry
Functionalization of 2¢-amino-LNA with additional nucleobases†
Tadashi Umemoto, Jesper Wengel and Andreas Stahl Madsen*
Received 16th January 2009, Accepted 16th February 2009
First published as an Advance Article on the web 19th March 2009
DOI: 10.1039/b901028a
Thymin-1-yl-acetic acid and adenin-9-yl-acetic acid have been coupled to the N2¢-atom of a
2¢-amino-LNA thymine nucleoside, and these “double-headed” LNA monomers have been
incorporated into oligodeoxyribonucleotides via their corresponding phosphoramidite derivatives.
Oligonucleotides containing these modified nucleotides show in most cases increased thermal stability
when forming duplexes with complementary DNA, even allowing multiple incorporations.
Incorporation of “double-headed” LNA monomers in both strands also led to stable duplexes,
however, no indication of Watson–Crick base pairing between the extra nucleobases could be found.
Introduction
Results and discussion
One of the most important biological recognition events is hy-
drogen bond formation between nucleobases leading to Watson–
Crick, Hoogsteen or wobble pairings. Such base pairings are
pivotal for central processes like replication and transcription,
and for formation of higher order structures of DNA and RNA.
Modified nucleotides containing an additional nucleobase1–5 are
of interest as the base pairing or stacking involving the additional
nucleobases could be exploited to create novel types of secondary
structures and possibly enhance affinity towards a target. Herein
we report the synthesis and properties of novel N2¢-functionalized
2¢-amino-LNA nucleotides possessing an additional adenine or
thymine nucleobase. The corresponding phenyl derivative was
included in this study as a planar hydrocarbon counterpart to the
natural nucleobases (Fig. 1). Functionalization of the N2¢-atom
of the locked bicyclic 2-oxa-5-azabicyclo[2.2.1]heptane skeleton
of 2¢-amino-LNA (2¢-amino-Locked Nucleic Acid)6 monomers
has previously been shown to present functionalities towards
the minor groove of duplexes formed with complementary
strands.7–11
O5¢-dimethoxytritylated 2¢-amino-LNA thymine nucleoside 112
was used as starting material for the synthesis of phospho-
ramidites 4a–c. Acetic acid derivatives 2a (thymin-1-ylacetic
acid), 2b ((6-N-benzoyladenin-9-yl)acetic acid),13 and 2c (phenyl-
acetic acid) were reacted with nucleoside 1 using 1-ethyl-3-
(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl)
as the condensation reagent to give amides 3a–c in excellent
yields. The 3¢-hydroxy functionality of amides 3a–c was subse-
quently phosphitylated to give phosphoramidite building blocks
4a–c in acceptable yields (Scheme 1), thus establishing a reliable
and straightforward synthetic route towards “double headed”
nucleoside derivatives.
Preparation of the oligonucleotides containing “double-
headed” LNA monomers TLT, TL or TL (Fig. 1) was carried
A
Ph
out by conventional automated DNA synthesis. Functionalized
phosphoramidites 4a–c were coupled in high yield (>95%) using
1H-tetrazole as activator and a coupling time of 20 min. The
synthesized oligonucleotides were purified by RP-HPLC, and
their constitution and purity (>80%) was verified by MALDI-
MS analysis and ion-exchange-HPLC, respectively. The sequences
of the “double-headed” LNA oligonucleotides are depicted in
Tables 1 and 2.
Nucleic Acid Center‡, Department of Physics and Chemistry, University of
Southern Denmark, 5230 Odense M, Denmark. E-mail: asm@ifk.sdu.dk;
Fax: +45 6615 8780; Tel: +45 6550 2599
† Electronic supplementary information (ESI) available: General experi-
mental; MALDI-MS of synthesized ONs (Table S1); full NMR assigment
of 3a–3c; NMR spectra of 3a–3c and 4a–4c. See DOI: 10.1039/b901028a
The influence of “double-headed” LNA monomers on the
thermal stability of DNA duplexes was studied with unmodified
complementary DNA strands (Table 1). No significant stability
difference was observed between the three different modifica-
tions, though it seems that the contribution to duplex stability
follows the trend of TL < TL < TLT, as judged from the
‡ The Nucleic Acid Center is funded by the Danish National Research
Foundation for studies on nucleic acid chemical biology.
A
Ph
Fig. 1 LNA, 2¢-amino-LNA and “double-headed” 2¢-amino-LNAs.
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Table 2 Thermal denaturation studies of duplexes with multiple incor-
porations of “double headed” LNA monomersa
Tm (DTm)/◦C
5¢-GTG AATL TLTGC C
43.0
(+4.5)
T
3¢-CAC TTA ACG G
5¢-GAC GTLTL TLTLTL TLTLTL GCA C
3¢-CTG CAA AAA AAA CGT G
67.5
(+16.0)
5¢-GAC GTLTTL TLATLTTL TLTTLTTL GCA C
62.0
(+10.5)
A
A
A
3¢-CTG CAA AAA AAA CGT G
a For conditions of thermal denaturation experiments see Table 1. TL
=
LNA thymine monomer. For reference duplexes 5¢-GTG AAT TGC C:3¢-
CAC TTA ACG G, Tm = 38.5 ◦C and 5¢-GAC GTT TTT TTT GCA C:
3¢-CTG CAA AAA AAA CGT G, Tm = 51.5 ◦C.
The effect of incorporation of “double-headed” LNA
monomers in both strands of a duplex was evaluated by thermal
denaturation studies using duplexes containing the different
“double-headed” LNA monomers in either downstream or up-
stream constitutions (Table 3, entries 1–3 and 4–6, respectively).
In both constructs, stable duplexes were formed, but the upstream
arrangement invariably led to thermally more stable duplexes than
the downstream arrangement, regardless of N2¢-functionalities.
Furthermore, incorporation of two modifications led to more
than additive increases in the thermal stability of the duplex
(e.g. compare Tm’s of the singly modified duplexes 5¢-GTG AA
Scheme 1 Synthesis of phosphoramidites for “double-headed” LNA.
DMTr = 4,4¢-dimethoxytrityl, T = thymin-1-yl, ABz = 6-N-benzoyl-
adenin-9-yl.
Table 1 Thermal denaturation studies of duplexes with a single incorpo-
ration of a “double-headed” LNA monomera
Tm (DTm)/◦C
B = TL
B = TL
B = TL
T
A
Ph
5¢-GTG AAB AGC C
3¢-CAC TTA TCG G
36.5
(+1.5)
35.0
( 0.0)
37.0
(+2.0)
TL AGC C:3¢-CAC TTA TCG G and 5¢-GTG AA TAGC
T
C:3¢-CAC TTA TLTCG G in Table 1 with Tm of the upstream
5¢-GTG AAT AGC C
3¢-CAC TBA TCG G
34.0
(-1.0)
32.0
(-3.0)
33.0
(-2.0)
arrangement of doubly modified duplex 5¢-GTG AA TL AGC
T
C:3¢-CAC TTA TLTCG G in Table 3). The differences in thermal
5¢-GTG AAT AGC C
3¢-CAC TTA BCG G
38.0
(+3.0)
35.5
(+0.5)
37.0
(+2.0)
stability of the doubly modified duplexes seem to follow the
trend of the singly modified duplexes, with TL being the least
A
stabilizing and TL being the most stabilizing. Accordingly, the
a Thermal denaturation temperatures (Tm values) of duplexes [Tm
values/◦C (DTm = Tm of modified duplex - Tm of unmodified DNA:DNA
duplex)] measured as the maximum of the first derivative of the melting
curve (A260 vs. temperature) recorded in 10 mM phosphate buffer ([Na+] =
110 mM, [Cl-] = 100 mM, [EDTA] = 0.1 mM, pH 7.0) using 1.0 mM
concentrations of the two complementary strands. For reference duplex
5¢-GTG AAT AGC C:3¢-CAC TTA TCG G, Tm = 35.0 ◦C.
T
duplex with an upstream arrangement of two TL monomers is
A
noted as the least stable duplex with an upstream constitution.
This is most likely explained by steric hindrance between the
Table 3 Thermal denaturation studies of duplexes with incorporation of
a “double-headed” LNA monomer in each stranda
thermal denaturation temperatures of the singly modified duplexes
(Table 1). However, the specific positioning of the modification
within the sequence context evidently exhibits a significant
Tm (DTm)/◦C
B = TL
B = TL
B = TL
T
A
Ph
influence on duplex stability (e.g. DTm of TL modified duplexes
T
5¢-GTG AAB AGC C
3¢-CAC TTLTA TCG G
38.0
(+3.0)
37.0
(+2.0)
38.0
(+3.0)
range from -1.0 ◦C to +3.0 ◦C).
Interestingly, multiple incorporations in the same strand were
well tolerated and let to thermally stable duplexes (Table 2). In
particular, only a minor difference between the thermal denatura-
tion temperatures of a duplex containing eight contiguous LNA
thymine monomers (TL) and a duplex containing eight contiguous
“double-headed” LNA monomers is notable. The obtained results
demonstrate that the N2¢-atom of 2¢-amino-LNA monomers is
a highly suitable site for attachment, allowing one or several
additional nucleobases to be oriented towards the minor groove of
thermally stable duplexes. Thus, “double-headed” LNA compares
favorably with previously reported “double-headed” nucleosides,
where single incorporations typically result in considerable desta-
bilization of the duplexes formed and multiple incorporations in
the same strand result in much less stable duplexes.1–5
5¢-GTG AAB AGC C
3¢-CAC TTLAA TCG G
39.0
(+4.0)
39.0
(+4.0)
38.0
(+3.0)
5¢-GTG AAB AGC C
39.5
(+4.5)
37.0
(+2.0)
36.0
(+1.0)
3¢-CAC TTLPhA TCG G
5¢-GTG AAB AGC C
3¢-CAC TTA TLTCG G
45.0
(+10.0)
40.5
(+5.5)
43.5
(+8.5)
5¢-GTG AAB AGC C
3¢-CAC TTA TLACG G
42.0
(+7.0)
39.5
(+4.5)
40.5
(+5.5)
5¢-GTG AAB AGC C
42.0
(+7.0)
40.5
(+5.5)
41.5
(+6.5)
3¢-CAC TTA TLPhCG G
a For conditions of thermal denaturation experiments see Table 1.
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two purine nucleobases, though changes in hydration pattern
in and around the minor groove might also play a role in
the effects observed. The observed changes in thermal affinity
contrast with previous results where any observed stabilizing effect
was attributed to stacking between the additional nucleobases.2,4
However, the variation of attachment site of the additional
nucleobases combined with the conformationally restricted LNA-
skeleton render direct comparison difficult.
Incorporation of both an additional thymine and adenine
nucleobase opens the possibility of additional hydrogen bonding.
The potential of additional hydrogen bond formation between
the two strands of a doubly modified duplex was evaluated by
simple molecular modeling. A model structure of 5¢-GTG AATL
T
AGC C:3¢-CAC TTA TLACG G, containing a thymine and adenine
modified “double-headed” 2¢-amino-LNA monomer in the two
strands, respectively, was constructed in MacroModel V9.1.14 The
duplex structure was subjected to Monte Carlo conformational
analysis, and the generated structures were energy minimized
using the AMBER force field15,16 as implemented in MacroModel
V9.1. Two protocols were employed, either allowing the N2¢-
functionalities to move freely in space or forcing the two extra
nucleobases to be positioned at a distance favoring hydrogen
bonding. Subsequently, the lowest energy structure obtained by
each of the protocols was subjected to a stochastic dynamics
simulation followed by energy minimization of structures taken
evenly distributed along the simulation trajectory. In both sim-
ulation protocols the global duplex conformation is found to
be an intermediate A/B-type duplex. This is in agreement with
previous studies for LNA-modified DNA-type duplexes, which
have shown that the LNA-skeleton is conformationally driving
the local duplex geometry from a B-type towards an intermediate
A/B-type duplex.17 The lowest energy structure obtained with
non-restricted N2¢-functionalities, indicates, in agreement with
previous results, that N2¢-functionalities are positioned in the
minor groove of the duplex. Furthermore, the functionalities are
found to be directed towards the 3¢-end of the appended strand,
without any interaction with each other (Fig. 2). However, forcing
hydrogen bonding between the additional nucleobases indicates
that the two functionalities potentially could reach each other
across the minor groove of the duplex (Fig. 2). Inspection of
the measured Tm values, however, does not give any clear-cut
indication of such inter-strand communication via base-pairing.
Thus, the energy gain associated with base pairing is most likely
to be too small to facilitate the necessary rearrangement of the
N2¢-functionalities.
Fig. 2 Illustrations of the lowest energy structures of doubly modified
duplex 5¢-GTG AA TL AGC C:3¢-CAC TTA TLACG G, without (to the
T
left) and with forced hydrogen bonding of the extra nucleobases, respec-
tively. Coloring scheme: nucleobases: green; sugar–phosphate backbone:
red; N2¢-thymin-1-ylacetyl: light blue; N2¢-adenin-9-ylacetyl: blue.
Experimental
Preparation of “double-headed” LNA phosphoramidites
General procedure for synthesis of 3.
1-(2-Amino-2-deoxy-5-O-4,4¢-dimethoxytrityl-2-N,4-C-methy-
lene-2-N-(thymin-1-ylacetyl)-b-D-ribofuranosyl)thymine
(3a).
A
mixture of 1-(2-amino-2-deoxy-5-O-4,4¢-dimethoxytrityl-
2-N,4-C-methylene-b-D-ribo-furanosyl)thymine12 (1, 286 mg,
0.50 mmol) and thymin-1-ylacetic acid (2a, 110 mg, 0.60 mmol)
was dried by co-evaporation with 1,2-dichloroethane (2 ¥ 2 ml),
and suspended in dry dichloromethane (5 ml). 1-Ethyl-3-(3-
dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl,
◦
115 mg, 0.60 mmol) was added to the suspension at 0 C. After
stirring the reaction mixture for 3 hours water (5 ml) was added.
The reaction mixture was diluted with dichloromethane (50 ml)
and washed with water (2 ¥ 50 ml). The aqueous phase was back
extracted with dichloromethane (50 ml). The combined organic
layer was dried with Na2SO4, then evaporated and the residue was
purified by silica-gel column chromatography (0–8% MeOH in
CH2Cl2 (v/v)) to afford a rotameric mixture (~1:1.5 by 1H NMR)
of nucleoside 3a as a colorless foam (334 mg, 90%). Physical data
for rotameric mixture: Rf = 0.4 (10% MeOH in CH2Cl2, (v/v));
Selected signals 1H NMR (400 MHz, DMSO-d6):18,19 d 11.50 (br
s, 1.5H, ex, N3-HA), 11.39 (br s, 1H, ex, N3-HB), 11.31 (br s, 2.5H,
ex, N3¢¢¢-HA + B), 7.54 (s, 1H, H6B), 7.53 (s, 1.5H, H6A), 7.22–7.47
(m, 25H, H6¢¢¢A + B, ArA + B), 6.09 (d, J = 4.2 Hz, 1.5H, ex, 3¢-OHA),
6.04 (d, J = 4.3 Hz, 1H, ex, 3¢-OHB), 5.63 (s, 1.5H, H1¢A), 5.44 (s,
1H, H1¢B), 4.76 (d, J = 16.7, 1.5H, COCH2,A,a), 4.57 (d, J = 16.7,
1.5H, COCH2,A,b), 4.49 (s, 2H, COCH2,B), 3.75 (s, 15H, OCH3),
1.76 (s, 7.5H, 5¢¢¢-CH3,A + B), 1.51 (s, 4.5H, 5-CH3,A), 1.49 (s, 3H, 5-
CH3,B); Selected signals 13C NMR (101 MHz, DMSO-d6): d 165.9
(COCH2,A), 165.6 (COCH2,B), 142.1 (C6¢¢¢A), 142.0 (C6¢¢¢B), 134.3
(C6B), 134.0 (C6A), 86.2 (C1¢A), 86.0 (C1¢B), 55.0 (OCH3,A + B), 48.3
Conclusion
The efficient synthesis of novel “double-headed” LNA nucleosides
and oligonucleotides thereof has been achieved. Incorporation of
one or more of these novel monomers into DNA strands generally
resulted in thermally very stable duplexes with unmodified DNA
complements. This demonstrates the suitability of the N2¢-atom
of 2¢-amino-LNA monomers as an attachment site for additional
nucleobases oriented towards the minor groove. These “double-
headed” LNA monomers are thus one step forward towards
increasing the information content of synthetic oligonucleotides,
and further structural studies and design optimization is war-
ranted.
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(COCH2,A), 47.9 (COCH2,B), 12.21 (5-CH3,A), 12.18 (5-CH3,B),
11.81 (5¢¢¢-CH3,B), 11.77 (5¢¢¢-CH3,A); HRMS MALDI FT-MS
m/z 760.2556 ([M + Na]+, calcd C39H39N5O10·Na+ 760.2589).
orated, and the resulting residue was purified by silica-gel column
chromatograpy (25% acetone in petroleum ether (v/v)), affording
a rotameric and diastereomeric mixture of phosphoramidite 4a as
a colorless foam (298 mg, 86%). 31P NMR (121 MHz, CDCl3):
d 150.9, 150.4, 150.2, 149.0 ppm. HRMS MALDI FT-MS m/z
(M + Na+) found 960.3646 calc. 960.3668.
1-(2-Amino-2-N-(6-N-benzoyladenin-9-ylacetyl)-3-O-(2-cyano-
ethoxy(diisopropylamino)phosphinoxy)-2-deoxy-5-O-4,4¢-dime-
thoxytrityl-2-N,4-C-methylene-b-D-ribofuranosyl)thymine (4b).
Phosphoramidite 4b was obtained from amide 3a (327 mg,
0.38 mmol) as a colorless foam (339 mg, 85%) in a similar manner
as phosphoramidite 4a except for the column chromatography
(50% acetone in petroleum ether (v/v)). 31P NMR (CDCl3): d
151.2, 150.7, 150.5, 149.1 ppm. HRMS MALDI-FT MS m/z
(M + Na+) found 1073.4035 calc. 1073.4045.
1-(2-Amino-3-O-(2-cyanoethoxy(diisopropylamino)phosphin-
oxy)-2-deoxy-5-O-4,4¢-dimethoxytrityl-2-N,4-C-methylene-2-N-
phenylacetyl-b-D-ribofuranosyl)thymine (4c). Phosphoramidite
4c was obtained from amide 3c (264 mg, 0.38 mmol) as a colorless
foam (220 mg, 65%) in a similar manner as phosphoramidite 4a
except for the column chromatography (50% acetone in petroleum
ether (v/v)). 31P NMR (CDCl3): d 150.8, 150.3, 149.5 ppm. HRMS
MALDI-FT MS m/z (M + Na+) found 912.3680 calc. 912.3708.
1-(2-Amino-2-N-(6-N-benzoyladenin-9-ylacetyl)-2-deoxy-5-O-
4,4¢-dimethoxytrityl-2-N,4-C-methylene-b-D-ribofuranosyl)thym-
ine (3b). A rotameric mixture (~1:1.3 by 1H NMR) of ade-
nine derivative 3b was obtained from nucleoside 112 (286 mg,
0.50 mmol) and N-6-benzoyl(adenin-9-yl)acetic acid13 (2b, 178 mg,
0.60 mmol) as a colorless foam (421 mg, 99%) in a similar manner
as thymine derivative 3a except for the column chromatography
(0–7% MeOH in CH2Cl2 (v/v)). Physical data for rotameric mix-
ture: Rf = 0.5 (10% MeOH in CH2Cl2, (v/v)); 1H NMR (DMSO-
d6):18,19 d 11.56 (br s, 1.3H, ex, NHA/6¢¢¢-NHA), 11.39 (br s, 1H,
ex, NHB/6¢¢¢-NHB), 11.17 (br s, 2.3H, ex, NHA + B/6¢¢¢-NHA + B),
8.72 (s, 2.3H, H2¢¢¢A + B), 8.47 (s, 1.3H, H8¢¢¢A), 8.38 (s, 1H, H8¢¢¢B),
7.53–7.59 (m, 6.9H, H6A + B, ArA + B), 6.18 (d, J = 4.3 Hz, 1.3H,
ex, 3¢-OHA), 6.08 (d, J = 4.3 Hz, 1H, ex, 3¢-OHB), 5.75 (s, 1.3H,
H1¢A), 5.53 (s, 1H, H1¢B), 5.48 (d, J = 17.0 Hz, 1.3H, COCH2,A,a),
5.30 (d, J = 17.0 Hz, 1.3H, COCH2,A,b), 5.28 (d, J = 17.2 Hz,
1H, COCH2,B,a), 5.20 (d, J = 17.2 Hz, 1H, COCH2,B,b), 3.74–
3.77 (m, 15.8H, OCH3,A + B, H5¢¢B), 1.54 (s, 3.9H, 5-CH3,A), 1.51 (s,
3H, 5-CH3,B); 13C NMR (DMSO-d6): d 165.5 (COPhA + B), 165.2
(COCH2,A), 165.1 (COCH2,B), 151.5 (C2¢¢¢A/B), 151.4 (C2¢¢¢A/B),
145.6 (C8¢¢¢A), 145.5 (C8¢¢¢B), 134.3 (C6B), 134.1 (C6A), 86.4 (C1¢A),
86.0 (C1¢B), 55.1 (OCH3,A + B), 44.6 (COCH2,A), 44.3 (COCH2,B),
12.29 (5-CH3,A), 12.26 (5-CH3,B); HRMS MALDI FT-MS m/z
873.2943 ([M + Na]+, calcd C46H42N8O9·Na+ 873.2967).
Synthesis of oligonucleotides containing “double-headed” LNA
Syntheses of modified ONs were performed on 0.2 mmol scale
using an automated DNA synthesizer, using standard conditions
for unmodified phosphoramidites. Incorporation of “double-
headed” 2¢-amino-LNA thymine monomers was carried out by a
manual coupling protocol, using extended coupling time (20 min)
and 1H-tetrazole as activator. Stepwise coupling yields were >99%
for unmodified phosphoramidites and approximately 95% for
modified phosphoramidites. After deprotection and cleavage from
the solid support (32% aq. NH3, 12 h, 55 ◦C), the oligonucleotides
were purified by RP-HPLC, and their purity (>80%) and compo-
sition verified by IE-HPLC and MALDI-MS analysis (Table S1,
see ESI†), respectively.
1-(2-Amino-2-deoxy-5-O-4,4¢-dimethoxytrityl-2-N,4-C-methyl-
ene-2-N-phenylacetyl-b-D-ribofuranosyl)thymine (3c). A rota-
1
meric mixture (~1:1.6 by H NMR) of phenyl derivative 3c was
obtained from nucleoside 1 (286 mg, 0.50 mmol) and phenylacetic
acid (2c, 82 mg, 0.60 mmol) as a colorless foam (303 mg, 88%) in
a similar manner as thymine derivative 3a except for the column
chromatography (0–5% MeOH in CH2Cl2 (v/v)). Rf = 0.6 (10%
MeOH in CH2Cl2, (v/v)); 1H NMR (DMSO-d6):18,19 d 11.50 (br s,
1.5H, ex, NHA), 11.39 (br s, 1H, ex, NHB), 7.57 (s, 1H, H6B),
7.54 (s, 1.5H, H6A), 5.99 (d, J = 4.2 Hz, 1.5H, ex, 3¢-OHA),
5.96 (d, J = 4.1 Hz, 1H, ex, 3¢-OHB), 5.53 (s, 1.5H, H1¢A), 5.40
(s, 1H, H1¢B), 3.77 (s, 3H, COCH2,A), 3.74 (s, 15H, OCH3,A + B),
3.63 (d, J = 15.8 Hz, 1H, COCH2,B,a), 3.59 (d, J = 15.8 Hz,
Thermal denaturation studies
1H, COCH2,B,b), 1.51 (s, 4.5H, 5-CH3,A), 1.47 (s, 3H, 5-CH3,B); 13
C
Melting temperatures (Tm values) were determined as the max-
imum of the first derivative of◦ the melting curve (A260 vs.
temperature; temperature ramp 1 C/min) and were recorded in
10 mM sodium phosphate buffer (100 mM NaCl, 0.1 mM EDTA,
adjusted to pH 7.0 by 10 mM NaH2PO4/5 mM Na2HPO4) using
1.0 mM concentrations of the two complementary strands. The
reported thermal denaturation temperatures are an average of at
least two measurements within 0.5 ◦C.
NMR (DMSO-d6): d 169.3 (COCH2,A), 169.2 (COCH2,B), 134.4
(C6B), 134.1 (C6A), 86.8 (C1¢A), 86.2 (C1¢B), 55.0 (OCH3,A + B), 39.9
(COCH2,A + B, overlap with DMSO-d6), 12.3 (5-CH3,A), 12.2 (5-
CH3,B); HRMS MALDI FT-MS m/z 712.2623 ([M + Na]+, calcd
C40H39N3O8·Na+ 712.2629).
General procedure for synthesis of 4.
1-(2-Amino-3-O-(2-cyanoethoxy(diisopropylamino)phosphin-
oxy)-2-deoxy-5-O-4,4¢-dimethoxytrityl-2-N,4-C-methylene-2-N-
(thymin-1-ylacetyl)-b-D-ribofuranosyl)thymine (4a). Amide 3a
(275 mg, 0.37 mmol) was dried by co-evaporation with dry
dichloroethane, then dissolved in dichloromethane (5 ml). N,N¢-
diisopropylethylamine (129 ml, 0.74 mmol) and O-(2-cyanoethyl)-
N,N¢-diisopropyl-aminophosphorochloridite (123 ml, 0.55 mmol)
were added at 0 ◦C. After stirring at rt for 2 h, absolute
EtOH was added (2 ml). The reaction mixture was diluted with
dichloromethane (50 ml) and washed with phosphate buffer (5 ¥
50 ml, pH 7.0). Organic layer was dried with Na2SO4, then evap-
Molecular modeling
Unmodified duplex 5¢-d(GTG AAT AGC C):3¢-d(CAC TTA
TCG G) was built having standard B-type helical geometry and
subsequently modified to duplex 5¢-d(GTG AATL AGC C):3¢-
T
d(CAC TTA TLACG G) in MacroModel V9.1.14 The charge
of the phosphodiester backbone was neutralized with sodium
ions, which were placed 3.0 A from the non-bridging oxygen
atoms. Monte Carlo conformational search was performed, with
torsional sampling of the N2¢-functionalities (for monomer TL
˚
,
T
1796 | Org. Biomol. Chem., 2009, 7, 1793–1797
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N2¢-C(O) through Ca-N1¢¢ were sampled; for monomer TLA, N2¢-
C(O) through Ca-N9¢¢ were sampled). Where applicable, hydrogen
bonding of additional nucleobases was ensured by restraining
2 T. Wu, K. Nauwelaerts, A. Van Aerschot, M. Froeyen, E. Lescrinier
and P. Herdewijn, J. Org. Chem., 2006, 71, 5423.
3 S. L. Pedersen and P. Nielsen, Org. Biomol. Chem., 2005, 3,
3570.
4 M. S. Christensen, C. M. Madsen and P. Nielsen, Org. Biomol. Chem.,
2007, 5, 1586.
5 C. Andersen, P. K. Sharma, M. S. Christensen, S. I. Steffansen, C. M.
Madsen and P. Nielsen, Org. Biomol. Chem., 2008, 6, 3983.
6 S. K. Singh, R. Kumar and J. Wengel, J. Org. Chem., 1998, 63,
10035.
7 M. D. Sørensen, M. Petersen and J. Wengel, Chem. Commun., 2003,
2130.
˚
˚
N3T–N1A to 2.87 A and 4-OT–6-NA to 2.83 A by a force constant
2
˚
of 100 kJ/molA . Sodium–oxygen distances were restrained by a
2
˚
force constant of 100 kJ/molA , and all atoms except the sugar
and N2¢-functionality moiety of modified monomers TL and
T
TL and the adjacent phosphodiester groups (including O3¢ of
A
the preceding nucleotide and O5¢ of the following nucleotide)
were frozen. 1000 structures were generated and subsequently
minimized using the Polak-Ribiere conjugate gradient method,
the all-atom AMBER force field15,16 and GB/SA solvation model20
as implemented in MacroModel V9.1. Non-bonded interactions
8 P. J. Hrdlicka, B. R. Babu, M. D. Sørensen and J. Wengel, Chem.
Commun., 2004, 1478.
9 P. J. Hrdlicka, B. R. Babu, M. D. Sørensen, N. Harrit and J. Wengel, J.
Am. Chem. Soc., 2005, 127, 13293.
10 M. Kalek, A. S. Madsen and J. Wengel, J. Am. Chem. Soc., 2007, 129,
˚
were treated with extended cut-offs (van der Waals 8.0 A and
9392.
˚
electrostatics 20.0 A). The lowest energy structures obtained
11 D. Lindegaard, A. S. Madsen, I. V. Astakhova, A. D. Malakhov, B.
R. Babu, V. A. Korshun and J. Wengel, Bioorg. Med. Chem., 2008,
94.
12 B. R. Babu, D. Lindegaard, T. S. Kumar, I. V. Astakhova, A. D.
Malakov, M. D. Sørensen, V. A. Korshun, P. J. Hrdlicka and J. Wengel,
in preparation.
13 T. Kofoed, H. F. Hansen, H. Ørum and T. Koch, J. Peptide. Sci., 2001,
7, 402.
14 MacroModel, version 9.1, Schro¨dinger, LLC, New York, NY, 2005.
15 S. J. Weiner, P. A. Kollman, D. A. Case, U. C. Singh, C. Ghio, G.
Alagona, S. Profeta and P. J. Weiner, J. Am. Chem. Soc., 1984, 106, 765.
16 S. J. Weiner, P. A. Kollman, D. T. Nguyen and D. A. Case, J. Comput.
Chem., 1986, 7, 230.
were then subjected to 3 ns of stochastic dynamics (simulation
temperature 300 K, time step 2.2 fs, SHAKE all bonds to
hydrogen), during which 300 structures were collected evenly
and subsequently minimized. During dynamics and the following
minimization all atoms were allowed to move freely, but the
previously applied atom-to-atom restraints were kept intact.
Acknowledgements
We thank the Danish National Research Foundation and the
Villum Kann Rasmussen Foundation for financial support.
17 K. E. Nielsen, S. K. Singh, J. Wengel and J. P. Jacobsen, Bioconjugate
Chem., 2000, 11, 228.
18 The integral of the H-1¢ signal of the least predominant rotamer (termed
B) is set to 1.0.
Notes and references
19 For full assignment, see ESI†.
1 T. Wu, M. Froeyen, G. Schepers, K. Mullens, J. Rozenski, R. Busson,
A. Van Aerschot and P. Herdewijn, Org. Lett., 2004, 6, 51.
20 W. C. Still, A. Tempczyk, R. C. Hawley and T. Hendrickson, J. Am.
Chem. Soc., 1990, 112, 6127.
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The Royal Society of Chemistry 2009
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