Functionalized 2-amino-α-L-LNA: Directed positioning of intercalators for DNA targeting

Article abstract of DOI:10.1021/jo802037v

Chemically modified oligonucleotides are increasingly applied in nucleic acid based therapeutics and diagnostics. LNA (locked nucleic acid) and its diastereomer α-L-LNA are two promising examples thereof that exhibit increased thermal and enzymatic stabil

Full text of DOI:10.1021/jo802037v

Functionalized 2′-Amino-r-L-LNA: Directed Positioning of
Intercalators for DNA Targeting
T. Santhosh Kumar,† Andreas S. Madsen,† Michael E. Østergaard,‡ Sujay P. Sau,‡
Jesper Wengel,† and Patrick J. Hrdlicka*,‡
Nucleic Acid Center, Department of Physics and Chemistry, UniVersity of Southern Denmark,
DK-5230 Odense M, Denmark, and Department of Chemistry, UniVersity of Idaho,
Moscow, Idaho 83844-2343
hrdlicka@uidaho.edu
ReceiVed September 18, 2008
Chemically modified oligonucleotides are increasingly applied in nucleic acid based therapeutics and
diagnostics. LNA (locked nucleic acid) and its diastereomer R-L-LNA are two promising examples thereof
that exhibit increased thermal and enzymatic stability. Herein, the synthesis, biophysical characterization,
and molecular modeling of N2′-functionalized 2′-amino-R-L-LNA is described. Chemoselective N2′-
functionalization of protected amino alcohol 1 followed by phosphitylation afforded a structurally varied
set of target phosphoramidites, which were incorporated into oligodeoxyribonucleotides. Incorporation
of pyrene-functionalized building blocks such as 2′-N-(pyren-1-yl)carbonyl-2′-amino-R-L-LNA (monomer
X) led to extraordinary increases in thermal affinity of up to +19.5 °C per modification against DNA
targets in particular. In contrast, incorporation of building blocks with small nonaromatic N2′-functionalities
such as 2′-N-acetyl-2′-amino-R-L-LNA (monomer V) had detrimental effects on thermal affinity toward
DNA/RNA complements with decreases of as much as -16.5 °C per modification. Extensive thermal
DNA selectivity, favorable entropic contributions upon duplex formation, hybridization-induced batho-
chromic shifts of pyrene absorption maxima and increases in circular dichroism signal intensity, and
molecular modeling studies suggest that pyrene-functionalized 2′-amino-R-L-LNA monomers W-Y having
short linkers between the bicyclic skeleton and the pyrene moiety allow high-affinity hybridization with
DNA complements and precise positioning of intercalators in nucleic acid duplexes. This rigorous positional
control has been utilized for the development of probes for emerging therapeutic and diagnostic applications
focusing on DNA targeting.
Introduction
assembling biomaterials.3 Chemical modification of oligonucle-
otides is often required to provide adequate protection from
enzymatic degradation, to facilitate strong binding to comple-
mentary nucleic acid targets, and to add functionality to
oligonucleotides. Incorporation of conformationally restricted
nucleotide monomers into oligonucleotides is a popular approach
toward this end.4,5 Locked nucleic acid (LNA, ꢀ-D-ribo con-
Oligonucleotides are widely used for modulation of gene
expression (e.g., antigene/antisense/siRNA),1 for detection of
nucleic acid targets,2 and as building blocks of novel self-
† University of Southern Denmark.
‡ University of Idaho.
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10.1021/jo802037v CCC: $40.75 2009 American Chemical Society
Published on Web 12/24/2008

Functionalized 2′-Amino-R-L-LNA Phosphoramidites
antisense ONs,16-18 triplex-forming ONs,19 modified
DNAzymes,20 and transcription factor decoy ONs.21
We have previously taken advantage of the known high-
affinity hybridizations of 2′-amino-LNA6,22 (Figure 1) to develop
a series of N2′-functionalized 2′-amino-LNAs, which precisely
position functional entities in the minor groove of nucleic acid
duplexes without compromising duplex stability.23 This has
resulted in the development of tools for applications within
therapeutics, diagnostics, and material science, including (a)
probes yielding brightly fluorescent duplexes upon hybridization
to DNA/RNA targets with quantum yields approaching unity,23d,k
(b)probesforsinglenucleotidepolymorphism(SNP)detection,23b,h
(c) nucleic acid architectures autosignaling their self-assembly,23b,h
and (d) artificial dinuclear ribonucleases.23j
Stimulated by these findings, we recently developed a
synthetic route to 2′-amino-R-L-LNA (R-L-ribo configuration,
monomer Q, Figure 1)6,24 and N2′-functionalized analogs
thereof (Figure 1). Appended functional entities were anticipated
to be positioned in the major groove of nucleic acid duplexes.24a
However, initial studies with N2′-pyrene-functionalized 2′-
amino-R-L-LNA suggest that the conjugated functional entity
is directed toward the duplex core instead.25,26 This has already
resulted in the development of promising tools for DNA
targeting,25a detection of single nucleotide polymorphisms,25b
and nucleic acid structural engineering.25d
FIGURE 1. Structures of LNA, 2′-amino-LNA, R-L-LNA (monomer
O), and 2′-amino-R-L-LNA monomers Q-Z.
figuration, Figure 1) is a very promising member of this class
of compounds. LNA6-8 exhibits increases in thermal affinity
toward DNA/RNA complements of up to +10 °C per modifica-
tion along with markedly improved enzymatic stability relative
to that of unmodified oligodeoxyribonucleotides (ONs).9,10
These properties render LNA with pronounced therapeutic and
diagnostic potential,11-14 which is underlined by ongoing phase
I/II clinical evaluations of LNA drug candidates against a variety
of diseases. One of the diastereoisomers of LNA, i.e.,
R-L-LNA6,15 (R-L-ribo configuration, monomer O, Figure 1),
shares the beneficial properties of LNA and has been used as
Herein, full experimental details on the synthesis of a
structurally varied set of N2′-functionalized 2′-amino-R-L-LNA
phosphoramidites and their incorporation into ONs are presented
(Figure 1). Results from biophysical and computational studies
(5) For recent representative examples, see: (a) Morita, K.; Takagi, M.;
Hasegawa, C.; Kaneko, M.; Tsutsumi, S.; Sone, J.; Ishikawa, T.; Imanishi, T.;
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M.; Nielsen, P. J. Org. Chem. 2006, 71, 7731–7740. (c) Honcharenko, D.;
Vargese, O. P.; Plashkevych, O.; Barman, J.; Chattopadhyaya, J. J. Org. Chem.
2006, 71, 299–314. (d) Varghese, O. P.; Barman, J.; Pathmasiri, W.; Plaskevych,
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Nielsen, P. J. Org. Chem. 2008, 73, 6891–6894.
(6) We define LNA, R-L-LNA, 2′-amino-LNA, and 2′-amino-R-L-LNA as
a oligonucleotide containing one or more 2′-O,4′-C-methylene-ꢀ-D-ribofuranosyl
monomer(s), 2′-O,4′-C-methylene-R-L-ribofuranosyl monomer(s), 2′-amino-2′-
deoxy-2′-N,4′-C-methylene-ꢀ-D-ribofuranosyl monomer(s), or 2′-amino-2′-deoxy-
2′-N,4′-C-methylene-R-L-ribofuranosyl monomer(s), respectively. Similar defi-
nitions are used for N2′-functionalized R-L-LNA derivatives.
(7) (a) Singh, S. K.; Nielsen, P.; Koshkin, A. A.; Wengel, J. Chem. Commun.
1998, 455–456. (b) Koshkin, A. A.; Singh, S. K.; Nielsen, P.; Rajwanshi, V. K.;
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3630. (c) Wengel, J. Acc. Chem. Res. 1999, 32, 301–310.
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C. A.; Westergaard, M.; Hansen, H. F.; Ørum, H.; Koch, T. Nucleic Acids Res.
2003, 31, 6365–6372.
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J. ChemBioChem 2007, 8, 2240–2248. (i) Lindegaard, D.; Madsen, A. S.;
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(2008), New Orleans, BIOL-032.
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J.; Hrdlicka, P. J. J. Org. Chem. 2006, 71, 4188–4201.
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4279–4281. (b) Kumar, T. S.; Wengel, J.; Hrdlicka, P. J. ChemBioChem 2007,
8, 1122–1125. (c) Andersen, N. K.; Wengel, J.; Hrdlicka, P. J. Nucleosides
Nucleotides Nucleic Acids 2007, 26, 1415–1417. (d) Kumar, T. S.; Madsen, A. S.;
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(8) Obika, S.; Nanbu, D.; Hari, Y.; Andoh, J.; Morio, K.; Doi, T.; Imanishi,
T. Tetrahedron Lett. 1998, 39, 5401–5404.
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(12) Frieden, M.; Ørum, H. IDrugs 2006, 9, 706–711.
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J. Org. Chem. Vol. 74, No. 3, 2009 1071

Kumar et al.
SCHEME 1. Synthesis of N2′-Functionalized 2′-Amino-r-L-LNA Phosphoramiditesa
a DMTr ) 4,4′-dimethoxytrityl, T ) thymin-1-yl, Py ) pyren-1-yl, EDC ·HCl ) 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride, HATU
) O-(7-azabenzotriazole-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate.
are discussed together with the suggested binding mode of the
appended functional entities.
to 90%. Disappearance of 1H NMR signals of the exchangeable
3′-OH protons upon D2O addition ascertained the N2′-func-
tionalized constitution of nucleosides 2S-2Z, which subse-
quently were converted to the corresponding phosphoramidites
3S-3Z using 2-cyanoethyl N,N′-(diisopropyl)-phosphorami-
dochloridite and Hu¨nig’s base. While amidites 3S-3Y were
obtained in good to excellent yields (60-90%), 3Z was only
obtained in 36% yield. The yield of 3X was improved using
bis(N,N-diisopropylamino)-2-cyanoethoxyphosphine in dichlo-
romethane with diisopropylammonium tetrazolide28 as activator
(71%).
Synthesis of ONs was performed in 0.2 µmol scale using an
automated DNA synthesizer. The corresponding phosphora-
midites for incorporation of R-L-LNA thymine monomer O
(obtained from commercial sources) and 2′-amino-R-L-LNA
thymine monomer Q (synthesized via a known protocol)24b were
incorporated into our preferred model system, i.e., a set of mixed
sequence 9-mer ONs, as previously described.15b,24b Standard
procedures were applied for incorporation of N2′-functionalized
2′-amino-R-L-LNA thymine monomers S-Z (Figure 1) except
for extended coupling times: 3S (10 min), 3V (10 min), 3W25a
(30 min), 3Y25b (30 min), 3X (30 min), and 3Z (15 min)) using
1H-tetrazole as catalyst resulting in stepwise coupling yields
of ∼99% for monomers S, V, X, Y, and Z and ∼95% for
monomer W (Figure 1 for structures). Following standard
workup and purification, the composition and purity (>80%)
of all modified ONs was verified by MALDI-MS (Table S3,
Supporting Information)29 and ion-exchange HPLC, respec-
tively. Please note that the unmodified reference DNA and RNA
strands are denoted D1/D2 and R1/R2, respectively, and ONs
Results and Discussion
Synthesis of N2′-Functionalized 2′-Amino-r-L-LNA.
Known O5′-tritylated bicyclic nucleoside 1,24b which is obtained
from commercially available diacetone-R-D-glucose in 5%
overall yield over 17 steps involving eight chromatographic
purification steps, was used as a suitable starting material for
the synthesis of N2′-functionalized 2′-amino-R-L-LNA phos-
phoramidites 3Q-3Z (Scheme 1). The targets were selected to
probe the available structural space in nucleic acid duplexes
and fall into two groups based on the nature of the N2′-moiety,
i.e., monomers with small nonaromatic units (monomers Q, S,
and V) or with aromatic units (monomers W-Z). Sodium
triacetoxyborohydride mediated reductive amination27 of sec-
ondary amine 1 with acetaldehyde or 1-pyrenecarbaldehyde
furnished tertiary amines 2S and 2W25a in 48% and 67% yield,
respectively. Chemoselective N-acylation of amino alcohol 1
was achieved using two different strategies. Treatment of
nucleoside 1 with slight excess of acetic anhydride followed
by selective O3′-deacylation using dilute methanolic ammonia
furnished nucleoside 2V in excellent 88% yield over two steps.
EDC-mediated coupling of amino alcohol 1 with 1-pyrenylcar-
boxylic acid, 1-pyrenylacetic acid, or 4-(1-pyrenyl)butyric acid
afforded nucleosides 2X, 2Y,25b,d and 2Z in 62%, 86%, and
63% yield, respectively. A HATU (O-(7-azabenzotriazole-1-
yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate)-medi-
ated coupling procedure successfully improved the yield of 2X
(27) Abdel-Magid, A. F.; Carson, K. G.; Harris, B. D.; Maryanoff, C. A.;
Shah, R. D. J. Org. Chem. 1996, 61, 3849–3862.
(28) Pedersen, D. S.; Rosenbohm, C.; Koch, T. Synthesis 2002, 802–808.
(29) See Supporting Information.
1072 J. Org. Chem. Vol. 74, No. 3, 2009

Functionalized 2′-Amino-R-L-LNA Phosphoramidites
TABLE 1. Thermal Denaturation Data for N2′-Functionalized 2′-Amino-r-L-LNA and Reference Strands against DNA Complementsa
Tm [∆Tm/mod] (°C)
ON
duplex
B )
T
O
Q
S
V
W
X
Y
Z
B1 5′-GBG ATA TGC
D2 3′-CAC TAT ACG
28.5 31.0 [+2.5] 26.5 [-2.0] 20.0 [-8.5] 17.5 [-11.0] 35.5 [+7.0] 38.5 [+10.0] 39.0 [+10.5] 29.0 [+0.5]
28.5 34.5 [+6.0] 29.0 [+0.5] 20.0 [-8.5] 16.5 [-12.0] 42.5 [+14.0] 47.5 [+19.0] 44.0 [+15.5] 34.5 [+6.0]
28.5 31.5 [+3.0] 27.5 [-1.0] 16.5 [-12.0] 14.5 [-14.0] 39.0 [+10.5] 42.5 [+14.0] 40.0 [+11.5] ND
28.5 32.0 [+3.5] 28.0 [-0.5] 17.0 [-11.5] 12.0 [-16.5] 35.0 [+6.5] 39.0 [+10.5] 38.5 [+10.0] 29.0 [+0.5]
28.5 36.5 [+8.0] 31.0 [+2.5] 22.5 [-6.0] 19.0 [-9.5] 44.0 [+15.5] 48.0 [+19.5] 45.0 [+16.5] 35.0 [+6.5]
B2 5′-GTG ABA TGC
D2 3′-CAC TAT ACG
B3 5′-GTG ATA BGC
D2 3′-CAC TAT ACG
D1 5′-GTG ATA TGC
B4 3′-CAC BAT ACG
D1 5′-GTG ATA TGC
B5 3′-CAC TAB ACG
D1 5′-GTG ATA TGC
B6 3′-CAC BAB ACG
28.5 36.0 [+3.8] 27.5 [-0.5] <10
28.5 36.0 [+2.5] 27.0 [-0.5] <10
<10
<10
ND
ND
53.5 [+12.5] 55.5 [+13.5] ND
ND 69.0 [+13.5] ND
B7 5′-GBG ABA BGC
D2 3′-CAC TAT ACG
a Thermal denaturation temperatures [Tm values in °C (∆Tm ) change in Tm value calculated relative to D1:D2, D1:R2, and R1:D2 reference
duplexes)] measured as the maximum of the first derivative of the melting curve (A260 vs temperature) recorded in medium salt buffer ([Na+] ) 110
mM, [Cl-] ) 100 mM, pH 7.0 (NaH2PO4/Na2HPO4)), using 1.0 µM concentrations of the two complementary strands. Tm values are averages of at least
two measurements; A ) adenin-9-yl DNA monomer, C ) cytosin-1-yl DNA monomer, G ) guanin-9-yl DNA monomer, T ) thymin-1-yl DNA
monomer, O ) R-L-LNA thymin-1-yl monomer (Figure 1). For structures of monomers O-Z see Figure 1. ND ) not determined.
TABLE 2. Thermal Denaturation Data for N2′-Functionalized 2′-Amino-r-L-LNA and Reference Strands against RNA Complementsa
Tm [∆Tm/mod] (°C)
ON
duplex
B )
T
O
Q
S
V
W
X
Y
Z
B1 5′-GBG ATA TGC
R2 3′-CAC UAU ACG
26.5 32.5 [+6.0] 27.5 [+1.0] 19.5 [-7.0] 19.0 [-7.5] 27.0 [+0.5] 32.5 [+6.0] 32.5 [+6.0] 26.5 [(0]
26.5 35.0 [+8.5] 28.5 [+2.0] 18.5 [-8.0] 17.0 [-9.5] 31.5 [+5.0] 36.5 [+10.0] 36.0 [+9.5] 33.5 [+7.0]
26.5 32.0 [+5.5] 28.0 [+1.5] 19.0 [-7.5] 16.5 [-10.0] 28.0 [+1.5] 33.5 [+7.0] 32.5 [+6.0] ND
24.5 31.0 [+6.5] 27.5 [+3.0] 19.5 [-5.0] 17.5 [-7.0] 23.5 [-1.0] 29.0 [+4.5] 30.5 [+6.0] 19.5 [-5.0]
24.5 34.5 [+10.0] 29.0 [+4.5] 21.5 [-3.0] 20.5 [-4.0] 32.0 [+7.5] 36.0 [+11.5] 36.5 [+12.0] 31.0 [+6.5]
B2 5′-GTG ABA TGC
R2 3′-CAC UAU ACG
B3 5′-GTG ATA BGC
R2 3′-CAC UAU ACG
R1 5′-GUG AUA UGC
B4 3′-CAC BAT ACG
R1 5′-GUG AUA UGC
B5 3′-CAC TAB ACG
R1 5′-GUG AUA UGC
B6 3′-CAC BAB ACG
24.5 35.5 [+5.5] 28.5 [+2.0] <10
26.5 44.0 [+5.8] 31.0 <10
<10
<10
ND
ND
39.0 [+7.3] 44.0 [+9.8] ND
ND 52.5 [+8.7] ND
B7 5′-GBG ABA BGC
D2 3′-CAC UAU ACG
a For conditions of thermal denaturation experiments see Table 1.
containing a single incorporation of a modified nucleotide in
the 5′-GBG ATA TGC context are named O1, Q1, S1, etc.
Similar conventions were used for ONs in the B2-B7 series
(Tables 1 and 2). In addition, the following descriptive
nomenclature is used: R-L-amino-LNA (Q-series), Et-R-L-
amino-LNA (S-series), Ac-R-L-amino-LNA (V-series), PyMe-
R-L-amino-LNA (W-series), PyCO-R-L-amino-LNA (X-series),
PyAc-R-L-amino-LNA (Y-series), and PyBu-R-L-amino-LNA
(Z-series).
Thermal Denaturation Studies. Experimental Setup. The
effect upon incorporation of one to three O-Z monomers
(Figure 1) into mixed sequence 9-mer ONs on thermal affinity
toward DNA and RNA targets (Tables 1 and 2, respectively)
was evaluated by UV thermal denaturation experiments using
medium salt buffer ([Na+] ) 110 mM) and compared to
unmodified DNA. The UV thermal denaturation curves of all
modified duplexes exhibited sigmoidal monophasic transitions
with hyperchromicities (9-15%) that are comparable to the
corresponding unmodified DNA:DNA or DNA:RNA duplexes
(Figure S1, Supporting Information).29 All changes in thermal
denaturation temperatures (Tm) of modified nucleic acid duplexes
are discussed relative to Tm values of unmodified reference
duplexesunlessotherwisementioned.Inaddition,theWatson-Crick
specificity of ONs with a single central incorporation of
monomer O-Z (B2-series) was evaluated by determining Tm
values of the duplexes with DNA/RNA strands with central
mismatches (Table 3).
Thermal Denaturation Studies. r-L-LNA and 2′-Amino-r-
L-LNA. Reference ONs. R-L-LNAs O1-O5 exhibit substan-
tially increased thermal affinity toward DNA (∆Tm up to +8.0
°C, Table 1) and RNA complements (∆Tm ) up to +10.0 °C,
Table 2). The corresponding 2′-amino-R-L-LNAs Q1-Q5
display notably smaller increases in thermal affinity (∆Tm up
to +2.5 °C with DNA, Table 1; ∆Tm up to +4.5 °C with RNA,
Table 2). Similar ∆Tm values for duplexes between R-L-amino-
LNA Q4 and DNA/RNA complements determined at different
ionic strengths were observed (Table S4, Supporting Informa-
tion),29 suggesting that the 2-oxo-5-azabicyclo[2.2.1]heptane
J. Org. Chem. Vol. 74, No. 3, 2009 1073

Kumar et al.
TABLE 3. Discrimination of Mismatched DNA/RNA Targets by N2′-Functionalized 2′-Amino-r-L-LNA
Tm [∆Tm] (°C)
DNA: 3′-CAC TBT ACG
RNA: 3′-CAC UBU ACG
ON
sequence
B )
A
C
G
T
A
C
G
U
D1
O2
Q2
5′-GTG ATA TGC
5′-GTG AOA TGC
5′-GTG AQA TGC
28.5 12.0 [-16.5]
34.5 13.0 [-21.5]
19.0 [-9.5]
11.5 [-17.0]
26.5 <10 [<-16.5] 22.0 [-4.5]
35.0 14.0 [-21.0] 28.5 [-6.5]
<10 [<-16.5]
14.5 [-20.5]
<10 [<-18.5]
<10 [<-17.0]
<10 [<-14.5]
27.0 [-4.5]
21.5 [-13.0] 14.5 [-20.0]
29.0 <10 [<-19.0] 17.5 [-11.5] <10 [<-19.0] 28.5 <10 [<-18.5] 22.5 [-6.0]
28.5 <10 [<-18.5] 17.0 [-11.5] <10 [<-18.5] 27.0 <10 [<-17.0] 25.5 [-1.5]
24.5 <10 [<-14.5] 14.5 [-10.0] <10 [<-14.5] 24.5 <10 [<-14.5] 28.0 [+3.5]
42.5 30.0 [-12.5]
47.5 31.0 [-16.5]
44.0 26.0 [-18.0]
34.5 17.0 [-17.5]
S2b 5′-GTG ASA TGC
V2b 5′-GTG AVA TGC
W2 5′-GTG AWA TGC
37.0 [-5.5]
40.0 [-7.5]
38.5 [-5.5]
39.0 [-3.5]
40.5 [-7.0]
34.0 [-10.0]
31.5 19.5 [-12.0]
36.5 21.0 [-15.5]
36.0 14.5 [-21.5]
30.5 [-1.0]
33.5 [-3.0]
33.0 [-3.0]
X2
Y2
Z2
5′-GTG AXA TGC
5′-GTG AYA TGC
5′-GTG AZA TGC
28.5 [-8.0]
25.0 [-11.0]
20.0 [-14.5] 20.0 [-14.5]
33.5 <10 [<-22.5] 17.5 [-16.0] <10 [<-22.5]
a For conditions of thermal denaturation experiments see Table 1. Tm values of fully matched duplexes are shown in bold. ∆Tm ) change in Tm value
relative to fully matched DNA:DNA or DNA:RNA duplex. b Tm values for duplexes involving S2 and V2 are measured using high salt buffer ([Na+] )
710 mM, [Cl-] ) 100 mM, pH 7.0 (NaH2PO4/Na2HPO4)).
skeleton of monomer Q is not protonated at physiological pH.
The increased binding affinity of R-L-LNA and 2′-amino-R-L-
LNA was accompanied by improved discrimination of singly
mismatched DNA and RNA targets relative to unmodified DNA
D1 (e.g., ∆Tm values for R-L-amino-LNA Q2 and D1 against
DNA mismatches, Table 3).
Information), suggesting that short linkers between the pyrene
and nucleoside moieties facilitate DNA selectivity. Although
PyMe/PyCO-R-L-amino-LNAs exhibit a similar degree of DNA
selectivity as acyclic intercalating nucleic acids (INAs),30 2′-
O-pyrenylmethyl uridines,31 or pyrene-functionalized 4′-C-
piperazinomethyl thymidines,32 they generally form stronger
duplexes with DNA targets, which renders them as highly
interesting probes for DNA-targeting applications.25a
Centrally modified PyMe-R-L-amino-LNAs W2 exhibit less
efficient discrimination of mismatched DNA/RNA targets than
the corresponding reference strand D1 (Table 3). Interestingly,
a change in linker chemistry from methylene to carbonyl (W
f X) results in higher affinity toward DNA/RNA complements
as well as significantly improved mismatch discrimination (Table
3). With the exception of T:T/U mismatches, PyCO-R-L-amino-
LNA X2 displays mismatch discrimination comparable to that
of reference strand D1. Increases in linker length result in
progressively improved discrimination of DNA/RNA mis-
matches (compare ∆Tm data for X2, Y2, and Z2, Table 3).
Accordingly, PyBu-R-L-amino-LNA Z2 exhibits superior dis-
crimination of RNA mismatches in general and of the chal-
lenging T:rG mismatch in particular (∆Tm ) -16.0 °C, data
for Z2, Table 3), relative to the already highly discriminative
R-L-LNA O2.
Thermal Denaturation Studies. N2′-Ethyl/Acetyl-Modified
2′-Amino-r-L-LNA. Et-R-L-amino-LNAs S1-S7 exhibit greatly
decreased thermal affinities toward DNA/RNA targets in general
and complementary DNA in particular (∆Tm/mod down to
-12.0 °C, Table 1). These effects are even more pronounced
with Ac-R-L-amino-LNAs V1-V7, which exhibit decreases in
Tm values down to -16.5 °C per modification (Table 1).
Accordingly, no duplex transitions could be observed for ONs
with two or three incorporations of S or V monomers and their
DNA/RNA targets. Interestingly, the large decreases in thermal
affinity toward DNA/RNA complements of Et/Ac-R-L-amino-
LNA generally did not compromise Watson-Crick specificity
(see data for S2 and V2, Table 3), which suggests that base-
pairing is preserved.
Thermal Denaturation Studies. N2′-Pyrene-Functionalized
2′-Amino-r-L-LNA. Incorporation of a single PyMe/PyCO/
PyAc-R-L-amino-LNA monomer W, X or Y, respectively, into
ONs resulted in extraordinary increases in thermal affinity
toward DNA complements (∆Tm from +6.5 to +19.5 °C, Table
1). Moderate increases were observed upon incorporation of
PyBu-R-L-amino-LNA monomer Z (∆Tm up to +6.5 °C, Table
1). The observed trends in thermal affinity of singly modified
strands toward DNA targets (X > Y > W . Z) suggest that
(a) alkanoyl linkers are thermally preferred over alkyl linkers
of the same length (X > W), and (b) shorter linkers are
thermally preferred (X > Y . Z). For a discussion on the
sequence-dependent variations of Tm values observed for these
ONs, the reader is directed to Supporting Information.29
Interestingly, additive increases in thermal affinity toward DNA
targets are observed upon multiple incorporations of PyAc-R-
L-amino-LNA Y monomers (e.g., compare Tm/mod values of
Y6:D1, Y4:D1, and Y5:D1, Table 1), whereas subadditive
increases are observed for the corresponding R-L-LNA O6, 2′-
amino-R-L-LNA Q6, or PyMe-R-L-amino-LNA X6. Thus,
PyAc-R-L-amino-LNA monomer Y lends itself as the building
block of choice for applications necessitating densely function-
alized ONs with maximal thermal affinity toward DNA targets.
PyCO/PyAc-R-L-amino-LNAs (X1-X6 and Y1-Y7, respec-
tively) exhibit prominent and additive increases in thermal
affinity toward RNA complements (∆Tm from +4.5 to +12.0
°C, Table 2). In contrast, minor destabilizations to moderate
increases were observed for PyMe/PyBu-R-L-amino-LNAs W1-
W5 and Z1-Z5, respectively, with the exception of Z4, which
exhibited a very pronounced decrease in thermal affinity toward
its RNA target (Table 2).
Accordingly, N2′-pyrene-functionalized 2′-amino-R-L-LNA
exhibit a marked DNA selectivity, i.e., a positive ∆∆Tm/mod
(DNA-RNA) ) ∆Tm/mod (DNA) - ∆Tm/mod (RNA). This is
particularly noteworthy as the parent R-L-LNA and 2′-amino-
R-L-LNA exhibit moderate RNA selectivity (∆∆Tm/mod (DNA-
RNA) ) -3.5 to -1.7 °C, Tables 1 and 2 or, more conveniently,
Table S5, Supporting Information29). PyMe/PyCO-R-L-amino-
LNAs exhibit the most pronounced DNA selectivity (∆∆Tm/
mod (DNA-RNA) ) +6.0 to +9.0 °C, Table S5, Supporting
It is noteworthy that an exchange of a centrally positioned
PyCO-R-L-amino-LNA monomer X with a corresponding Ac-
(30) (a) Christensen, U. B.; Pedersen, E. B. Nucleic Acids Res. 2002, 30,
4918–4925. (b) Filichev, V. V.; Hilmy, K. M. H.; Christensen, U. B.; Pedersen,
U. B. Tetrahedron Lett. 2004, 45, 4907–4910.
(31) Yamana, K.; Iwase, R.; Furutani, S.; Tsuchida, H.; Zako, H.; Yamaoka,
T.; Murakami, A. Nucleic Acids Res. 1999, 27, 2387–2392.
(32) Bryld, T.; Højland, T.; Wengel, J. Chem. Commun. 2004, 1064–1065.
1074 J. Org. Chem. Vol. 74, No. 3, 2009

Functionalized 2′-Amino-R-L-LNA Phosphoramidites
TABLE 4. Energetics Derived from Thermal Denaturation Curves of Duplexes between 2′-Amino-r-L-LNA Functionalized with Nonaromatic
Moieties and DNA/RNAa
complementary DNA
complementary RNA
∆G298 [∆∆G298
]
∆H [∆∆H]
(kJ/mol)
T298∆S [∆(T298∆S)]
(kJ/mol)
∆G298 [∆∆G298
]
∆H [∆∆H]
(kJ/mol)
T298∆S [∆(T298∆S)]
(kJ/mol)
ON
sequence
(kJ/mol)
(kJ/mol)
D1
O2
Q2
S2
5′-GTG ATATGC
5′-GTGAOATGC
5′-GTGAQATGC
5′-GTG ASATGC
5′-GTGAVATGC
-41
-327
-286
-38
-275
-237
-46 [-5]
-41 [0]
-33 [+8]
-29[+12]
-354[-27]
-312[+15]
-349[-22]
-295[+32]
-308[-22]
-271[+15]
-316[-30]
-266[+20]
-46[-8]
-40[-2]
-31[+7]
-30[+8]
-361[-86]
-302[-27]
-352[-77]
-303[-28]
-315[-78]
-261[-24]
-321[-84]
-273[-36]
V2
a Thermal denaturation curves were obtained as described in Tables 1 and 2. ∆∆G298, ∆∆H, and ∆(T298∆S), change in ∆G298, ∆H and (T298∆S)
values, respectively, calculated relative to D1:D2 and D1:R2 reference duplexes). Values in italics indicate deviation from (or lack of) monomer trend.
ND ) not determined. See Table S6 in Supporting Information for data from B1-B5 series.
R-L-amino-LNA monomer V (i.e., a formal change of pyrene
to methyl) was accompanied by a decrease in thermal affinity
toward complementary DNA of 31.0 °C (compare Tm values
of X2:D2 and V2:D2, Table 1). This suggests very different
binding modes for ONs modified with monomers S/V and W/Y,
respectively, which was underlined upon additional biophysical
characterization (vide infra).
Additional Biophysical Characterization of N2′-Functional-
ized 2′-Amino-r-L-LNA. Experimental Setup. To obtain ad-
ditional insight into the highly divergent thermal affinities of
N2′-functionalized 2′-amino-R-L-LNA, the following biophysi-
cal studies were performed: (a) determination of thermodynamic
parameters for duplex formation, (b) CD spectra, (c) UV-vis
spectra (shifts of pyrene absorption maxima), and (d) molecular
modeling studies.
Thermodynamic parameters for duplex formation were de-
termined by melting curve analysis assuming bimolecular
reactions and two-state equilibrium hypothesis. Quality of the
baseline permitting, thermodynamic parameters for two melting
curves per investigated duplex were determined, and an average
value is listed. In full agreement with expectations, formation
of all studied duplexes was favorable (∆G298 < 0 kJ/mol), with
favorable enthalpic (∆H < 0 kJ/mol) and unfavorable entropic
contributions (T298∆S < 0 kJ/mol). The thermodynamic data
rely on assumptions of two-state melting behavior and a heat
capacity change ∆Cp ) 0 upon hybridization, which may not
necessarily be fulfilled. However, apart from few exceptions
(see footnote a in Table 4), the observed enthalpic/entropic
contributions for hybridization of N2′-functionalized 2′-amino-
R-L-LNA to DNA/RNA targets clearly followed monomer- and
sequence-specific trends, which validates the utilized approach
(data shown for the representative B2-series in Tables 4 and 5;
for data and full discussion of B1-B5 series see Table S6,
Supporting Information29).
Duplexes between N2′-functionalized 2′-amino-R-L-LNA and
DNA/RNA complements were studied by force field simula-
tions. For this, DNA duplexes were built in silico and modified
with an N2′-functionalized 2′-amino-R-L-LNA monomer. A
starting B-type helix geometry was chosen as duplexes between
R-L-LNA and DNA complements adopt helix geometries that
are globally unperturbed relative to unmodified DNA:DNA
duplexes.33 ONs with centrally positioned modifications (B2-
series) were selected for the simulations to minimize the
influence of fraying on the helix geometry near the modified
nucleotides. The position of N2′-functionalities was explored
using a truncated Monte Carlo search,29 and the partially
constrained duplexes were subjected to stochastic dynamics
simulations using the all-atom AMBER force field34 and GB/
SA solvation model35 as implemented in the MacroModel V9.1
suite of programs.36
Integrated Structural Discussion. r-L-LNA and 2′-Amino-
r-L-LNA. Reference ONs. The markedly increased thermal
affinity of R-L-LNAs O1-O5 toward DNA/RNA relative to
unmodified ONs results from a more favorable enthalpic term
that largely is counterbalanced by an unfavorable entropic term,
i.e., ∆∆G298(O2DNA) ) ∆G298(O2:D2) - ∆G298(D1:D2) ) -5
kJ/mol; ∆∆H(O2DNA) ) ∆H(O2:D2) - ∆H(D1:D2) ) -27
kJ/mol; ∆(T298∆S) (O2DNA) ) T298∆S (O2:D2) - T298∆S (D1:
D2) ) -22 kJ/mol (Table 4). This suggests that high thermal
affinity of the conformationally restricted R-L-LNAs O1-O5
toward DNA/RNA complements is a result of a more favorable
stacking/hydrogen bonding geometry and/or duplex solvation
rather than preorganization of the single stranded probe.
Similarly, the hybridization of 2′-amino-R-L-LNAs Q1-Q5
to complementary RNA is also driven by favorable enthalpy
that is partially counterbalanced by unfavorable entropy,
although the individual contributions are less pronounced than
for R-L-LNAs O1-O5 (e.g., ∆∆H ) -86 and -27 kJ/mol for
O2RNA and Q2RNA, respectively, Table 4). The energetics for
hybridization of Q1-Q5 to DNA complements are sequence-
dependent and could not be fitted to a clear pattern (Tables 4
and S6, Supporting Information29).
The applied molecular modeling protocol successfully re-
produced expected global and local features of R-L-LNA duplex
O2:D2 (Figure S4, Supporting Information), providing cred-
ibility to the applied computational protocol. These features of
O2:D2 include a standard B-type global duplex geometry similar
to that of D1:D2 (Figure S3, Supporting Information) and very
characteristic local perturbations in the backbone needed to
accommodate the inverted configurations at the C2′-, C3′- and
C4′-positions of R-L-LNA monomer O.29,33 Interestingly, the
global helix structures of O2:D2 and Q2:D2 (Figure S5,
Supporting Information)29 are virtually identical, which is
validated by very similar circular dichroism spectra of Q7:D2
and O7:D2 (Figure S2, Supporting Information). Thus, different
solvation patterns rather than substantially altered helical
geometries likely account for the diverging energetics observed
(34) (a) Weiner, S. J.; Kollman, P. A.; Case, D. A.; Singh, U. C.; Ghio, C.;
Alagona, G.; Profeta, S.; Weiner, P. J. Am. Chem. Soc. 1984, 106, 765–784. (b)
Weiner, S. J.; Kollman, P. A.; Nguyen, D. T.; Case, D. A. J. Comput. Chem.
1986, 7, 230–252.
(35) Still, W. C.; Tempczyk, A.; Hawley, R. C.; Hendrickson, T. J. Am.
Chem. Soc. 1990, 112, 6127–6129.
(36) MacroModel, version 9.1, S; LLC: New York, NY, 2005.
(33) Nielsen, K. M. E.; Petersen, M.; Håkansson, A. E.; Wengel, J.; Jacobsen,
J. P. Chem. Eur. J. 2002, 8, 3001–3009.
J. Org. Chem. Vol. 74, No. 3, 2009 1075

Kumar et al.
FIGURE 4. Nucleotide numbering for duplexes studied by molecular
modeling; B ) thymidine (D1) or monomers O-Z (O2-Z2).
monomer Q,29 accounts for the unfavorable entropy observed
upon hybridization of Et-R-L-amino-LNA S2 with DNA/RNA
targets (Table 4). In a related manner, one part of the N2′-acetyl
moiety of monomer V (i.e., either the -CO- or -CH3) in V2:D2
is directed toward the major groove where it can interfere with
structural water, while the other part simultaneously protrudes
into the duplex core to disrupt π-π stacking (Figures 3 and
S7, Supporting Information).29
FIGURE 2. Circular dichroism spectra of D1:D2, S2:D2, and V2:D2.
To sum up, biophysical characterization and computer
simulations jointly suggest that N2′-functionalization of 2′-
amino-R-L-LNA, contrary to preliminary expectations,24a is not
suitable to position small nonaromatic moieties in the major
groove of duplexes with DNA or RNA complements.
Integrated Structural Discussion. N2′-Pyrene-Functional-
ized 2′-Amino-r-L-LNA. The very pronounced stabilization of
duplexes between PyMe-R-L-amino-LNA or PyAc-R-L-amino-
LNA and complementary DNA (e.g., ∆∆G298(W2DNA) ) -12
kJ/mol) results from highly favorable entropy (e.g.,
∆∆(T298∆S)(W2DNA) ) +32 kJ/mol, Table 5). Stabilization of
duplexes between PyCO-R-L-amino-LNA and DNA targets
(e.g., ∆∆G298(X2DNA) ) -18 kJ/mol) is to a greater extent
driven by favorable enthalpic factors. The moderate stabilization
of duplexes between PyBu-R-L-amino-LNA and DNA comple-
ments (e.g., ∆∆G298(Z2DNA) ) -5 kJ/mol) originates from
favorable enthalpy contributions that mostly were counterbal-
anced by unfavorable entropy components (∆∆H(Z2DNA) ) -9
kJ/mol, ∆(T298∆S)(Z2DNA) ) -4 kJ/mol, Table 5).
FIGURE 3. Side view representations of the lowest energy structure
of S2:D2 (left) and V2:D2 (right). For clarity, hydrogen atoms, sodium
ions and bond orders have been omitted. Coloring scheme: nucleobases,
green; sugar-phosphate backbone, red; ethyl moiety of monomer S
and acetyl moiety of monomer V, blue.
The observed stabilization of duplexes between PyMe/PyCO/
PyAc-R-L-amino-LNA with RNA complements results from
favorable enthalpy (∆∆H ) -53, -68, and -58 kJ/mol for
W2RNA, X2RNA, and Y2RNA, respectively). However, comparison
with 2′-amino-R-L-LNA reference strands instead of unmodified
DNA suggests that entropic contributions also aid duplex
formation with RNA targets (e.g., ∆(T298∆S) ) -17, -28, -17,
for these closely related ONs upon hybridization with DNA/
RNA complements.
Integrated Structural Discussion. N2′-Ethyl/Acetyl-Modi-
fied 2′-Amino-r-L-LNA. The dramatically destabilized duplexes
between Et/Ac-R-L-amino-LNAs and DNA/RNA targets
(∆∆G298 up to +12 kJ/mol) result from unfavorable entropic
components that are only partially counterbalanced by favorable
enthalpic components (e.g., ∆(T298∆S)(S2RNA) ) -84 kJ/mol
and ∆∆H(S2RNA) ) -77 kJ/mol, Table 4).
Intriguingly, very similar CD spectra are observed for S2:
D2, V2:D2, and the reference duplex D1:D2 suggesting that
incorporation of S and V monomers renders these duplexes
globally unperturbed while dramatically lowering stability
(Figure 2). In accordance with this, the lowest energy structures
of Et-R-L-amino-LNA duplex S2:D2 (Figures 3 and S6,
Supporting Information) and Ac-R-L-amino-LNA duplex V2:
D2 (Figures 3 and S7, Supporting Information) globally
resembled each other and R-L-amino-LNA duplex Q2:D2
(Figure S5, Supporting Information).29 In S2:D2 the ethyl group
of S5 protrudes from the major groove valley to become involved
in a steric clash with H5′ of A6 (for numbering scheme see
Figure 4). We speculate that unfavorable desolvation of the
apolar ethyl moiety (whereby fewer water molecules are
released) and interference with structural water along the sugar
-phosphate backbone,37,38 in a similar manner as proposed for
and -51 kJ/mol for W3RNA, X3RNA, Y3RNA, and Q3RNA
,
respectively, Table S6, Supporting Information).29
Thus, energetics suggest that N2′-pyrene-functionalized 2′-
amino-R-L-LNAs exhibit binding modes that rely on preorga-
nization, unlike 2′-amino-R-L-LNA modified with nonaromatic
moieties.
The pronounced DNA selectivity of PyMe/PyCO/PyAc-R-
L-amino-LNA suggests intercalation of the pyrene moieties as
a likely binding mode.30-32,39 The CD spectra of PyAc-R-L-
amino-LNA and duplexes with DNA/RNA support this hypoth-
esis as induced CD bands in the region of pyrene absorption (λ
) 320-360 nm, Figure 5), a feature indicative of intercalation,40
are observed upon hybridization. In addition, marked batho-
chromic shifts of pyrene absorption maxima of ONs containing
monomers W-Y upon hybridization with DNA/RNA targets
(37) Egli, M.; Tereshko, V.; Teplova, M.; Minasov, G.; Joachimiak, A.;
Sanishvili, R.; Weeks, C. M.; Miller, R.; Maier, M. A.; An, H.; Cook, P. D.;
Manoharan, M. Biopolymers 1998, 48, 234–252.
(38) Kielkopf, C. L.; Ding, S.; Kuhn, P.; Rees, D. C. J. Mol. Biol. 2000,
296, 787–801.
1076 J. Org. Chem. Vol. 74, No. 3, 2009

Functionalized 2′-Amino-R-L-LNA Phosphoramidites
TABLE 5. Energetics Derived from Thermal Denaturation Curves of Duplexes between N2′-Pyrene-Functionalized 2′-Amino-r-L-LNA and
DNA/RNAa
complementary DNA
complementary RNA
∆G298 [∆∆G298
]
∆H [∆∆H]
(kJ/mol)
T298∆S [∆(T298∆S)]
(kJ/mol)
∆G298 [∆∆G298
]
∆H [∆∆H]
(kJ/mol)
T298∆S [∆(T298∆S)]
(kJ/mol)
ON
sequence
(kJ/mol)
(kJ/mol)
D1
W2
X2
Y2
Z2
5′-GTG ATATGC
5′-GTGAWATGC
5′-GTG AXATGC
5′-GTG AYATGC
5′-GTG AZATGC
-41
-327
-286
-38
-275
-237
-53[-12]
-59[-18]
-54[-13]
-46 [-5]
-307[+20]
-348[-21]
-321 [+6]
-336 [-9]
-254[+32]
-289 [-3]
-267[+19]
-290 [-4]
-44 [-6]
-49[-11]
-48[-10]
ND
-328[-53]
-343[-68]
-333[-58]
ND
-284[-47]
-294[-57]
-285[-48]
ND
a See Table 4 for conditions.
FIGURE 5. Circular dichroism of spectra of Y7 (5′-GYG AYA YGC) and its duplexes with DNA/RNA complements and reference DNA:DNA
(D1:D2) and DNA:RNA (D1:R2).
TABLE 6. Pyrene Absorption Maxima for Single-Stranded Pyrene-Functionalized 2′-Amino-r-L-LNA and the Corresponding Duplexes with
DNA/RNA Complementsa
λmax [∆λmax] (nm)
B )
W
X
Y
Z
ON
sequence
ss
+DNA
+RNA
ss
+DNA
+RNA
ss
+DNA
+RNA
ss
+DNA
+RNA
B1 5′-GBG ATA TGC
B2 5′-GTG ABA TGC
B3 5′-GTG ATA BGC
B4 3′-CAC BAT ACG
B5 3′-CAC TAB ACG
348 350 [+2] 349 [+1] 349 351 [+2] 351 [+2] 350 351 [+1] 352 [+2] 346 348 [+2] 348 [+2]
347 351 [+4] 349 [+2] 348 353 [+5] 351 [+3] 346 351 [+5] 351 [+5] 346 347 [+1] 346 [(0]
348 351 [+3] 350 [+2] 350 354 [+4] 351 [+1] 348 351 [+3] 351 [+3] ND ND
ND
348 350 [+2] 348 [(0] 348 351 [+3] 349 [+1] 350 351 [+1] 351 [+1] 346 347 [+1] 346 [(0]
348 350 [+2] 349 [+1] 348 351 [+3] 351 [+3] 346 351 [+5] 352 [+6] 346 348 [+2] 347 [+1]
a Measurements were performed at room temperature on a spectrophotometer in the 300-400 nm range, using a quartz optical cell with a 1.0 cm path
length and the same conditions as for thermal denaturation experiments.
FIGURE 6. Absorption spectra of W2 (left panel) and Y2 (right panel) and their duplexes with complementary DNA (D2) and RNA (R2) targets.
(∆λmax ) 1-5 and 0-6 nm, respectively, Table 6) along with
hypochromic shifts (illustrated for W2 and Y2, Figure 6) suggest
strong electronic interactions between the pyrene and nucleobase
moieties in duplexes.40-42 A change in linker chemistry from
alkyl to alkanoyl (PyMe-R-L-amino-LNA W f PyCO-R-L-
amino-LNA X) resulted in small but consistently larger hybrid-
J. Org. Chem. Vol. 74, No. 3, 2009 1077

Kumar et al.
adjacent A6 moiety increase markedly in response to intercala-
tion (P from 97° to 127° and ꢁ from -147° to -105°, for Q2:
D2 and W2:D2, respectively, Tables S7 and S8, Supporting
Information),29 to facilitate efficient π-π stacking between the
pyrene and nucleobase moieties.
The lowest energy structures of duplexes between PyCO-R-
L-amino-LNA X2 or PyAc-R-L-amino-LNA Y2 and comple-
mentary DNA D2 (Figures 8 and S8 and S9, Supporting
Information)29 exhibited similar key features as W2:D2, i.e.,
intercalation of the pyrene moiety and efficient π-π overlapping
with flanking base pairs, similar sugar puckers and glycosidic
torsion angles for X5/Y5 and A6, and unwinding of the duplex
and widening of the grooves.29 Two minor structural differences
observed with PyAc-R-L-amino-LNA duplex Y2:D2 (Figures
8 and S9, Supporting Information) relative to W2:D2 (Figure
7) or X2:D2 (Figures 8 and S8, Supporting Information)
included increased stacking interactions with T13 and A14 and
an altered orientation of the pyrene moiety, i.e., H3py and H4py
of monomer Y face the major groove while facing the minor
groove in W2:D2 and X2:D2 (Figure S10, Supporting Informa-
tion).29
FIGURE 7. Three representations of the lowest energy structure of
W2:D2; side view (left), top view (upper right), and truncated top view
showing W5A6:T13A14 (lower right). Coloring scheme as in Figure 3
except that pyren-1-yl-methyl moiety of monomer W is in blue.
The binding mode of the pyrene moiety of PyBu-R-L-amino-
LNA was expected to be more ambiguous because (a) Z1-Z5
exhibited lower increases in thermal affinity toward DNA
complements in particular (Table 1), (b) Z2 displayed markedly
improved mismatch discrimination relative to PyMe/PyCO/
PyAc-R-L-amino-LNA (Table 3), and (c) more subtle hybrid-
ization-induced bathochromic shifts of pyrene absorption maxima
were observed (Table 6). In full agreement with these biophysi-
cal observations, molecular modeling suggested at least two
different binding modes. An intercalated binding mode was
observed that exhibited the hallmarks described above for W2-
Y2:D2 (Figure S11, Supporting Information). The model
structure suggested that the long and relatively bulky butanoyl
linker of PyBu-R-L-amino-LNA monomer Z (a) reduced π-π
overlap between the pyrene and the nucleobase moieties of Z5
and A6 to a minimum, while increasing overlap with T13 and
A14, (b) was wedged into the duplex core in between Z5 and A6
to locally perturb the duplex and introduce a kink, and (c)
oriented the pyrene moiety with the H3pyr and H4pyr sides facing
the major groove (Figure S10, Supporting Information).29
The second binding mode is more in line with biophysical
observations; the pyrene moiety is located at the floor of the
major groove and is involved in nonspecific contacts with the
Hoogsteen faces of A6, C11, A12, and T13 (Figures 8 and S12,
Supporting Information).29 Minor groove binders conjugated to
ONs are known to increase the strength and specificity of
hybridization.45,46 By analogy, major groove binding of the
pyrene moiety of monomer Z may explain the observed
increased mismatch discrimination of Z2 (Table 3). Thus,
biophysical characterization and computer simulations indicate
that PyBu-R-L-LNA may stabilize duplexes with DNA/RNA
complements by a wider variety of binding modes than PyMe/
PyCO/PyAc-R-L-LNA exhibiting shorter linkers between the
bicyclic skeleton and pyrene moiety.
ization-induced bathochromic shifts, and further extension of
the alkanoyl linker (PyCO-R-L-amino-LNA X f PyAc-R-L-
amino-LNA Y f PyBu-R-L-amino-LNA Z) progressively
reversed this trend. The very subtle hybridization-induced
bathochromic shifts of pyrene absorption maxima observed with
PyBu-R-L-amino-LNA indicate a nonintercalating binding mode
of the pyrene moiety of monomer Z (Table 6).
In full agreement with biophysical data, the lowest energy
structure of the duplex between PyMe-R-L-amino-LNA W2 and
complementary DNA D2 suggests precise intercalation of the
pyrene moiety (Figure 7). It is imperative to stress that the
utilized simulation protocol did not initiate from a structure
where the pyrene moiety was intercalated, i.e., the pyrene moiety
moved from an extrahelical to an intercalated position during
the simulation. As expected43 significant global unwinding,
concomitant lengthening of the duplex, and widening of the
minor groove was observed upon intercalation. The pyrene
moiety forms extensive π-π stacks with the nucleobase
moieties of W5 and the 3′-flanking A6 and to a lesser extent
with the nucleobase moieties of T13 and A14. The pseudorota-
tional phase angle P and glycosidic torsion angle ꢁ44 of PyMe-
R-L-amino-LNA monomer W change little relative to the 2′-
amino-R-L-LNA monomer in Q2:D2. However, P and ꢁ of the
(39) (a) Korshun, V. A.; Stetsenko, D. A.; Gait, M. J. J. Chem. Soc. Perkin
Trans. 1 2002, 1092–1104. (b) Dioubankova, N. N.; Malakhov, A. D.; Stetsenko,
D. A.; Gait, M. J.; Volynsky, P. E.; Efremov, R. G.; Korshun, V. A.
ChemBioChem 2003, 4, 841–847. (c) Kalra, N.; Babu, B. R.; Parmar, V. S.;
Wengel, J. Org. Biomol. Chem. 2004, 2, 2885–2887. (d) Donho, C.; Saito, I.
ChemBioChem 2005, 6, 1075–1081.
(40) Nakamura, M.; Fukunaga, Y.; Sasa, K.; Ohtoshi, Y.; Kanaori, K.;
Hayashi, H.; Nakano, H.; Yamana, K. Nucleic Acids Res. 2005, 33, 5887–5895.
(41) Dougherty, G.; Pilbrow, J. R. Int. J. Biochem. 1984, 16, 1179–1192.
(42) Okamoto, A.; Kanatani, K.; Saito, I. J. Am. Chem. Soc. 2004, 126, 4820–
4827.
(43) (a) Wang, A. H.-J.; Ughetto, G.; Quigley, G. J.; Rich, A. Biochemistry
1987, 26, 1152–1163. (b) Spielmann, H. P.; Wemmer, D. E.; Jacobsen, J. P.
Biochemistry 1995, 34, 8542–8553. (c) Gallego, J.; Reid, B. R. Biochemistry
1999, 38, 15104–15115. (d) Mukherjee, A.; Lavery, R.; Bagchi, B.; Hynes, J. T.
J. Am. Chem. Soc. 2008, 130, 9747–9755.
Closer scrutiny of the molecular arrangement in PyMe/PyCO/
PyAc-R-L-amino-LNA monomers W-Y reveals that the at-
(44) The following definitions of torsion angles are used: ꢁ (O4′-C1′-N1-
C2 or O4′-C1′-N9-C4 for pyrimidines or purines, respectively). The pseu-
dorotation phase angle P is given as tan P ) (ν4 + ν1- ν3 + ν0)/[2ν2(sin 36° +
sin 72°)], where ν0 (C4′-O4′-C1′-C2′), ν1 (O4′-C1′-C2′-C3′), ν2 (C1′-
C2′-C3′-C4′), ν3 (C2′-C3′-C4′-O4′), and ν4 (C3′-C4′-O4′-C1′). For
further information see: Saenger, W. Principles of Nucleic Acid Structure;
Springer-Verlag: Berlin, 1984.
(45) Afonina, I.; Kutyavin, I.; Lukhtanov, E.; Meyer, R. B.; Gamper, H. Proc.
Natl. Acad. Sci. U.S.A. 1996, 93, 3199–3204.
(46) Kutyavin, I. V.; Afonina, I. A.; Mills, A.; Gorn, V. V.; Lukhtanov, E. A.;
Belousov, E. S.; Singer, M. J.; Walburger, D. K.; Lokhov, S. G.; Gall, A. A.;
Dempcy, R.; Reed, M. W.; Meyer, R. B.; Hedgpeth, J. Nucleic Acids Res. 2000,
28, 655–661.
1078 J. Org. Chem. Vol. 74, No. 3, 2009

Functionalized 2′-Amino-R-L-LNA Phosphoramidites
FIGURE 8. Side view representations of the lowest energy structures of X2:D2, Y2:D2, and Z2:D2 (nonintercalated binding mode), respectively.
Coloring scheme as in Figure 3 except that pyrene moieties are shown in blue.
FIGURE 9. Illustration of directed positioning of pyrene moieties in the duplex core by N2′-functionalized 2′-amino-R-L-LNA.
tachment points of the nucleobase and pyrene moieties (i.e.,
C1′ and N2′, respectively) are efficiently locked relative to
each other (Figure 9) as a consequence of the 2-oxo-5-
azabicyclo[2.2.1]heptane skeleton. This, in concert with the short
linker between the bicyclic skeleton and pyrene moiety and the
strength of π-π stacking in aqueous environments, de facto
directs the pyrene moiety of monomers W-Y into the duplex
core to facilitate intercalation. This molecular arrangement leads
to π-π stacking with the T5:A14 and A6:T13 base pairs and a
reduction in buckle and propeller twist fluctuation in this
nucleotide step (results not shown) to form a highly stablilized
duplex segment. The observed thermodynamic data for PyMe/
PyCO/PyAc-R-L-amino-LNA are in agreement with this pre-
organized binding mode of the pyrene as favorable entropic
components were identified as important factors for duplex
stabilization (Table 5). Desolvation upon intercalation of the
highly apolar pyrene moiety is also likely to result in additional
favorable entropic contributions upon duplex formation. Since
the observed modeling structures of W2:D2, X2:D2, and Y2:
D2 are very similar, it is likely that differential solvation of the
single-stranded probes or of their duplexes with DNA/RNA
complements accounts for the observed differences in thermal
affinity toward nucleic acid targets. For example, less pro-
nounced desolvation of PyCO-R-L-amino-LNA X2 relative to
PyMe-R-L-amino-LNA W2 upon hybridization to complemen-
tary DNA may account for less favorable entropy (fewer water
molecules free upon hybridization) and more favorable enthalpy
(formation of hydrogen bonds with surrounding water mol-
ecules).
to +19.5 °C per modification are observed. Directed positioning
of intercalators inside nucleic acid duplex cores has many
potential interesting applications within nucleic acid based
diagnostics, therapeutics, and nanotechnology,47 including
detection of DNA/RNA complements and/or single nucleotide
polymorphisms by fluorescence,25b,30a,48-50 study of charge
transfer processes,51 positioning of metal ions within nucleic
acid duplex cores,52,53 or development of artificial nucleases.54
Unlike previously reported building blocks, N2′-intercalator-
functionalized 2′-amino-R-L-LNA effectively combines high-
affinity hybridization with DNA complements and precise
positioning of intercalators in nucleic acid duplexes. We propose
N2′-intercalator-modified 2′-amino-R-L-LNA monomers as
highly valuable monomers for established and emerging DNA-
targeting applications.
Experimental Section
(1S,3R,4S,7R)-1-(4,4′-Dimethoxytrityloxymethyl)-5-ethyl-7-hy-
droxy-3-(thymin-1-yl)-2-oxa-5-azabicyclo[2.2.1]heptane, 2S. Amino
alcohol 1 (0.40 g, 0.70 mmol) was coevaporated with anhydrous
1,2-dichloroethane (2 × 8 mL) and dissolved in anhydrous 1,2-
dichloroethane (8 mL). To this were added NaBH(OAc)3 (230 mg,
1.09 mmol) and CH3CHO (44 µL, 0.78 mmol), and after the
reaction mixture was stirred at room temperature for 40 h, it was
diluted with EtOAc (35 mL) and washed with saturated aqueous
NaHCO3 (2 × 15 mL). The organic phase was evaporated to
¨
(47) Persil, O.; Hud, N. V. Trends Biotechnol. 2007, 25, 433–436.
(48) Wilson, J. N.; Kool, E. T. Org. Biomol. Chem. 2006, 4, 4265–4274.
(49) Ko¨hler, O.; Jarikote, D. V.; Seitz, O. ChemBioChem 2005, 6, 69–77.
(50) Benvin, A. L.; Creeger, Y.; Fisher, G. W.; Ballou, B.; Wagonner, A. S.;
Armitage, B. A. J. Am. Chem. Soc. 2007, 129, 2025–2034.
(51) Shao, F.; Augustyn, K.; Barton, J. K. J. Am. Chem. Soc. 2005, 127,
17445–17452.
Conclusion
Herein we demonstrate that the 2-oxo-5-azabicyclo-
[2.2.1]heptane skeleton of 2′-amino-R-L-LNA, in concert with
short linkers, directs intercalators appended to the N2′-position
very effectively to the nucleic acid cores. Consequently, dramatic
increases in thermal affinity toward DNA complements of up
(52) Clever, G. H.; Kaul, C.; Carell, T. Angew. Chem., Int. Ed. 2007, 46,
6226–6236.
(53) Tanaka, K.; Shionoya, M. Coord. Chem. ReV. 2007, 251, 2732–2742.
(54) Nakano, S-I.; Uotani, Y.; Uenishi, K.; Fujii, M.; Sugimoto, N. J. Am.
Chem. Soc. 2005, 127, 518–519.
J. Org. Chem. Vol. 74, No. 3, 2009 1079

Kumar et al.
dryness, and the resulting residue was purified by silica gel column
chromatography (0-5% i-PrOH in CH2Cl2, v/v) to afford nucleoside
2S (200 mg, 48%). Rf ) 0.5 (10% MeOH in CH2Cl2, v/v); MALDI-
HRMS m/z 622.2524 ([M + Na]+, C34H37N3O7Na+ calcd 622.2522);
1H NMR (DMSO-d6)55 δ 11.26 (s, 1H, ex, NH), 7.49 (s, 1H, H-6),
7.21-7.44 (m, 9H, Ar), 6.87-6.92 (d, 4H, J ) 8.8 Hz, Ar), 5.91
(d, 1H, J ) 1.5 Hz, H-1′), 5.70 (d, 1H, ex, J ) 3.5 Hz, 3′-OH),
4.31 (d, 1H, J ) 3.5 Hz, H-3′), 3.74 (s, 6H, 2 × CH3O), 3.19-3.30
(m, 4H, H-2′, 2 × H-5′, H-5′′), 2.64-2.83 (m, 3H, CH2CH3, H-5′′),
1.83 (s, 3H, CH3Ar), 0.82 (t, 3H, J ) 7.3 Hz, CH3). 13C NMR
(DMSO-d6) δ 163.8, 158.0, 150.3, 144.8, 137.4, 135.4, 135.3, 129.7,
127.8, 127.6, 126.6, 113.1, 105.9, 90.0, 85.0, 74.4, 65.4, 60.8, 58.3,
54.9, 49.5, 14.8, 12.2. Anal. Calcd for C34H37N3O7: C, 68.10; H,
6.22; N, 7.01. Found: C, 67.96; H, 6.37; N, 6.54. Calcd with 1/8
i-PrOH: C, 68.00; H, 6.31; N, 6.92.
(70% EtOAc in petroleum ether, v/v); MALDI-HRMS m/z
822.3636 ([M + Na]+, C43H54N5O8P · Na+ calcd 822.3602); 31P
NMR (CH3CN + DMSO-d6) δ 149.8, 147.9.
(1S,3R,4S,7R)-7-[2-Cyanoethoxy(diisopropylamino)phosphinoxy]-
1-(4,4′-dimethoxytrityloxymethyl)-5-(pyren-1-yl)carbonyl-3-(thymin-
1-yl)-2-oxa-5-azabicyclo[2.2.1]heptane, 3X. Nucleoside 2X (0.31 g,
0.39 mmol) was dried by coevaporation with anhydrous 1,2-
dichloroethane (2 × 5 mL) and dissolved in anhydrous CH2Cl2
(10 mL). To this were added N,N′-diisopropylammonium tetrazolide
(112 mg, 0.66 mmol) and bis(N,N′-diisopropylamino)-2-cyanoet-
hoxyphosphine (0.21 mL, 0.66 mmol), and the reaction mixture
was stirred at room temperature for 12 h, whereupon it was diluted
with CH2Cl2 (20 mL) and washed with saturated aqueous NaHCO3
(10 mL) and brine (10 mL). The aqueous phase was back-extracted
with CH2Cl2 (30 mL), the combined organic phase was evaporated
to dryness, and the resulting residue was purified by silica gel
column chromatography (0-50% acetone in petroleum ether, v/v)
to afford amidite 3X as a white solid material (276 mg, 71%). Rf
) 0.5 (50% acetone in petroleum ether, v/v); MALDI-HRMS m/z
1022.3864 ([M + Na]+, C58H58N5O9PNa+ calcd 1022.3852; 31P
NMR (CH3CN + DMSO-d6) δ 154.2, 153.8, 153.3, 151.9.
Protocol for Synthesis of ONs. ONs containing 2′-amino-R-L-
LNA monomers Q-Z (see Figure 1 for structures) were synthesized
on a 0.2 µmol scale using succinyl-linked LCAA-CPG (long chain
alkyl amine controlled pore glass) columns with a pore size of 500
Å on an automated DNA synthesizer. Synthesis of R-L-LNA and
2′-amino-R-L-LNA was performed as previously described.15b,24b
For the incorporation of the N2′-functionalized 2′-amino-R-L-LNA
monomers (S-Z), standard procedures were used, i.e., trichloro-
acetic acid in CH2Cl2 as a detrytilation reagent; 0.25 M 4,5-
dicyanoimidazole (DCI) in CH3CN as activator; acetic anhydride
in THF as cap A solution; 1-methylimidazole in THF as cap B
solution, and 0.02 M iodine in H2O/pyridine/THF as the oxidizing
solution. Extended coupling times used for phosphoramidites 3S
(10 min), 3V (10 min), 3W (30 min), 3Y (30 min), 3X (30 min),
and 3Z (15 min) using 1H-tetrazole as catalyst resulted in stepwise
coupling yields of ∼99% for monomers S, V, X, Y, and Z and
∼95% for monomer W. Coupling yields were determined by trityl
monitoring. Removal of the nucleobase protecting groups of ONs
and cleavage from solid support was accomplished using standard
conditions (32% aqueous ammonia for 12-16 h at 55 °C).
Unmodified DNA and RNA strands were obtained from commercial
suppliers and, if necessary, further purified as described below.
Purification of all modified ONs (till minimum 80% purity) was
performed by two different methods: (a) if overall yield >90%,
precipitation of crude ONs (DMT-OFF, absolute EtOH, -18 °C,
12-16 h, followed by washing with absolute EtOH (2 × 300 µL),
or (b) purification of the ONs (DMT-ON) by RP-HPLC using the
system described below, followed by detritylation (80% aqueous
AcOH, 20 min, room temperature) and precipitation/washing as
outlined above. Purification of crude ONs (DMT-ONs) was
performed using a HPLC system equipped with an Xterra MS C18
(10 µm, 7.8 mm × 10 mm) precolumn and an Xterra MS C18 (10
µm, 7.8 mm × 150 mm) column using the representative gradient
protocol depicted in Table S1, Supporting Information. The
composition of all synthesized ONs were verified by MALDI-MS
analysis (Table S3, Supporting Information) recorded in negative
ion mode using 3-hydroxypicolinic acid as a matrix, whereas the
purity (>80%) was verified by ion-exchange HPLC system
equipped with a Dionex PA100 column (4 × 250 mm) at pH 8
using the representative protocol shown in Table S2, Supporting
Information.
(1S,3R,4S,7R)-1-(4,4′-Dimethoxytrityloxymethyl)-7-hydroxy-5-
(pyren-1-yl)carbonyl-3-(thymin-1-yl)-2-oxa-5-azabicyclo[2.2.1]hep-
tane, 2X. 1-Pyrenylcarboxylic acid (162 mg, 0.65 mmol), O-(7-
Azabenzotriazole-1-yl)-N,N,N′,N′-tetramethyluronium hexafluoro-
phosphate (HATU, 183 mg, 0.48 mmol), and N,N′-diisopropyleth-
ylamine (0.19 mL, 1.1 mmol) were dissolved in anhydrous DMF
(4.2 mL), and the mixture was allowed to stir for 6 h at room
temperature. To this was added a solution of nucleoside 1 (0.25 g,
0.44 mmol), which had been dried by coevaporation with anhydrous
toulene (2 × 10 mL) ahead of time, dissolved in anhydrous DMF
(4.2 mL). After stirring at room temperature for 12 h, the reaction
mixture was diluted with CH2Cl2 (50 mL) and washed with
saturated aqueous NaHCO3 (10 mL) and H2O (4 × 10 mL). The
aqueous phase was back-extracted with CH2Cl2 (2 × 30 mL), the
combined organic phase was evaporated to dryness, and the re-
sulting crude residue was adsorbed on silica gel and purified by
silica gel column chromatography (0-4% MeOH in CH2Cl2, v/v)
1
to afford a rotameric mixture (∼1:1.4 by H NMR) of nucleoside
2X as a white solid material (0.32 g, 90%). Rf ) 0.2 (50% acetone
in petroleum ether, v/v), MALDI-HRMS m/z 822.2786 ([M + Na]+,
C49H41N3O8 ·Na+ calcd 822.2746; selected signals 1H NMR (DMSO-
d6)56 δ 6.47 (d, 1H, ex, J ) 3.7 Hz, 3′-OHB), 6.43 (d, 1.4H, ex, J
) 3.8 Hz, 3′-OHA), 6.25 (s, 1H, H-1′B), 5.80 (s, 1.4H, H-1′A), 4.79
(d, 1H, J ) 3.7 Hz, H-3′B), 4.56 (d, 1.4H, J ) 3.8 Hz, H-3′A), 2.05
(s, 4.2H, CH3-A), 1.92 (s, 3H, CH3-A); 13C NMR (DMSO-d6) δ 169.6,
169.5, 164.2, 163.8, 158.3, 158.1, 150.3, 149.6, 144.8, 144.6, 135.4,
135.3, 135.2, 135.1, 134.6, 131.6, 131.4, 130.7, 130.5, 130.25,
130.18, 129.9, 129.8, 128.8, 128.53, 128.46, 128.1, 127.9, 127.8,
127.7, 127.3, 127.2, 126.92, 126.86, 126.8, 126.4, 126.2, 126.1,
125.9, 124.9, 124.5, 124.2, 123.8, 123.7, 123.6, 113.4, 113.2, 109.1,
108.5, 89.0, 88.8, 86.0, 85.6, 85.4, 72.4, 71.5, 64.9, 62.0, 60.3, 59.6,
55.1, 55.0, 53.9, 52.1, 12.6, 12.5. Anal. Calcd for
C49H41N3O8 ·1H2O: C, 71.96; H, 5.30; N, 5.14. Found: C, 71.63;
H, 4.95; N, 4.77.
(1S,3R,4S,7R)-7-[2-Cyanoethoxy(diisopropylamino)phosphinoxy]-
1-(4,4′-dimethoxytrityloxymethyl)-5-ethyl-3-(thymin-1-yl)-2-oxa-5-
azabicyclo[2.2.1]heptane, 3S. Nucleoside 2S (190 mg, 0.32 mmol)
was coevaporated with anhydrous 1,2-dichloroethane (2 × 5 mL)
and dissolved in a mixture of anhydrous EtN(i-Pr)2 in CH2Cl2
(2 mL, 20%, v/v). To this was added 2-cyanoethyl N,N′-
(diisopropyl)phosphoramidochloridite (0.14 mL, 0.63 mmol), and
the reaction mixture was stirred at room temperature for 1 h,
whereupon it was diluted with CH2Cl2 (20 mL). The organic
phase was washed with saturated aqueous NaHCO3 (10 mL),
and the aqueous phase was back-extracted with CH2Cl2 (25 mL).
The combined organic phase was evaporated to dryness, and
the resulting residue was purified by silica gel column chroma-
tography (0-50% EtOAc in petroleum ether, v/v) to afford
amidite 3S (160 mg, 63%) as a white solid material. Rf ) 0.5
Protocol for Thermal Denaturation Studies. Concentrations
of ONs were estimated using the following extinction coefficients
for DNA (OD/µmol): G (12.01), A (15.20), T (8.40), C (7.05); for
RNA (OD/µmol): G (13.70), A (15.40), U (10.00), C (9.00); and
for pyrene (22.4). ONs (1.0 µM each strand) were thoroughly
mixed, denatured by heating, and subsequently cooled to the starting
temperature of the experiment. Quartz optical cells with a path
(55) Assignments of 1H NMR signals of H5′ and H5′′ (and of the
corresponding 13C signals) may in principle be interchanged.
(56) The integral of the H1′ signal of the least predominant rotamer (termed
B) is set to 1.0.
1080 J. Org. Chem. Vol. 74, No. 3, 2009

Functionalized 2′-Amino-R-L-LNA Phosphoramidites
length of 1.0 cm were used. Thermal denaturation temperatures
(Tm values/°C) were measured on a UV-vis spectrometer equipped
with a Peltier temperature programmer and determined as the
maximum of the first derivative of the thermal denaturation curve
(A260 vs temperature) recorded in medium salt buffer (Tm buffer;
100 mM NaCl, 0.1 mM EDTA and pH 7.0 adjusted with 10 mM
NaH2PO4/5 mM Na2HPO4). For studies evaluating the dependence
of Tm on ionic strength, Tm values were also determined in low
and high salt buffers (composition as for medium salt buffer except
that 0 and 700 mM NaCl were used, respectively). The temperature
of the denaturation experiments ranged from at least 15 °C below
Tm to 20 °C above Tm (although not below 1 °C). A temperature
ramp of 1.0 °C/min was used in all experiments. Reported thermal
denaturation temperatures are an average of two measurements
within (1.0 °C.
from the COBRE Program of the National Center for
Research Resources, and The Danish National Research
Foundation. The Nucleic Acid Center is funded by the Danish
National Research Foundation for studies on nucleic acid
chemical biology. The Ph.D. school Nucleic Acid Based Drug
Design (NAC DRUG) supported by the Danish Agency for
Science Technology and Innovation is gratefully acknowl-
edged. The Hrdlicka research team is thanked for proofread-
ing of the manuscript preparation.
Supporting Information Available: General experimental
section; experimental section for synthesis of nucleosides 2 and
1
3 (V/W/Y/Z-series); copies of H NMR, 13C NMR, 31P NMR,
1
1H-1H COSY and/or H-13C HETCOR spectra for all novel
Protocol for Determination of Thermodynamic Para-
meters. Thermodynamic parameters were obtained by analysis of
the melting curves used to determine Tm values assuming bimo-
lecular reactions and two-state equilibrium hypothesis, using
software accompanying the utilized UV-vis spectrometer. The
graphs of ln Ka (affinity constant) as a function of 1/T were
approximated with straight lines facilitating parameter determination
(∆G, ∆H, and ∆S, Tables 4 and 5). Quality of the baseline
permitting, thermodynamic parameters for two melting curves per
investigated duplex were determined, and an average value was
listed. The changes in Gibbs free energy, ∆G, were determined at
temperatures close to the Tm value of the investigated duplexes to
minimize errors (T ) 298 K).
nucleosides; protocols for RP-HPLC and ion-exchange HPLC
purification of ONs; MALDI-MS of synthesized ONs (Table
S3); representative thermal denaturation curves (Figure S1); Tm
values of N2′-functionalized 2′-amino-R-L-LNA at various ionic
strengths (Table S4); discussion of sequence dependent variation
of Tm values; DNA-selectivity of N2′-functionalized 2′-amino-
R-L-LNA (Table S5); thermodynamic data for B1-B5 and full
discussion thereof (Table S6); protocol for acquisition and listing
of additional CD spectra (Figure S2); protocol for molecular
modeling studies; additional molecular modeling structures
(Figures S3-S9, S11, and S12), illustration of intercalation
(Figure S10); pseudorotational phase angle P and glycosidic
torsion angle ꢁ observed in simulated duplexes (Tables S7 and
S8). This material is available free of charge via the Internet at
http://pubs.acs.org.
Acknowledgment. We greatly appreciate financial support
from Idaho NSF EPSCoR, the BANTech Center at the
University of Idaho, a University of Idaho Research Office
and Research Council Seed Grant, NIH Grant P20 RR016448
JO802037V
J. Org. Chem. Vol. 74, No. 3, 2009 1081