Amino acids attached to 2′-amino-LNA: Synthesis and excellent duplex stability

Article abstract of DOI:10.1039/c0ob00532k

The synthesis of 2′-amino-LNA (the 2′-amino derivative of locked nucleic acid) has opened up a number of exciting possibilities with respect to modified nucleic acids. While maintaining the excellent duplex stability inferred by LNA-type oligonucleotides,

Full text of DOI:10.1039/c0ob00532k

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Organic &
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PAPER
Amino acids attached to 2¢-amino-LNA: synthesis and excellent duplex
stability†
Marie W. Johannsen, Lia Crispino, Michael C. Wamberg, Neerja Kalra and Jesper Wengel*
Received 3rd August 2010, Accepted 21st September 2010
DOI: 10.1039/c0ob00532k
The synthesis of 2¢-amino-LNA (the 2¢-amino derivative of locked nucleic acid) has opened up a
number of exciting possibilities with respect to modified nucleic acids. While maintaining the excellent
duplex stability inferred by LNA-type oligonucleotides, the nitrogen in the 2¢-position of
2¢-amino-LNA monomers provides an excellent handle for functionalisation. Herein, the synthesis of
amino acid functionalised 2¢-amino-LNA derivatives is described. Following ON synthesis, a glycyl unit
attached to the N2¢-position of 2¢-amino-LNA monomers was further acylated with a variety of amino
acids. On binding to DNA/RNA complements, the modified ONs induce a marked increase in thermal
stability, which is particularly apparent in a buffer system with a low salt concentration. The increase in
thermal stability is thought to be caused, at least in part, by decreased electrostatic repulsion between
the negatively charged phosphate backbones when positively charged amino acid residues are
appended. Upon incorporation of more than one 2¢-amino-LNA modification, the effects are found to
be nearly additive. For comparison, 2¢-amino-LNA derivatives modified with uncharged groups have
been synthesised and their effect on duplex thermal stability likewise investigated.
Introduction
In recent years, an abundance of modified oligonucleotides (ONs)
have been presented. These contain modifications of the sugar ring
and its substituents,1 the nucleobases2,3 or the backbone.4 Previous
research in our group has focused on locked nucleic acid (LNA,
Fig. 1). LNA is a nucleotide analogue where the sugar ring is
constrained as part of a bicyclic system.5,6 This locks the furanose
in the C3¢-endo conformation, thus mimicking the conformation
found in RNA. ONs modified with LNA nucleotides have a high
affinity for both complementary DNA and RNA strands, as evi-
Fig. 1 Structures of LNA and 2¢-amino LNA thymine monomers.
denced by increased duplex thermal denaturation temperatures. 2¢-
Substituted analogues of LNA have been developed, including 2¢-
modification of the nucleotide building blocks may increase the
stability towards nuclease degradation, affinity for complementary
strands, potency and biodistribution of therapeutic ONs.4,14–16
thio-LNA and 2¢-amino-LNA (Fig. 1).7,8 Unlike 2¢-amino-DNA,
which destabilises both DNA and RNA type helix formation,9,10 2¢-
amino-LNA has a higher affinity for complementary DNA/RNA
A large area of research is the conjugation of various functional
strands than unmodified ONs, though 2¢-amino-LNA is slightly
groups and molecules to ONs.17,18 Recently, many conjugations
less stabilising than LNA.8
have been performed using Huisgen–Sharpless–Meldal reactions
Pharmacokinetic challenges, including delivery, potentially
(more commonly known as “click”-chemistry)19 and other bio-
limit realisation of the many possible pharmacological uses
orthogonal chemical reactions. Conjugations can be carried out
of ONs.11–13 Increasing the structural diversity of ONs is one
both during and after ON synthesis, and may take place at the
possible way of achieving ONs with drug-like characteristics, and
5¢-end, 3¢-end or internally in the ON.15,17,18 A major obstacle to
delivery of ONs is cell penetration. Negatively charged proteo-
glycans on the cell membrane repel the polyanionic ONs, and
the hydrophobic nature of the cell membrane hampers entry of
hydrophilic ONs. Proposed solutions for this problem include
neutral backbone ONs20 and conjugation to various delivery
agents such as cholesterol21,22 or cell penetrating peptides (CPPs).
Nucleic Acid Center, Department of Physics and Chemistry, University of
Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark. E-mail:
jwe@ifk.sdu.dk; Fax: +45 6550 4385; Tel: +45 6550 2510
† Electronic supplementary information (ESI) available: MALDI TOF
mass spectrometry data for modified ONs, overview of yields for synthesis
of modified ONs, 1H and 13C NMR spectra for compounds 2a–b, 31P NMR
spectra for compounds 3a–b. See DOI: 10.1039/c0ob00532k
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CPPs are short sequences of amino acids which, when conjugated
to biopharmaceuticals, have been found to increase cellular uptake
and biological activity of their cargo.23–25
It is intriguing that ONs with several positive amino acid
residues (e.g. lysines) appended can have a neutral or positive
net charge while maintaining water solubility. Several studies
on cationic ONs, both with positively charged backbones and
with positively charged groups attached have been reported.15,26–28
Several ONs modified with cationic groups in the 2¢-position
exhibit efficient binding affinity to target RNA, enhanced chemical
stability and increased nuclease resistance.29
In this paper, we present various structures based on a 2¢-
amino-LNA/DNA mixmer scaffold. We have previously used
LNA to counter the destabilising effect of the incorporation of
2¢-amino-DNA monomers with and without modifications of the
2¢-amino group.10,30 The research described here instead focuses
on conjugation of amino acids and peptides to the thermally
stabilising 2¢-amino-LNA monomer. The amino group of 2¢-
amino-LNA nucleotides is an excellent site for functionalisation
of ONs.31–37 Conjugation to a 2¢-amino group is potentially more
selective and easy than conjugation to a 2¢-hydroxy group of RNA,
and the nitrogen atom furthermore allows functionalisation even
when the 2¢-nitrogen atom is partaking in the bicyclic ring skeleton
of an LNA nucleotide.
We report here the design and synthesis of various 2¢-amino-
LNA phosphoramidites functionalised at the N2¢ position by the
amino acid glycine (“gly”), acetic acid (“acetyl”), and palmitic
acid (“C16”), and incorporation of these into ONs. Conjugation
of palmitic acid to ONs has previously been investigated with the
intention of facilitating cellular uptake,38 but in the context of
the research presented here, a palmitic acid residue is used as an
uncharged substituent with an alkyl side chain.
A key aspect of the research reported herein is solid-phase
conjugation of amino acids to ONs. Addition of multiple amino
acids to the synthesised ONs was accomplished by solid phase cou-
pling to 2¢-amino-LNA monomers already functionalised with one
glycyl moiety. This conjugation approach is compatible with both
automated phosphoramidite-based ON synthesis and fluoren-
9-ylmethoxycarbonyl (Fmoc) based peptide coupling chemistry,
and therefore provides effective structural diversification while
allowing straight-forward monitoring of reaction yields.
The modified ONs (Fig. 2) were hybridised to complementary
DNA or RNA strands and thermal denaturation studies were car-
ried out to assess the duplex-forming ability of the functionalised
ONs.
Fig. 2 Structures of modified N2¢-acyl 2¢-amino-LNA monomers.
Scheme 1 (i) 2a: N-(Fmoc)glycine, DMF, HATU, DIPEA, rt, 1 h,
76%; 2b: palmitoyl chloride, CH2Cl2, pyridine, h, 68%;
◦C,
(ii) 2-cyanoethyl-N,N,N¢,N¢-tetraisopropylphosphane, N,N-diisopropyl-
ammonium tetrazolide, CH2Cl2, rt, 12 h (3a: 94%, 3b: 61%). T =
thymin-1-yl; DMT = 4,4¢-dimethoxytrityl.
0
2
Results and discussion
Chemistry
Synthesis of the 5¢-O-4,4¢-dimethoxytritylated 2¢-amino-LNA-
thymine nucleoside 1 was accomplished according to a literature
procedure.39 Selective N2¢-acylation of nucleoside 1 was carried
out in the presence of O-(7-azabenzotriazol-1-yl)-N,N,N¢,N¢-
tetramethyluronium hexafluorophosphate (HATU) as coupling
reagent to furnish N-(Fmoc)glycyl derivative 2a. The 2¢-N-
palmitoyl derivative 2b was obtained by reacting nucleoside 1 and
palmitoyl chloride without a coupling reagent (Scheme 1).
The 3¢-hydroxy group of nucleoside derivatives 2a–2b was
phosphitylated using standard conditions to afford the corre-
sponding phosphoramidite building blocks 3a–3b that were used
for automated solid-phase ON synthesis (Scheme 1). Synthesis
of the N-acetyl derivative will be published elsewhere,40 whereas
literature procedures were used to synthesise the N-trifluoroacetyl
protected 2¢-amino LNA phosphoramidite.8
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Table 1 Thermal stability studies, medium salt buffer
Medium salt (110 mM Na+)
3¢-d(CAC TAT ACG)-5¢
3¢-r(CAC UAU ACG)-5¢
DNA modification (charge difference)
Tm (◦C)
DTm/modification (◦C)
Tm (◦C)
DTm/modification (◦C)
none (DNA ref.)
2¢-amino-LNA (0)
acetyl (0)
C16 (0)
gly (+1)
(gly)2 (+1)
(gly)3 (+1)
(gly)4 (+1)
(gly)5 (+1)
30
36
36
31
38
38
38
37
36
37
39
41
41
41
45
—
47
Reference
+6
+6
+1
+8
+8
+8
+7
+6
+7
+9
+11
+11
+4
+5
—
+6
27
36
38
33
37
37
37
38
35
38
37
38
39
49
53
21
51
Reference
+9
+11
+6
+10
+10
+10
+11
+8
+11
+10
+11
+12
+7
gly-pro (+1)
gly-lys (+2)
gly-gly-lys (+2)
gly-lys(lys)2 (+4)
3 ¥ 2¢-amino-LNA (0)
3 ¥ acetyl (0)
3 ¥ C16 (0)
+9
-2
+8
3 ¥ gly (+3)
Thermal denaturation temperatures were measured using 1.0 mM of each strand of sequence 5¢-GTG AXA TGC-3¢ and complementary DNA or RNA as
shown, where X is the modified monomer. Likewise, 1.0 mM of each strand of sequence 5¢-GXG AXA XGC-3¢ and complementary DNA or RNA, in the
case of those entries prefaced by “3 ¥” (e.g. “3 ¥ acetyl”). All Tm values were measured in Na2HPO4/NaH2PO4 buffer with 110 mM Na+ concentration
at pH 7. The noted charge difference is relative to the DNA reference and anticipates protonated terminal amino groups of side chains.
Table 2 Thermal stability studies, low salt buffer
Low salt (10 mM Na+)
3¢-d(CAC TAT ACG)-5¢
Tm/◦C
3¢-r(CAC UAU ACG)-5¢
DNA modification (charge difference)
DTm/modification (◦C)
Tm/◦C
DTm/modification (◦C)
none (DNA ref.)
2¢-amino-LNA (0)
acetyl (0)
C16 (0)
gly (+1)
(gly)2 (+1)
(gly)3 (+1)
(gly)4 (+1)
(gly)5 (+1)
14
20
20
15
24
23
23
23
23
23
27
29
31
26
28
—
36
Reference
+6
+6
+1
+10
+9
+9
+9
+9
+9
+13
+15
+17
+4
+5
—
11
19
21
18
22
22
22
22
22
23
22
25
25
32
37
12
37
Reference
+8
+10
+7
+11
+11
+11
+11
+11
+12
+11
+14
+14
+7
gly-pro (+1)
gly-lys (+2)
gly-gly-lys (+2)
gly-lys(lys)2 (+4)
3 ¥ 2¢-amino-LNA (0)
3 ¥ acetyl (0)
3 ¥ C16 (0)
+9
+0.3
+9
3 ¥ gly (+3)
+7
Thermal denaturation temperatures were measured using 1.0 mM of each strand of sequence 5¢-GTG AXA TGC-3¢ and complementary DNA or RNA,
where X is the modified monomer. Likewise, 1.0 mM of each strand of sequence 5¢-GXG AXA XGC-3¢ and complementary DNA or RNA, in the case of
those entries prefaced by “3 ¥” (e.g. “3 ¥ acetyl”). All Tm values were measured in Na2HPO4/NaH2PO4 buffer with 10 mM Na+ concentration at pH 7.
The noted charge difference is relative to the DNA reference, and anticipates protonated terminal amino groups of side chains.
Based on DMT cation measurement, ON synthesis proceeded
in >90% stepwise coupling yields for modified amidites 3a–b,
and in >99% yields for unmodified DNA amidites. In the case
of the ONs with the modifications “(gly)2”, “(gly)3”, “(gly)4”,
“(gly)5”, “gly-pro”, “gly-lys”, “gly-gly-lys”, and “gly-lys(lys)2”,
further conjugation was carried out by solid-phase synthesis
after completion of sequences containing a 2¢-N-glycyl-2¢-amino-
LNA monomer (“gly”, see Fig. 2) (vide infra). In the case
of the remaining ONs, where no further modifications were
required,‡ the ONs were released from the support and purified.
The purity of the ONs were determined by ion exchange HPLC
(>80%), and their identity confirmed by MALDI-TOF mass
spectrometry (for ON sequences, see captions of Tables 1 and
2). For further synthetic details, see experimental section and
ESI.†
‡ “2¢-amino-LNA”, “acetyl”, “gly”, “3 ¥ 2¢-amino-LNA”, “3 ¥ acetyl”,
“3 ¥ gly”, “C16” and “3 ¥ C16”
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Scheme 2 (i) 20% v/v DEA in MeCN, 10 mL, 5 min, rt, (ii) 20% v/v piperidine in DMF, 1.0 mL, rt, 20 min, (iii) N-(Fmoc)-amino acid, DMF, HATU,
DIPEA, rt, 3 h, (iv) 28–30% NH3 in water, 1 mL, 55 ◦C, 12 h.
Solid-phase conjugation
yields were estimated to be 5–83%. There is trend that the
more solid-phase reactions have to be carried out, the lower the
overall yield, and further that the oligonucleotides with charged
modifications are isolated in lower yield that those with uncharged
modifications (a detailed breakdown of individual yields for
different ONs can be found in the ESI). The theoretical loading
of the resin used for ON synthesis was used as a starting point
and the optical density of the purified ON as an endpoint (see
experimental section and ESI for further details).†
For further acylation of an ON containing a 2¢-N-glycyl 2¢-
amino-LNA monomer incorporated in the middle of a DNA
nonamer, the Fmoc-protected glycyl-modified ON was kept on
the resin, the amino group of the glycyl moiety deprotected, and
then reacted further using standard Fmoc chemistry (Scheme 2,
example).
The 2-cyanoethyl phosphate triester is not stable to Fmoc-
deprotection conditions29,41 and was deprotected selectively using
diethyl amine (DEA) prior to Fmoc-removal. This eliminates the
risk of conjugate addition of the unprotected amino acid amine
groups to the acrylonitrile released from phosphate deprotection.
Subsequent peptide couplings furnished ONs conjugated with
di-, tri-, tetra- or pentapeptides at the N2¢-atom of the internally
positioned 2¢-amino-LNA monomer.
Thermal stability studies
We determined the thermal denaturation temperatures (Tm values)
of modified nonamer ONs with both DNA and RNA complemen-
tary strands in sodium phosphate buffers with concentrations of
Na+ of 10 mM (low salt buffer) and 110 mM (medium salt buffer).
The results of the thermal stability studies are summarised in
Tables 1–4.
It is possible to monitor the efficiency of the coupling process
by measuring the absorbance, and thus the concentration, of
the N-(9-fluorenylmethyl)piperidine released upon deprotection of
Fmoc-protected amino acids using piperidine.42,43 Most individual
peptide couplings proceed in good yields and the overall synthesis
Medium salt buffer – 110 mM Na+ concentration (Table 1).
For DNA:DNA duplexes, both the nonamers modified in the
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Table 3 Both strands modified, medium salt concentration
DNA reference
2 ¥ LNA
Tm/◦C
2 ¥ 2¢-amino-LNA
Tm/◦C
2 ¥ gly
Tm/◦C
Modification X
Tm/◦C
DNA reference
3 ¥ LNA
30
41 (+6)
62 (+6)
38 (+4)
45 (+8)
45 (+5)
36 (+6)
41 (+4)
38 (+8)
47 (+6)
1 ¥ 2¢-amino-LNA
3 ¥ 2¢-amino-LNA
1 ¥ gly
45 (+5)
55 (+5)
50 (+7)
58 (+6)
3 ¥ gly
Tm values measured in Na2HPO4/NaH2PO4 buffer with 110 mM Na+ concentration at pH 7 using 1.0 mM of each single strand. Numbers in brackets
denote change in Tm value per modification relative to the DNA:DNA reference duplex
Table 4 Both strands modified, low salt concentration
DNA reference
2 ¥ LNA
Tm/◦C
2 ¥ 2¢-amino-LNA
Tm/◦C
2 ¥ gly
Tm/◦C
Modification X
Tm/◦C
DNA reference
3 ¥ LNA
14
25 (+6)
45 (+6)
22 (+4)
34 (+10)
29 (+5)
20 (+6)
26 (+4)
24 (+10)
36 (+7)
1 ¥ 2¢-amino-LNA
3 ¥ 2¢-amino-LNA
1 ¥ gly
30 (+5)
38 (+5)
37 (+8)
44 (+6)
3 ¥ gly
Tm values measured in Na2HPO4/NaH2PO4 buffer with 10 mM Na+ concentration at pH 7 using 1.0 mM of each single strand. Numbers in brackets
denote change in Tm value per modification relative to the DNA:DNA reference duplex
central position with unconjugated 2¢-amino-LNA-T and those
with acetylated 2¢-nitrogen have DTm values of +6 ◦C (DTm values
are the differences in Tm values between modified duplexes and
the unmodified reference duplexes). ONs containing the parent
unconjugated 2¢-amino-LNA-T monomer are not expected to be
protonated to any significant extent at pH 7 as the 2¢-amino group
of the 2¢-amino-LNA-T nucleoside has a pKa value of 6.17.44
While the pKa values of the conjugated amino acid ONs have
not been determined experimentally, the terminal amino groups
are assumed to be positively charged at pH 7 as the pKa values
of unconjugated amino acid amino groups are >9 for all amino
acids used in this study.
bilisation through electrostatic interactions or extensive hydration
effects.
For the palmitic acid modification (“C16”), the Tm value
was 1 ◦C higher with a DNA complementary strand versus the
unmodified DNA/DNA duplex. Conjugation of 2¢-amino-LNA
monomers with a palmitic acid residue thus lead to significantly
less stabilisation than obtained by unconjugated 2¢-amino-LNA
or amino acid modified 2¢-amino-LNA monomers.
Upon incorporation of three 2¢-amino-LNA monomers in the
sequence 5¢-GXG AXA XGC-3¢ (entries “3 ¥ 2¢-amino-LNA”,
“3 ¥ acetyl” and “3 ¥ gly” in Tables 1 and 2), the affinity effects are
nearly additive as will be discussed later. When three palmitoyl-
conjugated monomers are incorporated into the nonamer (“3 ¥
C16”), no duplex formation can be seen.
The ON modified with a glycyl monomer at the 2¢-nitrogen
◦
(“gly”) (DTm = +8 C) is slightly more stable than unconjugated
2¢-amino-LNA (DTm = +6 ◦C). Attaching further glycyl groups by
solid-phase conjugation to make di- or tripeptides (“(gly)2” and
“(gly)3”) does not alter the melting temperature. For the tetra-
and pentapeptides there is a slight decrease in Tm, but further
studies are required to determine whether this trend will continue
for longer peptide chains.
When measuring the thermal stability of duplexes with an RNA
complementary strand, very similar results are seen. The relative
stabilisation incurred by modification with positively charged
◦
residues is a bit larger, “gly”-”(gly)3” has DTm values of +10 C,
and “(gly)4” even shows a DTm value of +11 ◦C. Here, the
slightly lower stabilisation seen for the longer peptide chains in
DNA:DNA duplexes is not apparent except for the pentagly-
cylated duplex (“(gly)5”) which has a DTm value of +8 ◦C. As
for the DNA:DNA duplexes, no steric influence is seen with
conjugation of proline, but unlike those, the lysine-modifed ONs
show no particular increase in stability compared to the singly
charged amino acids. For the modification with four potentially
protonated amino groups (“gly-lys(lys)2”), the DTm value is
+12 ◦C which is only 1 ◦C higher than the highest melting
temperature for a singly charged modification, and “gly-lys”,
with two positive charges, is no different from “gly”. The acetyl
modification is under these conditions as stable as the charged
modifications. The palmitic acid modification shows a DTm value
Steric factors do not appear to greatly influence the stabilisation,
since conjugation of the cyclic amino acid proline as the second
amino acid (“gly-pro”) has the same effect as glycine (“(gly)2”).
Stabilisation does appear to be charge-dependent, as conjugation
of a single lysine residue (“gly-lys”) raises the DTm to +9 ◦C, and
adding a lysine branch (“gly-lys(lys)2”, four protonated amino
groups) gives a DTm value of +11 ◦C.
For comparison, a 2¢-amino-LNA monomer was conjugated
with palmitic acid at the 2¢-nitrogen (“C16”). The C16 and
acetyl modifications are comparable in length to the modifications
“(gly)5” and “gly”, respectively. The palmitoyl residue especially
is obviously significantly more lipophilic and does not offer sta-
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of +6 ◦C compared to the unmodified DNA reference duplex,
again a destabilisation compared to unconjugated 2¢-amino-LNA
and the charged modifications. For ONs with three modified
monomers incorporated, the duplex formed between RNA and
the strand with three acetyl-modified 2¢-amino-LNA monomers
(“3 ¥ acetyl”) is more stable, by 2 ◦C, than the glycyl duplex (“3 ¥
gly”) making this the most stable of all duplexes, indicating that
favourable hydration or steric orientation of the acyl group may be
more important than electrostatic effects under these conditions.
The ON with three palmitoyl-modifications does form a duplex
with RNA, but it is severely destabilised.
investigated (Fig. 3), namely duplexes with a single incorporated
modification paired with an unmodified strand (type A), with
two modifications paired with an unmodified strand (type B),
with three modifications paired with an unmodified strand (type
C), with a single modification across from a strand with two
modifications (type D), and with three modifications across
from two modifications (type E). The results are summarised
in Tables 1 and 2 for duplex types A and C, while compar-
ison with the remaining duplex types is shown in Tables 3
and 4.
The relatively more pronounced stabilising effect with an RNA
counter strand is not unexpected, since a similar trend has been
observed for LNA modified ONs.5 This RNA-stabilising effect
does not appear to be amplified by the electrostatic stabilisation
from multiple positive charges.
Measurements have also been carried out in a low salt buffer
(10 mM Na+). These measurements do show very similar, but
much more pronounced trends compared to the measurements in
the medium salt buffer. For duplexes with a DNA counter strand,
unconjugated (“2¢-amino-LNA”) and acetylated (“acetyl”) 2¢-
amino-LNA-T modifications stabilise the duplex by +6 ◦C. In
comparison, the singly charged modifications show DTm values
of +9–10 ◦C, with no decrease in stabilisation even with longer
chains (“(gly)4” and “(gly)5”) or with a cyclic amino acid (“gly-
pro”). The charge dependence is evi◦dent with the modification
“gly-lys” giving a DTm value of +13 C and the branched lysine
chain “gly-lys(lys)2” giving a DTm value of +17 ◦C. This is a melting
temperature 12 ◦C higher than seen for the duplex with unmodified
2¢-amino-LNA. Although the effect of the multiply charged lysines
is once again stronger with a DNA than with an RNA counter
strand, the increased stabilisation and charge dependence seen
in the low salt buffer supports the hypothesis that stabilisation
is facilitated by electrostatic interactions between the positively
charged residues and the negatively charged ON backbone. Such
an effect is expected to be stronger in the low salt buffer where the
concentration of positive counter ions is lowered.
Again, multiple modifications are near additive. A slight de-
crease in the DTm per modification is seen when more modified
nucleotides are incorporated. This effect is strongest for duplexes
with a DNA complement, especially for the modification “gly”.
When three palmitoyl residues are incorporated (“3 ¥ C16”), no
melting transition is seen with a DNA complementary strand.
While a duplex does form with an RNA complementary strand,
no significant stabilisation is seen compared to an unmodified
DNA ON. In comparison to unconjugated 2¢-amino-LNA, the
palmitoyl residues have a destabilising effect.
Fig. 3 Duplex types measured for thermal stability. Sequences of
single strands are 5¢-GXG AXA XGC-3¢ and the complementary
5¢-GCA XAX CAC-3¢ where X = DNA-T, LNA-T, 2¢-amino-LNA-T or
2¢-N-glycylamino-LNA-T (these monomers are shown as droplets).
This study is relevant for several applications. Thus, while an-
tisense applications only require one modified strand in a duplex,
aptamers and siRNA constructs may benefit from modifications
on both strands, e.g. to stabilise basepairing regions such as
stems.
Melting studies were carried out first in a medium salt buffer
(Table 3). For all modifications, the stabilisation per modification
is close to additive, with only a slight decrease when multiple
modifications are present in each strand/duplex. In general,
glycyl-modified monomers generate the highest relative thermal
stability. The differences in stabilisation between the acylated and
unconjugated 2¢-amino-LNA are not great, but they are consistent
across the various duplex types. Furthermore, the increase in
stabilisation is sufficiently large for the charged modification
(“gly”) to surpass LNA in stabilisation for duplex types B
and C.
The thermal stability of duplexes with modifications in both
strands (Fig. 3) has also been studied in a low salt buffer as
summarised in Table 4. Here, DTm values per modification are
increased 1–2 ◦C for duplexes incorporating the charged glycyl
amino group compared to the medium salt buffer. In comparison,
the uncharged modifications (“LNA” and “2¢-amino-LNA”) show
the exact same DTm per modification as in the medium salt
buffer.
It is clear from the measurements described above that N2¢-
acylation of 2¢-amino-LNA monomers is an excellent choice
for appending amino acids and oligopeptides to ONs. When
comparing the effects of the palmitoyl unit with those of the
pentaglycyl units, it is clear that hydrophobicity of the conjugated
moiety can be unfavorable for duplex stability, an effect which
may originate from differences in hydration of the appended side
chains.
Mismatch discrimination studies. Thermal denaturation stud-
ies of selected modified ONs with one and three modifications
have been carried out in medium salt buffer (Table 5). These show
satisfactory discrimination of mismatches in the complementary
strand. ONs modified with glycyl (“gly”) generally show mismatch
discrimination that is as good as or better than for LNA-modified
ONs. Both these modifications are significantly more sensitive to
mismatches than unmodified DNA ONs. The palmitoyl modified
ONs are at least as good as unmodified DNA, but not consistently
improved for all types of mismatches like the “gly” and “LNA”
modifications. As mismatched duplexes would likely be too
unstable in a low salt buffer for melting transitions to be observed,
Modifications in both strands. Binding studies have also been
carried out to determine the influence of one or more modified
monomers in both strands. Five different duplex types were
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Table 5 Mismatch discrimination studies
Tm/◦C
3¢-d(CAC TBT ACG)-5¢
3¢-r(CAC UBU ACG)-5¢
DNA modification
B =
A
C
G
T
A
C
G
U
none (DNA ref.)
LNA
gly
C16
3 ¥ LNA
3 ¥ gly
28
34
37
30
43
46
12
15
20
16
23
31
19
24
22
17
31
28
13
17
17
16
26
26
27
35
38
33
52
51
<10
15
22
15
35
22
28
25
21
42
38
15
17
19
15
34
33
34
T
m values for matched and mismatched duplexes were measured in Na2HPO4/NaH2PO4 buffer at pH 7 (110 mM Na+) using 1.0 mmol of each ON strand.
Due to different instrumentation used, there are minor differences in the Tm values reported for some matched duplexes above and in Tables 1 and 3. The
ON “3 ¥ C16” is not reported due to the previously mentioned inability to form duplexes with complementary DNA.
mismatch discrimination was studied solely in the medium salt
buffer.
Experimental section
General
All reagents were used as purchased without further purification.
Conclusions
Reactions (except solid-phase conjugation of amino acids) were
conducted under an atmosphere of nitrogen. Petroleum ether,
used for column chromatography, was of distillation range 60–
80 ◦C. For column chromatography, silica gel 60 (0.040–0.063 mm)
from Merck was used. Progress in reactions was monitored by
thin-layer chromatography (TLC) on analytical silica gel TLC
plates (60 F254) from Merck with UV-light used for visualisation.
NMR spectra were recorded on a Varian Gemini 2000 NMR
spectrometer. The d values are in ppm relative to tetramethyl-
silane as internal standard for 1H NMR, deuterated solvent
CDCl3 (d 77.00) for 13C NMR, and relative to 85% H3PO4 as
external standard for 31P NMR. Assignments of NMR spectra,
when given, are based on 2D spectra and follow the standard
carbohydrate/nucleoside nomenclature (the carbon atom of the
C4¢-substituent is numbered C5¢¢). The assignments of methylene
protons, when given, may be interchanged. Coupling constants
(J-values) are given in Hertz. The NMR spectra of the N-
acylated 2¢-amino-LNA nucleosides revealed two conformations
of the amide bond (rotamers). High resolution MALDI spectra
were recorded in positive ion mode on a Fourier Transform Ion
Cyclotron Resonance Mass Spectrometer (IonSpec, Irvine, CA).
For accurate ion mass determinations, the [M + Na]+ ion was
peak matched using ions derived from the 2,5-dihydroxybenzoic
acid matrix. Electrospray mass spectrometry was performed using
a Q-Star Pulsar Mass Spectrometer (Sciex/Applied Biosystems)
equipped with a nano electrospray source. The instrument was
externally calibrated in the relevant range by observing cluster
ions (NaI)nNa+. Samples were dissolved to a concentration of
approximately 10 mM in methanol and sprayed from metal coated
borosilicate emitters.
In conclusion, we have synthesised phosphoramidite derivatives
of a 2¢-amino-LNA N2¢-acylated with glycine. N-Glycylated 2¢-
amino-LNA oligonucleotides were further modified by solid phase
conjugation to yield di-, tri-, tetra- and pentapeptide modifications
as well as an oligonucleotide with a lysine-branched chain. When
incorporated into a DNA strand, these amino acid and peptide
modifications increase stability of duplexes formed with both
DNA and RNA complements. The stabilising effect in general
becomes more profound when thermal denaturation studies are
carried out in a buffer with low counterion concentration. A
duplex containing 2¢-amino-LNA monomer(s) modified with
positively charged amino acids is more stable than DNA/DNA
or DNA/RNA duplexes and more stable than the corresponding
duplexes with unconjugated 2¢-amino-LNA monomer(s) incorpo-
rated. The stabilising effect was found to be nearly additive for
multiple incorporations of modified monomers in one or both
strands.
Furthermore, oligonucleotides containing 2¢-amino-LNA
monomers N-acylated with palmitic or acetic acid were synthe-
sised. Unlike the amino acid modifications, only negligible changes
in thermal stability were observed for these modifications when the
counterion concentration of the buffer was decreased. We propose
that the stabilising influence of the amino acid-modified monomers
is due to an electrostatic effect. The positively charged protonated
amino groups of one strand may interact with the negatively
charged phosphate backbone of the same strand, decreasing the
effective charge of that strand and thus the repulsive electrostatic
interaction with the negatively charged complementary strand.
It is also possible that positively charged amino groups of one
strand interact with negatively charged phosphate groups on
the complementary strand, creating an attractive electrostatic
interaction.
(1R,3R,4R,7S)-1-(4,4¢-Dimethoxytrityloxymethyl)-5-[N-(fluoren-
9-ylmethoxycarbonyl)glycyl]-3-(thymin-1-yl)-2-oxa-5-
azabicyclo[2.2.1]heptane (2a)
N2¢-Acylated 2¢-amino-LNA monomers are appealing as con-
stituents of biologically active ONs, e.g. antisense, aptamer or
siRNA constructs, as they offer an opportunity of increasing
molecular diversity and modulating physicochemical properties
while preserving high-affinity DNA and RNA-binding.
Fmoc-gly-OH (312 mg, 1.05 mmol, 1.2 eq), DMF (10 mL)
and HATU (400 mg, 1.05 mmol, 1.2 eq) were mixed, DIPEA
(0.275 mL, 1.58 mmol, 1.8 eq) was added and the reaction
mixture was stirred for 10 min at room temperature. A solution
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of nucleoside 1 (500 mg, 0.88 mmol) in DMF (10 mL) was added
dropwise, and the reaction was stirred for a further 60 min at
which point it was deemed complete by TLC. The reaction mixture
was diluted with ethyl acetate (300 mL), washed with H2O (2 ¥
450 mL), 5% NaHCO3 (2 ¥ 450 mL), and finally H2O (2 ¥ 450 mL).
The aqueous phases were back-extracted in portions with ethyl
acetate (300 mL in total). The combined organic phase was dried
(MgSO4), filtered, and the solvent removed in vacuo to yield the
crude product as a pale yellow foam (667 mg). The crude product
was purified by column chromatography on silica with 60–100%
ethyl acetate in petroleum ether, the relevant fractions combined
and the eluent removed in vacuo to yield a rotameric mixture
(~0.66 : 0.33 by 1H NMR) of nucleoside 2a as a pale yellow foam
(567 mg, 76%). dH (300 MHz, CDCl3, Me4Si) (subscript A = major
rotamer, subscript B = minor rotamer; integrals are shown only
for dominating rotamer) 9.83 (1H, br s, NHA), 9.43 (br s, NHB),
7.70–7.12 (17H, m, Ar), 7.59 (1H, s, 6-HA), 7.57 (s, 6-HB), 6.80–
6.76 (4H, m, Ar), 5.95 (1H, t, 3¢-OH), 5.50 (s, H-1¢B), 5.44 (1H, s,
H-1¢A), 5.16 (s, H-2¢B), 4.73 (1H, s, H-2¢A), 4.38 (1H, s, H-3¢), 4.26–
4.08 (5H, m, H-5¢, H-5¢, CH-Fmoc, CH2-Fmoc), 3.71 (6H, s, 2 ¥
OCH3), 3.61–3.45 (4H, m, CH2, H5¢, H-5¢), 1.97 (s, 5-MeB), 1.58
(3H, s, 5-MeA); dC (75 MHz, CDCl3) 168.0, 164.5, 158.6, 157.0,
150.7, 144.5, 144.4, 143.9, 143.7, 141.2, 135.5, 135.4, 135.3, 135.2,
134.6, 130.1, 130.0, 129.2, 128.1, 127.9, 127.8, 127.7, 127.1, 125.2,
119.9, 113.4, 113.2, 110.7, 88.7, 88.0, 87.1, 86.8, 86.7, 70.1, 69.1,
67.4, 67.3, 63.0, 59.2, 55.2, 51.6, 47.1, 46.9, 43.1, 42.9, 12.7, 12.6;
HR ESI MS m/z 873.3070 ([M + Na]+, C49H46N4O10Na+ calcd
873.3106).
22.7, 14.1, 12.5, 12.4; HR MALDI MS m/z 832.4475 ([M + Na]+,
C48H63N3O8Na+ calcd 832.4507).
(1R,3R,4R,7S)-7-[2-Cyanoethoxy(diisopropylamino)-
phosphinoxy]-1-(4,4¢-dimethoxytrityloxymethyl)-5-[N-(fluoren-9-
ylmethoxycarbonyl)glycyl]-3-(thymin-1-yl)-2-oxa-5-
azabicyclo[2.2.1]heptane (3a)
Nucleoside 2a (517 mg, 0.61 mmol) was co-evaporated
with anhydrous dichloroethane. Diisopropylammonium tetra-
zolide (209 mg, 1.22 mmol, 2 eq.) was added and the
solids dissolved in CH2Cl2 (7 mL). 2-Cyanoethyl-N,N,N¢,N¢-
tetraisopropylphosphane (388 mL, 1.22 mmol, 2.0 eq) was added
dropwise and the reaction mixture was stirred at room temperature
for 12 h. The reaction mixture was diluted with ethyl acetate
(2.5 mL), washed with NaHCO3 (20 mL), and the aqueous phase
was back-extracted with CH2Cl2 (3 ¥ 10 mL). The combined
organic phase was dried (Na2SO4), filtered, and the eluent removed
in vacuo, yielding a pale yellow foam (crude yield 866 mg).
The foam was dissolved in the minimal amount of ethyl acetate
(2 mL) and added dropwise to cold petroleum ether (200 mL,
◦
app. 0 C). The precipitating mixture was stirred for 10 min, the
resulting cream-coloured solid filtered off and dried under vacuum
overnight. The mother liquor was concentrated in vacuo, the
crystallization procedure repeated, and following TLC analysis,
the crystallization products were combined (601 mg, 94%). dP
(121.5 MHz, CDCl3) 150.0, 149.7, 149.4, 148.3; HR ESI MS m/z
1051.4390 ([M + H]+, C58H64N6O11P+ calcd 1051.4365).
(1R,3R,4R,7S)-7-(2-Cyanoethoxy(diisopropylamino)-
phosphinoxy)-1-(4,4¢-dimethoxytrityloxymethyl)-5-
(hexadecanoyl)-3-(thymin-1-yl)-2-oxa-5-azabicyclo[2.2.1]heptane
(3b)
(1R,3R,4R,7S)-1-(4,4¢-Dimethoxytrityloxymethyl)-5-
(hexadecanoyl)-7-hydroxy-3-(thymin-1-yl)-2-oxa-5-
azabicyclo[2.2.1]heptane (2b)
Nucleoside 1 (1.51 g, 2.64 mmol) was co-evaporated with anhy-
drous toluene (2 ¥ 2 mL) and subsequently dissolved in anhydrous
CH2Cl2 (25 mL). Anhydrous pyridine (1.0 mL, 12.4 mmol,
4.7 equiv.) was added, the solution was cooled to 0 ◦C, and
palmitoyl chloride (0.75 g, 2.73 mmol, 1.02 equiv.) was added
dropwise. The reaction mixture was stirred at 0 ◦C for 2 h,
whereupon it was diluted with CH2Cl2 (25 mL) and washed with
sat. aq. NaHCO3 (2 ¥ 25 mL). The organic phase was dried
(MgSO4) and concentrated to dryness under reduced pressure. The
residue was purified by silica gel column chromatography using
MeOH/pyridine/CH2Cl2 (4 : 1 : 95, v/v/v) as eluent to afford a
Nucleoside 2b (1.423 g, 1.76 mmol) and diisopropylammonium
tetrazolide (0.453 g, 2.65 mmol, 1.5 equiv.) were mixed and dis-
solved in anhydrous CH2Cl2 (25 mL). 2-Cyanoethyl-N,N,N¢,N¢-
tetraisopropylphosphane (1.060 g, 3.52 mmol, 2.0 equiv.) was
added dropwise and the resulting mixture was stirred at room
temperature for 12 h. It was diluted with CH2Cl2 (25 mL),
washed with sat. aq. NaHCO3 (2 ¥ 25 mL), dried (Na2SO4) and
concentrated under reduced pressure. The residue was purified
by silica gel column chromatography using toluene–EtOH/Et3N
(91 : 7 : 2, v/v/v) as eluent to afford the amidite 3b as off-
white foam (1.089 g, 61%). Rf 0.43 (EtOH–toluene 1 : 10); dP
(121.5 MHz, CDCl3) 149.8, 149.3, 148.8, 147.3; HR ESI MS m/z
1032.5501 ([M + Na]+, C57H80N5O9PNa+ calcd 1032.5586).
1
rotameric mixture (~0.45 : 0.55 by H NMR) of nucleoside 2b as
a white solid (1.45 g, 68%). Rf 0.52 (MeOH–CH2Cl2 8 : 92); dH
(300 MHz, CDCl3, Me4Si) (subscript A = major rotamer, subscript
B = minor rotamer; integrals are shown only for major rotamer)
9.86 (br s, NHB), 9.44 (1H, br s, NHA), 7.66 (1H, s, 6-HA), 7.60
(s, 6-HB), 7.49–7.20 (9H, m, Ar), 6.84 (4H, m, Ar), 5.53 (s, H-
1¢B), 5.44 (1H, s, H-1¢A), 5.20 (s, H-3¢B), 4.53 (1H, s, H-3¢A), 4.37
(1H, s, H-2¢A), 4.30 (s, H-2¢B), 3.78 (6H, s, 2 ¥ OCH3), 3.82–3.62
(4H, m, H-5¢, H-5¢), 2.50–2.37, 2.17–2.13 (2H, m, CH2), 1.61 (3H,
2 ¥ s, 5-MeA+B), 1.65–1.50 (2H, m, CH2), 1.24–1.22 (24H, m, CH2),
0.87 (3H, t, J = 6.4 Hz, Me); dC (75 MHz, CDCl3) 173.5, 173.2,
164.5, 164.2, 158.7, 158.6, 150.1, 149.7, 144.4, 144.3, 135.5, 135.3,
135.2, 134.7, 130.1, 130.1, 130.0, 128.1, 128.0, 127.1, 127.0, 113.3,
113.2, 110.5, 109.9, 88.7, 88.0, 86.9, 86.8, 86.7, 70.2, 68.9, 63.6,
59.1, 59.6, 55.2, 34.2, 33.9, 31.9, 29.7, 29.6, 29.5, 29.3, 25.0, 24.6,
Oligonucleotide synthesis and purification
The oligonucleotides were synthesised in the DMT-ON mode
on an automated DNA synthesizer using an ExpediteTM Nucleic
Acid Synthesis System model 8909 from Applied Biosystems.
Synthesis was carried out in 0.2–1.0 mmol scale using polystyrene
resin purchased from Glen Research. Phosphoramidites 3a–b were
dissolved in acetonitrile as a 0.05 M solution and coupled using
tetrazole as an activator. The coupling time was 15–20 min for
amidites 3a–b, while commercial DNA phosphoramidites were
subjected to 2 min coupling time. After completion of the coupling
steps, the cyanoethyl protecting groups were removed from the
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phosphate backbone (20% v/v diethylamine in acetonitrile, 10 mL,
5 min, rt). The resin was washed with acetonitrile (3 ¥ 5 mL)
and dried briefly. Oligomers needing no further modification were
deprotected and cleaved from the resin using standard conditions
modification relative to the Tm values of the reference duplexes.
Mismatch discrimination data were obtained on a Perkin Elmer
UV-visible spectrometer Lambda 20 equipped with a PTP-6 Peltier
Temperature Programmer.
◦
(28–30% aqueous ammonia, 1 mL, 12 h, 55 C) and purified by
reversed phase HPLC on a Waters 600 system (with Waters 2996
PDA detector) equipped with a Waters XterraTM MS C18 10 mm
7.8 ¥ 150 mm column using a 38 min linear gradient of 0–53%
MeCN in 0.05 M triethyl ammonium acetate (pH 7.4) at a flow rate
of 2.50 mL min-1. The purified oligonucleotides were subjected to
standard detritylation and then precipitated from ethanol (“C16”,
“3 ¥ C16”) or acetone (all others). The ONs were >80% pure by
ion-exchange HPLC (Merck Hitachi D-7000 LaChrom Interface
(with L-7400 UV detector) equipped with a Gen-Pak Fax 4.6 ¥
100 mm column). The composition of the oligonucleotides was
verified by MALDI-TOF MS, which was recorded on a Bruker
Daltonics Microflex LT using a 3-hydroxy picolinic acid matrix.†
Acknowledgements
The Danish National Research Foundation is thanked for funding
the Nucleic Acid Center, a research center for studies on nucleic
acid chemical biology. This work was furthermore supported by
the Sixth Framework Programme Marie Curie Host Fellowship
for Early Stage Research Training under contract number MEST-
CT-2004-504018.
Notes and references
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