RNA cleavage by 2,9-diamino-1,10-phenanthroline PNA conjugates

Article abstract of DOI:10.1080/15257770701542769

We report on the synthesis of 2,9-diamino-1,10-phenanthroline PNA conjugates as well as on their action in cleavage of a target RNA. Synthesis of the PNA conjugates are performed on solid support and the phenanthroline derivative is conjugated either to the amino-end or to a centrally positioned diaminopropionic acid in the PNA via a urea linker. Cleavage of the target RNA is achieved and compared to cleavage with the corresponding 2,9-dimethyl-1,10- phenanthroline and glycine conjugates. Copyright Taylor & Francis Group, LLC.

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RNA Cleavage by 2,9-Diamino-1,10-
Phenanthroline PNA Conjugates
Merita Murtola a , Dimitri Ossipov a , Jessica Sandbrink a & Roger
Strömberg a
a Department of Biosciences and Nutrition , Karolinska Institutet,
Novum , Huddinge, Sweden
Published online: 05 Dec 2007.
To cite this article: Merita Murtola , Dimitri Ossipov , Jessica Sandbrink & Roger Strömberg (2007)
RNA Cleavage by 2,9-Diamino-1,10-Phenanthroline PNA Conjugates, Nucleosides, Nucleotides and
Nucleic Acids, 26:10-12, 1479-1483, DOI: 10.1080/15257770701542769
To link to this article: http://dx.doi.org/10.1080/15257770701542769
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Nucleosides, Nucleotides, and Nucleic Acids, 26:1479–1483, 2007
Copyright ꢀ Taylor & Francis Group, LLC
C
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770701542769
RNA CLEAVAGE BY 2,9-DIAMINO-1,10-PHENANTHROLINE PNA
CONJUGATES
Merita Murtola, Dimitri Ossipov, Jessica Sandbrink, and Roger Stro¨ mberg
Department of Biosciences and Nutrition, Karolinska Institutet, Novum,
2
Huddinge, Sweden
We report on the synthesis of 2,9-diamino-1,10-phenanthroline PNA conjugates as well as on
2
their action in cleavage of a target RNA. Synthesis of the PNA conjugates are performed on solid
support and the phenanthroline derivative is conjugated either to the amino-end or to a centrally
positioned diaminopropionic acid in the PNA via a urea linker. Cleavage of the target RNA is
achieved and compared to cleavage with the corresponding 2,9-dimethyl-1,10-phenanthroline and
glycine conjugates.
Keywords PNA conjugate; solid phase synthesis; artificial nuclease; 2,9-diamino-1,10-
phenanthroline
Rationally designed oligonucleotide based artificial nucleases (OBANs) may
be used as artificial restriction enzymes in vitro or as novel therapeutic
agents for a wide variety of diseases.[1] In the present study, 2,9-diamino-
1,10-phenanthroline PNA conjugates have been synthesized and the evalua-
tion for their use as artificial ribonucleases in the presence of Zn2+ ions has
been initiated.
The 5-amino-2,9-dimethyl-1,10-phenanthroline Zn2+ complex has been
shown to work as an RNA cleaving group in several artificial ribonuclease
studies.[2] On the other hand, the Cu2+ complex of the 2,9-diamino-1,10-
phenanthroline has been reported to hydrolyze 2ꢁ,3ꢁ-cAMP substantially
faster than the Cu2+ complex of the 2,9-dimethyl-1,10-phenanthroline.[3]
We therefore decided to explore the 2,9-diamino-1,10-phenanthroline as
the cleaving group of PNA-based OBANs.
PNA was chosen as backbone because of its many desirable qualities.[4,5]
PNA oligomers can easily be synthesized by solid phase peptide synthe-
sis protocols[5] and readily can be modified with additional amino acids
Address correspondence to Roger Stro¨mberg, Bioorganic Chemistry, Department of Bio-
sciences and Nutrition, Karolinska Institutet, NOVUM, S-14157 Huddinge, Sweden. E-mail:
roger.stromberg@biosci.ki.se
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M. Murtola et al.
or other functional groups. PNAs are capable of binding target RNA in a
sequence-specific fashion.[4,5] PNAs are also chemically stable and highly re-
sistant to degradation in biological fluids.[4,5] In the present study we modi-
fied PNA with an internally placed diaminopropionic acid (dapa) and with
a glycine placed at the N-terminal, both serving as amino linkers for post
conjugation of the phenanthroline derivatives.
2,9-Diamino-1,10-phenanthroline (1)[6] was converted into the bis-
phenylcarbamate (2) in THF, by reaction with phenyl chloroformate
(Scheme 1). PNA was synthesized with Fmoc chemistry using a standard pro-
tocol (for the Applied Biosystems 433A peptide synthesizer). The dapa unit
was methyltrityl (Mtt) protected on the side chain. Synthesis of the different
PNA 2,9-diamino-1,10-phenanthroline conjugates is outlined in Scheme 1.
Synthesis of PNA 1 from the assembled and capped PNA-dapa hybrid was
carried out manually but on solid support as follows. The Mtt was first re-
moved from the dapa unit by treatment with 1% TFA in dichloromethane
(repeated 5 times). Subsequent conjugation with the diaminophenanthro-
line derivative was carried out with 5 eq. of bis-phenylcarbamate reagent (2)
and 20 eq. 4-methylmorpholine in N-methylpyrrolidone (NMP) overnight
at room temperature. After cleavage of the PNA-conjugate from support
with trifluoroacetic acid/triisopropylsilane/water (95:2.5:2.5), the crude
PNA was freeze dried, extracted with cold diethylether and then treated
for 10 minutes with 1.0 M NaOH to cleave off the remaining phenyl car-
bamate. Synthesis of PNA2 from the solid support bound Fmoc-glycine-
PNA also was carried out manually. After removal of the Fmoc protecting
group with 20% piperidine in NMP the resin bound PNA-glycine was re-
acted with 2 under the same condition as for PNA1. The Mtt protection
on dapa was removed (with 1% TFA as above) prior to cleavage from sup-
port in order to conveniently wash off the protecting group. PNA2 was
then obtained after cleavage from support and hydroxide treatment in
the same fashion as for PNA1. In order to compare the activity of the
2,9-diaminophenanthroline conjugate with a 2,9-dimethylphenanthroline
derivative we synthesized PNA3 using the same procedure as for PNA2 ex-
cept that the 5-phenoxycarbamoyl-2,9-dimethylphenanthroline reagent was
used and the final NaOH treatment was omitted. PNA4 was obtained by
removing Fmoc and Mtt protection (using the same reagents as above)
and then cleaving the PNA-glycine from support using the TFA/TIS/water
reagent.
All PNA conjugates were purified by RP-HPLC,[7] freeze dried three
times and characterized with Micromass LCT ESI-TOF mass spectrometry.
PNA1: Mobs 3545.0, Mcalc 3544.5, PNA2: Mobs 3542.0, Mcalc 3541.5, PNA3:
Mobs 3555.0, Mcalc 3554.5 and PNA4: Mobs 3306.0, Mcalc 3305.2 (Figure 1).
Kinetic cleavage studies have been initiated using equimolar amounts of
artificial nuclease and RNA target. Reactions were carried out at 37◦C with a
4 µM conc. of both PNA and substrate RNA (in 10 mM HEPES buffer (pH

RNA Cleavage
1481
SCHEME 1 Synthesis of PNA 2,9-diamino-1, 10-phenanthroline conjugates. Mtt = 4-methyltrityl, dapa
= diaminopropionic acid, NMP = N-methylpyrrolidone, TIS = triisopropylsilane.
7.4) containing 0.1 M NaCl and 100 µM Zn(NO3)2). The target RNA was
chosen to form a A4 bulge opposite to the diaminopropionic acid (dapa)
unit and with a closing G-T wobble base pair on one side. The initial re-
sults from the cleavage studies give the following half-lifes, PNA2 60 hours,
PNA3 140 hours, and PNA4 43 hours (cleavage with PNA1 as yet not com-
pleted). With PNA2 cleavage appears to be more site selective than with

1482
M. Murtola et al.
FIGURE 1 a) Schematic presentation of the PNA conjugates and the artificial ribonuclease substrate
RNA; b) ion exchange-HPLC chromatograms from the analysis of reaction mixtures of PNA and RNA
substrate. RNA substrate retention time is 29 minutes and the other peaks are fragments formed in the
reaction. A) PNA2, after 53 hours B) PNA3, after 143 hours C) PNA4, after 32 hours.
PNA3 (Figure 1b). Interestingly PNA4 gives considerably higher selectivity
than both PNA2 and PNA3 in cleavage of the target RNA (Figure 1b). It ap-
pears that under these conditions and linked in this way the glycine moiety
actually excerts higher cleavage activity than the phenanthroline derivatives.
This is unlikely to be general as it is bound to be highly dependent on po-
sitioning of the cleaving groups relative to cleaved phosphates, but further
investigation are need to clarify this. The result with PNA4 suggest that, for
the purpose of developing more selective cleavers, this finding is worth a
look at in more detail.
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20 min was used. Retention times were 35 min for PNA1 and 32 min for PNA2. PNA3 and PNA4
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rate of 4 ml/min and a linear gradient of 10% B for 20 min, then 10–37% B for 20 min was used.
Retention times were 32 min for PNA3 and 27 min for PNA4.