Synthesis and characterization of polyamine-based biomimetic catalysts as artificial ribonuclease

Article abstract of DOI:10.1081/NCN-100001441

Several polyamine derivatives (I-V) conjugated with or without an intercalative moiety were prepared as ribonuclease mimics. Although no DNA-cleaving activity was observed for all compounds tested, mimics I, III, and V bearing an intercalative moiety alon

Full text of DOI:10.1081/NCN-100001441

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SYNTHESIS AND CHARACTERIZATION OF POLYAMINE-
BASED BIOMIMETIC CATALYSTS AS ARTIFICIAL
RIBONUCLEASE
Kazuo Shinozuka a , Yoshimitu Nakashima a , Kazuhiro Shimizu a & Hiroaki Sawai a
a Department of Chemistry, Faculty of Engineering , Gunma University , Kiryu City,
376-8515, Japan
Published online: 17 Aug 2006.
To cite this article: Kazuo Shinozuka , Yoshimitu Nakashima , Kazuhiro Shimizu & Hiroaki Sawai (2001) SYNTHESIS AND
CHARACTERIZATION OF POLYAMINE-BASED BIOMIMETIC CATALYSTS AS ARTIFICIAL RIBONUCLEASE, Nucleosides, Nucleotides
and Nucleic Acids, 20:1-2, 117-130
To link to this article: http://dx.doi.org/10.1081/NCN-100001441
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS, 20(1&2), 117–130 (2001)
SYNTHESIS AND CHARACTERIZATION
OF POLYAMINE-BASED BIOMIMETIC
CATALYSTS AS ARTIFICIAL RIBONUCLEASE
Kazuo Shinozuka,∗ Yoshimitu Nakashima, Kazuhiro Shimizu,
and Hiroaki Sawai
Department of Chemistry, Faculty of Engineering, Gunma University,
Kiryu City 376-8515, Japan
ABSTRACT
Several polyamine derivatives (I–V) conjugated with or without an in-
tercalative moiety were prepared as ribonuclease mimics. Although no DNA-
cleaving activity was observed for all compounds tested, mimics I, III, and V
bearing an intercalative moiety along with the primary amine and/or imidazole
moieties exhibited potent RNA-cleaving activity at near physiological pH. The
RNA-cleaving reactions of the compounds show characteristic bell-shaped pH
dependency, and the optimal pH values for III and V were well correlated to
the pKa values of their active sites, primary amine, and imidazole moieties.
The design and synthesis of a small synthetic molecule that mimics the ac-
tive center of naturally occurring ribonuclease and is capable of cleaving RNA
molecules are an interesting subject. Such artificial nuclease would find several sig-
nificant applications in the development of new types of therapeutic agents and in
the study of molecular biology. For example, the conjugation of an artificial ribonu-
clease and a nucleic acid binding group, such as an antisense oligonucleotide, will
result in a sequence-specific ribonuclease that is capable of cleaving certain mRNA
in a sequence-specific manner an-selective manner (2) and will be useful in the study
of molecular biology as an artificial restricted RNase that is not known in nature (3).
∗Address correspondence to Kazuo Shinozuka. E-mail: sinozuka@chem.gunma-u.ac.jp
117
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C
Copyright 2001 by Marcel Dekker, Inc.
www.dekker.com

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SHINOZUKA ET AL.
Meanwhile, Breslowetal. initiatedaseriesofstudiestoresolvethemechanism
of pancreatic ribonuclease, RNase A, which possesses two histidine groups around
the catalytic center (4). Through their studies they proposed that in RNase A, a
protonated imidazole moiety of His-119 and a free imidazole moiety of His-12 act
as general acid and general base catalysts, respectively, and promote the hydrolysis
of the phosphodiester bonds of RNA (4).
Several small organic molecules capable of cleaving RNA in a hydrolytic man-
ner via a possible acid–base catalytic mechanism have also been reported, including
simple diamine, guanidine derivatives, imidazole conjugated cyclodextrin (5). More
recent reports from several different research groups are quite interesting in terms
of RNase mimicking molecule (6–9). These researchers synthesized short peptides
containing histidine residues (6) or small molecules containing histamine residues
along with an intercalative molecule (7–9). These molecules exhibited potent RNA-
cleaving activity and the mechanism of the action of the molecules was assumed to
be an acid–base catalytic reaction like that of the natural enzyme mentioned above.
We have also undertaken the study of artificial ribonuclease and reported the
preliminary results of the synthesis and the RNA-cleaving activity of polyamine-
derived novel artificial ribonuclease (I–IV) in a previous paper (10). These com-
pounds were designed to mimic the functions of RNase A. However, one of the
imidazole moieties of the natural RNase active center was substituted by a primary
amine in the mimics. Thus, the mimics bear a primary amine and/or an imidazole
group as catalytic functional groups on certain length of linker arms (Fig. 1). Among
the compounds, I and III exhibited potent RNA-cleaving activity with 1.0 mM of
concentration at pH 7.4 when 16s/23s ribosomal RNA (16s/23s rRNA) was used
as the substrate (6,10). Although the observed cleavage of the RNA molecule was
assumed to be hydrolytic in nature, a detailed study to elucidate the mechanism had
not been conducted at that time. This situation is also true for most of the analogous
RNase mimicking molecules mentioned above.
Figure 1. Structure of the RNase mimic compounds.

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119
In this paper we wish to describe the results of our further investigation about
the mechanism of RNA-cleaving reaction conducted by these polyamine-derived
nuclease mimics, as well as the structure–activity relationships. Also, the details of
the synthesis of I–IV and the new compound V having two imidazole moieties as
active sites are reported.
Preparation of Artificial Ribonuclease I–IV. The syntheses of artificial
ribonuclease I–IV mimicking RNase A are outlined in Scheme 1 (10). Reaction
of acridine carbonyl chloride (1), readily obtained from treatment of acridine car-
boxylic acid with thionyl chloride, with excess amount of tris(2-aminoethyl)amine
gave mimic I as a major product (66.5%) along with some di- and a very little
of tri-substituted by-products. The desired product was separated from the by-
products by silica gel column chromatography. Monomethoxytritylated imidazole
carboxylic acid ethyl ester (2) (11) was also readily reacted with the same amine
to give compound 3, which was then converted to mimic II by brief treatment with
80% acetic acid to remove the trityl group. The treatment of 3 with equimolar
of acridine carbonyl chloride (1) in CH2Cl2 gave compound 4 in moderate yield
(52.5%) along with a di-substituted by-product which was easily removed from
the desired product by silica gel column chromatography. Compound 4 was de-
tritylated in the same manner as above to give mimic III. Condensation of 4 with
Boc-β-alanine in CH2Cl2 in the presence of dicyclohexylcarbodiimide (DCC) (12)
gave compound 5. The treatment of 5 with 30% HBr in glacial acetic acid effected
Scheme 1. Preparation of RNase mimics I–IV.

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SHINOZUKA ET AL.
Scheme 2. Preparation of RNase mimic V.
complete deprotection (13) to afford mimic IV. Mimic V bearing two imidazole
groups was also prepared by the condensation of 4 and monomethoxytritylated
imidazole carboxylic acid (6) (14) in the presence of carbonyl diimidazole as a
condensation reagent. Subsequent removal of the protecting group as depicted in
Scheme 2 gave mimic V. All the mimic compounds were purified by either silica gel
column chromatography, recrystallization, or a combination of both. These details
are described in the Experimental Section.
RNA Cleaving Activity. 16s/23s ribosomal RNA (16s/23s rRNA) and a
plasmid DNA (pBR322) were used as the substrates in the cleavage reaction since
the nucleic acids possess double-stranded regions and are expected to provide
the binding sites for the intercalative moiety of some of the mimic compounds
(6,7). The cleaving activity of the mimics was monitored by denaturing agarose
gel electrophoretic analysis through the experiments. Figure 2 displays typical
degradation of the RNA substrate (0.1 OD unit) generated by mimics I–V (1 mM)
under near physiological condition at 37◦C. The substrate was simply mixed with
the mimics (1 mM) and allowed to react for 1 h in PBS buffer (pH 7.4). The
reaction mixture was subjected to electrophoresis through denaturing agarose gel.
The fainting in the main bands in lanes 3–7 along with the generation of smeary
ladder bands was observed by the following ethidium bromide staining of the gel.
This is due to the cleavage of intact RNA. As shown in Figure 2, mimics I, III,
Figure 2. Degradation of 16s/23s rRNA promoted by the RNase mimic compounds. Lane 1: 0-h
negative control; lane 2: 1-h negative control (incubation of the RNA without the mimic compound);
lane 3: 1-h reaction with mimic I; lane 4: 1-h reaction with mimic II; lane 5: 1-h reaction with mimic
III; lane 6: 1-h reaction with mimic IV; lane 7: 1-h reaction with mimic V. The details of the reaction
conditions are described in the Experimental Section.

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121
and V exhibited potent RNA-cleaving activity within 1-h-reaction. The cleavage
efficiency of the substrate RNA was estimated by photodensitometric reading of
the remaining intact RNA on the gel. The experiment was repeated three times and
the average amounts of cleavage resulted in the experiments are none (0-h) – 0;
none (1-h) – 6.6; I – 41.0; II – 11.7; III – 56.9; IV – 14.3; V – 22.5. The results
thus obtained for mimics I–IV were in good agreement with the previous results
(10). Among the compounds, mimic III was the most active. Marked difference in
activity was observed between mimics II and III in which the intercalative moiety is
absent in mimic II, otherwise, both compounds have the same functional moieties.
The results indicate that the presence of an intercalative moiety, which presumably
acts as the binding group to the RNA, is essential to exhibit the activity (7).
The difference in activity between mimics III and IV is rather interesting since
the compounds have exactly the same active sites, primary amine, and imidazole
moieties, along with the intercalative moiety. In mimic IV, however, a linker moiety
of the primary amine moiety is elongated by the insertion of extra methylene bridges
compared to mimic III. Therefore, the relative geometry between the primary amine
moiety and the imidazole moiety between III and IV is different. The activity of
mimic V bearing two imidazole moieties both connected to the same length of
linker group in mimic IV is also somewhat lower compared to mimic III. These
results may reflect the fact that the RNA-cleaving activity was brought about by the
cooperative action of both active sites. In addition, the active sites should occupy
a precise spatial position to accomplish favorable interaction with target site to
promote efficient cleavage of the target, as was suggested before (6,7).
None of the mimics exhibited cleaving activity on the double-stranded DNA
(pBR 322) substrate in the same experiment even after prolonged incubation (48 h,
data not shown), indicating that the presence of 2ꢁ-OH in the substrate is essential
for the cleavage (15).∗ Thus, the cleaving activity of mimics I, III, and V is specific
for RNA substrate. It should be noted that the cleavage of the RNA by the mimics
was not affected by the presence of a radical trapping agent such as DMSO and
DUBCO (data not shown).
Nucleic Acid Binding Activity. Nucleic acid binding activity of mimics I,
III, IV, and V was determined by a spectrophotometrical titration method at pH 7.4.
Double-strandedDNA(calfthymus)wasusedasthenucleicacidcomponentinstead
of RNA to avoid possible degradation of the nucleic acid during the experiment.
The absorption of the mimic compounds at 360 nm, which corresponds to the λmax
of the acridine moiety in the mimics, steadily decreased as the amount of the DNA
was increased (data not shown). The observed hypochromic effect indicates that the
interaction between the mimic compounds and the DNA proceeds upon the mixing
∗ Takagi et al. reported the cleavage of double-stranded DNA by a Cu(II) complex of polyamine-linked
DNA-intercalator1 which is an analogous compound of mimic I. We have also observed a cleavage
of pBR322 DNA by mimic I under the presence of an equal amount of Cu(II) ion. The magnitude of
the cleavage was, however, modest to low in our results (data not shown).

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SHINOZUKA ET AL.
Figure 3. Scatchard plots of mimic III by UV-vis spectrophotometric titration.
of these (16). The decrease in the absorption of III at 360 nm, where the DNA has
no absorption, was converted to Scatchard plots, as shown in Figure 3 as a typical
example, and the binding constant (k) was calculated to be 2.2 × 104 M−1 by the
Scatchard equation (17,18). The binding constants of I, IV, and V were obtained
in a similar manner and are listed in Table 1.
As indicated in Table 1, it is confirmed that the compounds bearing an in-
tercalative moiety have potent binding ability to the double-stranded nucleic acid.
It would be safe to assume that the compounds also have nearly the same magni-
tude of binding ability toward the rRNA (19). Mimics III–V showed almost the
same binding activity that was slightly smaller than that of mimic I. This may be
attributed to the presence of two primary amine groups in mimic I since the groups
are expected to exist in protonated form (see the following section) and can interact
with the negatively charged backbone of nucleic acid more strongly than the other
mimics (20).
The protonation constants of the active sites of mimics I, III, and V were
determined by a potentiometric titration method. HCl-Salts of the mimics were used
for the titration in the presence of 0.1 M NaCl. The titrated data were analyzed by a
computer program, SUPERSQUAD (21), to calculate the log values of the protonation
constants (pKa values) and these are listed in Table 2.
The pKa values for the proposed active site in III, the imidazole and the
primary amine moieties, thus obtained are 6.48 and 9.57, respectively. Based on
this, it is evident that at the pH of the cleaving reaction (pH 7.4), the imidazole exists
Table 1. Nucleic Acid Binding Con-
stant (K) of the Mimics Estimated by
the Scatchard Plots
Mimic Compound
K(M−1
)
n
I
1.0 × 105 0.1
2.2 × 104 0.2
1.3 × 104 0.1
1.1 × 104 0.1
III
IV
V

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Table 2. The pKa Values of Active Sites in Mimics I, III, and V
123
Mimic Compound
I
III
V
Active site
and its pKa
−NH2, 8.84
−NH2, 9.71
−Im, 6.48
−Im, 6.07
−Im, 7.53
−NH2, 9.57
as an unprotonated form whereas the primary amine exists as a protonated form
predominantly. ThepKa valuesoftheactivesitesofmimicsIandVweredetermined
in a similar manner and are also given in Table 2. The pKa values of the imidazole
groups in mimic V indicate that these exist in almost equimolar equilibrium between
the protonated and unprotonated forms at near-neutral to slightly acidic pH range.
Contrary to this, the pKa values of two primary amine groups in mimic I were
considerably higher than 7.4. This means both of the amines in mimic I exist as
predominantly protonated form at the pH of the nucleic acid cleaving reaction
described in the earlier section.
It is well known that the rate of cleavage versus pH shows a characteristic bell-
shaped relationship in the case of RNase A-mediated cleavage of RNA (Breslow
(5); (22)). This is accounted for by the involvement of two essential imidazole
moieties of histidine residues (His-12 and His-119) acting as general base and acid
catalysts (4). Therefore, we examined the pH dependency of the RNA-cleaving
reaction to further elucidate the mechanism of the mimics I, III, and V (Fig. 4).
As shown in Figure 4B, the total amount of the cleavage as a function of the
pH of the reaction exhibited a bell-shaped profile in the case of compound III as
it was reported previously on the relevant compounds (7). The optimal pH for the
reaction of III estimated from Figure 4B is about 7. The results strongly suggest that
the reaction really proceeds via an acid–base catalytic mechanism. Furthermore,
considering the result obtained in the foregoing section, the primary amine and
the imidazole in mimic III are assumed to predominantly exist as the protonated
and unprotonated forms, respectively, at the pH. Thus, in mimic III the protonated
primary amine and the unprotonated imidazole of III act as acid–base catalysts,
Figure 4. The pH dependent activity of the RNase mimic compounds.

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SHINOZUKA ET AL.
respectively, in the RNA-cleaving reaction. Essentially the same result was obtained
for compound V in which the optimal pH value was also about 7 (Fig. 4C) and two
imidazole groups of V were supposed to exist in an almost equimolar equilibrium
between the protonated and unprotonated forms at that pH (see Tab. 2).
Thus, the reaction mechanism of mimics III and V could be accounted for by
a general base and general acid hydrolysis mechanism like a natural RNase A-type
enzyme. In this mechanism, a free amine or imidazole group acts as the general
base, which deprotonates 2ꢁ-OH of the target. On the other hand, a protonated
imidazole group acts as the general acid that would transfer its proton to negatively
charged oxygen on the phosphodiester linkage being cleaved. This is summarized
in Scheme 3. It may be worth noting that the RNA-cleaving activity of mimic III
at near optimal pH (pH 6.9) for the two mimics was higher than that of mimic V
(data not shown).
In the case of compound I, some discrepancies exist between the optimal pH
value for the RNA cleaving reaction and the pKa values (8.84 and 9.71, see Tab. 1)
of the primary amine moieties. At the optimal pH of I (Fig. 4A, pH 7.4), both
of the primary amine moieties of compound I are expected to exist as protonated
form. Therefore, these could act as a general acid catalyst, but not as a general base
catalyst. At this stage we do not understand the exact reason behind this observa-
tion. It may be possible that 1,3-bis[tris(hydroxymethyl)methylamino]propane, the
buffer component used for the study of pH dependence of RNA cleaving reaction,
is involved in the reaction since the compound is a polyimine derivative and, at
least, one of its imino functions would be able to act as the base component in
Scheme 3. Proposed mechanism of RNA cleavage by RNase mimic III (and V) (Breslow et al. J.
Am. Chem. Soc. (4)).

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125
the reaction (23) whereas the primary amine moieties of compound 1 may act as
the general acid. In any case, the pH dependence of the reaction also supports the
general base and acid type of reaction mechanism in the RNA-cleaving reaction
mediated by compound I and, therefore, the reaction is hydrolytic in nature.
Conclusion. We have successfully synthesized several polyamine deriva-
tives mimicking the active center of pancreatic ribonuclease (RNase A). Among
them, mimics I, III, and V bearing an intercalative moiety along with the active sites
exhibited potent RNA-cleaving activity at near-physiological condition. In particu-
lar, mimic III cleaved almost 60% of the substrate. Meanwhile, these mimics did not
show any detectable DNA-cleaving activity under the same conditions. The result
indicates that the existence of a nucleic acid binding function in the mimic com-
pound as well as the 2ꢁ-OH group in the substrate molecule are the indispensable
factors for the derivatives to show nucleic acid-cleaving activity. The RNA-cleaving
reaction mediated by the above compounds showed characteristic bell-shaped pH
dependency. Thus, the reaction of these compounds proceeds through a general
base–acid type of hydrolytic mechanism as that of natural RNase. Considering this
with the results of the pKa analysis of the mimic’s active sites, one of the two active
sites existing as a protonated form may act as the acid catalyst. Meanwhile, the
other active site existing as an unprotonated form may act as the base catalyst at
that pH.
The study also indicates that the RNA-cleaving activity was brought about
by the cooperative action of the two active sites, therefore, consideration should be
taken to design RNase mimic compounds in order to achieve precise interaction of
the active sites to their target sites.
The current results will help to design potent RNA-cleaving agents. In our
group, a study to incorporate such an agent into a molecule that is able to recognize
RNA in sequence-specific manner (24),∗ is now in progress and the results will be
reported elsewhere.
EXPERIMENTAL
Ribosomal RNA(16s/23s) and calf thymus DNA were purchased from
Boehringer Mannhaim. 1H NMR spectra were measured on a Varian Gemini-200
spectrometer. All chemical shifts (δ) are reported in ppm down-field from internal
tetramethylsilane (TMS) or 3-(trimethylsilyl)propionic acid sodium salt (TMSP).
J values are given in Hz. Melting points were determined on a Yanagimoto MP-
500D electrothermal apparatus and uncorrected.
∗ A recent report of Ushijima et al. describes a site-specific cleavage tRNA mediated by β-oligoDNA
functionalized at the 5ꢁ-terminus with polyamine derivatives26 which are essentially same as mimic I
and III described in this article.

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2-[(Acridin-9-yl)carbonyl]amino-2ꢀ,2ꢀꢀ-diaminotriethylamine
(I). 9-
Acridine carboxylic acid (400 mg, 1.8 mmol) was treated with excess thionyl
chloride in benzene, then the mixture was evaporated to dryness. The residue
was dissolved in dichloromethane and the solution was added by dropwise to a
stirred solution of tris(2-aminoethyl)amine (1.3 g, 3.7 mmol) in dichloromethane
under cooling in an ice-bath. After 30 min of stirring, the mixture was evapo-
rated in vacuo. The residue was purified by silica gel chromatography, using the
mixture of dichloromethane:methanol:conc. ammonia (4:1:0.1) to afford 421 mg
(1.19 mmol, 66.5%) of mimic I, m.p. 55.6–55.8◦C (Found: C, 65.04; H, 7.28; N,
18.74. C20H25N5O · 1H2O requires C, 65.05; H, 7.37; N, 18.96%); δH(200 MHz;
D2O) 2.63–2.77 (8H, m, (−(CH2)2−(NH2)2), 2.83 (2H, t, J = 5.5, −CH2−CH2−
NH−CO−), 3.77 (2H, t, J = 5.5, −CH2−CH2−NH−CO−), 7.60–8.02 (8H, m,
Acr).
Purified mimic I (100 mg, 0.27 mmol) was dissolved in dichloromethane
(20 ml). HCl gas was bubbled through the solution for 20 min under cooling in
an ice-bath. The deposited, very hygroscopic crystals were collected by filtration
and washed with dichloromethane, then quickly dried under reduced pressure in
a desiccator overnight. The yield of mimic I · 5HCl salt thus obtained was almost
quantitative(93.3%)(Found:C, 43.59;H, 5.76;N, 12.78. C20H25N5O · 5HCl · 1H2O
requires C, 43.71; H, 5.86; N, 12.75%).
2-[1-H-(Imidazol-4-yl)acetyl]amino-2ꢀ,2ꢀꢀ-diaminotriethylamine
(II).
1-Dimethoxytrityl-1-H-imidazole-4-aceticacidethylester(11)(355mg, 0.78mmol)
was treated with excess of tris(2-aminoethyl)amine at room temperature for 2 days.
The whole mixture was applied for silica gel column chromatography and the prod-
uct was eluted by a mixed solvent of dichloromethane:methanol:conc. ammonia
(4:1:0.25). The isolated imidazole acetic acid conjugate of tris(2-aminoethyl)amine
(1) (332 mg, 76.5%) was treated with 80% acetic acid for 30 min, then the mixture
was evaporated to dryness. The residue was applied on silica gel chromatography,
and the product was eluted with the mixture of dichloromethane:methanol:conc.
ammonia (1:1:0.3) to afford 140 mg (91.7%) of mimic II. The compound was
further purified as a hydrochloride salt, m.p. 163.0–164.0◦C (Found: C, 32.71; H,
6.62; N, 20.51. C11H22N6O · 4HCl · 0.5H2O requires C, 32.42; H, 6.68; N, 20.64%);
δH(200 MHz; D2O) 2.82 (2H, t, J = 7, −CH2−CH2−NH−CO−), 2.91–3.21 (8H,
m, (−(CH2)2−NH2)2), 3.40 (2H, t, J = 7−CH2−CH2−NH−CO−), 7.37 (1H, s,
H-5 of Im), 8.68 (1H, s, H-2 of Im).
2-[1-H-(Imidazol-4-yl)acetyl]amino-2ꢀ-[(acridin-9-yl)carbonyl]amino-
2ꢀꢀ-aminotriethy lamine (III). 9-Acridine carboxylic acid (170 mg, 0.76 mmol)
was treated with excess thionyl chloride in benzene (10 mL), then the mixture
was evaporated to dryness. The residue was dissolved in dichloromethane and
the solution was added dropwise to a stirred mixture of imidazole acetic acid
conjugate of tris(2-aminoethyl)amine (1) (423 mg, 0.76 mmol) and triethylamine
(3.8 mmol) in dichloromethane under cooling in ice-bath. After 30 min of stirring,
the insoluble matter was removed by filtration and the filtrate was evaporated in
vacuo. The residue was purified by silica gel chromatography, using the mixture of

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127
dichloromethane:methanol:conc. ammonia (95:5:1) to afford compound (2)
(304 mg, 52.5%). Compound 2 was treated with 80% acetic acid for 30 min,
then evaporated to dryness. Silica gel chromatography of the residue, using the
same mixture as above and the recrystallization from ethanol afforded mimic
III (131 mg, 71.5%), m.p. 75.0–75.5◦C (Found: C, 62.59; H, 6.52; N, 20.41.
C25H29N7O2 · 1H2O requires C, 62.88; H, 6.54: N, 20.53%); δH(200 MHz; D2O)
2.58–2.79 (6H, m, −(CH2)2−NH2 and −CH2−CH2−NH−CO−Im), 2.81 (2H, t,
J = 5, −CH2−CH2−NH−CO−Acr), 2.88 (2H, s, −CH2Im), 3.28 (2H, t, J = 6,
−CH2−CH2−NH−CO−CH2−Im), 3.77 (2H, t, J = 5, −CH2−CH2−NH−CO−
Acr), 6.53 (1H, s, H-5 of Im), 7.27 (1H, s, H-2 of Im), 7.63–8.25 (8H, m, Acr).
2-(β-Alanyl)amino-2ꢀ-[1-H-(imidazol-4-yl)acetyl]amino-2ꢀꢀ-[(acridin-9-
yl)carbonyl] aminotriethy lamine (IV). Compound 2 (145 mg, 0.19 mmol) was
reacted with N-(tert-butoxycarbonyl) −β-alanine (Boc-β-alanine) (49 mg, 0.26
mmol) in dichloromethane in the presence of dicyclohexylcarbodiimide (DCC)
(160 mg) for 24 h. The reaction was quenched by the addition of methanol and
the mixture was allowed to stand at 4◦C overnight. The deposited urea was re-
moved by filtration and the filtrate was evaporated in vacuo. The obtained crude
mixture of compound 3 was treated with 30% HBr in acetic acid. Addition of
ether to this mixture caused precipitation and the supernatant was removed by
decantation. After repetition of the same treatment, the precipitates were dis-
solved in water (10 mL) and extracted with ethylacetate (20 mL × 3). The sep-
arated aqueous layer was adjusted the pH at around 11 with triethylamine and
allowed to stand at 4◦C overnight. The solution was concentrated in vacuo and the
residue was applied for silica gel column chromatography, using the mixture of
dichloromethane:methanol:conc. ammonia (1:1:0.1) to afford mimic (IV) (82 mg,
83.2%), m.p. 67.5–68.0◦C (Found: C, 61.47; H, 6.58; N, 20.27. C28H34N8O3 · 1
H2O requires C, 61.28; H, 6.62; N, 20.43%); δH(200 MHz; CD3OD) 2.49 (2H, t,
J = 6.5, H2N−(CH2)2−CO−NH−CH2−CH2−), 2.64–2.82 (4H, m, H2N−CH2−
and−CH2−CH2−NH−CO−CH2−Im), 2.85(2H, t, J = 6, −CH2−CH2−NH−CO
−Acr), 3.02 (2H, t, J = 6.5, H2N−(CH2)2−CO−NH−CH2−), 3.21–3.39 (4H, m,
HN−CH2−CH2− and −CH2−NH−CO−CH2−Im), 3.29 (2H, s, −CH2−Im),
3.72 (2H, t, J = 6, −CH2−NH−CO−Acr), 6.86 (1H, s, H-5 of Im), 7.51 (1H,
s, H-2 of Im), 7.65–8.20 (8H, m, Acr).
2,2ꢀ-Bis-[1-H-(imidazol-4-yl)acetyl]amino-2ꢀꢀ-[(acridin-9-yl)-carbonyl]-
aminotriethy lamine (V). Mono-methoxytritylated imidazole carboxylic acid
sodium salt (6) (14) (159 mg, 0.40 mmol) and carbonyl diimidazole (326 mg,
3.0 mmol) were dissolved in dry THF (20 mL) and the solution was stirred at
0◦C for 2 h under argon atmosphere. To this solution the THF solution (20 mL)
of compound 4 (200 mg, 0.267 mmol) was added and the whole mixture was
stirred at 0◦C for 3 h, then at room temperature for 12 h under argon atmosphere.
After quenching the reaction by the addition of dry ethanol (1 mL), the mixture
was evaporated to dryness. The residue was dissolved in CH2Cl2 (50 mL) and the
organic portion was washed with water (30 mL × 2). The obtained organic layer
was dried over MgCl2, then evaporated to a thick oil, which was supposed to contain

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SHINOZUKA ET AL.
compound 7. The oil was treated with trifluoroacetic acid (2 mL) for 20 min, then
evaporated to dryness. The residue was purified by silica gel column chromatog-
raphy, using the mixture of dichloromethane:methanol:conc. ammonia (4:1:0.2) to
afford mimic V (39 mg, 17.1%), m.p. 75.8–76.2◦C (Found: C, 53.98; H, 6.26; N,
18.73. C30H33N9O3 · 5.5H2O requires C, 54.03; H, 6.66; N, 18.91%); δH(200 MHz;
D2O) 2.69–2.79 (4H, m, 2 × −CH2−CH2−NH−CO−Im), 2.88 (2H, t, J = 6,
−CH2−CH2−NH−CO−Acr), 3.11–3.28 (4H, m, 2 × −CH2−CH2−NH−
CO−Im), 3.18 (4H, s, 2 × −CH2−Im), 3.64(2H, t, J = 6, −CH2−CH2−NH−CO−
Acr), 6.75 (2H, s, 2 × H-5 of Im), 7.44–7.98 (10H, m, 2 × H-2 of Im and Acr).
The Assay of RNA Cleaving Activity. 16s/23s rRNA (0.1 unit A260) was
allowed to react with or without mimic compounds (1 mM) in 10.4 µL of PBS
buffer (50 mM, pH 7.4) containing 120 mM of NaCl and 2.7 mM of KCl at
37◦C for an appropriate period. After incubation, the whole reaction mixture was
subjected to electrophoresis through denaturing (18% formaldehyde) agarose gel
(1.0%), followed by ethidium bromide staining. The amount of remaining intact
RNA was estimated by photodensitometric reading of the gel by ATTO AE-6900 M
DensitographconnectedwithApplecomputersystem. Theexperimentwasrepeated
three times and the average values of the intact RNA were calculated from the results
obtained by the above procedure.
Nucleic Acid Binding Activity. Nucleic acid-binding activity of the mimics
was determined by UV- vis spectrophotometrical titration method using calf thymus
DNA in a Tris–acetate buffer (pH 7.4). The binding constants (K) of the mimics
with DNA were estimated by the Scatchard plot method (17) based on the result of
the titration. A fixed amount (0.1 mM) of the mimics was titrated with DNA over
the range of DNA concentration of 0–2.5 mM. The UV spectra were measured after
equilibration. The differences in the absorbance at 360 nm in the acridine moiety
of the mimics were applied to the following Scatchard equation r/Cf = K(n −r)
(19). Here, K is the association constant of the mimic with DNA, r is the number of
mimics bound per DNA nucleotide residue, where r = Cb/Cp, Cf is the molar con-
centration of the free mimic, and n is the number of binding sites per DNA. Cb is the
concentrationoftheboundmimicandCpistheinputconcentrationoftheDNA(18).
The Measurement of pKa Values. The acid–base equilibria were deter-
mined by potentiometric titration of 1.0 mM of mimic–HCl salts and I = 0.10
(NaCl) at 25.0◦C, using a Fisher Accumet pH meter. The titration points were ob-
tained from separately prepared solutions to which were added desired quantity
of 0.10 M NaOH solutions. The titrated data were analyzed by a computer pro-
gram, SUPERSQUAD (21), to calculate the log values of the protonation constants (pKa
values).
pH Dependence of the RNA Cleavage Reaction. 16s/23s rRNA (0.1 unit
A
260) was allowed to react in the presence of each mimic compound (1 mM) at
37◦C for 1 h. MES buffer (50 mM) containing 120 mM of NaCl and 2.7 mM of KCl
was used for the reactions at pH of 5.4, 5.9, and 6.4. 1,3-Bis[tris(hydroxymethyl)
methylamino]-propane–HCl buffer (50 mM) ) containing 120 mM of NaCl and
2.7 mM of KCl was used for the reactions at pH of 6.9, 7.4, 7.9, 8.4, and 8.9.

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129
The total volume of each reaction mixture was 10.4 µL. After the reaction, the
whole reaction mixtures were subjected to electrophoresis through denaturing (18%
formaldehyde) agarose gel (1.0%), followed by ethidium bromide staining quanti-
tation of RNA cleavage was done by photodensitometrically using ATTO AE-6900
M Densitograph connected with Apple computer system as described above. The
experiment was repeated three times.
ACKNOWLEDGMENTS
A part of this work was financially supported by a Grant-in-Aid for Scientific
Research from the Ministry of Education Japan.
REFERENCES
1. Cohen, J.S., Ed. Oligodeoxynucleotides. Antisense Inhibition of Gene Expression.
CRC Press: Florida, 1989; Demesmaeker, A.; Haner, R.; Martin, P.; Moser, H.E. Acc.
Chem. Res. 1995, 28, 366.
2. Shibahara, S.; Mukai, S.; Nishihara, T.; Inoue, H.; Ohtsuka, E.; Morisawa, H. Nu-
cleic Acids Res. 1987, 15, 4403; Corey, D.; Shults, P. Science 1987, 238, 1401;
Reynolds, M.A.; Beck, T.A.; Say, P.B.; Schwartz, D.A.; Dwyer, B.P.; Daily, W.J.;
Vaghefi, M.M.; Meltzer, M.D.; Klem, R.E.; Arnold, Jr., L.J. Nucleic Acids Res. 1996,
24, 760; Hovinen, J.; Guzaev, A.; Azhayeva, E.; Azhayeva, A.; Lonnberg, H. J. Org.
Chem. 1995, 60, 2205.
3. Blackburn, P.; Moore, S. In The Enzymes; Boyer, P.D., Ed.; Accademic Press: New
York, 1982; Vol. 15, 317.
4. Breslow, R.; Huang, D.-L.; Anslyn, E. Proc. Natl. Acad. Sci., USA. 1989, 86, 1746.
Anslyn, E.; Breslow, R. J. Am. Chem. Soc. 1989, 111, 4473; Breslow, R.; Hung, D.-L.
J. Am. Chem. Soc. 1990, 112, 9621; Breslow, R.; Xu, R. Proc. Natl. Acad. Sci., USA.
1993, 90, 1201; Breslow, R. Proc. Natl. Acad. Sci., USA. 1993, 90, 1208; Breslow,
R.; Xu, R. J. Am. Chem. Soc. 1993, 115, 10705; Breslow, R.; Dong, S.D.; Webb, Y.;
Xu, R. J. Am. Chem. Soc. 1996, 118, 6588.
5. Komiyama, M.; Yoshinari, K. J. Chem. Soc., Chem. Commun. 1989, 1880; Yoshinari,
K.; Komiyama, M. Chem. Lett. 1990, 519; Yoshinari, K.; Yamazaki, K.; Komiyama,
M. J. Am. Chem. Soc. 1991, 113, 5899; Springs, B.; Haake, P. Tetrahedron Lett.
1977, 32, 3223; Bats, J. W.; Durner, G. Angw. Chem., Int. Ed. Engl. 1992, 31, 207;
Anslyn, E.; Breslow, R. J. Am. Chem. Soc. 1989, 111, 5972; Anslyn, E.; Breslow, R.
J. Am. Chem. Soc. 1989, 111, 8931; Breslow, R. Acc. Chem. Res. 1991, 24, 317.
6. Tung, C.-H.; Wei, Z.; Leibowitz, M.J.; Stein, S. Proc. Natl. Acad. Sci., USA. 1992,
89, 7114.
7. Podyminogin, M.A.; Vlassov, V.V.; Giege. R. Nucleic Acids Res. 1993, 21, 5950.
8. Vlassov, V.V.; Zuber, G.; Felden, B.; Behr, J.-P.; Giege, R. Nucleic Acids Res. 1995,
23, 3161.
9. Lorente, A.; Espinosa, J.F.; Fernandez-Saiz, M.; Lehn, J.-M.; Wilson, W.D.; Zhong,
Y.Y. Tetrahedron Lett. 1996, 37, 4417.

ORDER
REPRINTS
130
SHINOZUKA ET AL.
10. Shinozuka, K.; Shimizu, K.; Nakashima, Y.; Sawai, H. Bioorg. Med. Chem. Lett.
1994, 4, 1979.
11. Bashkin, J.K.; Gard, J.K.; Modak, A.J. Org. Chem. 1990, 55, 5125.
12. Seehan, J.C.; Hess, G.P. J. Am. Chem. Soc. 1955, 77, 1067.
13. Ben-Ishai, D.; Berher, A. J. Org. Cherm. 1952, 17, 1564; McKay, F.C.; Albertson,
N.F. J. Am. Chem. Soc. 1957, 79, 6180.
14. Polushin, N.P.; Chen, B.; Anderson, L.W.; Cohen, J.S. J. Org. Chem. 1993, 58, 4606.
15. Ihara, T.; Inenaga, A.; Takagi, M. Chem. Lett. 1994, 1053.
16. Wilson, W.D.; Lopp, I.G. Biopolymers 1979, 18, 3025.
17. Scatchard, G. Ann. N.Y. Acad. Acu. 1949, 51, 660.
18. Rao, K.E.; Dasgupta, D.; Sasisekharan, V. Biochemistry 1988, 27, 3018.
19. Wilson, W.D.; Ratmeyer, L.; Zhao, M.; Strekowski, L.; Boykin, D. Biochemistry 1993,
32, 4098.
20. Adlam, G.; Blagbrough, I.S.; Taylor, S.; Latham, H.C.; Haworth, I.S.; Rodger,
A. Bioorg. Med. Chem. Lett. 1994, 4, 2435; Rodger, A.; Taylor, S.; Adlam, G.;
Blagbrough, I.S.; Haworth, I.S. Bioorg. Med. Chem. 1995, 3, 861.
21. Gans, P.; Sabatini, A.; Vacca, A. J. Chem. Soc., Dalton Trans. 1985, 1195.
22. Deakyne, C.A.; Allen, L.C. J. Am. Chem. Soc. 1979, 101, 3951.
23. Chandler, A.J.; Kirby, A.J. J. Chem. Soc. Chemm. Commun. 1992, 769.
24. Ushijima, K.; Gouzu, H.; Hosono, K.; Shirakawa, M.; Kagoshima, K.; Takai, K.;
Takaku, H. Biochim. Biophys. Acta 1998, 1379, 217.
Received August 2, 2000
Accepted September 7, 2000

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