Intercalating nucleic acids (INAs) with insertion of N-(pyren-1-ylmethyl)-(3R,4R)-4-(hydroxymethyl)pyrrolidin-3-ol. DNA (RNA) duplex and DNA three-way junction stabilities

Article abstract of DOI:10.1039/b210335b

N-(Pyren-1-ylmethyl)-(3R,4R)-4-(hydroxymethyl)pyrrolidin-3-ol was synthesised from (3R,4R)-4-(hydroxymethyl)pyrrolidin-3-ol and (3R,4S)-4-[(1S)-1,2-dihydroxyethyl]pyrrolidin-3-ol using alkylation with 1-(chloromethyl)pyrene or reductive amination with pyrene-1-carbaldehyde and NaCNBH₃. The incorporation of N-(pyren-1-ylmethyl)azasugar moiety into oligodeoxynucleotides (ODN) as a bulge to form an intercalating nucleic acid (INA) induced a slight destabilization of INA-DNA duplex, whereas the INA-RNA duplex was strongly destabilized and 9°C difference per modification in thermal stability between INA-DNA over INA-RNA duplexes was observed. The stabilization of a DNA three way junction (TWJ) was improved when the intercalator moiety was inserted into the junction region as a bulge.

Full text of DOI:10.1039/b210335b

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Intercalating nucleic acids (INAs) with insertion of
N-(pyren-1-ylmethyl)-(3R,4R)-4-(hydroxymethyl)pyrrolidin-3-ol.
DNA (RNA) duplex and DNA three-way junction stabilities
Vyacheslav V. Filichev and Erik B. Pedersen*
Nucleic Acid Center†, Department of Chemistry, University of Southern Denmark,
DK-5230 Odense M, Denmark. E-mail: EBP@chem.sdu.dk;
Fax: ϩ45 6615 8780; Tel: ϩ45 6550 2555
Received 21st October 2002, Accepted 7th November 2002
First published as an Advance Article on the web 29th November 2002
N-(Pyren-1-ylmethyl)-(3R,4R)-4-(hydroxymethyl)pyrrolidin-3-ol was synthesised from (3R,4R)-4-
(hydroxymethyl)pyrrolidin-3-ol and (3R,4S)-4-[(1S)-1,2-dihydroxyethyl]pyrrolidin-3-ol using alkylation with 1-
(chloromethyl)pyrene or reductive amination with pyrene-1-carbaldehyde and NaCNBH3. The incorporation of N-
(pyren-1-ylmethyl)azasugar moiety into oligodeoxynucleotides (ODN) as a bulge to form an intercalating nucleic
acid (INA) induced a slight destabilization of INA–DNA duplex, whereas the INA–RNA duplex was strongly
destabilized and 9 ЊC difference per modification in thermal stability between INA–DNA over INA–RNA duplexes
was observed. The stabilization of a DNA three way junction (TWJ) was improved when the intercalator moiety was
inserted into the junction region as a bulge.
often believed. It has actually been shown that recognising the
1. Introduction
structure of nucleic acids could be achieved by forming a
The stabilized formation of the secondary structures of nucleic
three way junction (TWJ) without disruption of the stem.10
acids with chemically modified nucleosidic units is the main
Formation of the TWJ has been confirmed in NMR studies on
goal at the transcriptional (antigene) and translational (anti-
complexes with two unpaired bases at the branch point and a
sense) level of the gene expression.1 The increased affinity and
sequence selectivity are desirable properties to achieve a good
recognition for therapeutic targets. It is known that there are
some differences in the three-dimensional structures of DNA–
preferred coaxial base stacking interaction was indicated.11
Based on these findings we started out investigations devoted
to the synthesis of pyren-1-yl units linked to different sugar-
mimicking fragments which were used for insertions into ODNs
DNA and DNA–RNA duplexes. By the chemical modification
for hybridization studies.12
of nucleosidic glycons some oligonucleotide analogues having
Recently we synthesised the enantiomerically pure 1-aza
better increased affinity towards single stranded RNA (ssRNA)
analogue of 2-deoxy--ribofuranose having a nitrogen instead
and towards single stranded DNA (ssDNA) were discovered,
of the anomeric carbon and a methylene group instead of the
for example locked nucleic acids (LNA),2 hexitol nucleic acids
ring oxygen.13 The corresponding 1Ј-aza-C-pseudonucleosides
(HNA)3 and others.4 On the other hand all nucleic acids are
in which a carbon in the heterocyclic base is linked to the
stabilized both by hydrogen bonding interactions and by base
nitrogen of (3R,4R)-4-(hydroxymethyl)pyrrolidin-3-ol were
stacking.1 Therefore polyaromatic units with hydrophobicity in
also obtained.14 In continuation of this we now investigate the
conjunction with a large surface area can be used in place of the
synthesis of N-pyrenemethyl type pseudonucleoside of the
enantiomerically pure 1-aza analogue of 2-deoxy--ribo-
natural nucleic bases for mimicking base stacking in Watson–
Crick/Hoogsteen base pairing.5 The design and the synthesis of
furanose and its insertion into several ODNs to form INAs in
various oligonucleotides containing planar polycyclic aromatic
order to investigate their thermal stability when forming
chromophores such as pyrene, anthracene, dansyl and others,
duplexes with complementary ssDNA and ssRNA or when
which have a long fluorescence lifetime and possibilities to
forming DNA TWJs.
form π-stacking interactions in aqueous solutions, have been
the subject of active research in recent years.6 However there are
2. Results and discussion
only a few articles focused on the possible discrimination
between ssDNA and ssRNA with the modification or sub-
stitution of nucleic bases in the complementary sequences.
Bleczinski et al. observed the DNA/RNA discrimination by
insertion of cholic acid and its deoxy derivatives into the
5Ј-termini of short oligonucleotides.7 Recently we have found
that intercalating nucleic acids (INAs) containing 1-O-(pyrenyl-
methyl)glycerol insertions have a strong preference for hybrid-
ization with DNA over RNA,8 but little is known about the
requirements of the backbone structure for the selectivity
between DNA and RNA.
The synthesis of 1Ј-aza pyrenemethyl pseudonucleoside 4
started from the previously published enantiomerically pure
1-aza analogues of 2-deoxy--hexofuranose 1 or 2-deoxy--
ribofuranose 2.13 To find the most efficient synthesis of the
target compound 4, we used pyrene substrates having
chloromethyl and carbaldehyde functionalities that could be
coupled with the secondary amines 1 and 2. We found that
alkylation by 1-(chloromethyl)pyrene in the presence of Et3N
led to a lower yield (57% and 46% for 3 and 4, respectively)
compared to reductive amination with pyrene-1-carbaldehyde
and NaCNBH3 (63% and 57%, respectively, Scheme 1). Surpris-
ingly low overall yield 40% was observed in the synthesis of 4
using oxidative cleavage of the diol 3 followed by reduction of
the obtained aldehyde. This may be due to low stability of the
pyren-1-ylmethyl azasugars 3 and 4, which should be stored in a
refrigerator under nitrogen or used immediately to avoid low
yields in subsequent reactions.
Single stranded oligonucleotides are often written as single
stretches, but because of self pairing, branched structures
(hairpins, internal loops, junctions) have been observed.9 For
this reason, the number of possible targets is lower than it is
† A research center funded by The Danish National Research
Foundation for studies on nucleic acid chemical biology
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 1 0 0 – 1 0 3
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 3
100

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Table 1 Sequences and hybridization data of synthesised ODNs in DNA–DNA (RNA) duplexes
No.
X
Y
T m/ЊC (DNA–DNA)
∆T m/ЊC
T m/ЊC (DNA–RNA)
∆T m/ЊC
∆∆T m DNA–RNA
A
B
C
D
—
G
I
—
—
—
I
43.0
32.2
41.8
39.4
42.2
32.6
32.2
21.8
Ϫ10.8
Ϫ1.2
Ϫ1.2
Ϫ9.6
Ϫ10.0
Ϫ10.2
1.2
8.8
9.0
I
I = inserted nucleoside analogue 4, ∆T m = decrease in T m per modification, ∆∆T m DNA–RNA = discrimination in T m between DNA–DNA and DNA–
RNA duplexes per modification
Table 2 Hybridization data (T m/ЊC) for the DNA three-way junction
F1
(X = 0)
F2
(X = A)
F3
(X = I)
E1
E2
E3
38.6
<18.0
<18.0
39.4
20.2
19.4
48.6
24.2
<18.0
I = inserted nucleoside analogue 4.
INA with incorporation of the N-(pyren-1-ylmethyl)-
azasugar as the bulge resulted in lowering of the melting tem-
perature with 1.2 ЊC per modification towards ssDNA (Table 1).
The corresponding reference duplex in entry B containing a
bulging deoxynucleotide (dG) had a considerably lower T m
=
32.2 ЊC (T m = Ϫ10.8 ЊC). For the INA–RNA duplexes the
pyrene containing sequence C and the reference B decreased
the stability of the INA–RNA duplex with 10 ЊC and 9.6 ЊC,
respectively, compared to the perfectly matched duplex (entry
A). Consequently, INA with pyrene azasugar incorporated as
the bulge has better hybridization affinity towards the comple-
mentary ssDNA than towards ssRNA. The differences in melt-
ing temperatures for ssDNA and ssRNA seems to be additive
with respect to the number of pyrene moieties in the targeting
ODN. These results are also in agreement with other investi-
gations where 1-O-(pyren-1-ylmethyl)glycerol was inserted
twice as bulges. A larger discrimination up to 25.8 ЊC between
INA–DNA and INA–RNA was then observed.8 In that case
the INA–DNA structure is stabilized compared to the wild type
duplex in contrast to our case where a slight decrease is
observed for T m. The flexibility of the bulge may be an import-
ant factor to obtain both duplex stabilization and discrimin-
ation. In order to understand the nature of the difference in the
thermal stability between DNA and RNA duplexes observed,
further experiments are in progress using NMR spectroscopy
for structural examination of the duplexes. The synthesis of
different linkers and planar aromatic moieties is also in pro-
gress. The RNA/DNA discrimination displayed may be applied
for purification or detection of DNA targets in a mixture with
the very same sequences of RNA.
Scheme 1 (a) 1-(chloromethyl)pyrene, Et3N, DMF; (b) pyrene-1-
carbaldehyde, NaCNBH3, DMF–EtOH (3 : 1); (c) 1) NaIO4, 2) NaBH4;
(d) DMTCl, pyridine; (e) NC(CH2)2OP(NPri2)2, N,N-diisopropyl-
ammonium tetrazolide, CH2Cl2.
The 4,4Ј-dimethoxytriphenylmethyl (DMT) protected
phosphoramidite 6 is required for the oligonucleotide synthesis.
The primary alcohol 4 was treated with an excess of DMTCl in
pyridine with further purification on a silica gel column to give
compound 5 in 61% yield. The synthesis of the final phosphor-
amidite by treatment with 2-cyanoethyl-N,N-isopropylchloro-
phosphoramidite in the presence of the excess of Hunig’s
base12 failed. To obtain the required phosphoramidite 6 we
used an alternative method with 2-cyanoethyl-N,N,NЈ,NЈ-tetra-
isopropylphosphorodiamidite and N,N-diisopropylammonium
tetrazolide.15 The compound 6 was obtained in 57% yield.
The phosphoramidite 6 was incorporated into different
oligonucleotide sequences to give INAs on an automated solid
phase DNA synthesizer using an increased coupling time
(24 min) and repeating the cycle twice. The coupling efficiencies
for the pyrene azasugar derivative 6 were approximately
80–85% compared to approximately 99% for commercial
phosphoramidites (2 min coupling).
As a second target in our investigation we chose a DNA three
way junction (TWJ) composed of two arms linked to a stem
(Table 2). Here we observed a considerable stabilization when
the pyrene azasugar intercalator was inserted in the INA (F3)
The synthesised INAs were used in the hybridization studies
of INA–DNA and INA–RNA duplexes (Table 1) and INA–
DNA three way junction (TWJ) (Table 2).
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 1 0 0 – 1 0 3
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compared to the ODN having dA at the same position (F2) or
without an insertion in the ODN (entry F1). To be sure that
hybridization in the arms is important for the stability of the
complex; we prepared ODNs with mismatches in either arm of
TWJ (entry E2 and E3). In both cases it resulted in a large
lowering of the hybridization affinity.
mg, 1.18 mmol) and NaCNBH3 (74 mg, 1.18 mmol) were
added. The reaction mixture was stirred at room temp. under
nitrogen overnight. Concentrated HCl was added until pH < 2.
Solvent was evaporated under reduced pressure and co-
evaporated with toluene (2 × 5 cm3). The residue was purified
using silica gel column chromatography with CH2Cl2–MeOH
(0–20%, v/v) affording the compound 3 (110 mg, 63%).
3. Conclusion
4.1.2 N-(Pyren-1-ylmethyl)-(3R,4R)-4-(hydroxymethyl)-
pyrrolidin-3-ol (4). Method A. Azasugar 2 (100 mg, 0.86 mmol)
was dissolved in DMF (10 cm3) and 1-(chloromethyl)pyrene
(257 mg, 1.03 mmol) and Et3N (0.140 cm3, 1.03 mmol) were
added. The reaction mixture was stirred at room temp. under
nitrogen overnight. The solvent was evaporated under reduced
pressure and co-evaporated with toluene (2 × 5 cm3). The
residue was dissolved in H2O–CH2Cl2 (1 : 1, 40 cm3) and
the water layer was extracted with CH2Cl2. The combined
organic fractions were dried (Na2SO4), evaporated in vacuo and
chromatographed on a silica gel column with CH2Cl2–MeOH
(0–20%, v/v) affording the title compound 4 (130 mg, 46%): Rf
0.17 (10% MeOH–CH2Cl2); δH (CDCl3) 2.08 (1 H, m, H-4), 2.29
(1 H, m, H-5), 2.57 (1 H, dd, J 2.8 and 10.2, H-2), 2.85 (2 H, m,
H-2 and H-5), 3.43 (2 H, s, CH2OH), 3.47 (2 H, br s, 2(OH)),
4.08 (1 H, m, H-3), 4.19 (2 H, s, CH2pyren-1-yl), 7.90–8.40 (9 H,
m, Harom); δC (CDCl3) 49.8 (C-4), 55.9 (C-5), 57.5 (C-2), 62.3
(CH2pyren-1-yl), 64.2 (CH2OH), 73.9 (C-3), 123.3, 124.4,
124.6, 124.8, 125.1, 125.9, 127.3, 127.6, 127.8, 129.5, 130.7,
130.9, 131.1 (pyren-1-yl); m/z (FAB) 332.1631 [M ϩ H]ϩ,
C23H24NO3 requires 332.1651.
Method B. Azasugar 2 (1.18 g, 10.1 mmol) was dissolved in
DMF–EtOH (3 : 1, 150 cm3) and pyrene-1-carbaldehyde (3.47
g, 15.1 mmol) and NaCNBH3 (950 mg, 15.1 mmol) were added.
The reaction mixture was stirred at room temp. under nitrogen
overnight. Concentrated HCl was added until pH<2. The
solvent was evaporated under reduced pressure and co-
evaporated with toluene (2 × 5 cm3). The residue was dissolved
in H2O–CH2Cl2 (1 : 1, v/v, 150 cm3) and the water layer was
extracted with CH2Cl2 (3 × 75 cm3). The combined organic
fractions were dried (Na2SO4) and evaporated under dimin-
ished pressure. The residue was purified using silica gel column
chromatography with CH2Cl2–MeOH (0–20%, v/v) affording
the compound 4 as an oil which crystallised on standing (1.9 g,
57%), mp 104–105 ЊC.
Thus we prepared and used N-(pyren-1-ylmethyl)-(3R,4R)-4-
(hydroxymethyl)pyrrolidin-3-ol (4) in the synthesis of several
INAs and investigated the hybridization affinity of INA–DNA,
INA–RNA duplexes and DNA TWJ region. When the N-
(pyren-1-ylmethyl)azasugar was inserted as a bulge we observed
good discrimination between stabilities of INA–DNA and
INA–RNA duplexes and the increased stability of a DNA
three-way junction. We believe that these findings will be
helpful in molecular design of targets and probes for DNA
diagnostics.
4. Experimental
4.1 General
NMR spectra were recorded on a Bruker AC-300 FT NMR
spectrometer at 300 MHz for 1H NMR and at 75.5 MHz for 13
C
NMR. Internal standards used in 1H NMR spectra were TMS
(δ: 0.00) for CDCl3, CD3OD; in 13C NMR were CDCl3 (δ: 77.0),
CD3OD (δ: 49.0). Accurate ion mass determination was per-
formed on a Kraton MS-50-RF equipped with FAB source.
The [M ϩ H]ϩ ions were peakmatched using ions derived from
the glycerol matrix. Thin layer chromatography (TLC) analyses
were carried out with use of TLC plates 60 F254 purchased from
Merck and were visualized in UV light (254 and/or 343 nm)
and/or with a ninhydrin spray reagent (0.3 g ninhydrin in 100
cm3 butan-1-ol and 3 cm3 HOAc) for azasugars and their
derivatives. The silica gel (0.063–0.200) used for column
chromatography was purchased from Merck. ODNs were
synthesised on an Assembler Gene Special DNA-Synthesizer
(Pharmacia Biotech). Purification of 5Ј-O-DMT-on and 5Ј-O-
DMT-off ODNs were accomplished using a Waters Delta
Prep 4000 Preparative Chromatography System. The modified
ODNs were confirmed by MALDI-TOF analysis on a Voyager
Elite Biospectrometry Research Station from PerSeptive
Biosystems. All solvents were distilled before use. The reagents
used were purchased from Aldrich, Sigma or Fluka. The
reagents for Gene Assembler were purchased from Cruachem
(UK).
Method C. A cooled solution of compound 3 (110 mg, 0.304
mmol) in EtOH (4 cm3) was added to a solution of NaIO4 (71.6
mg, 0.335 mmol) in H2O (1.5 cm3) under stirring. After 30 min
NaBH4 (12.3 mg, 0.335 mmol) was added. After 30 min the
resulting solution was acidified with 2 M HCl until pH<2 under
vigorous stirring. The solvent was removed in vacuo. The
residue was dissolved in H2O–CH2Cl2 (1 : 1, v/v, 20 cm3) and
extracted with CH2Cl2 (4 × 15 cm3). The combined organic
layers were dried (Na2SO4), evaporated under diminished
pressure to dryness affording compound 4 (40 mg, 40%).
4.1.1 N-(Pyren-1-ylmethyl)-(3R,4S )-4-[(1S )-1,2-di-
hydroxyethyl]pyrrolidin-3-ol (3). Method A. Azasugar 1 (50 mg,
0.34 mmol) was dissolved in DMF (5 cm3); 1-(chloro-
methyl)pyrene (103 mg, 0.41 mmol) and Et3N (0.057 cm3, 0.41
mmol) were added. The reaction mixture was stirred at room
temp. under nitrogen overnight. The solvent was evaporated
under reduced pressure and co-evaporated with toluene (2 × 5
cm3). The residue was chromatographed on a silica gel column
with CH2Cl2–MeOH (0–20%, v/v) as eluent affording the pure
product 3 (70 mg, 57%): Rf 0.20 (10% MeOH–CH2Cl2);
δH(CD3OD) 2.36 (1 H, m, H-4), 2.95 (1 H, m, H-5), 3.08 (1 H,
dd, J 2.8 and 10.5, H-2), 3.22 (1 H, dd, J 5.4 and 12.0, H-5),
3.34 (1 H, m, H-2), 3.50–3.65 (3 H, m, CH[OH]CH2OH), 4.42
(1 H, m, H-3), 4.65 (2 H, s, CH2pyren-1-yl), 4.88 (3 H, br s,
3 × OH), 7.90–8.40 (9 H, m, Harom); δC(CD3OD) 50.7 (C-4), 56.7
(C-5), 57.2 (C-2), 62.7 (CH2pyren-1-yl), 65.7 (CH2OH), 71.8
(CH[OH]), 72.7 (C-3), 123.8, 125.5, 125.8, 125.9, 126.6, 126.8,
127.3, 128.0, 128.2, 129.1, 129.4, 129.9, 131.2, 131.9, 132.5,
133.2 (pyren-1-yl); m/z (FAB) 362.1748 [M ϩ H]ϩ, C23H24NO3
requires 362.1756.
4.1.3 N-(Pyren-1-ylmethyl)-(3R,4R)-4-[(4,4Ј-dimethoxy-
triphenylmethoxy)methyl]pyrrolidin-3-ol (5). Compound
4
(139 mg, 0.42 mmol) was dissolved in anhydrous pyridine
(10 cm3) and DMTCl (178 mg, 0.53 mmol) was added. The
mixture was stirred for 24 h under nitrogen at room temp.
MeOH (1 cm3) was added to quench the reaction and the
solvents were evaporated under reduced pressure and
co-evaporated with toluene (2 × 5 cm3). The residue was
re-dissolved in H2O–CH2Cl2 (1 : 1, v/v, 20 cm3), and the mixture
was washed with saturated aqueous NaHCO3. The organic
layer was dried (Na2SO4), and concentrated under reduced
pressure. Purification using silica gel column chromatography
(5–40% EtOAc–cyclohexane, v/v) gave the title compound 5 as
a foam (160 mg, 61%) which was used in the next step without
further purification: Rf 0.45 (49% EtOAc–49% cyclohexane–2%
Et3N, v/v/v); δH (CDCl3) 2.20 (1 H, m, H-4), 2.34 (1 H, m, H-5),
Method B. Azasugar 1 (70 mg, 0.48 mmol) was dissolved in
DMF–EtOH (3 : 1, 10 cm3) and pyrene-1-carbaldehyde (270
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 1 0 0 – 1 0 3
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2.53 (1 H, br s, OH), 2.62 (1 H, dd, J 5.6 and 9.9, H-2), 2.72 (1
H, dd, J 2.5 and 9.8, H-2), 3.06 (3 H, m, CH2ODMT and H-5),
3.71 (6 H, s, OCH3), 4.01 (1 H, m, H-3), 4.21 (2 H, s, CH2pyren-
1-yl), 6.78 (4 H, m, DMT), 7.10–7.40 (9 H, m, DMT), 7.90–8.40
(9 H, m, Harom); δC(CDCl3) 48.8 (C-4), 55.2 (OCH3), 56.1 (C-5),
58.0 (C-2), 61.9 (CH2pyren-1-yl), 64.5 (CH2OH), 74.9 (C-3),
85.9 (C–Ar3), 113.0, 123.8–132.3 (DMT and pyren-1-yl), 144.9,
158.4 (DMT); m/z (FAB) 634.2740 [M ϩ H]ϩ, C44H42NO5
requires 634.2722.
All oligonucleotides containing pyrenylmethylazasugar deriv-
ative 6 were confirmed by MALDI-TOF analysis (entry C:
found 4005.65, calcd. 4005.76; entry D: 4398.02, calcd. 4398.87;
entry F3: found 4903.05, calcd. 4904.89).
4.3 Melting experiments
Melting temperature measurements were performed on a
Perkin-Elmer UV/VIS spectrometer fitted with a PTP-6 Peltier
temperature-programming element. The absorbance at 260 nm
was measured from 18 ЊC to 85 ЊC in 1 cm cells. The melting
temperature was determined as the maximum of the derivative
plots of the melting curve. The oligodeoxynucleotides were
dissolved in a medium salt buffer (pH = 7.0, 1 mM EDTA, 10
mM Na2HPO4 × 2H2O, 140 mM NaCl) to a concentration of
1.0 µM for each strand.
4.1.4 N-(Pyren-1-ylmethyl)-(3R,4R)-3-O-[2-cyanoethoxy-
(diisopropylamino)phosphino]-4-[(4,4Ј-dimethoxytriphenyl-
methoxy)methyl]pyrrolidine (6). Compound 5 (140 mg, 0.22
mmol) was dissolved under nitrogen in anhydrous CH2Cl2 (5
cm3). N,N-Diisopropylammonium tetrazolide (61 mg, 0.42
mmol) was added followed by dropwise addition of 2-cyano-
ethyl-N,N,NЈ,NЈ-tetraisopropylphosphorodiamidite (0.140 cm3,
0.44 mmol). After 2.0 h analytical TLC showed no more
starting material and the reaction was quenched with H2O
(1 cm3) followed by addition of CH2Cl2(10 cm3). The mixture
was washed with saturated aqueous NaHCO3 (2 × 10 cm3). The
organic phase was dried (Na2SO4) and the solvents were
removed under reduced pressure. The residue was purified using
silica gel column chromatography with cyclohexane–EtOAc
(0–20%, v/v). Combined UV-active fractions were evaporated
in vacuo affording 6 (158 mg, 57%) as foam that was
co-evaporated with dry acetonitrile (3 × 30 cm3) before using
it in ODN synthesis. Rf 0.85 (49% EtOAc–49% cyclohexane–2%
Et3N, v/v/v); δH (CDCl3) 0.93 (6 H, m, CH3 [Pri]), 1.04 (6 H, m,
CH3 [Pri]), 2.30 (2 H, m, H-4 and H-5), 2.48 (2 H, m, CH2CN),
2.64 (1 H, m, H-2), 2.78 (1 H, m, H-2), 2.98 (2 H, m, OCH2-
CH2CN), 3.08 (1 H, m, H-5), 3.50 (4 H, m, CH [Pri]
and CH2ODMT), 3.65 (6 H, s, OCH3), 4.01 (1 H, m, H-3), 4.20
(2 H, m, CH2pyren-1-yl), 6.68 (4 H, m, DMT), 7.05–7.40 (9 H,
m, DMT), 7.85–8.40 (9 H, m, Harom); δP(CDCl3) 148.2 (s), 149.0
(s) in the ratio 2 : 1.
Acknowledgements
We thank Mr U. B. Christensen for the great help in the
synthesis and purification of ODNs.
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4.2 Synthesis and purification of modified and unmodified
oligodeoxynucleotides
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The oligodeoxynucleotides were synthesised on a Pharmacia
Gene Assembler® Special synthesizer in 0.2 µmol-scale (7.5
µmol embedded per cycle, Pharmacia primer supportTM) using
commercially available 2-cyanoethylphosphoramidites and 6.
The synthesis followed the regular protocol for the DNA
synthesizer. The coupling time for 6 was increased from 2 to 24
min and the cycle was repeated twice. The 5Ј-O-DMT-on
ODNs were removed from the solid support and deprotected
with 32% aqueous NH3 (1 cm3) at 55 ЊC for 24 h and then
purified on preparative HPLC using a Hamilton PRP-1
column. The solvent systems were buffer A [950 cm3 0.1 M
NH4HCO3 and 50 cm3 CH3CN (pH = 9.0)] and buffer B [250
cm3 0.1 M NH4HCO3 and 750 cm3 CH3CN (pH = 9.0)] which
were used in the following order: 5 min A, 30 min linear
gradient of 0–70% B in A, 5 min liner gradient of 70–100% B in
A. Flow rate was 1 cm3 minϪ1. The purified 5Ј-O-DMT-on
ODNs eluted as one peak after approximately 30 min [UV
control 254 nm and 343 nm (for pyrene containing ODNs)].
The fractions were concentrated in vacuo followed by treatment
with 10% aqueous HOAc (1 cm3) for 20 min and further
purification on HPLC under the same conditions to afford
detritylated ODNs which eluted at 23–28 min. The purity of
oligos synthesised was 99–100% according to the preparative
HPLC. The resulted solutions were evaporated in vacuo and
co-evaporated twice with water to remove volatile salts to afford
ODNs, which were used in melting temperature measurements.
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O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 1 0 0 – 1 0 3
103