Synthesis of new OBAN's and further studies on positioning of the catalytic group.

Article abstract of DOI:10.1039/b403652b

Two new zinc ion dependent oligonucleotide based artificial nucleases (OBAN's) have been synthesized. These consist of 2'-O-methyl modified RNA oligomers conjugated to 5-amino-2,9-dimethylphenanthroline (neocuproine)via a urea linker. OBAN 4 carries the c

Full text of DOI:10.1039/b403652b

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Synthesis of new OBAN’s and further studies on positioning
of the catalytic group†
Hans Åström and Roger Strömberg*
Division of Organic and Bioorganic Chemistry, MBB, Scheele Laboratory,
Karolinska Institutet, S-17177, Stockholm, Sweden. E-mail: Roger.Stromberg@mbb.ki.se;
Fax: ϩ46-8-311052; Tel: ϩ46-8-7287749
Received 10th March 2004, Accepted 19th April 2004
First published as an Advance Article on the web 7th June 2004
Two new zinc ion dependent oligonucleotide based artificial nucleases (OBAN’s) have been synthesized. These consist
of 2Ј-O-methyl modified RNA oligomers conjugated to 5-amino-2,9-dimethylphenanthroline (neocuproine) via a
urea linker. OBAN 4 carries the catalytic group on a linker extending from the C-4 of an internal cytosine moiety.
OBAN 5 has two neocuproine units attached, each to linkers extending from the C-5 position of uridine moieties, one
placed internally and the other at the at the 5Ј-end of the oligonucleotide. The key step in the synthesis of the OBAN
systems is conjugation of the catalytic group to the respective amino linkers of the modified oligonucleotides. This is
achieved by first converting the 5-amino-2,9-dimethylphenanthroline to the phenylcarbamate. The reaction of this
neocuproine phenylcarbamate with the oligonucleotide carrying one or two primary aliphatic amines in aqueous
buffer (at pH 8.5) leads to nearly quantitative formation of the urea-linked conjugates. Both OBAN systems were
found to cleave RNA in the bulged out regions formed from the non-complementary part of the target sequences,
in the presence of Zn() ions. Differences in efficiency between these and previously reported systems are discussed.
via a linker that is intended to place the catalytic group in close
proximity to the targeted cleavage site. We have recently
Introduction
Oligonucleotide based artificial nucleases (OBAN’s) with built
developed some OBAN systems where the catalytic group is
in activity to degrade complementary RNA sequences are of
the Zn() complex of 5-amino-2,9-dimethylphenanthroline.9
current interest in life science and biotechnology.1 OBAN’s may
Cleavage of simple phosphate esters was first demonstrated
be regarded as a development of traditional antisense method-
by use of a copper complex of the closely related 2,9-di-
ology where gene expression can be regulated at the level of
methylphenanthroline.10 There have also been a couple of
mRNA by hybridising synthetic oligonucleotides to natural
reports that copper and zinc complexes of neocuproine
complementary mRNA sequences by means of Watson–Crick
derivatives differently linked to deoxyoligoribonucleotides11 or
base-pairing.2 Translation may then be inhibited3,4 and
PNA12 (zinc only) can be active in cleavage of RNA, although
typically catalytic turnover through action of the host RNAse
catalytic turnover was not shown. In our studies, aimed at
H is beneficial.4 Chemical modifications of the nucleoside
moieties and/or the internucleosidic phosphate linkage are in
developing more efficient OBAN systems, we have recently
developed 2Ј-O-methyl modified OBAN systems with tethered
Zn() 5-amino-2,9-dimethylphenanthroline moieties and have
general necessary to avoid degradation of the antisense oligo-
mer2 but in most cases these give duplexes where the target
shown that these provide turnover of substrate and thus
RNA is not cleaved by RNase H2. The basis for creation of
perform real enzyme catalysis in the cleavage of RNA.9
OBAN’s, is that the catalytic activity is built into the antisense
For the further development of these artificial enzymes we
oligomer by attachment of a catalytic group via a linker. Thus, a
have begun studies on the influence of linker and linker position
system with catalytic turnover that is independent on host
as well as of the structure of target RNA on the efficiency
enzymes can be achieved. Apart from the potential use of
of cleavage. We have varied the target so that it forms bulges (or
OBAN’s as mRNA targeting drug substances, these artificial
enzymes could also become useful as tools in molecular biology.
In the cleavage of RNA, the nature of the target has been
not), of 0–5 nucleotides, when bound to the OBAN. We have
also started to evaluate different attachment points for the
catalytic group. OBAN 1 from our initial study9 has the
proven to be important. The susceptibility of the RNA to
catalytic group linked to C-5 of a deoxyuridine placed centrally
in a 2Ј-O-methyl modified RNA sequence (Fig. 1). This OBAN
cleavage is sequence dependent.5 Single stranded RNA and
RNA-bulges have proven to be more susceptible to cleavage
showed the highest activity, especially when the target formed
than duplexes.6,7 This is suggested to be due to the 2Ј-hydroxyl
a 3–4-nt bulge upon binding to the OBAN.9
in an RNA duplex not being in a suitable orientation for intra-
molecular transesterification, and a conformational change is
In the present study our efficient method for the conjugation
of ligand to oligonucleotides in an aqueous environment is
energetically unfavourable since the duplex must be partially
described. The method gives consistently nearly quantitative
disrupted.7 RNA bulges are thus interesting as targets for
conversions and isolated yields of 80% are generally achieved
artificial nucleases and a bulge in the RNA target can be created
after purification by RP HPLC. The current study is also a
upon binding of the antisense oligonucleotide by partial
continuation of our efforts to evaluate linkers and synthesis
of two new OBAN’s are described. These carry 5-amino-2,9-
dimethylphenanthroline ligands but linked from other positions
non-complementarity of the base sequence.8,9
An oligonucleotide based artificial nuclease (OBAN) consists
of a recognition element, a modified antisense oligonucleotide,
and with one new linker arm. This involves synthesis of a novel
and one or several attached groups that catalyse the hydrolytic
building block with a linker from the N-4 position of a cytidine
cleavage of the RNA target. The catalytic groups are anchored
moiety, which has a different directionality and length com-
pared to previous linkers. We have also started to evaluate the
possibility of concerted action by two neocuproine ligands.
The latter system with the ligands extending from the C-5
† Oligonucleotide based artificial nuclease (OBAN) systems. Part. 2.
For Part 1 see ref. 9.
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 4
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 9 0 1 – 1 9 0 7
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made according to the method developed by Reese and
co-workers.14
The derivative 6 was then treated with 4-monomethoxytrityl
chloride in pyridine overnight to produce the 5Ј-O-MMT
protected dervative 7. Subsequent treatment with 8 M ethylene-
diamine in ethanol afforded the 4-aminoethyl cytidine
derivative 8. We found it more convenient to carry out the
synthesis in this order i.e. monomethoxytritylation before
substitution. When carrying out the synthesis by reacting 6 with
ethylenediamine, i.e., without prior tritylation, the conversion
to product was satisfactory but due to the high polarity of the
compound the purification became somewhat cumbersome.
Hence, introduction of the monomethoxytrityl group first gives
a more convenient synthesis of 8. Compound 8 was isolated,
after chromatography, in 54% yield over the two steps. The
free primary amine of derivative 8 was then protected as the
trifluoroacetate (9). The protection was performed by reaction
of 8 with 1.1 eq. ethyl trifluoroacetate in ethanol at 0 ЊC,
producing compound 9 in 97% isolated yield.15 Interestingly the
NMR spectrum of 9 as recorded from a solution in deuterated
chloroform gave doubled signals, presumably from restricted
rotation around the amide bond, but change of the solvent
to DMSO-d6 gave only the averaged signals. A plausible
explanation could be that the isomerisation rate is lower for the
rotamers in chloroform due to a slightly stonger internal
hydrogen bond between the 4-NH and the carbonyl oxygen of
the trifluoroacetate in the less polar solvent. From 9 the
H-phosphonate building block 10 was synthesized by use of the
imidazole, PCl3, triethylamine method.16 The H-phosphonate
10 was isolated in 67% yield, after chromatography.
Synthesis of the phenylcarbamate reagent of 5-amino-2,9-di-
methyl-1,10-phenanthroline
Fig. 1 Illustration of the 1–5-nt bulge systems. Substrate RNA’s are
written from the 5Ј-end (from left to right). OBAN 1,2 and 4 had either
modification A, B or C incorporated at position X1. OBAN 3 had
The ligand used, 5-amino-2,9-dimethyl-1,10-phenanthroline
was synthesized as described in Scheme 2. 1,10-dimethyl
phenanthroline 11 was nitrated to 5-NO2-2,9-dimethyl-1,10-
phenanthroline (12) using HNO3 in oleum at 168 ЊC.17 The
purification of 12 was complicated due to the multiple by-
products that formed during the reaction. Nevertheless,
satisfactory amounts of pure 12 could be isolated by silica gel
chromatography. 12 was then reduced to 5-amino-2,9-dimethyl
1,10 phenanthroline (13) by catalytic hydrogenation;18 13
was reacted with phenyl chloroformate at –20 ЊC overnight
whereupon the phenylcarbamate 14 formed as a precipitate.
modification
A added at X2. OBAN 5 had two A monomers
incorporated at positions X1 and X2.
of deoxyuridines, one at the 5Ј-end and one internally placed
(Fig. 1). Results from clevavage of RNA with these systems is
compared to the previously developed systems.
Results and discussion
The systems studied here (OBAN 4 and 5) together with
previous constructs (OBAN 1–3) are shown in Fig. 1 Assembly
of the modified oligonucleotide leading to OBAN 4 was carried
out by standard H-phosphonate methodology on a solid
support.13 A modified deoxycytidine H-phosphonate monomer
was used to produce the linker-bearing residue.
Synthesis of the N 4-aminoethyl cytidine building block
This new building block was synthesised from uridine
(Scheme 1) via the N 4-(4-nitrophenoxy) derivative 6 which was
Scheme 2 Synthetic route for the phenylcarbamate reagent 14.
A method for conjugation of the neocuproine derivatives to
oligonucleotides bearing a free primary amine
Conjugation of the 5-aminoneocuproine ligand to the amino-
linker-containing oligonucleotides was carried out on the
fully deprotected oligonucleotides in aqueous solution. The
conjugation was performed in sodium tetraborate buffer (aq.)
at pH 8.5. The reaction of carbamate 14 with the aminolinker-
containing oligonucleotides gave virtually quantitative form-
Scheme 1 Synthetic route for the 4-aminoethyl cytidine H-phos-
phonate building block used for synthesis of OBAN 4.
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 9 0 1 – 1 9 0 7
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Table 1 Rate constants for cleavage of the RNA substrates in Fig. 1 by OBAN’s 1 to 5
Previously reported systemsa
Present systems
OBAN 1
OBAN 29
kobs/106 sϪ1
OBAN 39
kobs/106 sϪ1
OBAN 4
OBAN 5
Bulge size
kobs/106 sϪ1
kobs/106 sϪ1
kobs/106 sϪ1
5
4
3
2
9.1 0.29 7.2 0.2b
17.1 0.49
7.5 0.7
4.8 0.3
6.2 0.4
7.5 0.4
5.8 0.1
5.1 0.1
4.0 0.1
2.4 0.1
9.5 0.5
13.4 0.5
9.3 0.9
6.7 0.3
4.4 0.1
3.3 0.2
4.6 0.5
n.d.
14.1 0.29
3.3 0.29
Experiments carried out in 10 mM HEPES (pH 7.4), 0.1 M NaCl, substrate RNA’s and OBAN’s were present at 4 µM, all reactions were incubated
at 37 ЊC. Observed 1st order rate constants were calculated as described in the Experimental section.a Data from reference 9, except for the 5A bulge.
b Result obtained with 5-nt bulge containing 5 adenosine residues (AAAAA) instead of the AAUAA bulge.
ation of the desired conjugates within 2 hours of incubation at
ambient temperature (Fig. 2). After purification by RP-HPLC
the conjugates were isolated in about 80% yield. Thus, this
is a highly efficient method for conjugation to oligonucleotides
with aminolinkers in aqueous solution. Post synthetic intro-
duction of the catalytic group gives the advantage that a given
oligonucleotide precursor can be reacted with different catalytic
groups to produce a plethora of conjugates, and without a need
to alter the protocols for oligonucleotide synthesis.
bulges. As for our previous systems, both the 1-nt bulge and
the fully complementary sequence displayed very low rates of
cleavage over the time measured and were not further
evaluated. For the bulges evaluated, cleavage was exclusively
observed in the bulged regions and the bulge size preference for
OBAN 4 was indeed shifted towards smaller bulges (Fig. 3,
Table 1). The 4-nt bulge is still cleaved with the highest rate but
with a lower rate than for OBAN 1. However, the rate differ-
ences between the 2–3-nt bulges and the 4-nt bulge are smaller
for OBAN 4 and for the 2-nt bulge the rate is double that for
OBAN 1 with the 2-nt bulge. Thus, OBAN 4 is a more efficient
cleaver for the 2-nt bulged target and in this respect comparable
to OBAN 2. The linker of the latter does however lead to a
higher selectivity for smaller vs. larger bulges.
Fig. 2 HPLC chromatograms showing conjugation of the neocu-
proine unit to the oligonucleotide precursors of OBAN 4 and 5 after 2
h. The reactions were carried out in aqueous sodium tetraborate buffer
(pH 8.5). The large residual peak in the chromatograms originates from
carbamate 14. Retention times for non-conjugated OBAN 4 and 5 are
31.13 and 31.23 respectively.
Studies on the cleavage of RNA substrates by OBAN 4 and 5
OBAN 4 in the present study consisted of an 11-mer 2Ј-O-Me
modified oligonucleotide carrying a linker at the C-4 position
of a central cytosine and OBAN 5 was a 12-mer, conjugated to
2 neocuproines both via the C-5 of separate deoxy uridine
residues, one additional at the 5Ј-end and one centrally placed
(Fig. 1). Both OBAN’s were incubated individually at pH 7.4
with 100 µM Zn2ϩ and 6 different substrate RNA’s containing
non complementary regions of 0–5 nt to produce 6 different
OBAN-RNA complexes for each OBAN. The main goal with
the cleavage study was to compare rates of RNA degradation
with those obtained for OBAN 1–3. Therefore these conditions
were singled out. In our previous study a maximum rate was
observed for a 4-nt bulge with OBAN 1, closely followed by
cleavage of the 3-nt bulge. With OBAN 4 we introduce a linker
that is one carbon shorter, tethered to the same nucleoside
position but with a different directionality. From a 3D-model
of the 5-nt bulge it appeared likely that this linker and linker
position would be better positioned for cleavage of smaller
Fig. 3 HPLC chromatograms showing reaction mixtures of OBAN 4
and RNA substrates after 24 h, from the 5-nt bulge forming complex
(top) to the complementary duplex (bottom). All reactions were carried
out at 37 ЊC with a 4 µM conc. of both OBAN 4 and substrate RNA, in
a 10 mM HEPES buffer (pH 7.4) containing 0.1 M NaCl and 100 µM
Zn(NO3)2. HPLC analysis (see Experimental section) is carried out at
pH 6.5.
The sites cleaved by OBAN 4 in the 4-nt bulge system were
studied in more detail by LC-MS analysis. As can be seen in
Fig. 4, cleavage takes place exclusively in the bulged out region
and the extent of cleavage is quite similar between the first 4
phosphates in the bulge (5Ј to 3Ј direction) while the phosphate
between the 4th and the 5th nucleoside is degraded less readily.
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 9 0 1 – 1 9 0 7
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differences in cleavage rates and patterns. A major factor is
likely to be how well the catalytic group is positioned which
is also displayed in the differences in substrate (bulge-size)
preferences. What is quite clear though is that the difference in
absolute numbers is remarkably small. The difference between
the highest and lowest rate constants for all systems in cleavage
of substrates forming 2–5-nt bulges is just short of an order of
magnitude. One plausible explanation for the similarities in rate
could be that the bulge region is quite flexible and can be
reached quite readily. This flexibility also means that the
intramolecularity will be relatively modest. The phosphate–
metal interaction is not likely to be strong enough to fix
the bulge in a single conformation. This is also supported by the
observation of more than a single cleavage site as well as that
there is some correlation between site selectivity and overall
rate. There are numerous possibilities to vary linkers and linker
positions and these may well lead to improvements. However, it
does not seem unlikely that these improvements will still be
somewhat modest. This reasoning is further justified by com-
parison with other reported Zn() based OBAN’s. Komiyama
achieved 5% cleavage in 3 h using a binuclear (TPBA) OBAN in
25 eq. excess to substrate RNA and 50 eq. Zn().20 Putnam and
Bashkin degraded 25% of a 28-mer and 18% of a 159-mer
RNA substrate in 15 and 10 h respectively using a 50 eq. excess
of a neocuproine tagged OBAN and the same concentration of
Zn().11 Whitney et al. conjugated neocuproine to PNA back-
bones and cleaved 12–30% (internal and external phosphates)
of an RNA substrate based on human telomerase in 12–24 h
with 50 eq. OBAN and 100 eq. of Zn().12 More recently,
Niitymäki et al. demonstrated catalytic turnover using an
azacrown Zn() complex.21 Substrate was present in 2 fold
excess and Zn() in 20% excess to OBAN giving a half-life for
cleavage of 20 h. With equimolar amounts of our fastest
systems (see Table 1), i.e, with approximately 80–90% substrate
saturation and about 70–80% Zn() saturation of the
neocuprine complex, we obtain half-lives of 11–14 h. Since the
neocuproine complex does not complex strongly to zinc() ions
the rates at lower zinc concentration are then obviously
somewhat lower and the maximum rate at full saturation
higher. In any case, the rates for most published systems display
a very modest distribution, even though different catalytic
groups have been used. A clear possibility to overcome this
could be to introduce oligonucleotide modifications that
specifically rigidify the bulged out region of the RNA substrate.
If this is achieved together with a fairly rigid linker and a
productive positioning a much higher intramolecularity is likely
to be obtained. This is quite some task to be undertaken, and it
will also require good molecular modelling as well as refine-
ment based on structural studies. Our efforts in this direction
have only begun, but at least a modest bulge stabilisation
of RNA bulges has so far been achieved by introduction
of 2Ј-deoxy-2Ј-C-naphtyl tubercidine in oligonucleotides.22
We are currently investigating additional bulge stabilising
modifications and their use in OBAN systems.
Fig. 4 LC-MS analysis of the fragments formed in cleavage of the 4-nt
bulge system using OBAN 4. The reaction was incubated for 24 h at pH
7.4. Equimolar (4 µM) concentrations of OBAN 4 and substrate RNA
were used in a buffer containing 0.1 M NaCl, 10 mM HEPES and 100
µM Zn(NO3)2. The peak to the far right is intact RNA substrate and the
numbered peaks correspond to specific cleavage sites at the bulge.
M(Ϫ1) for fragments were detected using ESI-TOF MS. 1: 1530 ϩ
3206, 2: 2221 ϩ 2516, 3: 2548 ϩ 2187, 4: 1859 ϩ 2823, 5: 1201 ϩ 3536.
Apparently, the linker–catalyst system is equally close in space
to first 4 phosphates but still reaches the last phosphate in the
bulge. It is notable that there is no selectivity, which is in
contrast to the pattern with OBAN 1 that predominantly causes
cleavage at positions 3 and 4 (49 and 31% of total cleavage
respectively).9 Fig. 3 indicates that the 5-nt and the 3-nt bulge
systems also display cleavage patterns that are essentially
non-selective.
It has been shown in studies on dinucleotides that cleavage of
ApA in the presence of Cu() bi- or terpyridyl chelates can be
considerably faster than cleavage of dinucleotides with other
base combinations.19 This apparent base selectivity has been
attributed to a π–π interaction between the adenines and the
bi or terpyridyl system. If a π–π interation would also be
influential in the present case we could obtain a substantial
influence on the clevage rate of the 5-nt bulge if the uridine
residue in the middle of the bulge is changed to an adenosine.
Hence, an oligoribonucleotide forming a 5-nt all adenosine
bulge was incubated together with OBAN 1 under indentical
conditions to the that for the AAUAA bulge forming substrate.
The first order rate constant for the 5-A system was lower and
quite similar to that for the AAUAA system. (7.10 0.2 × 10Ϫ6
sϪ1 and 9.1 0.2 × 10Ϫ6 sϪ1 respectively). This indicates that
there is no strong influence from a π–π interaction between
the neocuproine and the adenosine containing bulge. The
directionality and length of the linker is clearly more important
for positioning of the neocuproine complex.
OBAN 5 carries two catalytic groups in the same oligo-
nucleotide and each respectively linked as in OBAN 1 and 3. As
the catalytic groups of both OBAN 1 and OBAN 3 reach and
cleave the bulged regions of the substrate RNA a combination
of these could give an additive or even synegistic effect. How-
ever, this was not to be. Instead, the RNA cleavage activity of
OBAN 5 with dual catalytic groups was lower than for either
of the monofunctionalised OBAN’s or at best comparable
to OBAN 3. One reason for this could be if binding of the
substrate is inhibited, but thermal melting studies confirmed
that OBAN 5 also formed stable complexes with the RNA
substrates (e.g. T m = 48.2 ЊC for the 4-nt bulge). A plausible
explanation for the low activity with OBAN 5 could be that the
two catalytic groups clash sterically, which makes it more
difficult for either catalyst to reach the phosphates in a product-
ive orientation. Another possibility is the involvement of an
equilibrium where the two ligands also coordinate the same
metal ion forming a sandwich complex, which is likely to be
inactive. It is however, quite clear that the positions of the
catalytic groups are unsuitable and the system perhaps becomes
too flexible to obtain a concerted or additive action.
Experimental
Materials
All solvents used were of analytical quality (p.a) and were
purchased from Merck (CH2Cl2, MeOH, DMSO, THF) and
LabScan (pyridine anhydrous) Reagents were purchased from:
Merck (Pd/C, oleum), Lancaster (ethylenediamine, imidazole),
Aldrich (MMT-Cl) and Acros (phenyl chloroformate).
The ethyl ester of trifluoroacetic acid is synthesised by mixing
stoichiometic amounts of ethanol and trifluoroacetic acid.
The ester is formed rapidly and can easily be distilled of at
60–62 ЊC.15 N 4-(4-Nitrophenoxyl)-2Ј-deoxy cytosine 6 was
synthesized according to a published procedure.11 The oligo-
nucleotide precursor to OBAN 5 was purchased from TriLink
BioTechnologies (San Diego, CA, USA) and purified as
We now have five different oligonucleotides with attached
zinc chelates that are cleaving RNA targets. There are marked
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 9 0 1 – 1 9 0 7
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described below. All RNA substrates were purchased from
Dharmacon (Boulder, CO, USA). All purchased oligonucleo-
tides were purified by reversed phase HPLC (Hypersil ODS,
250 × 10 mm) using a linear gradient of 0–25% CH3CN in
50 mM triethylammoniumacetate acetate (pH 6.5). The oligo-
nucleotides were collected, lyophilized, dissolved in water and
lyophilized again. Buffers, metal salts and water used for the
solutions in the cleavage studies were all of molecular biology
grade from Sigma (NaCl, HEPES) or Fluka (Zn(NO3)2, H2O).
for 1 h and then diluted with 30 mL 2.0 M TEAB aq. pH 7.5.
The organic layer was further exracted with 30 mL TEAB
buffer. The organic layer was then collected, dried over Na2SO4,
concentrated and purified on a silica gel column using CH2Cl2–
MeOH (4 : 1 with 0.1% triethylamine). Fractions containing
pure product were pooled and concentrated. After drying under
vacuum, 0.58 g (0.072 mmmol) of 10 corresponding to an
isolated yield of 92% was obtained. TLC analysis of compound
10 gave: Rf 0.24 (CH2Cl2–MeOH, 4 : 1 with 0.1% triethylamine).
1H NMR (DMSO-d6): 7.92 (t, 1H, NH C5), 7.62 (d, 1H, C6),
7.4–7.23 (m, 12.5H, Ar, PH), 6.91 (d, 2H, tr), 6.17 (t, 1H, 1Ј),
5.85 (s, 0.5H, PH), 5.56 (d, 1H, C5), 4.68 (m, 1H, 3Ј), 4.05 (m,
1H, 4Ј), 3.74 (s, 3H, O-Me), 3.4 (m, 4H, CH2, CH2), 3.28–3.18
(m, 2H, 5Ј), 2.50 (m, 1H, 2Ј), 2.14 (m, 1H, 2Ј). High resolution
MS ESI-TOF analysis in negative mode gave m/z = 701.2012.
Calculated m/z = 701.1988.
N4-(2-aminoethyl)-5Ј-O-(4-monomethoxytrityl)-2Ј-deoxy-
cytidine 8. 1.1 g (3.16 mmol) 6 was dissolved in 35 mL dry
pyridine and the solvent was evaporated; 6 was then dissolved
in another 35 mL portion of dry pyridine and the solution was
chilled on an ice-water bath. MMT-Cl (1.07 g, 3.5 mmol) was
added under vigorous stirring. The reaction mixture was kept
stirring overnight. TLC analysis in CH2Cl2 : MeOH 9 : 1 then
revealed that all starting material was consumed. 50 µL MeOH
was added and the solvent was evaporated under reduced
pressure. The crude tritylated derivative was dissolved in 8 M
ethylenediamine (in ethanol), 7.9 mL (63.2 mmol). The reaction
mixture was stirred for 2 h after which TLC analysis in CH2Cl2–
MeOH (4 : 1, with 0.1% triethylamine) showed disapperance of
the starting material and a product with Rf 0.2. The reaction
mixture was, after concentration, partitioned between CH2Cl2
and NaHCO3, 100 mL of each. The organic phase was washed
two times with NaHCO3 and then with 100 mL water. The
organic phase was then dried over Na2SO4 and concentrated.
The product was purified by chromatography on silica,
CH2Cl2–MeOH (4 : 1, with 0.1% triethylamine). Fractions
containing pure product were pooled, dried over Na2SO4 and
concentrated. 0.93 g (1.72 mmol) of 8 corresponding to a yield
5-Nitro-2,9-dimethyl-1,10-phenanthroline11,23 12. 2,9-di-
methyl-1,10-phenanthroline (11), 2g (9.2 mmol), was dissolved
in 9.2 ml oleum. 4.8 ml HNO3 was added and the reaction
mixture was heated under stirring to 125 ЊC (the HNO3
addition should be carried out at a rate low enough to keep the
temperature below 170 ЊC). After addition was complete the
mixture was left stirring for 30 min. Then the mixture was
poured onto 130 g of crushed ice. The reaction mixture was
treated with 30% NaOH until the solution was neutral. Then
dilute nitric acid was added to make the solution slightly
acidic, upon which compound 12 precipitated. The crystals
were filtered off and washed with water. After drying was the
crude product placed in a glass filter funnel and dissolved in
chloroform to wash off insoluble byproducts. The filtrate was
concentrated and purified on a silica gel column CH2Cl2:MeOH
(19:1 with 1% triethylamine), Rf 0.33. Fractions containing pure
product were pooled, dried over Na2SO4 and evaporated. 0.6 g
(2.7 mmol) of 12 corresponding to an isolated yield of 29% was
obtained. 1H NMR (CDCl3): δ = 8.92 (d, 1H), 8.61 (s, 1H), 8.28
(d, 1H), 7.65 (dd, 2H), 3.01 (s, 1H), 2.99 (s, 1H).
1
of 54% was obtained. H NMR (DMSO-d6): 7.71 (t, 1H, NH
C5), 7.58 (d, 1H, C6), 7.24–7.40 (m, 12H, Ar), 6.88 (d, 2H tr),
6.18 (t, 1H, 1Ј), 5.6 (d, 1H, C5), 5.3 (d, 1H, 3Ј-OH), 4.25 (m,
1H, 3Ј), 3.87 (m, 1H, 4Ј), 3.75 (s, 3H, O–Me), 3.22 (m, 4H,
5Ј and N4-CH2), 2.66 (t, 2H, CH2-N), 2.16 (m, 1H, 2Ј), 2.04 (m,
1H, 2Ј). MS (ES/TOF): m/z = 585.2.
5-Amino-2,9-dimethyl-1,10-phenanthroline8,2313. 12 (34.2 mg,
0.14 mmol) was dissolved in 2 ml MeOH and a catalytic
amount of Pd/C was added. The reaction mixture was kept
under stirring and a H2 balloon was placed on top of the flask.
After 10 min all starting material was consumed and a new
product with (Rf 0.33) (CHCl3 : MeOH 19 : 1) had been formed.
The reaction mixture was filtered and washed with MeOH. The
methanol was then evaporated. The crude product was then
purified on silica CHCl3 : MeOH 4 : 1 containing 0.01% TEA.
Fractions containing pure product were pooled and concen-
trated under reduced pressure. 25.7 mg (0.12 mmol) product
corresponding to a yield of 85% was isolated. 1H NMR
(CD3OD): δ = 8.46 (d, 1H), 7.99 (d, 1H), 7.57 (d, 1H), 7.44 (d,
1H), 6.89 (s, 1H), 2.82 (s, 3H), 2.78 (s, 3H).
N4-[2-(2,2,2-trifluoroacetamido)ethyl]-5Ј-O-(4-methoxy-
trityl)-2Ј-deoxycytidine 9. 0.93 g (1.72 mmol) of 8 was dissolved
in 40 mL ethanol and the mixture was chilled to 0 ЊC. 204.6 µL
(1.72 mmol) of ethyl trifluoroacetate was added under stirring.
After 1 h TLC analysis in CH2Cl2 : MeOH 20 : 1 containing
0.05% triethylamine showed completion of reaction and one
product with an Rf of 0.3. The reaction mixture was then
concentrated, the residue was redissolved in 50 mL CH2Cl2,
washed with 50 mL NaHCO3, and 50 mL water. The organic
phase was then dried over Na2SO4 and concentrated. The crude
product was purified by chromatography on silica, using
CH2Cl2–MeOH (20 : 1, with 0.05% triethylamine). Fractions
containing pure product were pooled dried over Na2SO4 and
concentrated. 1.06 g (1.66 mmol) of 9, corresponding to an
isolated yield of 97% was obtained. 1H NMR (DMSO-d6) 9.59
(t, 1H, NH, CF3CONH), 7.90 (t, 1H, NH, C4), 7.65 (d, 1H,
C6), 7.41–7.25 (m, 12H, Ar), 6.91 (d, 2H, tr), 6.22 (t, 1H, 1Ј),
5.60 (d, 1H, C5), 5.38 (d, 1H, 3Ј-OH), 4.29 (m, 1H, 3Ј), 3.91 (m,
1H, 4Ј), 3.75 (s, 3H, O-Me), 3.36–3.44 (m, 4H, CH2, CH2),
3.24–3.22 (m, 2H, 5Ј), 2.21–2.19 (m, 1H, 2Ј), 2.07–2.03 (m, 1H,
2Ј). ES/ TOF): m/z = 639.2
Phenyl N-(2,9-dimethyl[1,10]phenanthrolin-5-yl)carbamate23-
14. 21.2 mg (0.084 mmol) of 13 was dissolved in 3 ml THF and
17.4 µl triethylamine (0.0921 mmol) was added. The mixture
was concentrated to dryness under reduced pressure. 3 mL
THF was added together with 17.4 µl triethylamine (0.0921
mmol). Phenyl chloroformate 15.8 µl (0.092 mmol) was then
added to the mixture under vigorous stirring. After 30 min TLC
(THF) showed disappearence of the starting material and
formation of a new product (Rf 0.76). The reaction mixture
was then filtered (to remove triethylammonium hydrochloride).
The solid on the filter was washed with 1 ml dry THF. The
combined filtrates were pooled and placed in a freezer at
Ϫ20 ЊC overnight. The precipitated product was washed with
cold, dry THF and then dried under reduced pressure. The first
crop yielded 10 mg (2.91ؒ10Ϫ5 mol, 35%) of 14. 1H NMR
(CDCl3): δ = 9.01 (d, 1H, H8), 8.85 (s, 1H, NH), 8.18 (d, 1H,
N4-[2-(2,2,2-trifluoroacetamido)ethyl]-5Ј-O-(4-methoxytrityl)-
2Ј-deoxycytidine 3Ј-H-phosphonate 10. Imidazole 0.573 g (8.4
mmol) was dissolved in 30 mL dry CH2Cl2 and the mixture
was chilled to Ϫ10 ЊC (acetone–dry ice). First PCl3 (0.239 mL,
2.74 mmol) and then triethylamine (1.22 mL, 8.81 mmol) were
added drop-wise under vigorous stirring. The mixture was
stirred at Ϫ10 ЊC for 30 min and then cooled to Ϫ78 ЊC. 0.5 g
(0.078 mmol) 9 dissolved in 10 mL CH2Cl2 was added during a
period of 30 min. The reaction mixture was stirred at Ϫ78 ЊC
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 9 0 1 – 1 9 0 7
1905

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H3), 8.02 (s, 1H, H6), 7.58 (d, 1H, H4), 7.44–7.22 (m, 6H, H7,
HPh), 3.12 (s, 3H,CH3-C2), 2.81 (s, 3H, CH3-C9) MALDI–
TOF MS: m/z = 344.
to remove traces of particles and chelating resin. The aliquots
were then diluted to 50 µL with HPLC buffer A (see below)
before analysis. The buffers used in the RP HPLC analysis
A: 0.1 M triethylammoniumacetate, pH 6.5 and B: 0.1 M
triethylammoniumacetate, pH 6.5 containing 50% MeCN. The
gradient was used was as follows: 0–5 min: 100% A; 5–40 min
0–30% B; 30–60 min 30–100% B; and the flowrate was 0.2 mL
minϪ1. A Jones Genesis C18 4 µm (250 × 2.1 mm) column was
used. LC-MS analysis and calculation of rate constants (first
order observed rate constants, i.e., the sum of rate constants for
all cleavage sites meaning that the individual constants in, e.g.,
Oligonucleotide synthesis. The oligonucleotide precursor to
OBAN
4 was synthesised with 2Ј-O-Me ribonucleotide
H-phosphonate building blocks and the linker containing
building block 10 using standard H-phosphonate method-
ology.13 Purification by ion exchange HPLC was carried out at
50 ЊC on a Dionex NucleoPac PA–100 (9 × 250) column, using
a linear gradient of 0–90 mM LiClO4 in 20 mM sodium acetate
(aq., pH 6.5) containing 30% CH3CN. The collected fractions
were lyophilized and then purified on RP-HPLC as described
above for the purchased oligonucleotides. The integrity of the
oligonucleotide was confirmed by ESI-TOF MS m/z = 3715.
the system from Fig. 4 for cleavage sites 1–4 is 0.21–0.22ؒkobs
)
were carried out as described before.9
Acknowledgements
Conjugation of oligonucleotides to 5-amino 2,9-dimethyl
phenanthroline: OBAN 4. 1 mg (2.9 µmol) of carbamate 14 was
dissolved in 56 µL dry DMSO. To this solution was added: 28
µL H2O, 300 µL sodium tetraborate buffer (0.1 M, pH 8.5) and
finally a 16 µL (0.1 µmol) water solution of the OBAN 4
precursor. The vial containing the reaction mixture was placed
on a shaker, oscillating at low speed; 2 µL aliquots were
withdrawn from the reaction mixture, filtrated, diluted with
water to 100 µl and analysed by reversed phase HPLC. The
reaction was incubated overnight (although reaction appears to
be over within 2 h). The reaction mixture was then filtered and
OBAN 4 was purified by RP HPLC (as described for all
oligonucleotide in the materials section). 0.08 µmol OBAN 4
(as determined by UV) corresponding to a yield of 80% was
isolated after two lyophilizations. ES-TOF MS analysis in
negative mode gave m/z = 3965.
We gratefully acknowledge financial support from the Swedish
Research Council.
References
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OBAN 5. 1 mg (2.9 µmol) of carbamate 14 was dissolved in
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Conditions for RNA cleavage studies. All kinetic runs were
performed at t = 37 ЊC and pH 7.4 (10 mM HEPES buffer
containing 0.1 M NaCl and 100 µM Zn(NO3)2). Reactions were
performed with equimolar concentrations of substrate and
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