Toward the development of metal-free synthetic nucleases: Cleavage of a model substrates by 1,4-diazabicyclo[2.2.2]octane derivatives

Article abstract of DOI:10.1081/NCN-200026052

Artificial ribonucleases of A_nBCL series were synthesized by solid-phase method. They consist of a hydrophobic alkyl radical A (n = 3-12 carbon atoms), an "RNA-binding domain" B (bisquaternary salt of 1,4-diazabicyclo[2.2.2]octane), a "catalyti

Full text of DOI:10.1081/NCN-200026052

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Toward the Development of Metal‐Free Synthetic
Nucleases: Cleavage of a Model Substrates by
1
,4‐Diazabicyclo[2.2.2]Octane Derivatives
a
a
Lyudmila S. Koroleva & Vladimir N. Silnikov
a
Novosibirsk Institute of Bioorganic Chemistry , Lavrentev Ave. 8, Novosibirsk, 630090,
Russia
Published online: 23 Aug 2006.
To cite this article: Lyudmila S. Koroleva & Vladimir N. Silnikov (2004) Toward the Development of Metal‐Free Synthetic
Nucleases: Cleavage of a Model Substrates by 1,4‐Diazabicyclo[2.2.2]Octane Derivatives, Nucleosides, Nucleotides and Nucleic
Acids, 23:6-7, 989-992, DOI: 10.1081/NCN-200026052
To link to this article: http://dx.doi.org/10.1081/NCN-200026052
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
Vol. 23, Nos. 6 & 7, pp. 989–992, 2004
Toward the Development of Metal-Free Synthetic
Nucleases: Cleavage of a Model Substrates by
1
,4-Diazabicyclo[2.2.2]Octane Derivatives
*
Lyudmila S. Koroleva and Vladimir N. Silnikov
Novosibirsk Institute of Bioorganic Chemistry, Novosibirsk, Russia
ABSTRACT
Artificial ribonucleases of A BCL series were synthesized by solid-phase method.
n
They consist of a hydrophobic alkyl radical A (n = 3–12 carbon atoms), an ‘‘RNA-
binding domain’’ B (bisquaternary salt of 1,4-diazabicyclo[2.2.2]octane), a ‘‘catalytic
domain’’ C (histidine residue) and a ‘‘linker’’ L that joins the domains B and C. The
effect of the alkyl radical on the catalytic properties of the chemical catalyst was
studied using three activated phosphate ester substrates: p-nitrophenyl phosphate, bis-
p-nitrophenyl phosphate, and thymidine-3’-p-nitrophenyl phosphate.
Key Words: Metal-free artificial nucleases; p-nitrophenyl phosphate derivatives;
1
,4-diazabicyclo[2.2.2]octane derivatives.
INTRODUCTION
There is much current interest in the design and development of small compounds
that can mimic important enzymatic reactions under physiological conditions. One of the
goals of these studies is the preparation of artificial ribonucleases, which cut a specific
RNA at the target site and thus are applicable to molecular biology, therapy, and others.
*
Correspondence: Lyudmila S. Koroleva, Novosibirsk Institute of Bioorganic Chemistry,
Lavrentev Ave. 8, Novosibirsk 630090, Russia; E-mail: kls_2002@mail.ru.
9
89
DOI: 10.1081/NCN-200026052
1525-7770 (Print); 1532-2335 (Online)
www.dekker.com
Copyright D 2004 by Marcel Dekker, Inc.

990
Koroleva and Silnikov
Recently we have developed a series of artificial ribonucleases (ABLC series) containing
four functional domains—catalytic domain C, linker group L, RNA-binding di- or
[1]
polycation B and hydrophobic radical A. The optimal structure parameters and the
[2]
role of B, C, L domains were determined in series of experiments. According to
these results, still faster RNA hydrolysis should be possible if all these domains are
presented in artificial ribonucleases. However the role and the optimal structure of
hydrophobic radical A have remained not studied. In this study, we have synthesized a
series of compounds (Fig. 1) with different alkyl residues to optimize the structure of
this domain.
Figure 1. Solid-phase synthesis of artificial ribonucleases of ABCL series. 6a–h: a: n = 2,
b: n = 3; c: n = 5; d: n = 6; e: n = 8; f: n = 9; g: n = 11; h: R—cyclohexyl.

Metal-Free Synthetic Nucleases
RESULTS AND DISCUSSION
The synthesis of chemical ribonucleases is presented in Fig. 1. Merrifield resin was
991
a
im
used as a polymer carrier. Treatment of Merrifield resin with excess N -Boc-N -DNP-
L-Histidine and EDiPA results in compound 2. Subsequent attachment of the linker
group was carried out by N-acylation of amino acid’s a-amino groups by 4-bromobutyryl
chloride. Compounds 5 were synthesized by alkylation of 1,4-diazabicyclo[2.2.2]octane
using different alkyl halides. Mono-quantery salts of 1,4-diazabicyclo[2.2.2]octane
were joined to compound 4. Thus compounds differing in length of alkyl residue
were obtained.
Kinetic studies were performed by using three activated phosphate ester substrates:
p-nitrophenyl phosphate (pNPP), bis-p-nitrophenyl phosphate (BNPP), and thymidine-
3
cleavage of phosphomono-and phosphodiesters.
’-p-nitrophenyl phosphate (TNPP) which is considered a reference substrate for the
[3]
Pseudo-first-order rate constants
were determined for the three phosphate ester substrates.
Catalytic activity of RNase mimetics was determined on the surface of polymer
carrier. Thus, the influence of micelle formation was excluded. The kinetic measure-
ments were followed spectroscopically, monitoring the release of p-nitrophenolate ion
at 400 nm. The rate constants were obtained by curve fitting of the initial data of
the kinetic curve. Kinetics parameters were calculated by Origin program. Eq. 1
described experimental data in the best way.
Ã
0
AðtÞ ¼ a þ b expðt=k Þ
ð1Þ
A (t)—absorption of p-nitrophenolate at 400 nm, k’ = 1/kobs, a, b—constants.
The rate constants of the model substrates hydrolysis as a function of the alkyl
fragment length are represented in Fig. 2. The hydrolysis efficiency by artificial
ribonucleases depends on substrate structures. Hydrolysis rate of p-nitrophenylphosphate
weakly depends on structure of alkyl residue of catalyst. The highest value of kobs in this
Figure 2. Dependence hydrolysis rate on the number of carbons in alkyl residue
in RNasemimetics.

992
Koroleva and Silnikov
experiment is observed for compounds A BLC and A BLC. In the case of bis-p-
6
10
nitrophenylphosphate an increase of hydrolysis rate for all catalysts is observed. The
influence of alkyl residue length becomes more marked. The most active derivatives are
ꢀ
4
A BLC, A BLC and A BLC. The rate constants are changed in the range from 3.9*10
6
9
12
ꢀ1
ꢀ
1
ꢀ4
min to 8.3*10 min . For thymidine-3’-p-nitrophenyl phosphate further growth of
efficiency is observed, but in this case compound A BLC possesses maximum activity.
ꢀ1
1
0
ꢀ4
ꢀ4
ꢀ1
The rate constants are changed in the range from 4.8*10 min to 9.5*10 min .
Catalysts of A BLC series cleave phosphodiester bonds more efficiently than
n
phosphomonoesters. Even in the case of simple model substrates, the influence of alkyl
residue structure on efficiency of cleavage is displayed. In this study conditions
excluding micelle formation are used, therefore it can be considered that the role of
alkyl residues consists in promoting a suitable spatial complex structure based on
hydrophobic interactions.
ACKNOWLEDGMENTS
This work is supported by RFBR (02-04-48664-a), Grants BRHE Rec-008, Well-
come Trust (CRIG 063630) and Russian Science Support Foundation.
REFERENCES
1.
2.
3.
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Zenkova, M.A.; Shishkin, G.V.; Vlassov, V.V. Artificial ribonucleases: synthesis
and RNA cleaving properties of cationic conjugates bearing imidazole residues.
Tetrahedron 1999, 55, 503–512.
Konevetz, D.A.; Mironova, N.L.; Beck, I.E.; Zenkova, M.A.; Shishkin, G.V.;
Vlassov, V.V.; Silnikov, V.N. Chemical ribonucleases: 4. An analysis of the domain
structure of chemical ribonucleases based on 1,4-diazabicyclo[2.2.2]octane. Russ. J.
Bioorgan. Chem. 2002, 28 (4), 331.
Madhavaiah, C.; Verma, S. Kinetic evaluation of metalated diglycine conjugate as a
functional mimetic of phosphate ester hydrolase. Bioconjug. Chem. 2001, 12, 855–
860.
Received December 2, 2003
Accepted April 23, 2004