ABSTRACT
Considerable effort has been directed towards studying the structure and
function of nucleic acids and several approaches rely on the attachment of
reporter groups or reactive functional groups to nucleic acids. We report here
the selective modification of 2
'
-amino groups in oligoribonucleotides, through their reaction with
aliphatic isocyanates, to give the corresponding 2
'
-urea derivatives in >95% yield. Furthermore, such modification with (2-isocyanato)ethyl 2-pyridyl disulfide enables subsequent coupling to other thiols
(such as those contained in peptides and proteins) or to thiol-reactive electrophiles. A modified decamer was not significantly
destabilized by the 2
'
-urea group, compared with a 2
'
-amino group, as demonstrated by a mere 1.3
oC drop in the melting temperature of the duplex.
The conjugation of reporter groups and reactive groups to oligonucleotides
represents a useful approach for studying the structure and function of nucleic
acids (for recent reviews see
1
-
3
). The attachment of such groups onto oligonucleotides can be achieved either by
their incorporation during chemical synthesis or by post-synthetic modification. In the latter case it is often desirable to have
an oligonucleotide with a reactive functional group, such as an amine or a
thiol, which must be introduced during oligonucleotide synthesis. One of the
advantages of post-synthetic labelling is that there is a wide choice of compounds that can
be attached to the oligonucleotides and the choice of compound is not
restricted by the availability of phosphoramidites. Furthermore, the functional
groups to be incorporated might be incompatible with the conditions used in
oligonucleotide synthesis.
Although there are numerous examples of post-synthetic labelling at the ends of oligonucleotides, relatively few
examples have been reported for modification at internal positions (
4
-
10
). One of these approaches utilizes the incorporation of 2'-amino groups into oligonucleotides (
11
,
12
), which can be selectively reacted with aromatic isothiocyanates to form 2'-thiourea-modified oligomers (
6
,
7
). This method was recently utilized for the site-specific introduction of two thiol groups into the hammerhead ribozyme to
probe its tertiary structure by disulphide-mediated cross-linking (
13
,
14
). The drawback of this method is that an aromatic thiourea substituent could
cause structural perturbations in a complex three-dimensional RNA structure due to both its size and inflexibility. To avoid
these potential complications in our continued efforts towards establishing
tertiary interactions within ribozymes, we were interested in incorporating
smaller and more flexible aliphatic groups at internal 2' positions of such catalytic oligoribonucleic acids. We report here that
aliphatic isocyanates, such as methylbenzylisocyanate and (2-isocyanato)ethyl 2-pyridyl disulfide (isocyanate
1
), react selectively and in high yield with 2'-amino groups in oligoribonucleotides and describe the preparation
and characterization of such a modified oligoribonucleotide.
The aliphatic isocyanate
1
(Fig.
1
) was synthesized using a slightly modified procedure from our previously
published method for the efficient synthesis of isocyanates from aliphatic
amines (
15
). When the 2'-amino-containing oligoribonucleotide
I
was reacted with isocyanate
1
(Figure.
2
), under the same conditions as previously utilized for modification with
aromatic isothiocyanates (
13
), complete conversion to oligomer
II
was obtained. However, a control oligomer lacking a 2'-amino group (GCCGACCGACAUU) also yielded minor products, indicating
non-specific side reactions of the isocyanate with the oligoribonucleotide.
The reaction conditions (pH, temperature and concentration of isocyanate) were
therefore optimized such that the 2'-amino group was selectively modified without concurrent side
reactions. Interestingly, the yield of the 2'-modified oligomer
II
increased upon lowering the temperature, presumably due to repression of
competing isocyanate hydrolysis. Thus, incubation of oligomer
I
(1 mM) with isocyanate
1
(15 mM) at pH 8.6 and 0oC gave 96% conversion to oligomer
II
, whereas an oligomer lacking the 2'-amino group was unchanged, as monitored by HPLC (Fig.
3
). Selective modification of oligomer
I
with the secondary isocyanate
R
-(+)-[alpha]-methylbenzylisocyanate was achieved in >95% yield (data
not shown). For further characterization of the 2'-urea-derivatized oligomer
II
, its preparation was scaled up, followed by HPLC purification. Scaling up the
reaction required either a second addition of isocyanate
1
or the reaction to be performed at lower temperatures (-8oC) to give comparable yields to that of the analytical reactions.
The simplest experimental approach for derivatizing oligonucleotides is to
attach the reporter or reactive functional groups at either the 3'- or 5'-terminus, for which a variety of methods are available
(for reviews see
1
-
3
). However, it is often desirable to introduce probes at internal positions in
nucleic acids to investigate their interactions with other macromolecules. The
probes can be linked either to the nucleotide bases or the sugar-phosphate backbone, which minimizes disruption of base pairing
interactions. One method has been to label 2'-hydroxyl groups by the use of modified phosphoramidites during
oligomer synthesis. Sproat and co-workers have stabilized RNA molecules against nuclease degradation by
alkylation of 2'-hydroxyl groups (
16
,
17
), as well as linking an ethylene group containing a terminal thiol for post-synthetic labelling (
18
). The 2'-hydroxyl group has also been utilized for tethering anthracene at
internal positions in oligomers (
19
) and for installing an amino group, through a five carbon atom tether, for post-synthetic labelling (
20
). Modified phosphoramidites have also been used for the incorporation of amides
(
21
,
22
) at the 2'-position of oligoribonucleotides.
Alternatively, oligomers containing 2'-amino groups (
11
,
12
) can be post-synthetically modified by reaction with aromatic isothiocyanates, to yield
the corresponding 2'-thiourea derivatives (
6
,
7
,
13
). Attempts to extend this approach to aliphatic groups by the reaction of 2'-amino-containing oligomers with succinimidyl esters (
7
) or aliphatic isothiocyanates (data not shown) have met with limited success.
We have shown here that the 2'-amino group in oligoribonucleotides reacts readily with aliphatic
isocyanates to yield a 2'-urea derivative. Characterization of oligomer
II
by enzymatic digestion and comparison of the modified nucleotide with an
authentic sample is fully consistent with the structure depicted in Figure
2
. Thus, post-synthetic labelling of the sugar-phosphate backbone in oligoribonucleic acids can be accomplished by
reaction of 2'-amino groups with either aromatic isothiocyanates or aliphatic
isocyanates and complements the labelling of nucleotide bases by the
convertible nucleoside approach of Verdine and co-workers (
8
,
10
).
An important criterion for practical oligomer labelling is the effect of the
newly introduced functionality on duplex stability. It has been determined that
the introduction of 2'-amino groups into RNA oligomers results in a moderate decrease in
duplex stability; the melting temperature of an RNA duplex was 4.3oC lower after introduction of the modification, corresponding to the energy
of a hydrogen bond (
7
). We have shown here that subsequent modification of the 2'-amino group with isocyanate
1
results in a further decrease of only 1.3oC. These destabilizing effects are of similar magnitude to those reported
for other internal labels at either the sugar or base moieties of
oligonucleotides (
4
,
7
,
21
), with the exception of intercalating agents, which can stabilize duplex
structure (
19
).
To further the scope of this labelling strategy to molecules containing
functional groups other than aliphatic isocyanates we have used isocyanate
1
, which serves as a molecular adapter for introducing an activated (protected)
thiol. Reduction of the disulphide in oligomer
II
yielded the free thiol
III
, which can participate in nucleophilic substitution and addition reactions with
a variety of functional groups (
23
), as illustrated here by reaction with bromobimane (
24
) (Figs
6
and
7
). Thiols can also be used to form disulphides, which is useful for thiol-mediated cross-linking (
13
,
25
,
26
) and for the reversible incorporation of labels into oligonucleotides. The
thiol-protecting group in isocyanate
1
serves to activate the thiol for formation of mixed disulphides after its
incorporation into oligoribonucleotides (
27
). This approach has found use in the formation of oligonucleotide conjugates
with proteins (
28
) and peptides (
29
,
30
) and is demonstrated here by the efficient reaction of glutathione with
oligomer
II
to form oligomer conjugate
V
(Figs
6
and
7
).
We have shown that aliphatic isocyanates react selectively with 2'-amino groups in oligoribonucleotides to form 2'-urea derivatives in yields >95%. The introduction of
this modification causes only a minor destabilization of the duplex structure.
This strategy, coupled with our recent method for the efficient preparation of
aliphatic isocyanates containing sensitive functionalities (
15
), provides a general approach for the site-specific labelling of RNA using compounds that contain aliphatic amines.
In addition, the use of isocyanate
1
expands the range of functionalities for incorporating labels into oligomers to
thiols and thiol-reactive electrophiles.
Syntheses were carried out under a positive pressure of argon. 2'-Aminouridine was purchased from Amersham International,
trichloromethyl chloroformate and
R
-(+)-[alpha]-methylbenzylisocyanate from Aldrich Chemical Co.,
bromobimane from Sigma and glutathione (reduced form) from Merck. Snake venom
phosphodiesterase (
Crotalus durissus
) and calf spleen alkaline phosphatase were purchased from Boeringer Mannheim.
Flash column chromatography was performed on silica gel 60 (Merck) with a
particle size of 0.04-0.063 mm.
1
H NMR and
13
C NMR spectra were recorded in DMSO-d
6
on a Bruker AM 360L instrument at 360.13 and 90.55 MHz respectively. Chemical
shifts are reported in p.p.m. and coupling constants (J) in Hz. High
resolution, accurate mass spectra (HRMS) were recorded on a on VG Analytical
Autospec-T tandem mass spectrometer using electron impact ionization.
Oligoribonucleotides were prepared by automated chemical synthesis using
phosphoramidites from MilliGen/Biosearch, except for the incorporation of
trifluoroacetyl-protected 2'-amino-modified nucleotides (
11
). Deprotection and purification of oligoribonucleotides was performed as
previously described (
31
). Concentrations of oligomers were calculated using a molar extinction
coefficient of 6600 M
-1
cm
-1
/nucleotide, except for preparations of solutions for melting experiments (see
below).
HPLC analyses were carried out on a Waters Associates System with Model 6000A
pumps, a Model 680 Automated Gradient Controller, a Model 730 Data Module and a
Model 481 LC Spectrophotometer. Separations by HPLC were performed using
reverse phase ODS Hypersil (5 [mu]m; Shandon). Solvent gradients for analytical HPLC were run at 2 ml/min.
Elution was performed with a linear gradient of 100 mM triethylammonium
acetate, pH 7.0, containing from 0 to 16% CH
3
CN over 15 min, followed by an increase to 70% CH
3
CN over 5 min, which was then maintained for 10 min, with a subsequent return to
the original conditions (0% CH
3
CN) over 3 min. For preparative HPLC purification of modified oligomers, the
following modifications to the analytical conditions were made: flow-rate was 4 ml/min, 100 mM triethylammonium bicarbonate, pH 7.0, was used
instead of 100 mM triethylammonium acetate, pH 7.0, and during the elution the
conditions were left at 16% CH
3
CN for 5 min.
We observed that
1
slowly hydrolyses (~30% after 4 weeks) when stored concentrated at -20oC. However, when it was stored desiccated at -20oC, as 1 mg aliquots in 100 [mu]l CH
2
Cl
2
, the isocyanate was still intact after 3 months. The solvent was removed
in vacuo
prior to reaction with the oligomers.
Substance
2
.
A suspension of 2'-aminouridine (0.100 g, 0.411 mmol) in DMF (0.5 ml) was treated with
a solution of
1
in DMF (0.5 ml) and stirred at 25oC for 30 min. The solvent was removed
in vacuo
and the product was purified by flash column chromatography (5% MeOH/CH
2
Cl
2
) to yield
2
as a colourless oil (0.177 g, 94%).
1
H NMR (DMSO-d
6
; DMSO-d
5
as an internal standard at [delta] 2.50 p.p.m.): [delta] 2.81 (2H, t, J=6.6, SCH
2
), 3.21 (2H, m, NHC
H
2
), 3.56 (2H, m, H5'), 3.90 (1H, m, H4'), 4.02 (1H, m, H3'), 4.29 (1H, m, H2'), 5.16 (1H, t, J=5.0, 5'-OH), 5.65 (1H, d, J=8.1, H5), 5.81 (1H,
d, J=14.6, H1'), 5.82 (1H, s, 3'-OH), 6.13 (1H, d, J=8.7, 2'-NH), 6.58 (1H, t, J=5.7, CH
2
N
H
), 7.24 (1H, m, ArH), 7.79 (2H, m, ArH), 7.86 (1H, d, J=8.1, H6), 8.45 (1H, m,
ArH), 11.25 (1H, s, H3).
13
C NMR (DMSO-d
6
; DMSO-d
5
as an internal standard at [delta] 39.5 p.p.m.): [delta] 38.4, 38.7, 55.3, 61.9, 70.9, 86.4, 86.8, 102.1, 119.3, 121.2,
137.8, 141.0, 149.6, 151.1, 157.5, 159.3, 163.3. HRMS: 456.1006 (calculated
456.1011 for C
17
H
21
N
5
O
6
S
2
).
Reactions of isocyanates with oligoribonucleotides on an analytical scale were
carried out in an ice bath in a cold room (6oC). A solution of oligomer
I
(2 mM in 2.5 [mu]l 70 mM borate buffer, pH 8.6) was treated sequentially with DMF (2.0 [mu]l) and
1
in DMF (150 mM, 0.5 [mu]l) or
R
-(+)-[alpha]-methylbenzylisocyanate in DMF (100 mM, 0.5 [mu]l) and incubated for 3.5 h. For HPLC analyses, 1.5 [mu]l aliquots of reaction mixture were diluted
into 20 [mu]l triethylammonium acetate buffer (100 mM, pH 7.0) and extracted with CH
2
Cl
2
(2 * 150 [mu]l) to remove the organic material.
Large scale preparation of oligomer
II
was carried out in an ice bath. To a solution of oligomer
I
[5'-AGCGA(2'-NH
2
U)GCGA] (2 mM in 233 [mu]l 70 mM borate buffer, pH 8.6) was added
1
in DMF (30 mM, 233 [mu]l), followed by purification of the modified oligomer by HPLC. This reaction
resulted in 78% conversion to product, but a second addition of the isocyanate
or carrying out the reactions at -8oC resulted in comparable yields to those obtained in the analytical
reactions.
Enzymatic digestion of the oligoribonucleotides was essentially performed as
described by Connolly (
33
). A solution of the oligoribonucleotides (0.2 mM) in Tris buffer (56 [mu]l, 50 mM, pH 8.0) containing MgCl
2
(10 mM) at 37oC was treated sequentially with snake venom phosphodiesterase (6 [mu]l, 0.003 U/[mu]l) (incubation for 5 h) and alkaline phosphatase (6 [mu]l, 1 U/[mu]l) (further incubation at 37oC for 0.5 h) followed by HPLC analysis of the digest.
Absorption measurements (260 nm) for generating melting curves were recorded
with a Uvicon 820 spectrophotometer (Kontron, Zürich, Switzerland) at 1oC intervals. The molar extinction coefficients were calculated for
oligomer
I
[5'-AGCGA(2'- NH
2
U)GCGA; 107 * 10
3
M
-1
cm
-1
) and the complementary sequence (5'-UCGCAUCGCU; 92 * 10
3
M
-1
cm
-1
) using extinction coefficients of nucleotides and dinucleotides (
34
). The additional contribution of the thiopyridyl moiety to the extinction
coefficient of oligomer
II
was estimated to be 3000 M
-1
cm
-1
by comparing the integrated areas under the peaks of 2'-NH
2
U and
2
in the HPLC traces of the enzymatic digests of oligomers
I
and
II
respectively. The oligomers (2 [mu]M) in sodium cacodylate buffer (10 mM, pH 7.0) containing EDTA (1 mM) and
NaCl (1 M) were incubated for 3 min at 56oC and cooled slowly to 25oC in order to ensure complete hybridization of the two strands before
recording the absorbance versus temperature.
We thank S.Alefelder, P.Heaton, J.Thomson and T.Tuschl for critical reading of
the manuscript, E.Jares-Erijman for help in obtaining melting curves, K.Eckart for HRMS analyses
and J.Ficner, U.Kutzke and B.Seeger for expert technical assistance. Supported
by the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie.
SThS acknowledges a long-term post-doctoral fellowship from the European Molecular Biology
Organization.
(2-Isocyanato)ethyl 2-pyridyl disulphide (
1
).
(2-Isocyanato)ethyl 2-pyridyl disulphide was synthesized using a slightly modified
procedure from that previously reported (
15
).
S
-(2-Pyridyldithio)cysteamine hydrochloride 3 (0.100 g, 0.449 mmol) was
partitioned between CH
2
Cl
2
(1.5 ml) and 1 M NaOH (1 ml). The organic phase was separated and dried (Na
2
SO
4
) (note that this material decomposes upon concentration of the solution) and
added drop-wise to a stirred solution of trichloromethyl chloroformate (0.022 g, 0.11
mmol) in CH
2
Cl
2
(1 ml) at 0oC over a period of 1 min. After stirring for 2 min at 0oC, the suspension was partitioned between 1 M HCl (5 ml) and
CH
2
Cl
2
(10 ml), after which the organic phase was separated and washed successively
with 1 M HCl (1 ml) and 1 M NaOH (1 ml). After drying the organic phase (Na
2
SO
4
), the solvent was removed
in vacuo
to yield
1
as a pale yellow oil (0.024 g, 25% based on the starting amine), which was ~98% pure as determined by
1
H NMR analysis.
Reactions with bromobimane.
To a solution of oligomer
II
(2 mM in 4 [mu]l 50 mM HEPES buffer, pH 8.0, 5 mM EDTA) was added DTT (10 mM, 3 [mu]l), followed by incubation under argon at 37oC for 1.5 h, after which HPLC analysis indicated that reduction was
complete. An aliquot (2 [mu]l) of this solution was added to a solution of bromobimane (20 mM in 2 [mu]l 50% CH
3
CN-25 mM HEPES, pH 8.0, 2.5 mM EDTA) and incubated in the dark at 25oC for 1 h, followed by HPLC analysis.
Reactions with glutathione.
The disulphide-containing oligomer
II
(0.9 mM) was incubated with the reduced form of glutathione (1.8 mM) in borate
buffer (5 [mu]l, 35 mM, pH 8.6) under argon at 25oC for 2.5 h, followed by HPLC analysis.
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