Multicomponent synthesis of artificial nucleases and their RNase and DNase activity

Article abstract of DOI:10.3762/bjoc.7.131

The synthesis of new, artificial ribonucleases containing two amino acid residues connected by an aliphatic linker has beendeveloped. Target molecules were synthesized via a catalytic three-component Ugi reaction from aliphatic diisocyanides. Preliminaryinvestigations proved unspecific nuclease activity of the new compounds towards single-stranded RNA and doublestrandedcircular DNA .

Full text of DOI:10.3762/bjoc.7.131

Multicomponent synthesis of artificial nucleases and
their RNase and DNase activity
Anton V. Gulevich1, Lyudmila S. Koroleva2,3, Olga V. Morozova2,
Valentina N. Bakhvalova4, Vladimir N. Silnikov*2
and Valentine G. Nenajdenko*1
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2011, 7, 1135–1140.
1Department of Chemistry, Moscow State University, 119992,
Leninskie Gory, Moscow, Russia, 2Institute of Chemical Biology and
Fundamental Medicine, Siberian Branch of Russian Academy of
Sciences, 8 Lavrentyev Ave., 630090 Novosibirsk, Russia,
3Novosibirsk State University, 2 Pirogova St., 630090 Novosibirsk,
Russia and 4Institute of Systematic and Ecology of Animals, Siberian
Branch of Russian Academy of Sciences, 11 Frunze Street, 630091
Novosibirsk, Russia
doi:10.3762/bjoc.7.131
Received: 03 May 2011
Accepted: 14 June 2011
Published: 19 August 2011
This article is part of the Thematic Series "Multicomponent reactions".
Guest Editor: T. J. J. Müller
Email:
Vladimir N. Silnikov* - Silnik@niboch.nsc.ru;
Valentine G. Nenajdenko* - nen@acylium.chem.msu.ru
© 2011 Gulevich et al; licensee Beilstein-Institut.
License and terms: see end of document.
* Corresponding author
Keywords:
DNA; isocyanide; multicomponent reaction; organocatalysis;
peptidomimetic; RNA
Abstract
The synthesis of new, artificial ribonucleases containing two amino acid residues connected by an aliphatic linker has been
developed. Target molecules were synthesized via a catalytic three-component Ugi reaction from aliphatic diisocyanides. Pre-
liminary investigations proved unspecific nuclease activity of the new compounds towards single-stranded RNA and double-
stranded circular DNA.
Introduction
RNA cleavage can serve as a molecular tool for biological tics showed evident advantages due to their lower cytotoxicity
research [1], as well as for development of anticancer drugs and elevated potential penetration into living eukaryotic cells.
[2,3] and new therapeutics against RNA-containing viruses. Moreover, a few dipeptides [12] were shown to induce inter-
Recently, a number of synthetic RNA-cleaving molecules (arti- feron production, thus providing antivirus defence. Therefore,
ficial ribonucleases) had been developed and tested in vitro the development of new peptidomimetics with ribonuclease
[4-11]. Among numerous artificial ribonucleases, peptidomime- activity is an important task in organic and biomolecular chem-
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Beilstein J. Org. Chem. 2011, 7, 1135–1140.
istry. In this paper we present the rational design, multicompo- phatic diisocyanides 3, amines (with an easily removable
nent synthesis, and RT-qPCR quantitation of nuclease activity protective group) and aldehydes. We used an organocatalytic
of novel amino acids derivatives.
three-component modification of the Ugi reaction, recently
developed by List et al. [25]. The reaction results in diamines 4,
thus avoiding the acid residue removal stage.
Results and Discussion
Recently, a number of artificial peptide ribonucleases modeling
known catalytic centers of natural RNases A and T1 have been
described [13-15]. RNA cleavage was shown to be more effi-
cient in the presence of aliphatic hydrophobic linkers [16].
However, the potential role of the alkyl chain of the catalyst
remains unclear. Interaction of hydrophobic residues in peptides
was suggested to result in formation of RNase mimetics in solu-
tion thus enhancing their ribonuclease activity. To prove this
suggestion, symmetric aliphatic diamides 1 and 2 containing
natural amino acid residues have been synthesized (Figure 1).
The compounds showed high ribonuclease activity with model
oligoribonucleotides and an HIV-1 recombinant RNA fragment
96 nucleotides long [17].
Scheme 1: Multicomponent approach to target molecules.
The starting diisocyanides 3 were obtained in good overall
yields from commercially available diamines containing 6, 7, 8,
10 or 12 carbon atoms by the standard formylation–dehydra-
tion protocol (Scheme 2).
Figure 1: Novel artificial RNases based on amino acids.
Previously, compounds 1 and 2 have been synthesized from the
corresponding diamines by condensation with protected natural
amino acids and subsequent deprotection [18]. This approach is
significantly limited by using available natural amino acids (R
is a natural amino acid residue). Consequently, the develop-
ment of new simple, atom-economic methods for the synthesis
of this class of potential biologically active compounds is of
great importance in bioorganic and medicinal chemistry. The
development of multicomponent approaches is especially
important because multicomponent reactions (MCR) could be
Scheme 2: Synthesis of starting diisocyanides 3a–3e.
adapted to a high throughput synthesis of libraries of com- We found that these diisocyanides 3 participate successfully in
pounds.
the catalytic three-component reaction via a modified List
procedure [25]. Diamides 4 with benzyl protective groups were
It is known that isocyanide-based MCR are very efficient for synthesized in moderate to good yields under mild conditions.
synthesis of peptides and peptide molecules [19-24]. We There is no obvious dependence of yield on the length of the
proposed that the desired compounds 5, containing two amide carbon chain in 3; aliphatic aldehydes gave better results in
bonds and variable substituents, can be synthesized by the Ugi comparison to aromatic aldehydes (Scheme 3). Obviously, the
reaction with subsequent removal of diamine residue suggested approach is an efficient and short method to form the
(Scheme 1). Original substrates for the synthesis could be ali- skeleton of the target diamides. The benzyl groups can be easily
1136

Beilstein J. Org. Chem. 2011, 7, 1135–1140.
removed from compounds 4 by the standard hydrogenolysis Currently, real-time PCR is the better method for the
procedure. For example, using Pd/C as catalyst we obtained the quantitation of the target nucleic acids because of its high
target peptidomimetics 5 in up to 90% yield. Thus, we synthe- specificity and sensitivity of up to a few genome equivalents
sized a number of racemic peptidomimetics 4 and 5, containing in a complex mixture [26]. In the present work, ribonuclease
aliphatic or aromatic groups as well as various aliphatic linkers. activity of the new synthesized compounds was studied in
With these diamides in hand we began the investigation of their vitro by cleavage of the total cellular and the tick-borne
biological activity.
encephalitis virus (TBEV) full-length genomic RNA isolated
from infected mouse brain, with subsequent detection by
RT-qPCR (TBEV is a human pathogenic member of the
Flavivirudae family of RNA-containing viruses of positive
polarity).
Complete cleavage of 2 µg of cellular RNA including 105
genome equivalents of the TBEV full-length RNA was
observed after incubation of the total RNA from the virus-
infected mouse brain with 2.5 mM aqueous solutions of pepti-
domimetics 5a–g for 2 hours at 37 °C. Denaturating elec-
trophoresis in SDS-agarose gel revealed complete cleavage of
the total RNA (Supporting Information File 1, Figure S1) and
RT-real time PCR showed complete destruction of the TBEV
RNA (Figure 2).
Compounds 5e and 5g, the most hydrophobic among synthe-
sized substances, might potentially penetrate through cellular or
viral membranes and therefore the dependence of RNA
cleavage on the concentration of the peptidomimetics was
studied in detail. The concentration of compounds 5e and 5g,
optimal for RNA cleavage, was determined by varying the
Scheme 3: Synthesis of new aRNAses. Conditions: a. RCHO (3
eqiuv), BnNH2 (3 equiv), PhP(OH)2 (1 equiv), r.t.; b. Pd/C, 5%,
HCOONH4, MeOH/H2O, reflux.
Figure 2: Results of RT-qPCR of the TBEV RNA cleavage products in presence of 2.5 mM peptidomimetics 5a–g at 37 °C for 2 hours in H2O.
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Beilstein J. Org. Chem. 2011, 7, 1135–1140.
concentration from 2.5 × 10−3 to 2.5 × 10−7 M (Supporting
Information File 1, Figure S2 A and B). Dependence of optimal
RNA cleavage on the concentration of peptidomimetics was not
evident: Complete RNA cleavage of 2 μg RNA was observed at
a concentration of 2.5 mM for both 5e with an aliphatic
substituent and 5g with an aromatic one.
Figure 4: Results of electrophoresis in 2% TBE-agarose gel with
ethidium bromide of RT-qPCR products from Figure 3. (Cleavage of
total RNA from 10% brain suspensions from ICR mice infected with
TBEV). Lanes: 1 - negative control; 2 and 3 - compound 5e incubated
with RNA in RPMI 1640 and in H2O, respectively; 4 and 5 - compound
5g incubated with RNA in RPMI 1640 and in H2O, respectively; 6 and
7 - compound 5c incubated with RNA in RPMI 1640 and in H2O, res-
pectively; 8 and 9 - compound 5d incubated with RNA in RPMI 1640
and in H2O, respectively.
Ribonuclease activity of the compounds 5c–e and 5g at 2.5 mM
concentration was assayed in cultural medium RPMI 1640 and
compared cleavage in H2O. All artificial RNases cleaved RNA
more efficiently in water than in RPMI 1640 (Figure 3 and
Figure 4). Results of RT-real time PCR (Figure 3) and elec-
trophoresis RT-qPCR products in 2% TBE-agarose gel
(Figure 4) showed varying degrees of destruction of the TBEV Generally, all synthesized compounds were shown to be able to
RNA, respectively.
cleave completely single-stranded RNA but not single-stranded
cDNA or hybrid of RNA with cDNA after reverse transcription
To analyze DNase activity of the novel compounds, both irrespective of the structures of their substituents and the length
double-stranded circular recombinant plasmid DNA with cloned of polymethylene linkers. Double-stranded circular plasmid
full-length TBEV copy of genome and single-stranded cDNA DNA was partially destroyed possibly because of single-
after reverse transcription of the TBEV RNA from the infected stranded DNA breaks. The mechanism has been previously
mouse brain with random N6 primer was used. No destruction shown for several artificial metal-free [27] and metal-depen-
of single-stranded cDNA was observed after incubation with dent nucleases [28]. A further study of the TBEV RNA
2.5 mM solutions of compounds 5e or 5g for 2 hours at 37 °C cleavage, both in extracellular virions and within infected cells
(Supporting Information File 1, Figure S3). However, these as well as specific cleavage of only viral RNA, is required.
compounds could partly cleave double-stranded plasmid DNA Further investigations of developed artificial RNases are in
(Supporting Information File 1, Figure S4).
progress.
Figure 3: Results of RT-qPCR of the TBEV RNA cleavage products in the presence of 2.5 mM peptidomimetics after incubation in H2O and in cultural
medium RPMI 1640.
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Beilstein J. Org. Chem. 2011, 7, 1135–1140.
Conclusion
Compound 4a: The reaction was carried out in CH2Cl2, yield
New artificial nucleases based on diamides containing two 73%; colorless oil; Rf 0.4 (hexane/EtOAc 1:1); 1H NMR (400
amino acid residues connected by aliphatic linkers were synthe- MHz, CDCl3) δ 0.86 (d, J = 7.1 Hz, 6H, 2 × CH3), 0.94
sized by a catalytic three-component Ugi-type reaction and (d, J = 7.1 Hz, 6H, 2 × CH3), 1.29–1.36 (m, 4H,
subsequent deprotection. To quantitative cleavage of any ( C H 2 C H 2 C H 2 N H C O ) 2 ) , 1 . 4 4 – 1 . 5 2 ( m , 4 H ,
nucleic acids, including single-stranded RNA and cDNA as (CH2CH2CH2NHCO)2), 1.6 (br s, 2H, 2 × NH), 2.06–2.15 (m,
well as double-stranded circular plasmid DNA, high-throughput 2H, 2 × CH(CH3)2), 2.95 (d, J = 4.3 Hz, 2H, 2 × CH),
RT-qPCR was developed and used. All synthesized compounds 3.17–3.30 (m, 4H, (CH2CH2CH2NHCO)2), 3.68 (AB-system, J
were shown to be able to cleave completely single-stranded = 13.1 Hz, 4H, 2 × CH2Ph), 7.20–7.35 (m, 12H, Ph, 2 × NH);
RNA but not single-stranded cDNA or hybrid of RNA with 13C NMR (100 MHz, CDCl3) δ 173.3, 139.6, 128.6, 128.1,
cDNA after reverse transcription irrespective of the structure of 127.3, 67.9, 53.5, 38.6, 31.2, 29.7, 26.5, 19.6, 17.7; IR (cm−1)
their substituents and the length of the polymethylene linker.
1640 (CONH), 3300 (br, CONH); ESI-MS (m/z): [M + H]+
calcd for C30H46N4O2, 495.3621; found, 495.3698.
Experimental
General procedure for the synthesis of diiso-
Cleavage of the benzyl group
A solution of HCOONH4 (1 g in 5 mL H2O) was added to a
solution of the corresponding amide 4 (1 mmol) in 10 mL of
MeOH. The catalyst, Pd/C, (100 mg , 5%) was added and the
mixture heated under reflux for 5 h. The mixture was concen-
trated and treated with aqueous K2CO3. The product was
extracted with CH2Cl2 (3 × 30 mL), the organic layer dried
(K2CO3) and concentrated in vacuo. The residue was purified
by column chromatography (CH2Cl2/MeOH 10:1). The
resulting product (colorless oil or white solid) can be converted
into corresponding hydrochloride by treatment with gaseous
HCl in MeOH.
cyanides 3
A solution of the corresponding diamine (0.1 mol) in ethyl
formate (100 mL) was heated under reflux for 5 h. The reaction
mixture was concentrated in vacuo. The resulting formamide
(without additional purification) was suspended in anhydrous
dichloromethane (200 mL) and triethylamine (51 g, 0.5 mol)
added. The mixture was cooled to 0 °C and POCl3 (0.21 mol,
32 g) added dropwise at such a rate that the reaction tempera-
ture remained below 0 °C. The mixture was stirred for 2 h. The
reaction mixture was poured into ice–water (500 mL)
containing K2CO3 (100 g) maintaining the temperature below
25 °C. The resulting emulsion was stirred for 1 h at rt. The
organic layer was separated, the aqueous layer extracted with
CH2Cl2 (2 × 50 mL), and the combined organic layers were
dried (K2CO3), purified by flash chromatography and concen-
trated in vacuo. The diisocyanide was obtained as a dark oil.
Compound 5a: Yield 67%; colorless oil; Rf 0.6 (CH3CN/
EtOH/NH3 80:12:8); 1H NMR (400 MHz, CDCl3) δ 0.81 (d,
J = 7.1 Hz, 6H, 2 × CH3), 0.97 (d, J = 7.1 Hz, 6H, 2 × CH3),
1.30–1.40 (m, 4H, (CH2CH2CH2NHCO)2), 1.44–1.52 (m, 4H,
(CH2CH2CH2NHCO)2), 2.26–2.36 (m, 2H, 2 × CH(CH3)2),
1,6-Diisocyanohexane (3a): Yield 66%, dark oil, Rf 0.8 3.17–3.30 (m, 6H, 2 × CH, (CH2CH2CH2NHCO)2), 7.26–7.33
(hexane/EtOAc 2:1); 1H NMR (400 MHz, CDCl3) δ 1.39–1.47 (m, 2H, 2 × NH); 13C NMR (100 MHz, CDCl3) δ 174.3, 60.2,
(m, 4H, (CH2 CH2 CH2 NC)2 ), 1.55–1.73 (m, 4H, 38.6, 31.2, 29.7, 26.5, 19.6, 17.7; IR (cm−1) 1638 (CONH),
(CH2CH2CH2NC)2), 3.30–3.40 (m, 4H, (CH2CH2CH2NC)2); 3290 (br, NH, NH2); ESI-MS (m/z): [M + H]+ calcd for
13C NMR (100 MHz, CDCl3) δ 155.6 (t, J = 5.9 Hz, NC), 41.1 C16H34N4O2, 314.2682; found, 314.2670.
(t, J = 6.6 Hz, (CH2CH2CH2NC)2), 28.5, 25.2; IR (cm−1) 2150
(NC); Anal. calcd for C8H12N2: C, 70.55; H, 8.88; found: C,
Supporting Information
70.34; H, 8.62.
Supporting Information File 1
General procedure for the synthesis of 4a–g
The corresponding isocyanide 3 (3 mmol) and phenyl phos-
phinic acid (3 mmol, 441 mg) were added to a mixture of the
aldehyde (9 mmol) and benzylamine (9 mmol, 963 mg) in
CH2Cl2 or MeOH (30 mL). The mixture was stirred for 48 h at
rt, the solvent removed in vacuo and the residue purified by
column chromatography (hexane/ethyl acetate 1:1). The pro-
duct (colorless oil or white solid) can be converted into the
corresponding hydrochloride by treatment with gaseous HCl in
MeOH.
General information, procedures, spectral data of all
compounds, results of bioassay, and copies of selected
NMR spectra.
[http://www.beilstein-journals.org/bjoc/content/
supplementary/1860-5397-7-131-S1.pdf]
Acknowledgements
The study was partly supported by SB RAS-83, 88 and RFBR-
09-04-01483.
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