A method for synthesis of an artificial ribonuclease

Article abstract of DOI:10.1081/NCN-100002561

5-Amino-2,9-dimethyl-1,10-phenanthroline-oligonucleotide conjugates have been synthesized. A 2′-O-methyl octaribonucleotide carrying a 2′-aminoethoxymethyl linker in a central position was produced. Reaction of the aminoneocuproine phenyl carbamate with t

Full text of DOI:10.1081/NCN-100002561

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A METHOD FOR SYNTHESIS OF AN ARTIFICIAL
RIBONUCLEASE
Hans Åström a & Roger Strömberg b
a Division of Organic and Bioorganic Chemistry, MBB, Scheele Laboratory, Karolinska
Institutet, Stockholm, S-171 77, Sweden
b Division of Organic and Bioorganic Chemistry, MBB, Scheele Laboratory, Karolinska
Institutet, Stockholm, S-171 77, Sweden
Published online: 07 Feb 2007.
To cite this article: Hans Åström & Roger Strömberg (2001) A METHOD FOR SYNTHESIS OF AN ARTIFICIAL RIBONUCLEASE,
Nucleosides, Nucleotides and Nucleic Acids, 20:4-7, 1385-1388
To link to this article: http://dx.doi.org/10.1081/NCN-100002561
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS, 20(4–7), 1385–1388 (2001)
A METHOD FOR SYNTHESIS OF AN ARTIFICIAL
RIBONUCLEASE
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Hans Astro¨m and Roger Stro¨mberg∗
Division of Organic and Bioorganic Chemistry, MBB, Scheele
Laboratory, Karolinska Institutet, S-171 77 Stockholm, Sweden
ABSTRACT
5-Amino-2,9-dimethyl-1,10-phenanthroline-oligonucleotide conjugates have
been synthesized. A 2ꢀ-O-methyl octaribonucleotide carrying a 2ꢀ-amino-
ethoxymethyl linker in a central position was produced. Reaction of the amino-
neocuproine phenyl carbamate with the fully deprotected oligonucleotide in
aqueous solution gave virtually quantitative conversion into the conjugate. Pre-
liminary cleavage studies in presence of zinc ions show nuclease activity to-
wards RNA targets.
Oligonucleotide based artificial nucleases for hydrolytic cleavage of RNA
have potential for use in the next generation of antisense reagents. Such artificial
nucleases are generally designed to cleave target sequences using a tethered cat-
alytic group (1). These compounds have the advantage that catalytic turnover can be
achieved without the need of cellular enzymes, like RNase H. The catalytic group
can be an acid-base catalyst or a metal chelate. In the present study conjugates of 5-
amino-2,9-dimethyl-1,10-phenanthroline and a modified oligonucleotide have been
synthesized and evaluated for use as artificial ribonucleases in the presence of Zn2+.
Use of the same phenanthroline derivative in a different oligonucleotide based artifi-
cial nuclease construct has recently been reported (2). The initial design is based on
a 2ꢀ-O-methyl modified 8-mer (5ꢀ-GAGUACUC-3ꢀ) oligonucleotide (1) carrying a
2ꢀ-aminoethoxymethyl linker in a central position (Fig. 1a). The amino linker is used
to attach 5-amino-2,9-dimethyl-1,10-phenanthroline via a urea linkage (Fig. 1b).
∗Corresponding author.
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Copyright 2001 by Marcel Dekker, Inc.
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ASTROM AND STROMBERG
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Figure 1. b shows a schematic presentation of the conjugate part of 1. a shows the oligonucleotides
used in this study, 2 and 3 are substrates for the nuclease. c shows HPLC analysis of the conjugation
reaction: The top cromatogram is non conjugated. The second is reaction mixture without oligomer.
The third spectra shows the crude reaction mixture after incubation for 2 hours. The bottom chro-
matogram is from mixing the above reaction mixture with non conjugated oligomer. The first peak in
the third chromatogram was isolated and identified as the peak corresponding to 1.
A synthetic route for the preparation of the nucleases has successfully been
developed. The catalytic group could be conjugated in aqueous media to the al-
ready assembled and deprotected oligonucleotides in a virtually quantitative yield
by reacting the free amino group of the linker with the phenyl carbamate of the
amino phenanthroline derivative. Neocuproine (4) was first converted into the
5-NH2-analogue (Scheme 1). 5-NO2-2,9-dimethyl-1,10-phenanthroline (5) was

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SYNTHESIS OF AN ARTIFICIAL RIBONUCLEASE
1387
Scheme 1. Synthetic route to the neocuproine reagent.
synthesized from 4 using SO3/H2SO4, HNO3, 168◦C (3). The nitro derivative
5 was then reduced to 5-amino-2,9-dimethyl 1,10 phenanthroline (6) by catalytic
hydrogenation on palladium/carbon. The amino derivative 6 was then subsequently
converted into the phenyl carbamate 7 by reaction with phenyl chloroformate in
THF in the presence of triethyl amine (Scheme 1).
The building block bearing the linker arm was synthesized from uridine
(Scheme 2) via a previously developed route (Katcevica D., Rozners, E.,
Bizdena, E. and Stro¨mberg, R., unpublished results). Uridine was first protected
with the 1,1,3,3- tetraisopropyldisiloxyanylidene (TIPDS) group. Reaction of the
3ꢀ,5ꢀ-TIPDS-uridine with DMSO/acetic anhydride/acetic acid (4) gave after silica
gel chromatography and crystallization from hexane-ether the desired product 8.
Coupling of 8 with N-protected aminoethanol (made from reaction of
aminoethanol with S-Ethyl trifluorothioacetate) using NIS/TfOH (5) activation
gave the desired product 9. The silyl protection was removed using fluoride ion
and the deprotected compound was converted into building block 10 suited
for use in the H-phosphonate method for oligonucleotide synthesis (6). This
was achieved by standard monomethoxytritylation in pyridine followed by
phosphonylation with the PCl3/imidazole reagent (7). The oligonucleotides were
then made by machine assisted solid phase synthesis using a standard
protocol.
The conjugation reaction was performed in sodium tetraborate buffer (pH
8.5) and seemed to be quantitative as judged from analysis by reversed phase
HPLC (Fig. 1c). The oligonucleotide conjugate was purified by reversed phase
HPLC giving homogeneous products and mass spectrometry analysis confirmed
the identity of the conjugate.
Scheme 2. Coupling of protectd aminoethanol to 5.

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The thermal stability of the 2ꢀ-O-Me modified oligonucleotide 1 was deter-
mined by means of thermal melting of complex with complementary sequence 2.
In order to determine the effect of the catalytic group on the duplex-stability, the
melting temperature was checked both before and after conjugation with 7. The
melting point was determined to 44◦C and 39◦C respectively.
Kinetic cleavage studies have been initiated using the 5-aminoneocuproine-
1 nuclease to cleave complementary oligonucleotide substrate 2 and sequence 3
that contains an additional adenosine unit in the middle of the sequence. In both
cases cleavage was detected in presence of 10-mM Zn2+ ions at pH 7. In the case
of sequence 3 the cleavage appears to be more sequence selective. In the case of
5-aminoneocuproine-1 nuclease cleaving substrate 2 the estimated first order rate
constant is 2 × 10−4 min−1.
EXPERIMENTAL
Synthesis of the 5-aminoneocuproine-1 conjugate. Carbamate 7 (250 µg,
0.73 µmol) was dissolved in 14 µl dry DMSO. To this solution was added:
7 µl H2O, 75 µl Sodium tetraborate buffer (0.1-M, pH 8.5) and finally a 4 µl
(37 nmol) solution of oligonucleotide 1. The vial containing the reaction mixture
was agitated, 2 µl aliquotes were withdrawn from the reaction mixture, filtrated,
diluted to 100 µl water and analysed with reversed phase (RP) HPLC. The reaction
was incubated overnight although it appeared complete in 2 h. The reaction mixture
was then filtered and purified on RP HPLC. 18.3 nmol product corresponding to a
yield of 50% was isolated. Both analytical and preparative HPLC was performed
on a Hypersil ODS column (5 µm, 4.6 × 25 mm) using 5 min isocratic elution with
0.1-M triethylammonium acetate (aq.), then a linear gradient to the same buffer
containing 20% MeCN in 35 min and finally from 20 to 50% MeCN in 20 min.
Flow rate was 1 ml/min and temperature 50◦C.
REFERENCES
1. Trawick, B.N.; Daniher, A.T.; Bashkin, J.K. Chem. Rev. 1998, 98, 939–960.
2. Putnam, W.C.; Bashkin, J.K. Chem. Comm. 2000, 767–768.
3. Smith, G.F.; Cagle, F.W. JR. J. Org. Chem. 1947, 781–784.
4. Pojer, P.M.; Angyal, S.J. Tetrahedron Lett. 1976, 3067–3068.
5. Veeneman, G.H.; Van der Marel, G.A.; Van der Elst, H.; Van Boom, J.H. Tetrahedron
1991, 47, 1547–1562.
6. (a) Garegg, P.J.; Lindh, I.; Regberg, T.; Stawinski, J.; Stro¨mberg, R.; Henrichson,
C. Tetrahedron Lett. 1986, 27, 4051–4054. (b) Garegg, P.J.; Lindh, I.; Regberg, T.;
Stawinski, J.; Stro¨mberg, R.; Henrichson, C. Tetrahedron Lett. 1986, 27, 4055–4058.
(c) Froehler, B.C.; Matteucci, M.D. Tetrahedron Lett. 1986, 27, 469–472.
7. (a) Garegg, P.J.; Regberg, T.; Stawinski, J.; Stro¨mberg, R. Chemica. Scripta 1986, 26,
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