Twisted intercalating nucleic acids - Intercalator influence on parallel triplex stabilities

Article abstract of DOI:10.1002/ejoc.200600168

Phosphoramidites of several new twisted intercalating nucleic acid (TINA) monomers and the previously discovered (R)-1-O-[4-(1-pyrenylethynyl) phenylmethyl]glycerol (1) were synthesized and used in DNA synthesis. Stabilization of Hoogsteen-type triplexes was observed in cases of insertion of the novel (R)-1-O-[3-(naphthalen-1-ylethynyl)phenylmethyl]glycerol (2) as a bulge into homopyrimidine oligodeoxynucleotides (ONs), whereas phenylethynyl and 4-(biphenylylethynyl) derivatives of TINAs resulted in destabilization of parallel triplexes relative to the wild-type triplex. It was concluded that TINA monomers should possess at least two fused phenyl rings attached through the triple bond at the 4-position of bulged (R)-1-O-(phenylmethyl)glycerol in homopyrimidine ONs in order to stabilize parallel triplexes. Slight destabilization of DNA/DNA Watson-Crick type duplexes (ΔT_m = 1.0-4.5°C) was detected for 2 inserted as a bulge, while RNA/DNA duplexes and duplexes with other TINA analogues were considerably destabilized (ΔT_m > 6.0°C). In cases of double insertion of 1 opposite to base inversions in dsDNA, the thermal stabilities of the triplexes were higher than that of the wild-type triplex, which is a new solution to overcome the problem of targeting homopurine stretches with single base pair inversions. A DNA three-way junction was considerably stabilized (ΔT _m in a range of 10.0-15.5°C) upon insertion of TINA monomers in the junction point as a bulge. Wiley-VCH Verlag GmbH & Co. KGaA, 2006.

Full text of DOI:10.1002/ejoc.200600168

FULL PAPER
DOI: 10.1002/ejoc.200600168
Twisted Intercalating Nucleic Acids – Intercalator Influence on Parallel
Triplex Stabilities
Vyacheslav V. Filichev,*[a] Hatem Gaber,[a] Thomas R. Olsen,[a] Per T. Jørgensen,[a]
Carsten H. Jessen,[a] and Erik B. Pedersen[a]
Keywords: Parallel triplex / DNA / Intercalator / Sonogashira reaction
Phosphoramidites of several new twisted intercalating nu-
cleic acid (TINA) monomers and the previously discovered
(R)-1-O-[4-(1-pyrenylethynyl)phenylmethyl]glycerol (1) were
synthesized and used in DNA synthesis. Stabilization of
Hoogsteen-type triplexes was observed in cases of insertion
of the novel (R)-1-O-[3-(naphthalen-1-ylethynyl)phenyl-
methyl]glycerol (2) as a bulge into homopyrimidine oligo-
deoxynucleotides (ONs), whereas phenylethynyl and 4-(bi-
phenylylethynyl) derivatives of TINAs resulted in destabili-
zation of parallel triplexes relative to the wild-type triplex. It
was concluded that TINA monomers should possess at least
two fused phenyl rings attached through the triple bond at
the 4-position of bulged (R)-1-O-(phenylmethyl)glycerol in
homopyrimidine ONs in order to stabilize parallel triplexes.
Slight destabilization of DNA/DNA Watson–Crick type du-
plexes (∆Tm = 1.0–4.5 °C) was detected for 2 inserted as a
bulge, while RNA/DNA duplexes and duplexes with other
TINA analogues were considerably destabilized (∆Tm
Ͼ
6.0 °C). In cases of double insertion of 1 opposite to base in-
versions in dsDNA, the thermal stabilities of the triplexes
were higher than that of the wild-type triplex, which is a new
solution to overcome the problem of targeting homopurine
stretches with single base pair inversions. A DNA three-way
junction was considerably stabilized (∆Tm in a range of 10.0–
15.5 °C) upon insertion of TINA monomers in the junction
point as a bulge.
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2006)
ethynyl)phenylmethyl]glycerol (1) into the middles of homo-
pyrimidine oligodeoxynucleotides (twisted intercalating nu-
cleic acids, TINAs) can give rise to extraordinarily high
thermal stability in Hoogsteen-type triplexes and duplexes,
whereas Watson–Crick-type duplexes of the same nucleo-
tide content were destabilized.[9] Even at pH = 7.2, a TFO
with two bulged insertions of 1 separated by three bases
formed a stable triplex (Tm = 43.0 °C), whereas the native
oligonucleotide was unable to bind to the target dsDNA.
We believe that this technology, along with the development
of modified nucleic bases designed for the recognition of
inversion sites, should be applicable for sequence-selective
dsDNA targeting (Scheme 1).
Introduction
DNA triple helix formation is a subject of intensive re-
search for gene targeting and alteration.[1–3] Triple helixes
are formed when a single-stranded triplex-forming oligonu-
cleotide (TFO) binds to a purine-containing strand of
dsDNA through specific major groove interactions. Gen-
erally, the third-strand affinity of a TFO is low, due to the
requirement for the formation of pH-sensitive C+–G–C
Hoogsteen base triplexes under physiological conditions in
the parallel (pyrimidine) binding motif. Modification of
TFOs has been attempted in order to improve their binding
affinities to their targets and to alleviate restrictions in the
dsDNA sequence with the design of new triplex nucleo-
bases. Oligonucleotides containing modified nucleic acid
components – such as peptide nucleic acids (PNAs),[4]
locked nucleic acids (LNAs),[5,6] 2Ј-(aminoethyl)oligoribo-
nucleotides (2Ј-AE-RNAs),[7] and N3ЈǞP5Ј phosphoramid-
ates[8] – and inducing increased binding affinity are among
the most successful chemically modified TFOs. Recently, we
have found that bulge insertions of (R)-1-O-[4-(1-pyrenyl-
Scheme 1. TINA monomers.
[a] Nucleic Acid Center, Department of Chemistry, University of
Southern Denmark,
In order to evaluate how the structures of TINA mono-
mers influence the thermal stabilities of parallel triplexes
and antiparallel duplexes we synthesized several new TINA
monomers. These analogues were incorporated into oligo-
deoxynucleotides (ONs) and the thermal stabilities of their
Campusvej 55, 5230 Odense M, Denmark
Fax: +45-6615-8780
E-mail: filichev@chem.sdu.dk
Supporting information for this article is available on the
WWW under http://www.eurjoc.org or from the author.
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Twisted Intercalating Nucleic Acids – Influence on Triplex Stabilities
FULL PAPER
complexes were compared with those of the previously in- case a yield of 28% of compound 15 was obtained in a dry
vestigated TINAs. The synthesis of the phosphoramidite of DMF/1,4-dioxane (5:2) mixture as medium after heating at
the monomer 1, previously obtained by post-synthetic Son- 80 °C for 3 d. The low reactivity of phenylacetylene in reac-
ogashira coupling,[9] is described and the compound was tions with 12 and 13 [Pd(PPh3)2Cl2, CuI, iPr2NEt, THF,
used in a DNA synthesis. It was found that positioning of reflux under N2] resulted in difficulties with isolation of the
the intercalator through a triple bond attached at the 4- products on silica gel because of the similar mobilities of
position of the phenyl ring in (R)-1-O-(phenylmethyl)glyc- the products, byproducts and phenylacetylene, so com-
erol gave higher thermal stability than that through the 3- pounds 16 and 17 were finally purified by semipreparative
position in the case of Hoogsteen-type triplexes.
HPLC on a C18 column in 23% and 18% yields, respec-
tively. It has been shown that application of microwave[10]
or ultrasound[11] irradiation may result in increased rates
and yields in Sonogashira coupling reactions. Attempts to
obtain compound 17 with ultrasound irradiation using the
above reagents were unsuccessful, but the yield of 17 was
increased up to 33% through application of microwave irra-
diation (2×25 min, 120 °C) for the reaction mixture (also
containing PPh3 to improve the stability of the palladium
catalyst) in a solution of Et2NH/DMF.[11]
The isopropylidene protection groups of 14–17 were
cleaved in 80% aq. AcOH and the primary alcohols were
protected with DMTCl in CH2Cl2 in the presence of Et3N,
followed by silica gel purification to afford compounds 18–
21 in moderate yields over two steps (65–77%). Phosphityl-
ation of 18–21 by treatment with 2-cyanoethyl-N,N,NЈ,NЈ-
tetraisopropylphosphordiamidite and diisopropylammo-
nium tetrazolide in CH2Cl2 afforded the required phos-
phoramidites 22–25, which were used in the synthesis of
ONs.
Results and Discussion
Synthesis of Phosphoramidites of TINA Monomers
As the planned synthetic route for phosphoramidites of
TINA monomers was intended to be over only five steps,
we decided to synthesize these phosphoramidites. In this
way the previously developed post-synthetic Sonogashira
reaction on the CPG support[9] can be avoided for repetitive
synthesis of the same modification (although for screening
of large numbers of different modifications we still prefer
the post-synthetic approach). Compounds 9 and 10 were
treated with 3- or 4-bromobenzyl bromide under Dean–
Stark conditions (toluene, KOH), affording compounds 11–
13 in quantitative yields (Scheme 2). The degree of conver-
sion in the Sonogashira coupling reaction depended on the
reactivity of the corresponding acetylenes: thus, under the
same conditions [Pd(PPh3)2Cl2, CuI, iPr2NEt, THF, reflux
The insertion of the monomer 1 into ONs had previously
under N2], use of 1-ethynylnaphthalene resulted in the for- been achieved through post-synthetic Sonogashira modifi-
mation of compound 14 in 92% yield after 24 h, whereas cation of ONs immobilized on a CPG solid support com-
with 4-ethynylbiphenyl only very low conversion of starting prising (R)-1-O-(4-iodobenzyl)glycerol through treatment
material was observed by TLC even after 4 d. In the latter with 1-ethynylpyrene.[9] Such post-synthetic derivatization
Scheme 2. Reagents and conditions: a) 4(3)-Bromobenzyl bromide, KOH, toluene, reflux. b) For compounds 14, 16, 17: Ar-1-ylethynyl,
Pd(PPh3)2Cl2, CuI, iPr2NEt, THF, reflux. For compound 15: 4-ethynylbiphenyl, Pd(PPh3)2Cl2, CuI, DMF/NEt3, 80 °C. For compound
17: phenylacetylene, Pd(PPh3)2Cl2, CuI, PPh3, Et2NH/DMF, microwave. c) 80% aq. CH3COOH, room temp. d) DMTCl, NEt3, CH2Cl2,
room temp. e) NC(CH2)2OP(NiPr2)2, diisopropylammonium tetrazolide, CH2Cl2, room temp.
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V. V. Filichev et al.
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of ONs is a good method for screening of different ana- teen-type base pairing in the sequences possessing different
logues, but it is a time-consuming process with already dis- TINAs – both in a parallel triplex fashion toward the du-
covered compounds for routine DNA preparation. More- plex D1 and in a parallel dsDNA fashion toward ON15 –
over, we had observed that post-synthetic Sonogashira was studied (Table 1). The thermal stabilities of single mis-
modification was not efficient with more than two inser- matched parallel triplexes (D2–D4) are listed in Table 2.
tions of (R)-1-O-(4-iodobenzyl)glycerol in the middle of an
We also studied the hybridization affinities of TINAs
ON,[9] so the synthesis of the phosphoramidite of the containing 1, 2, 4, and 6 toward mixed purine/pyrimidine
monomer 1 was needed for routine incorporation into sequences of ssDNA and ssRNA in Watson–Crick-type du-
DNA. Because of the observed low reactivities of bro- plexes (Table 3) with sequences and conditions similar to
mobenzyl derivatives 11–13 in Sonogashira reactions with those previously described.[9]
arylethynyl compounds and problems with isolation of
As can be seen from the Tm data in Table 1, stabilization
products we decided to synthesize the phosphoramidite 28 of parallel triplexes and parallel duplexes [relative to the
in three steps from (R)-1-O-(4-iodobenzyl)glycerol (26,[9] wild-type complexes (ON1)] was observed for the newly
Scheme 3). This last compound was treated with a Sonoga- synthesized monomer 2 (ON3) and for the previously de-
shira reaction mixture containing 1-ethynylpyrene and scribed TINA monomers 1 (ON2) and 3 (ON10) when in-
Pd(PPh3)4 as a catalyst to afford (R)-3-[4-(pyren-1-ylethyn- serted as bulges in the middle of the sequence at pH = 6.0.
yl)benzyloxy]propane-1,2-diol, which was further used Incorporation of the monomer 2 at the 5Ј-end (ON4) gave
without chromatographic purification. Compound 27 was better stabilization of Hoogsteen-type triplexes and du-
obtained in 57% yield (calculated from 26) after protection plexes than 3Ј-end incorporation did (ON5). Interestingly,
of the primary alcohol with DMTCl in dry pyridine and at pH = 7.2 the TFO possessing intercalator 2 at the 5Ј-end
silica gel purification. Overnight treatment of compound 27 (ON4) formed the most stable triplex among all the TFOs
with
2-cyanoethyl-N,N,NЈ,NЈ-tetraisopropylphosphordi- incorporating single insertion of 2. Earlier we had observed
amidite in the presence of diisopropylammonium tetrazol- the contrary effect for the monomer 1.[9] On comparison of
ide in dry CH2Cl2, followed by silica gel column chromatog- the triplex and parallel duplex thermal stabilities of ON3
raphy, gave the final phosphoramidite 28, which was used and ON10 it can be concluded that attachment of a naph-
in a DNA synthesis.
thalene-1-ylethynyl substituent at the 4-position of the
phenyl ring in (R)-1-O-(phenylmethyl)glycerol inserted as a
bulge in the middle of TFO is more efficient than attach-
ment at the 3-position. This conclusion can also be applied
to other aromatic structures, since the same tendency can
be seen for ON11 and ON12, with biphenyl-4-ylethynyl
substituents at 3- and 4-positions, respectively. It is believed
that the acetylene bond improves the intercalating proper-
ties,[12,13] as can also be seen on comparison of the Tm val-
ues for triplexes with ON14 and ON15, with attachment of
the acetylene bond to the phenyl ring resulting in a 2.5 °C
increase in triplex thermal stability. Use of a longer linker,
as in the case of monomer 6, resulted in a slightly decreased
thermal stability relative to the monomer 7. These data sup-
port the general impression that aromatic structures con-
taining more than two fused phenyl rings are best attached
to the 4-positions in (R)-1-O-(phenylmethyl)glycerol com-
ponents inserted as a bulge in the middle of a TFO. Ethynyl
substitution also improves intercalating properties, and
therefore thermal stability, for Hoogsteen-type helixes, and
the terminal acetylene bond is also an attractive functional
group for further post-synthetic chemical modifications
through, for example, “click-chemistry”.[14]
Scheme 3. Reagents and conditions: a) Pyren-1-ylethynyl, Pd-
(PPh3)4, CuI, DMF/NEt3, room temp. b) DMTCl, pyridine, room
temp. c) NC(CH2)2OP(NiPr2)2, diisopropylammonium tetrazolide,
CH2Cl2, 0 °C to room temp.
Thermal Stability Studies
As previously described, a TFO with bulged insertion of
The thermal stabilities of parallel triplexes, DNA/DNA 1 in the middle of the sequence significantly discriminated
and DNA/RNA duplexes, and DNA three-way junctions between matched and mismatched dsDNA (ON2,
with the synthesized ONs were assessed by thermal dena- Table 2),[9] and the same effect was observed with the
turation experiments. The melting temperatures (Tm [°C]), monomer 2 (ON3, Table 2). We decided to evaluate TINA
determined as the first derivatives of melting curves, are monomers in their ability to behave in a universal base
listed in Tables 1, 2, 3, and 4. For comparison we have in- mode.[15] The triplexes formed with ON17 and dsDNA
cluded data for ON2, ON10, ON12, ON15, ON22 and (D1–D4) had very close values of Tm, although these were
ON30, incorporating the previously published TINA very low in comparison with the perfectly matched wild-
monomers 1, 3, 5 and 8.[9] The pH dependence of Hoogs- type triplex (ON1/D1). We believe that this is a result of a
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Twisted Intercalating Nucleic Acids – Influence on Triplex Stabilities
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Table 1. Tm [°C] data for triplex and duplex melting, taken from UV melting curves (λ = 260 nm).
Triplex[a]
3Ј-CTGCCCCTTTCTTTTTT
5Ј-GACGGGGAAAGAAAAAA
(D1)
Parallel duplex[b]
5Ј-GACGGGGAAAGAAAAAA
(ON16)
pH = 6.0
pH = 7.2
pH = 6.0
ON1[c]
ON2[c]
ON3
5Ј-CCCCTTTCTTTTTT
5Ј-CCCCTT1TCTTTTTT
5Ј-CCCCTT2TCTTTTTT
5Ј-2CCCCTTTCTTTTTT
5Ј-CCCCTTTCTTTTTT2
5Ј-2CCCCTT2TCTTTTTT
5Ј-CCCCTTTC2TTTTTT
5Ј-CCCCTTT2CTTTTTT
5Ј-CCC2CTTTCTTTTTT
5Ј-CCCCTT3TCTTTTTT
5Ј-CCCCTT4TCTTTTTT
5Ј-CCCCTT5TCTTTTTT
5Ј-CCCCTT6TCTTTTTT
5Ј-CCCCTT7TCTTTTTT
5Ј-CCCCTT8TCTTTTTT
27.0
46.0
33.0
36.5
33.5
43.0
29.0
33.5
32.5
35.0
23.5
26.0
20.5
23.5
26.0
Ͻ5.0
28.0
11.0
11.5
6.0
22.0
Ͻ5.0
10.5
8.5
13.5
Ͻ5.0
Ͻ5.0
Ͻ5.0
Ͻ5.0
Ͻ5.0
19
33.5
19.5
26.0
25.0
27.5
24.5
22.5
27.5
22.0
10.0
17.0
9.5
ON4
ON5
ON6
ON7
ON8
ON9
ON10[c]
ON11
ON12[c]
ON13
ON14
ON15[c]
11.0
Ͻ5.0
[a] c = 1.5 µ of ON1–15 and 1.0 µ of each strand of dsDNA (D1) in 20 m sodium cacodylate, 100 m NaCl, 10 m MgCl2, pH =
6.0 and 7.2; duplex Tm = 58.5 °C (pH = 6.0) and 57.0 °C (pH = 7.2). [b] c = 1.0 µ of each strand in 20 m sodium cacodylate, 100 m
NaCl, 10 m MgCl2, pH = 6.0. [c] Oligonucleotides and Tm data are from ref.[9]
Table 2. Tm [°C] data for mismatched parallel triplex,[a] taken from UV melting curves (λ = 260 nm).
Sequence
3Ј-CTGCCCCTTXCTTTTTT
5Ј-GACGGGGAAYGAAAAAA
D1:
X·Y = T·A
D2:
A·T
D3:
C·G
D4:
G·C
ON1[b]
ON2[b]
ON3
ON17
ON18
ON19
ON20
5Ј-CCCCTTTCTTTTTT
27.0
46.0
33.0
Ͻ5.0
27.0
15.0
Ͻ5.0
34.5
19.5
8.5
Ͻ5.0
28.5
17.5
5Ј-CCCCTT1TCTTTTTT
5Ј-CCCCTT2TCTTTTTT
5Ј-CCCCTT2CTTTTTT
5Ј-CCCCTT11CTTTTTT
5Ј-CCCCTT11TCTTTTTT
5Ј-CCCCTT111CTTTTTT
10.0
11.0
11.0
32.0[c]
35.0
30.5[c]
28.0
30.0[c]
28.5
Ͻ5.0
30.0[c]
26.0
Ͻ5.0
Ͻ5.0
Ͻ5.0
3Ј-CTGCCX1CTTTCTTX2TTT
5Ј-GACGGY1GAAAGAAY2AAA
D5:
D6:
X1·Y1 = G·C;
X2·Y2 = T·A
14.0
X1·Y1 = C·G;
X2·Y2 = A·T
Ͻ5.0
ON18
ON19
5Ј-CCCCTT11CTTTTTT
5Ј-CCCCTT11TCTTTTTT
15.0
12.5
[a] c = 1.5 µ of ON1–3, ON17–20 and 1.0 µ of each strand of dsDNA (D1–6) in 20 m sodium cacodylate, 100 m NaCl, 10 m
MgCl2, pH = 6.0. [b] Oligonucleotides and Tm data are from ref.[9] [c] Reduced hyperchromicity for triplex transition relative to ON2.
Table 3. Tm [°C] data for Watson–Crick duplexes,[a] taken from UV melting curves (λ = 260 nm).
DNA
RNA
5Ј-AGCUUGCUUGAG
ON33
5Ј-AGCTTGZCTTGAG
ON29:
Z = 0
ON21
3Ј-TCGAACGAACTC
47.5[b]
32.0 (ON30: Z = 1)[b]
43.0 (ON31: Z = 2)
38.0 (ON32: Z = 6)
36.0 (ON30)[b]
40.5[b]
ON22[b]
ON23
ON24
ON25
ON26
ON27
ON28
3Ј-TCGAAC1GAACTC
3Ј-TCGAACGAACTC1
3Ј-TCGAAC11GAACTC
3Ј-TCGAAC2GAACTC
3Ј-TCGAAC4GAACTC
3Ј-TCGAAC6GAACTC
3Ј-TCGAAC7GAACTC
39.5
54.0
31.5
46.5
36.0
41.0
40.5
30.5
45.5
24.0
34.5
28.5
35.0
33.0
36.5 (ON30)
40.0 (ON31)
41.0 (ON32)
[a] c = 1.0 µ of each oligonucleotide in 140 m NaCl, 10 m sodium phosphate buffer, 1 m EDTA, pH = 7.0. [b] Oligonucleotides
and Tm data are from ref.[9]
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Table 4. Tm [°C] data for DNA three-way junction,[a] taken from D4) with ON19 were targeted. To determine the limits of
UV melting curves (λ = 260 nm).
bulged insertions of 1 in the sequence we synthesized oligo-
nucleotide ON20, with triple insertions of 1, and no triplex
formation was detected. From our experience we can
recommend not synthesizing TFOs with the content of
TINA monomer exceeding 30% in the sequence: the high
content of polyaromatic structures results in self aggrega-
tion of the oligonucleotide and inability to target dsDNA.
Bulged insertions of 1 as next-nearest neighbors are also
ON37
ON38
ON39
ON40
inefficient for binding to dsDNA at neutral conditions.[9]
We also studied the hybridization affinities of TINAs
possessing the new bulged intercalating monomers 2, 4, 6,
and 7 in mixed purine/pyrimidine sequences toward ssDNA
and ssRNA in Watson–Crick-type duplexes (Table 3). De-
stabilization of TINA/DNA and TINA/RNA duplexes rela-
tive to the wild-type duplexes was observed for all TINA
monomers as a bulge in the middle of the sequence. Incor-
(Q = A)
(Q = 1)
(Q = 6)
(Q = 7)
ON34
ON35
ON36
36.5
16.5
16.5
52.0
31.5
29.5
48.5
28.5
30.0
46.5
34.0
24.0
[a] c = 1.0 µ of each oligonucleotide in 140 m NaCl, 10 m so-
dium phosphate buffer, 1 m EDTA, pH = 7.0. Tm of the hairpin
(ON34) is 70.0 °C.
helix distortion by the 1-O-methylglycerol linker, which is poration of 1 at the 5Ј-end of the sequence ON23 resulted
too short to mimic the 2Ј-deoxyribofuranose of nucleotides. in stabilization of duplexes toward DNA (∆Tm = +6.5 °C)
On the other hand, the linker has an optimum shape to be and RNA (∆Tm = +5.0 °C), which can be ascribed to the
adopted by the helix when inserted as a bulge. In order stacking of an aromatic structure on the adjacent nucleo-
to stabilize parallel triplexes with single base inversion in base.[21,22] Incorporation of two bulged TINA monomers 1
dsDNA, we synthesized ON18, possessing two monomers in the middle of the Watson–Crick duplex produced further
1 instead of thymidine in the middle of the TFO. Two 1-O- destabilization of the duplex relative to the single bulged 1
methylglycerol linkers can mimic the 2Ј-deoxyribofuranose (ON22/ON29). The placement of the monomer 1 opposite
backbone better than the single insertion. As a result, the double bulged 1 resulted in a formation of a duplex
slightly increased triplex thermal stability, but decreased hy- (ON24/ON30) with increased thermal stability (∆Tm
=
perchromicity for triplex melting were detected for ON18 +5.0 °C) relative to the duplex ON24/ON29. When 3-
with all possible combinations of base pairs in dsDNA rela- (naphthalen-1-ylethynyl)phenyl residue 2 was positioned in
tive to the native triplex (ON1/D1). The observed thermal the middle of the sequence, it gave the least destabilized
stability difference, in a range of 1.5–2.0 °C, is a good indi- duplex among the TINA monomers tested. Interestingly,
cation that double insertion of 1 in the middle of TFO can the placement of two intercalators 2 opposite each other in
be used to bind to dsDNA with single purine/pyrimidine the Watson–Crick helix gave a duplex (ON25/ON31) with
inversion. Since reduced hyperchromicity could be an indi- a Tm value indicating a thermal stability lower than those of
cation of missing base-pairing in some part of the sequence, the corresponding duplexes with unmodified DNAs (ON25/
we checked the thermal stability of ON18 toward dsDNAs ON29 and ON21/ON31). This contrasts with the results ob-
with base inversions three bases away from the incorpora- served with TINA monomers 1 and 6 and is believed to
tion of intercalators (D5 and D6, Table 2). In both cases a be a consequence of the attachment of the naphthalen-1-
lowering of Tm for triplex (∆Tm Ͼ 18.0 °C) was observed. ylethynyl moiety to the 3-position of the phenyl substituent
That confirms that the whole sequence binds to the dsDNA in (R)-1-O-(phenylmethyl)glycerol.
and represents discrimination of binding to matched
As another target in our investigation we chose a DNA
dsDNA over dsDNA containing the mismatch several bases three-way junction (TWJ) composed of two arms linked to
away from the intercalator. The ability to use double inser- a stem (ON34, Table 4).[23] Here we observed considerable
tion of 1 in TFO as a universal base should be especially stabilization with all TINA monomers 1 (ON38), 6 (ON39),
applicable for targeting of dsDNA with TA inversion.[16] and 7 (ON40) upon insertion at the junction point relative
Success in the synthesis of compounds for recognition of to the oligonucleotide with dA at the same position
TA is still limited because such modified bases must avoid (ON37). Interestingly, despite a huge difference (∆Tm
=
the steric clash with the 5-methyl group of thymidine.[17]
22.5–25.5 °C) in the abilities of bulged monomers 1, 6, and
Unlike in the case of TA, there are several analogues 7 to stabilize parallel triplexes, the difference in Tm for TWJ
available for recognition of CG,[18–20] and the thermal sta- stabilization with the same monomers was quite small (∆Tm
bility of this type of triplexes can be further increased by = 3.5–5.5 °C). Unlike in the case of the parallel triplex, the
incorporation of TINA monomer 1 in another part of the use of a longer linker in the monomer 6 (ON39) resulted in
sequence as a bulge. Double bulged insertion of 1 in the stronger stabilization of TWJ in relation to the structure 7
TFO in the sequence ON19 also resulted in a decreased Tm (ON40). To be sure that hybridization in the arms is impor-
of the parallel triplex relative to the ON2/D1, although the tant for the stability of the complex, we prepared oligonu-
triplex was more stable than the wild-type triplex (ON1/ cleotides with mismatches in either arm of the TWJ (ON35
D1). In this case, however, a drop in Tm in the range of 6.5– and ON36). In both cases this resulted in a large lowering
9.0 °C was observed when single mismatched dsDNAs (D2– of the hybridization affinity (∆Tm in a range of 18.5–
3964
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© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2006, 3960–3968

Twisted Intercalating Nucleic Acids – Influence on Triplex Stabilities
FULL PAPER
CH2CHCH2), 4.54 (s, 2 H, OCH2Ar), 7.21 (m, 2 H, Ar), 7.40 (d,
J = 7.5 Hz, 1 H, Ar), 7.49 (s, 1 H, Ar) ppm. 13C NMR (CDCl3): δ
= 25.3, 26.7 (2×CH3), 66.7 [CH2OC(CH3)2], 71.3 (CH2CHCH2),
72.6 (CH2Ar), 74.6 (CH2OCH2Ar), 109.4 [C(CH3)2], 122.5, 126.0,
129.9, 130.5, 130.7, 140.3 (Ar) ppm. MS (EI): m/z = 302
[C13H17O381Br]+, 300 [C13H17O379Br]+.
22.5 °C). This finding confirms the applicability of TINAs
for targeting of DNA hairpins, which may also be useful
in the design of oligonucleotides targeting partially melted
dsDNA, as can occur during DNA replication or transcrip-
tion, such as, for example, in the formation of the “open
complex” initiated by RNA polymerase on dsDNA at the
transcription start.
(S)-4-[2-(4-Bromobenzyloxy)ethyl]-2,2-dimethyl-1,3-dioxolane (12):
This compound was synthesized in a quantitative yield (2.7 g) by
the same procedure as applied for compound 11, from 4-bro-
mobenzyl bromide and 2-[(S)-2,2-dimethyl-1,3-dioxolan-4-yl]etha-
nol (10). 1H NMR (CDCl3): δ = 1.35 (s, 3 H, CH3), 1.40 (s, 3
H, CH3), 1.88 (m, 2 H, CHCH2CH2), 3.57 (m, 3 H, OCH2CH,
CH2CH2O), 4.06 (dd, J = 6.1, 8.1 Hz, 1 H, OCH2CH), 4.21 (m, 1
H, CH2CHCH2), 4.45 (s, 2 H, OCH2Ar), 7.15–7.50 (m, 4 H, Ar)
Conclusions
Several phosphoramidites of twisted intercalating nucleic
acid monomers were synthesized and used in DNA synthe-
sis. In thermal denaturation studies of parallel triplexes sta-
bilized by bulged TINA insertion it was found to be prefer-
able to have more than two fused phenyl rings attached
through a triple bond at the 4-position (rather than connec-
ppm. 13C NMR (CDCl3):
δ = 25.9, 27.0 (2×CH3), 34.0
(CHCH2CH2), 67.3 (CHCH2O), 69.7 (CH2CHCH2), 72.4 (CH2Ar),
73.9 (CH2OCH2Ar), 108.7 [C(CH3)2], 121.5, 129.3, 131.6, 137.5
(Ar) ppm. MS (EI): m/z
=
316 [C14H19O381Br]+, 314
tion at the 3-position) of the (R)-1-O-(phenylmethyl)glyc- [C14H19O379Br]+.
erol component in homopyrimidine ONs. Double insertion
(S)-4-(4-Bromobenzyloxymethyl)-2,2-dimethyl-1,3-dioxolane (13):
This compound was synthesized in a quantitative yield (3.0 g) by
the same procedure as applied for compound 11, from 4-bro-
mobenzyl bromide and [(S)-2,2-dimethyl-1,3-dioxolan-4-yl]meth-
of (R)-1-O-[4-(pyren-1-ylethynyl)phenylmethyl]glycerol (1)
instead of the single nucleotide in the middle of the triplex-
forming oligonucleotide was found to be useful to provide
a universal base with similar thermal stabilities for all pos-
sible matched base-pairing in the dsDNA. Extraordinary
stabilization of a DNA three-way junction was observed
upon insertion of the TINA monomers in the junction re-
gion as a bulge.
1
anol (9). H NMR (CDCl3): δ = 1.36 (s, 3 H, CH3), 1.42 (s, 3 H,
CH3), 3.51 (m, 2 H, CHCH2OCH2), 3.70 (dd, J = 6.3, 8.0 Hz, 1
H, [CH2OC(CH3)2]), 4.04 (dd, J = 6.3, 8.0 Hz, 1 H, [CH2OC-
(CH3)2]), 4.29 (m, 1 H, CH2CHCH2), 4.51 (s, 2 H, OCH2Ar), 7.23
(d, J = 6.4 Hz, 2 H, Ar), 7.46 (d, J = 6.4 Hz, 2 H, Ar) ppm. 13C
NMR (CDCl3): δ = 25.4, 26.7 (2×CH3), 66.7 [CH2OC(CH3)2], 71.2
(CH2CHCH2), 72.7 (CH2Ar), 74.7 (CH2OCH2Ar), 109.4
[C(CH3)2], 121.5, 129.0, 129.2, 131.4, 131.5, 137.0 (Ar) ppm. MS
(EI): m/z = 302 [C13H17O381Br]+, 300 [C13H17O379Br]+.
Experimental Section
General Remarks: NMR spectra were recorded with a Varian Gem-
General Procedure for Sonogashira Coupling: A mixture of aryl bro-
mide (1.42 mmol), Pd(PPh3)2Cl2 (20 mg, 0.03 mmol), CuI (20 mg,
0.11 mmol), iPr2NEt (1.0 mL, 5.7 mmol), and the aromatic struc-
ture containing the terminal acetylene moiety (3.00 mmol) was
heated at reflux under N2 in dry THF (10 mL) for 24 h. The reac-
tion mixture was poured into H2O (50 mL) and extracted with
CH2Cl2 (3×25 mL), and the combined organic layers were washed
with H2O (2×25 mL), dried (MgSO4), filtered, and concentrated
in vacuo. The residue was purified by silica gel column chromatog-
raphy with EtOAc/petroleum ether (1:4, v/v) as an eluent, affording
the target compound. In the cases of compounds 16 and 17 the
final purification was carried out by semipreparative HPLC on a
Delta Pak C18 column (19×300 mm). Buffer A was 100% CH3CN,
buffer B was 10% CH3CN in H2O, the crude product was dissolved
in CH3CN (2 mL) and injected onto the column, and the elution
program was: 0–25 min linear gradient from 50% to 85% of A in
B; 25–30 min with 85% of A in B; 30–35 min linear gradient from
85% to 100% of A in B; flow was 5 mLmin–1, monitoring at
260 nm. The fractions at 36–40 min were concentrated to half the
volume and the product was extracted with CH2Cl2 (2×25 mL).
The combined organic layers were washed with H2O (2×25 mL),
dried (MgSO4), and filtered, and the solvents were evaporated in
vacuo to give the pure products 14, 16, or 17.
ini 2000 spectrometer at 300 MHz for 1H and 75 MHz for 13C.
1
The internal standard used in the H NMR spectra was TMS (δ =
0.00 ppm) in CDCl3, in 13C NMR it was CDCl3 (δ = 77.0 ppm),
and in 31P NMR it was external H3PO4 (δ = 0.00 ppm). Accurate
ion mass determinations were performed with a 4.7 Tesla Ultima
Fourier Transform (FT) mass spectrometer (Ion Spec, Irvine, CA).
The [M+Na]+ ions were peak-matched by use of ions derived from
the 2,5-dihydroxybenzoic acid matrix. EI-MS was performed with
a Finnigan SSQ 710 instrument. Thin layer chromatography (TLC)
analyses were carried out with use of 60 F254 TLC plates purchased
from Merck and were visualized under UV light (254 nm). The
silica gel (0.040–0.063 mm) used for column chromatography was
purchased from Merck. Solvents used for column chromatography
were distilled prior to use, while reagents were used as purchased.
Unmodified ONs were purchased from DNA Technology A/S
(Århus, Denmark) and from TAG Copenhagen A/S (Copenhagen,
Denmark).
(S)-4-(3-Bromobenzyloxymethyl)-2,2-dimethyl-1,3-dioxolane (11):
Pulverized KOH (12.5 g, 220 mmol) and 3-bromobenzyl bromide
(2.5 g, 10 mmol) were added to a solution of [(S)-2,2-dimethyl-1,3-
dioxolan-4-yl]methanol (9, 1.3 g, 10 mmol) in dry toluene
(125 mL). The mixture was heated at reflux under Dean–Stark con-
ditions for 16 h and cooled, and H2O (100 mL) was added. After
separation of the phases, the organic layer was washed with H2O
(3×100 mL), dried (MgSO4), and concentrated under reduced
pressure to give viscous oil 11 in a quantitative yield (3 g). 1H NMR
(CDCl3): δ = 1.37 (s, 3 H, CH3), 1.42 (s, 3 H, CH3), 3.50 (m, 2 H,
CHCH2OCH2), 3.71–3.78 (dd, J = 6.5, 7.6 Hz, 1 H, [CH2OC-
(S)-2,2-Dimethyl-4-[3-(naphthalen-1-ylethynyl)benzyloxymethyl]-
1,3-dioxolane (14): Yield 480 mg, 92%. 1H NMR (CDCl3): δ = 1.37
(s, 3 H, CH3), 1.44 (s, 3 H, CH3), 3.52 (m, 2 H, CHCH2OCH2),
3.76 [dd, J = 6.3, 8.2 Hz, 1 H, CH2OC(CH3)2], 4.07 [dd, J = 6.3,
8.2 Hz, 1 H, CH2OC(CH3)2], 4.34 (m, 1 H, CH2CHCH2), 4.61 (s,
2 H, OCH2Ar), 7.20–8.43 (m, 11 H, Ar) ppm. 13C NMR (CDCl3):
1 H, [CH2OC(CH3)2]), 4.31 (m, 1 H, δ = 25.4, 26.8 (2×CH3), 66.8 [CH2OC(CH3)2], 71.2 (CH2CHCH2),
(CH3)2]), 4.06 (m,
Eur. J. Org. Chem. 2006, 3960–3968
© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
3965

V. V. Filichev et al.
FULL PAPER
73.0 (CH2Ar), 74.7 (CH2OCH2Ar), 87.6, 94.1 (CϵC), 109.5
all the phenylacetylene to have been consumed, an additional quan-
tity of phenylacetylene (93 mg, 0.91 mmol) was injected into the
[C(CH3)2], 120.8, 123.5, 125.2, 126.2–133.2, 138.3 (Ar) ppm. HR-
MALDI-MS: m/z calcd. for C25H24NaO3 [M +Na]+ 395.1617; vial with the reaction mixture, followed by further stirring at 120 °C
+
found 395.1614.
under nitrogen in the microwave cavity for 25 min. The reaction
mixture was cooled and poured into aq. HCl (0.1 , 10 mL) and
extracted with diethyl ether (3×10 mL), and the combined organic
layer was washed with saturated aq. NaHCO3 (10 mL) and H2O
(10 mL). The combined water phase was re-extracted with diethyl
ether (2×10 mL), the combined organic layers were concentrated
under diminished pressure, and the residue was purified by silica
gel column chromatography with EtOAc/petroleum ether (1:4,
v/v), affording compound 17 together with impurities. The final
purification of 17 was carried out by semipreparative HPLC as
described in the general procedure for Sonogashira coupling. Yield
98 mg, 33%.
(S)-2,2-Dimethyl-4-[2-(4-phenylethynylbenzyloxy)ethyl]-1,3-dioxol-
ane (16): Yield 321 mg, 23% by the general procedure for Sonoga-
shira coupling described above. H NMR (CDCl3): δ = 1.36 (s, 3
1
H, CH3), 1.41 (s, 3 H, CH3), 1.90 (m, 2 H, CHCH2CH2), 3.59 (m,
3 H, OCH2CH, CH2CH2O), 4.06 [m, 1 H, CH2OC(CH3)2], 4.22
(m, 1 H, CH2CHCH2), 4.51 (s, 2 H, OCH2Ar), 7.20–7.60 (m, 9
H, Ar) ppm. 13C NMR (CDCl3): δ = 25.7, 26.9 (2×CH3), 33.8
(CHCH2CH2), 67.2 (CHCH2O), 69.5 (CH2CHCH2), 72.3 (CH2Ar),
73.7 (CH2OCH2Ar), 89.2, 89.3 (CϵC), 108.7 [C(CH3)2], 122.4,
123.2, 128.2, 127.4–131.6, 137.3, 138.6 (Ar) ppm. HR-MALDI-
MS: m/z calcd. for C22H24NaO3 [M + Na]+ 359.1617; found
+
359.1622.
General Procedure for Isopropylidene Group Cleavage and DMT
Protection: The 2,2-dimethyl-4-substituted-1,3-dioxolane was
stirred in aq. AcOH (80%, 10–20 mL) at room temp. for 24 h and
the mixture was then concentrated under reduced pressure. The
residue was co-evaporated twice with toluene/EtOH (25 mL, 1:1,
v/v), giving an oily product which was dried in vacuo overnight
and used in the next step without further purification. 4,4Ј-Dimeth-
oxytrityl chloride (1.1 equiv.) was added to a solution of diol
(1 equiv.) and dry Et3N (2 equiv.) in dry CH2Cl2 (10 mL). The reac-
tion mixture was stirred at room temp. under nitrogen overnight,
quenched with MeOH (2 mL) and concentrated in vacuo. The resi-
due was purified by silica gel column chromatography with NEt3
(2%, v/v)/EtOAc (30%)/petroleum ether. UV-active fractions were
collected and concentrated under diminished pressure, affording
the DMT-protected diols 18–21.
(S)-4-[3-(Biphenyl-4-ylethynyl)benzyloxymethyl]-2,2-dimethyl-1,3-di-
oxolane (15): 4-Ethynylbiphenyl (0.51 g, 2.8 mmol), Pd(PPh3)2Cl2
(40 mg, 0.057 mmol), CuI (27 mg, 0.14 mmol), and Et3N
(0.594 mL, 4.26 mmol) were added to a solution of compound 11
(0.43 g, 1.42 mmol) in dry DMF (50 mL) and 1,4-dioxane (20 mL).
The reaction mixture was heated at 80 °C under nitrogen for 3 d,
and was then allowed to cool to room temp., diluted with CH2Cl2
(150 mL), and washed with a aq. solution of EDTA ammonium
salt (0.3 , 100 mL) and H2O (5×100 mL). The organic layer was
dried (Na2SO4), filtered, and concentrated in vacuo, and the resi-
due was purified by silica gel column chromatography with EtOAc/
petroleum ether (1:8, v/v), affording compound 15 (0.156 g, 28%).
1H NMR (CDCl3): δ = 1.38 (s, 3 H, CH3), 1.49 (s, 3 H, CH3), 3.53
(m, 2 H, CHCH2OCH2), 3.76 [t, J = 7.2 Hz, 1 H, CH2OC(CH3)2],
4.08 [t, J = 7.2 Hz, 1 H, CH2 OC(CH3 )2 ], 4.31 (m, 1 H,
CH2CHCH2), 4.58 (s, 2 H, OCH2Ar), 7.30–7.60 (m, 13 H, Ar)
ppm. 1 3 C NMR (CDCl3 ): δ = 25.4, 26.8 (2 × CH3 ), 66.8
[CH2OC(CH3)2], 71.3 (CH2CHCH2), 73.0 (CH2Ar), 74.7 (CH2O-
CH2Ar), 89.4, 89.9 (CϵC), 109.5 [C(CH3)2], 122.1, 123.4, 127.0–
132.0, 138.3, 140.3, 141.0 (Ar) ppm. HR-MALDI-MS: m/z calcd.
(S)-1-O-(4,4Ј-Dimethoxytrityl)-3-O-[3-(naphthalen-1-ylethynyl)phen-
1
ylmethyl]glycerol (18): Yield 350 mg, 65%. H NMR (CDCl3): δ =
2.44 (d, J = 4.7 Hz, 1 H, OH), 3.23 (m, 2 H, CHCH2OCH2), 3.53
(m, 2 H, CH2ODMT), 3.74 (s, 6 H, 2 × OCH3), 4.02 (m, 1 H,
CHOH), 4.56 (s, 2 H, OCH2Ph), 6.78 (d, J = 8.5 Hz, 4 H, DMT),
7.20–8.43 (m, 20 H, DMT + Ph + naphthalene) ppm. 13C NMR
(CDCl3): δ = 55.1 (OCH3), 64.2 (CH2ODMT), 70.0 (CH2CHOH),
71.7 (CHOH), 72.9 (CH2Ph), 86.1 (CAr3), 87.6, 94.1 (CϵC), 113.1,
120.8, 123.5, 125.2–133.2, 135.9, 138.4, 144.8, 158.4 (Ph, naphtha-
+
for C27H26NaO3 [M+Na]+ 421.1774; found 421.1761.
(S)-2,2-Dimethyl-4-(3-phenylethynylbenzyloxy)methyl-1,3-dioxolane
(17). Method A: Yield 105 mg, 18% by the general procedure for
Sonogashira coupling described above. 1H NMR (CDCl3): δ = 1.39
(s, 3 H, CH3), 1.45 (s, 3 H, CH3), 3.50 (m, 2 H, CHCH2OCH2),
3.73 [dd, J = 6.5, 8.0 Hz, 1 H, CH2OC(CH3)2], 4.05 [t, J = 6.5,
8.0 Hz, 1 H, CH2OC(CH3)2], 4.30 (m, 1 H, CH2CHCH2), 4.58 (s,
2 H, OCH2Ar), 7.29–7.54 (m, 9 H, Ar) ppm. 13C NMR (CDCl3):
δ = 25.3, 26.7 (2×CH3), 66.7 [CH2OC(CH3)2], 71.2 (CH2CHCH2),
73.0 (CH2Ar), 74.7 (CH2OCH2Ar), 89.1, 89.4 (CϵC), 109.4
[C(CH3)2], 122.5, 123.2, 127.5, 128.2, 128.3, 131.6, 138.2 (Ar) ppm.
+
lene, DMT) ppm. HR-MALDI-MS: m/z calcd. for C43H38NaO5
[M + Na]+ 657.2611; found 657.2601.
(S)-3-O-[3-(Biphenyl-4-ylethynyl)phenylmethyl]-1-O-(4,4Ј-dimeth-
1
oxytrityl)glycerol (19): Yield 240 mg, 71%. H NMR (CDCl3): δ =
2.50 (brs, 1 H, OH), 3.24 (m, 2 H, CHCH2OCH2), 3.63 (m, 2 H,
CH2ODMT), 3.76 (s, 6 H, 2×OCH3), 4.01 (m, 1 H, CHOH), 4.53
(s, 2 H, OCH2Ph), 6.81 (d, J = 8.5 Hz, 4 H, DMT), 7.17–7.62 (m,
22 H, DMT + Ph + biphenyl) ppm. 13C NMR (CDCl3): δ = 55.1
(OCH3), 64.3 (CH2ODMT), 69.9 (CH2CHOH), 71.7 (CHOH),
72.9 (CH2Ph), 86.1 (CAr3), 89.4, 89.9 (CϵC), 113.1, 122.1, 123.4,
126.8–133.0, 135.9, 138.3, 140.2, 144.8, 158.4 (Ph, biphenyl, DMT)
HR-MALDI-MS: m/z calcd. for C21H22NaO3 [M + Na]+
+
345.1461; found 345.1447. Method B: Compound 13 (270 mg,
0.90 mmol), Pd(PPh3)2Cl2 (35 mg, 0.05 mmol), CuI (12 mg,
0.06 mmol), PPh3 (50 mg, 0.20 mmol) and phenylacetylene
(115 mg, 1.13 mmol) were dissolved in diethylamine (1.5 mL,
13.60 mmol) and DMF (0.5 mL), and the reaction mixture was
stirred at 120 °C under nitrogen in the microwave cavity for 25 min.
The reaction was checked by analytical HPLC on a Delta Pak C18
column (3.9×300 mm) with the following program: 0–5 min elu-
tion with 30% buffer A in B, 5–10 min linear gradient from 30%
to 15% of A in B, 10–15 min of 15% of A in B, 15–20 min of linear
gradient from 15% to 0% of A in B, 20–25 min of the linear gradi-
ent from 0% to 30% of A in B, where buffer A was 20% of CH3CN
in H2O and buffer B was 80 % of CH3CN in H2O, flow was
1 mLmin–1, monitoring at 260 nm. Since HPLC analysis showed
ppm. HR-MALDI-MS: m/z calcd. for C45H40NaO5 [M+ Na]+
+
683.2768; found 683.2740.
(S)-1-(4,4Ј-Dimethoxytrityloxy)-4-[4-(phenylethynyl)phenylmeth-
oxy]butan-2-ol (20): Yield 230 mg, 77%. 1H NMR (CDCl3): δ =
1.79 (m, 2 H, CH2CH2O), 2.70 (brs, 1 H, OH), 3.13 (m, 2 H,
CH2CH2O), 3.59 (m, 2 H, CH2ODMT), 3.77 (s, 6 H, 2×OCH3),
3.90 (m, 1 H, CHOH), 4.46 (s, 2 H, OCH2Ph), 6.81 (d, J = 8.5 Hz,
4 H, DMT), 7.10–7.60 (m, 18 H, DMT + Ph) ppm. 13C NMR
(CDCl3): δ = 33.5 (CH2CH2O), 55.2 (OCH3), 67.3 (CH2OCH2Ph),
68.0 (CH2ODMT), 69.5 (CH2Ph), 72.8 (CHOH), 86.0 (CAr3), 89.2,
89.3 (CϵC), 113.1, 122.5, 123.3, 126.7–131.6, 136.1, 138.4, 144.9,
3966
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© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Twisted Intercalating Nucleic Acids – Influence on Triplex Stabilities
158.5 (Ph, DMT) ppm. HR-MALDI-MS: m/z calcd. for
FULL PAPER
(pyren-1-ylethynyl)phenylmethyl]glycerol as an oil (3.1 g). The oil
was co-evaporated with pyridine (20 mL) and then dissolved in
anh. pyridine (50 mL), cooled in an ice/water bath, and 4,4Ј-di-
methoxytrityl chloride (1.45 g, 4.41 mmol) was added under argon.
The reaction mixture was stirred at room temp. for 16 h and an
extra portion of 4,4Ј-dimethoxytrityl chloride (0.5 g, 1.5 mmol) was
then added. After 24 h, TLC showed no more starting material and
the reaction mixture was quenched with MeOH (2 mL), diluted
with EtOAc (150 mL), and extracted with satd. aq. NaHCO3
(2 × 100 mL). The water phase was extracted with EtOAc
(2×50 mL) and the combined organic layers were dried (Na2SO4),
filtered, and concentrated under diminished pressure. The residue
was co-evaporated twice with toluene/EtOH (25 mL, 1:1, v/v). The
residue was adsorbed from EtOAc (50 mL) on silica gel (2.0 g) and
purified by silica gel dry column vacuum chromatography with
EtOAc (0–100%, v/v) in cyclohexane to afford 27 (1.75 g, 60%) as
C40H38NaO5 [M+Na]+ 621.2611; found 621.2606.
+
(S)-1-O-(4,4Ј-Dimethoxytrityl)-3-O-[4-(phenylethynyl)phenylmeth-
yl]glycerol (21): Yield 126 mg, 61%. 1H NMR (CDCl3): δ = 2.46
(br s, 1 H, OH), 3.22 (m, 2 H, CHCH2OCH2), 3.58 (m, 2 H,
CH2ODMT), 3.75 (s, 6 H, 2×OCH3), 3.95 (m, 1 H, CHOH), 4.53
(s, 2 H, OCH2Ph), 6.81 (d, J = 8.5 Hz, 4 H, DMT), 7.14–7.54 (m,
18 H, DMT + Ph) ppm. 13C NMR (CDCl3): δ = 55.2 (OCH3), 64.3
(CH2ODMT), 69.9 (CH2CHOH), 71.6 (CHOH), 72.9 (CH2Ph),
86.1 (CAr3), 89.2, 89.4 (CϵC), 113.1, 122.5, 123.2, 126.7–131.6,
135.9, 138.2, 144.8, 158.4 (Ph, naphthalene, DMT) ppm. HR-
+
MALDI-MS: m/z calcd. for C39H36NaO5 [M +Na]+ 607.2455;
found 607.2456.
General Procedure for Phosphitylation: DMT-protected diol
(1 equiv.) was dissolved under nitrogen in anh. CH2Cl2 (10–50 mL).
Diisopropylammonium tetrazolide (1.6 equiv.) was added, followed
by dropwise addition of 2-cyanoethyl tetraisopropylphosphordiam-
idite (2 equiv.) with external cooling with an ice/water bath. After
24 h, analytical TLC showed no more starting material and the
reaction was quenched with H2O (10–30 mL). The layers were sep-
arated and the organic phase was washed with H2O (10–30 mL),
the combined water layers were washed with CH2Cl2 (25 mL), the
organic phase was dried (Na2SO4) and filtered, and the solvents
were evaporated in vacuo. The residue was purified by silica gel
column chromatography with NEt3 (0.5–2%, v/v)/EtOAc (0–25%)/
cyclohexane and the combined UV-active fractions were concen-
trated in vacuo to afford the final compound, which was used in
DNA synthesis after drying under diminished pressure.
1
a yellow foam. H NMR (CDCl3): δ = 2.48 (d, 1 H, J = 5.0 Hz,
OH), 3.24 [m, 2 H, CH(OH)CH2 OCH2 ], 3.31 (m, 2 H,
CH2ODMT), 3.78 (s, 6 H, 2×OCH3), 4.00 (m, 1 H, CHOH), 4.58
(s, 2 H, CH2Ar), 6.80 (d, 4 H, J = 8.5 Hz, DMT), 7.10–7.45 (m,
11 H, DMT), 7.72 (d, 2 H, J = 8.0 Hz, phenyl), 8.00–8.30 (m, 9
H, pyren-1-yl) ppm. 13C NMR (CDCl3): δ = 55.2 (OCH3), 64.3
(CH2ODMT), 70.0 [CH(OH)CH2OCH2], 71.7 (CHOH), 72.9
(CH2-phenyl), 86.1 [C(Ar)3], 88.7, 94.9 (CϵC), 117.7, 127.7, 138.5,
139.4 (phenyl), 113.1, 124.5–131.8, 136.0, 144.8, 158.5 (DMT,
+
pyren-1-yl) ppm. HR-MALDI-MS: m/z calcd. for C49H40NaO5
[M+Na]+ 731.2768; found 731.2739.
(S)-2-O-[(2-Cyanoethoxy)(diisopropylamino)phosphanyl]-1-O-(4,4Ј-
dimethoxytrityl)-3-O-[4-(1-pyrenylethynyl)phenylmethyl]glycerol
(28): (S)-1-O-(4,4Ј-Dimethoxytrityl)-3-O-[4-(1-pyrenylethynyl)-
phenylmethyl]glycerol (27, 1.7 g, 2.4 mmol) was dissolved under ar-
gon in anh. CH2Cl2 (50 mL). Diisopropylammonium tetrazolide
(0.620 g, 3.6 mmol) was added, followed by dropwise addition of
2-cyanoethyl tetraisopropylphosphordiamidite (1.150 g, 3.8 mmol)
with external cooling with an ice/water bath. After 24 h, analytical
TLC showed no more starting material and the reaction was
quenched with H2O (30 mL). The layers were separated, the or-
ganic phase was washed with H2O (30 mL), and the combined
water layers were washed with CH2Cl2 (25 mL). The organic phase
was dried (Na2SO4) and filtered, silica gel (1.5 g) and pyridine
(0.5 mL) were added, and the solvents were removed under reduced
pressure. The residue was purified by silica gel dry column vacuum
chromatography with NEt3 (0.5%, v/v)/EtOAc (0–25%)/cyclohex-
ane and the combined UV-active fractions were concentrated in
vacuo to afford the final compound 28 (1.8 g, 83%) as a foam that
was used in DNA synthesis. 32P NMR (CDCl3): δ = 150.3,
(S)-2-O-[(2-Cyanoethoxy)(diisopropylamino)phosphanyl]-1-O-(4,4Ј-
dimethoxytrityl)-3-O-[3-(naphthalen-1-ylethynyl)phenylmethyl]gly-
cerol (22): Yield 290 mg, 83 %. 32P NMR (CDCl3) δ = 150.2,
150.4 ppm in a 5:4 ratio. HR-MALDI-MS: m/z calcd. for
C52H55N2NaO6P+ [M+Na]+ 857.3690; found 857.3684.
(S)-3-O-[3-(Biphenyl-4-ylethynyl)phenylmethyl]-2-O-[(2-cyano-
ethoxy)(diisopropylamino)phosphanyl]-1-O-(4,4Ј-dimethoxytrityl)-
glycerol (23): Yield 115 mg, 57%. 32P NMR (CDCl3) δ = 150.3,
150.4 ppm in a ratio 3:2. HR-MALDI-MS: m/z calcd. for
C54H57N2NaO6P+ [M+Na]+ 883.3846; found 883.3865.
(S)-2-[(2-Cyanoethoxy)(diisopropylamino)phosphanyloxy]-1-(4,4Ј-di-
methoxytrityloxy)-4-[4-(phenylethynyl)phenylmethoxy]butane (24):
Yield 260 mg, 90%. 32P NMR (CDCl3): δ = 149.3, 149.7 ppm in a
3:2 ratio. HR-MALDI-MS: m/z calcd. for C49H55N2NaO6P+
[M+Na]+ 821.3690; found 821.3721.
(S)-2-O-[(2-Cyanoethoxy)(diisopropylamino)phosphanyl]-1-O-(4,4Ј-
dimethoxytrityl)-3-O-[4-(phenylethynyl)phenylmethyl]glycerol (25): 150.5 ppm in a 3:2 ratio. HR-MALDI-MS: m/z calcd. for
Yield 139 mg, 82%. 32P NMR (CDCl3): δ = 150.3, 150.4 ppm in a
5:4 ratio. HR-MALDI-MS: m/z calcd. for C48H53N2NaO6P+
[M+Na]+ 807.3533; found 807.3498.
C58H57N2NaO6P+ [M+Na]+ 931.3846; found 931.3814.
Synthesis and Purification of TINAs: ONs were synthesized with an
Expedite Nucleic Acid Synthesis System Model 8909 from Ap-
(S)-1-O-(4,4Ј-Dimethoxytrityl)-3-O-[4-(pyren-1-ylethynyl)phenyl- plied Biosystems with 4,5-dicyanoimidazole as an activator. An in-
methyl]glycerol (27): A solution of (R)-3-(4-iodobenzyloxy)pro- creased deprotection time (100 s) and coupling time (2 min) for
pane-1,2-diol (26,[9] 1.18 g, 4.2 mmol) in DMF (40 mL) and Et3N 0.075  solutions of the phosphoramidites 22–25 and 28 in 1:1 mix-
(5.8 mL) was bubbled with argon for 30 min. Afterwards, 1-ethyn-
ylpyrene (1.05 g, 4.65 mmol) was dissolved under argon, and CuI
(56 mg, 0.3 mmol) and Pd(PPh3)4 (125 mg, 0.11 mmol) were added
to the solution. The reaction mixture was stirred at room temp.
under argon overnight, followed by the addition of CH2Cl2
(150 mL) and extraction with an aq. solution of EDTA ammonium
salt (0.3 , 2×75 mL). The organic layer was washed with H2O
(3×75 mL), dried (Na2SO4), and filtered, and the solvents were
evaporated to dryness in vacuo. The residue was co-evaporated
twice with toluene/EtOH (30 mL, 1:1, v/v), affording (R)-1-O-[4-
tures of dry MeCN/CH2Cl2 were applied. The 5Ј-DMT-on ONs
were cleaved from the solid support (room temp., 2 h) and depro-
tected (55 °C, overnight) by treatment with 32% aqueous ammo-
nia. Purification of 5Ј-O-DMT-on TINAs was accomplished by re-
versed-phase semipreparative HPLC on a Waters Xterra MS C18
column. Buffer A [0.05  triethylammonium acetate in H2O (pH
= 7.0)] and buffer B (75% CH3CN in H2O). Flow 2.5 mLmin–1.
Gradients: 2 min 100% A, linear gradient to 70% B in 38 min,
linear gradient to 100% B in 7 min, 100% B in 3 min and then
100% A in 10 min. The fractions containing the ONs eluted at 25–
Eur. J. Org. Chem. 2006, 3960–3968
© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
3967

V. V. Filichev et al.
FULL PAPER
[2] M. P. Knauert, P. Glazer, Hum. Mol. Genet. 2001, 10, 2243–
2251.
[3] M. Faria, C. Giovannageli, J. Gene Med. 2001, 3, 299–310.
[4] P. E. Nielsen, M. Egholm, R. Berg, O. Buchardt, Science 1991,
254, 1497–1500.
[5] S. Obika, Y. Hari, T. Sugimoto, M. Sekiguchi, T. Imanishi,
Tetrahedron Lett. 2000, 41, 8923–8927.
[6] B.-W. Sun, B. R. Babu, M. D. Sørensen, K. Zakrzewska, J.
Wengel, J.-S. Sun, Biochemistry 2004, 43, 4160–4169.
[7] M. J. J. Blommers, F. Natt, W. Jahnke, B. Cuenoud, Biochemis-
try 1998, 37, 17714–17725.
30 min and were concentrated, and the DMT group was cleaved by
treatment with aq. AcOH (80%, 100 µL) over 20 min, followed by
dilution with aq. NaOAc (1 , 150 µL) and precipitation of the
ONs from chilled EtOH (550 µL). The modified ONs were con-
firmed by MALDI-TOF analysis with a PerSeptive Biosystems
Voyager Elite Biospectrometry Research Station (see Supporting
Information). The purities of the final ONs were found to be Ͼ
85% by analytical ion-exchange chromatography with a Merck Hit-
achi LaChrom system on a GenPak-Fax column (Waters).
Melting Temperature Measurements: Melting temperature measure-
ments were performed with a Perkin–Elmer Lambda 35 UV/Vis
spectrometer with a PTP-6 temperature programmer. The triplexes
were formed by first mixing the two strands of the Watson–Crick
duplex, each at a concentration of 1.0 µ in the corresponding
buffer solution. The solution was heated to 80 °C for 5 min and
then cooled to room temp., and the third (TFO) strand was added
and then kept at 15 °C for 30 min. The duplexes were formed by
mixing the two strands, each at a concentration of 1.0 µ, in the
corresponding buffer solution, followed by heating to 70 °C for
5 min and then cooling to room temp. The melting temperatures
(Tm [°C]) were determined as the maxima of the first derivative
plots of the melting curves obtained by measuring absorbance at
260 nm against increasing temperature (1.0 °C per 1 min). Use of
a lower rate of temperature increase (0.5 °C per 1 min) resulted in
the same curves. Experiments were also performed at 373 nm for
ONs possessing monomer 1. All melting temperatures are within
the uncertainty of 0.5 °C as determined by repetitive experiments.
[8] S. M. Gryaznov, D. H. Lloyd, J. K. Chen, R. G. Schulz, L. A.
Dedionisio, L. Ratmeyer, W. D. Wilson, Proc. Natl. Acad. Sci.
USA 1995, 92, 5798–5802.
[9] V. V. Filichev, E. B. Pedersen, J. Am. Chem. Soc. 2005, 127,
14849–14858.
[10] M. Erdelyi, A. Gogoll, J. Org. Chem. 2001, 66, 4165–4169.
[11] A. R. Gholap, K. Venkatesan, R. Pasricha, T. Daniel, R. J. La-
hoti, K. V. Srinivasan, J. Org. Chem. 2005, 70, 4869–4872.
[12] B. C. Froehler, S. Wadwani, T. J. Terhorst, S. R. Gerrard, Tetra-
hedron Lett. 1992, 33, 5307–5310.
[13] N. Colocci, P. B. Dervan, J. Am. Chem. Soc. 1994, 116, 785–
786.
[14] V. D. Bock, H. Hiemstra, J. H. van Maarseveen, Eur. J. Org.
Chem. 2006, 51–68.
[15] D. Loakes, Nucleic Acids Res. 2001, 29, 2437–2447.
[16] D. Guianvarc’h, R. Benhida, J. L. Fourrey, R. Maurisse, J. S.
Sun, Chem. Commun. 2001, 1814–1815.
[17] D. M. Gowers, K. R. Fox, Nucleic Acids Res. 1999, 27, 1569–
1577.
[18] C.-Y. Huang, C. D. Cushman, P. S. Miller, J. Org. Chem. 1993,
58, 5048–5049.
Supporting Information (see footnote on the first page of this arti-
cle): MALDI-TOF-MS of synthesized ONs (Table S1).
[19] I. Prevot-Halter, C. J. Leumann, Bioorg. Med. Chem. Lett.
1999, 9, 2657–2660.
[20] R. T. Ranasinghe, D. Rusling, V. E. C. Powers, K. R. Fox, T.
Brown, Chem. Commun. 2005, 2555–2557.
[21] R. X.-F. Ren, N. C. Chaudhuri, P. L. Paris, I. S. Rumney, E. T.
Kool, J. Am. Chem. Soc. 1996, 118, 7671–7678.
[22] K. M. Guckian, B. A. Schweitzer, R. X.-F. Ren, C. J. Sheilis,
P. L. Paris, D. C. Tahmassebi, E. T. Kool, J. Am. Chem. Soc.
1996, 118, 8182–8183.
Acknowledgments
The Nucleic Acid Center is funded by the Danish National Re-
search Foundation for studies on nucleic acid chemical biology.
[23] V. V. Filichev, E. B. Pedersen, Org. Biomol. Chem. 2003, 1, 100–
103.
[1] T. G. Uil, H. J. Haisma, M. G. Rots, Nucleic Acids Res. 2003,
31, 6064–6078.
Received: February 27, 2006
Published Online: July 3, 2006
3968
www.eurjoc.org
© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2006, 3960–3968