RNA Hydrolysis by Heterocyclic Amidines and Guanidines: Parameters Affecting Reactivity

Article abstract of DOI:10.1002/ejoc.202100950

2-Aminobenzimidazole 10, although a weak catalyst in the monomeric state, is a successful building block for effective artificial ribonucleases. In an effort to identify new building blocks with improved catalytic potential, RNA cleavage by a variety of h

Full text of DOI:10.1002/ejoc.202100950

European Journal of Organic Chemistry
Accepted Article
Accepted Article
Title: RNA Hydrolysis by Heterocyclic Amidines and Guanidines:
Parameters Affecting Reactivity
Authors: Friederike Danneberg, Hauke Westemeier, Philip Horx, Felix
Zellmann, Kathrin Dörr, Elisabeth Kalden, Mirco Zeiger,
Abdullah Akpinar, Robert Berger, and Michael W. Göbel
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To be cited as: Eur. J. Org. Chem. 10.1002/ejoc.202100950
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01/2020

10.1002/ejoc.202100950
European Journal of Organic Chemistry
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RNA Hydrolysis by Heterocyclic Amidines and Guanidines:
Parameters Affecting Reactivity
Friederike Danneberg,[a] Hauke Westemeier,[b] Philip Horx,[b] Felix Zellmann,[a] Kathrin Dörr,[a] Elisabeth
Kalden,[a] Mirco Zeiger,[a] Abdullah Akpinar,[a] Robert Berger,[b] Michael W. Göbel*[a]
Dedicated to the memory of Prof. Dr. Klaus Hafner
Abstract: 2-Aminobenzimidazole 10, although a weak catalyst in the
monomeric state, is a successful building block for effective artificial
ribonucleases. In an effort to identify new building blocks with
ribonucleic acids.[12] Hydrolytic cleavage of RNA in most cases
results from nucleophilic attack of the 2’ hydroxy group forming a
2’,3’ cyclic phosphate and a free 5’ OH of the second
fragment.[11] The intramolecular nature of this attack accounts for
the large rate increase when compared to analogous
intermolecular reactions. The unmodified bisguanidine 1,
however, failed as a catalyst for RNA cleavage.[13] The role of
guanidines in such reactions may be threefold: as a general
base to deprotonate 2’ OH, as an electrophile/general acid to
improved catalytic potential, RNA cleavage by
a variety of
heterocyclic amidines and guanidines has been studied. In addition
to pKa values and steric effects, the energy difference between
tautomeric forms seems to be another important parameter for
catalysis. This information is available from quantum chemical
calculations on higher levels, but semiempirical methods are
sufficient to get a first estimate. According to this assumption,
imidazoimidazol 18, characterized by isoenergetic tautomeric forms,
is superior to 2-aminoimidazol 6, the best candidate among the
simple compounds. By far the largest effects are seen with 2-
aminoperimidine 24 which rapidly cleaves RNA even in the
micromolar concentration range. The impressive reactivity, however,
is related to a tendency of compound 24 to form polycationic
aggregates which are the actual catalysts.
stabilize the pentavalent transition structure at the phosphorus
[6t]
atom
and as a general acid for leaving group protonation (5’
OH). With pKa values around 14, normal guanidines will stay
protonated at pH 7 and, in consequence, behave as poor
general acids or bases in neutral aqueous solution. Better
catalysts, therefore, can be expected from guanidine analogs
with pKa values shifted towards neutrality. Consistent with that
idea, acyl guanidines with structures similar to 1 have been
reported to cleave RNA and RNA models more effectively.[5n,5o]
An alternative approach to lower the pKa values of amidines and
guanidines is to incorporate them into heterocyclic structures
(Figure 1).
Introduction
Guanidines are important functional groups for the molecular
[1]
recognition of phosphorylated compounds, both in nature and
in host-guest chemistry.[2] Phosphoric acid diesters, forming
anions at physiological pH, are protected against nucleophilic
attack by their negative charge. As a result, they are practically
inert in the absence of powerful catalysts.[3] While nature has
met the challenge of developing highly effective phosphoryl
transfer enzymes,[4] artificial systems are still far away from this
level of sophistication.[5-10] Thus, from the perspective of
biomimetic chemistry, creating synthetic phosphoryl transfer
catalysts is a challenging task.[11]
Bis- and oligoguanidines related to 1 (Figure 1) have been
shown not only to bind phosphates but also to accelerate
nucleophilic displacement reactions quite effectively.[5,6]
Catalysts of this type have been applied already to manipulate
[a]
Dr. F. Danneberg, Dr. F. Zellmann, Dr. K. Dörr, Dr. M. Zeiger,
A. Akpinar, E. Kalden, Prof. Dr. M. W. Göbel
Institut für Organische Chemie und Chemische Biologie
Goethe-Universität Frankfurt
Max-von-Laue-Str. 7, D-60438 Frankfurt am Main (Germany)
E-mail: M.Goebel@chemie.uni-frankfurt.de
H. Westemeier, Dr. P. Horx, Prof. Dr. R. Berger
Fachbereich Chemie
Figure 1. Compounds tested previously as RNA cleaving catalysts.
[b]
Philipps-Universität Marburg
Hans-Meerwein-Straße, 35032 Marburg (Germany)
robert.berger@uni-marburg.de
We previously investigated derivatives of 2-aminobenz-
imidazoles (2, pKa ≈ 7.0) and 2-aminopyridines (3, pKa ≈ 6.5).
Compound 2 is active and finally led us to the development of
the tris(2-aminobenzimidazole) 4.[14] Conjugates
molecule with DNA and PNA cleave complementary RNA
5 of this
Supporting information for this article is given via a link at the end of the
document.
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10.1002/ejoc.202100950
European Journal of Organic Chemistry
FULL PAPER
strands with high sequence specificity and substrate half-lives in
the range of 3.5-20 h.[12,15] Surprisingly, compound 3 failed as
RNA cleaving catalyst, in spite of similar pKa values. We
discussed the lack of rotational symmetry around the bond
connecting the heterocycle and the exocyclic nitrogen as a
possible reason:[14b] Binding to phosphate ions requires two
parallel NH groups of the protonated heterocycle to form
hydrogen bonds. Unfavorable conformational equilibria thus may
prevent substrate binding and catalysis (Figure 1).
The dye labeled RNA 13 (Figure 3) was incubated for 20 h at
37 °C and pH 7 with test compounds in concentrations of 10 mM.
Afterwards, fluorescently labeled RNA fragments were
separated with an ALFexpress II sequencer and the
corresponding signals integrated as reported before.[14,15] Such
reaction mixtures are far from being ideal solutions. Instead they
can be heavily aggregated, depending on the nature of the
heterocycle (see below). Standard loading buffers may
precipitate the sample when it is transferred to the separation
gel thus preventing analysis. In addition, many runs showed
increased cleavage between pyrimidines and adenosines, a
pattern typical for minor contaminations with natural RNases.
Exact quantification of weak cleavage activities is hardly
possible under such conditions. Therefore, all data shown in
Table 1 were finally obtained from assays using enantiomeric L-
RNA 14,[15a] known to be stable against natural RNases. Achiral
compounds 6–12, in contrast, are unable to distinguish between
both enantiomeric forms.[14a] A freshly purified sample of enantio
RNA 14 still contained detectable traces of cleavage fragments
in the 14mer ribo part, adding up to 0.9 % of the total peak area.
When incubated at pH 7 for 20 h at 37 °C the combined area of
fragments rose to 1.03 – 1.06 %. This value was unchanged in
the presence of compounds 7–9 or 11 (10 mM) or by addition of
DMSO (20 %). Increased cleavage was only seen with
compounds 10, 12 and in particular with 2-aminoimidazole 6. 2-
Aminopyridine 7, although the pKa (6.7 [16]) comes close to the
ideal value of 7, did not show noticeable RNA cleavage. This
observation clearly disproves our initial attempt to explain the
catalytic incompetence of compound 3. The lacking activity of
compounds 8 and 11 at pH 7 results from their unfavorable pKa.
However, melamine 9 remained almost inactive even at pH 6.1
and 5.1. Matching the buffer pH and the pKa of the catalyst is
important: When aminoimidazole 6 was tested at pH 6.1, the
amount of RNA cleavage dropped from 3.1 % to 0.8 %.
Results and Discussion
To test the conformation hypothesis, we have investigated
the RNA cleaving potential of some simple heterocycles (6–12,
Figure 2), all containing the structural element of amidines or
guanidines. Conformations unfavorable for catalysis as in the
case of
3 do not exist in compounds 6–12. Cleavage
experiments look simple but should be interpreted with caution:
Figure 2. Structures of compounds 6–12.
Table 1. Catalytic potential, pKa and ΔrH°298 of compounds 6–12.
[g]
pH
cleavage [a]
pKa
ΔrH°
298
6 x 0.5 H2SO4
7.0
6.1
7.0
7.0
7.0
6.1
5.1
7.0
7.0
7.0
3.1 ± 0.5 %
0.8 ± 0.2 %
8.5 [c]
40.6 kJ mol-1
7[b]
8[b]
9[b]
-
-
-
-
6.7 [d]
3.4 [d]
5.2 [e]
71.0 kJ mol-1
65.1 kJ mol-1
63.4 kJ mol-1
< 0.1 %
0.3 ± 0.1 %
-
10[b]
11[b]
12 [b]
7.5 [e]
3.7 [f]
7.3 [e]
15.7 kJ mol-1
34.9 kJ mol-1
43.7 kJ mol-1
0.2 ± 0.1 %
[a] 150 nM of 14, 10 mM of heterocycle, 37 °C, 20 h. Total RNA
cleavage after subtraction of background reaction. All experiments
were run at least in triplicate. [b] 20 % DMSO was added to the buffer
to keep the heterocycles in solution. [c] ref. [17]. [d] ref. [16]. [e] ref.
[18]. [f] determined as described in ref. [14b]. [g] energy difference
between tautomers, calculated on the semiempirical AM1 level.
An alternative view on catalytic efficiencies starts to ask for
the protonation state of the reacting phosphate esters during
RNA hydrolysis. Computational and experimental methods have
Figure 3. RNA substrates 13–15 and accessory oligonucleotides 16–17. The
RNA part of 15 is built from enantiomeric nucleotides.
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10.1002/ejoc.202100950
European Journal of Organic Chemistry
FULL PAPER
determined values of 8–9 as first pKa and approximately 14 as
second pKa of equatorial phosphorane hydroxy groups.[19] It is
thus reasonable to consider protonation of dianionic
phosphoranes in the pH region of 7–8: Breslow suggested a
proton transfer from 2’-OH to the phosphorane to occur in the
mechanism of RNase A.[20] Detailed QM/MM simulations have
supported this idea and assigned the role of the proton shuttle to
His12.[21] A mechanism proposed by Cleland for the pH-
independent nonenzymatic cleavage of RNA assumes a water
molecule acting simultaneously as a general acid and base to
transfer a proton from 2’-OH to the pentavalent phosphorane.[22]
While computational studies have emphasized the importance of
Imidazoimidazole 18 [25] exists in structurally identical tautomeric
forms. This property and the pKa value of 7.4 made 18 an
interesting candidate that turned out to be even twice as active
as aminoimidazole 6. Unfortunately, the compound slowly
degraded in aqueous buffer. All our attempts to attach side
chains for conjugation with oligonucleotides failed due to the low
stability of the intermediates. In contrast, no degradation was
[25b,26]
observed with compounds 19
and 20–23 (Figure 5). The
energy difference between tautomeric forms of compound 20,[27]
21 and 23 is also zero due to symmetry. The benzene rings,
however, shifted the pKa to unfavorable values, increased steric
hindrance and also reduced solubilities in water. As
a
this proton transfer step,[23a] the role of water as a proton shuttle
consequence, even at pH 5 and in the presence of 40 % DMSO
less than 2 mM of compounds 20–22 were soluble. No RNA
cleavage was seen at pH 7 or 6.1 and only minor effects at pH
5.0. In contrast, compound 19 is characterized by a pKa value of
6.5 and tautomers of similar energy. Although hardly active at
pH 6.9, it is a good RNA cleaver at pH 5.9 and outperforms
[23b]
has been supported by some
but not all authors.[23c]
However, as Lönnberg pointed out, it may well be that
heterocyclic guanidine analogs function as proton shuttles.[6r,11c,d]
Proton inventory studies have shown that even weakly acidic
guanidinium ions can contribute to catalysis by protonation of
the phosphorane.[5j] Heterocyclic guanidines acting as proton
shuttles would be tautomerized to form an exocyclic imine as
shown in Figure 4. This mechanism is feasible in such cases
only when the energy difference of both tautomers is not too
large. Fast semiempirical calculations are sufficient to get a first
estimate of the energy (heat of formation) differences of
tautomers (Table 1; see below for a comparison with more
advanced methods) and to find a weak correlation with the
catalytic potential of the heterocycles: Smaller values (15.7–
43.7ꢀkJꢀmol-1) are obtained for the active compounds 6, 10 and
12, larger energy differences (63.4–71.0ꢀkJꢀmol-1) for
compounds 7–9. Compound 11,[24] however, has a smaller
energy difference than 6 and 12 but is nevertheless inactive due
to its unfavorable pKa. Furthermore, the sterically less hindered
aminoimidazole 6 is a much better catalyst when compared to
compound 10 by a factor of 3. We could not determine the pKa
[28]
of compound 23
by our UV-spectroscopic method but the
corresponding hydrochloride is a strongly acidic compound
outside of the useful pKa range.
the benzimidazole analog 10, in spite of its less favorable ΔrH°
298
Figure 5. Guanidine analogs 18–23.
value. This shows that the energy difference alone cannot
predict the catalytic potential but may be one criterion in addition
to pKa values, steric effects and the tendency of compounds to
aggregate.
Table 2. Catalytic potential, pKa and ΔrH°298 of compounds 18–23.
[d]
[e]
pH
cleavage [a]
pKa
ΔrH°
298
18 [b]
7.0
6.9
5.9
7.0
6.1
5.0
7.0
7.0
7.0
5.9 ± 1 %
< 0.1 %
0.95 ± 0.3 %
7.4
0 kJ mol-1
19 [b,c, 20% DMSO]
6.5
4.7
17.2 kJ mol-1
20 [c, 40% DMSO]
-
-
0 kJ mol-1
< 0.1 %
21 [c, 40% DMSO]
22 [c, 40% DMSO]
23 [b]
-
-
-
4.5
3.7
low
0 kJ mol-1
2.8 kJ mol-1
0 kJ mol-1
[a] 150 nM of 14, 10 mM of 18 or 23, 2 mM of 20–22, 37 °C, 20 h.
Total RNA cleavage after subtraction of background reaction. All
experiments were run at least in triplicate. [b] The hydrochloride salt
was used. [c] DMSO was added to the buffer to increase the solubility
of heterocycles. Even then the solutions of 20–22 remained cloudy. [d]
determined by spectrophotometric titration (Supporting Information).
[e] energy difference between tautomers, calculated on the
semiempirical AM1 level.
Figure 4. Heterocyclic guanidine analogs, when acting as proton shuttles, are
converted into tautomeric forms that may be unfavorable as shown in the case
of compound 7.
To further test the tautomer hypothesis, the catalytic potential
of guanidine analogs which are expected to have a low or even
zero energy difference was investigated (Figure 5, Table 2).
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[30]
2-Aminoperimidine 24 [29] turned out in our systematic search
2-Methylaminoperimidine 25
was active in the same
as a promising candidate with a ΔrH° value of only 20.5 kJ
concentration range as 24 but, possibly due to steric hindrance,
RNA degradation was much slower. Almost no reaction was
seen with 2-dimethylaminoperimidine 26. To form stable ion
pairs with phosphates, protonated guanidine analogs must have
two parallel NH groups not available in compound 26.[30] The
structure also does not allow proton shuttling as shown in Figure
4. To synthesize conjugates of 2-aminoperimidines with
oligonucleotides, attachment as 2-alkylamino derivatives in
analogy to compound 4 would be attractive. The diminished
activity of such derivatives, however, motivated us to investigate
other linking modes. As expected, for all perimidines 27–31
tautomeric forms with similar energy differences were found
(Table 3).
298
mol-1 (Figure 6 and Table 3). In fact, compound 24 appeared to
be the most effective catalyst among all monomeric guanidine
analogs we have tested so far. At the optimal concentration of
250 µM it almost attained the activity of trisbenzimidazole 4.
Increasing the concentration of 24 into the mM range caused
reduced cleavage, presumably by massive aggregation (Figure
7). This result prompted us to take a closer look on 24 and its
derivatives 25–31.
RNA cleavage experiments were conducted near the pH
optimum for each compound: pH 7 for the less basic derivatives
27 and 29 and at pH 8 for compounds 28, 30, and 31. Most
perimidine derivatives have activities comparable to those of 24
(Figure 8). Ester 30 and in particular the bromo derivative 27 are
distinctly weaker catalysts. The importance of the reaction pH
24
100
90
80
70
60
50
40
30
20
10
0
27
28
29
30
31
Figure 6. 2-Aminoperimidine 24 and derivatives 25–31.
Table 3. pKa and ΔrH°298 values of compounds 24–31.
24
25
27
28
29
30
31
[a]
pKa
ΔrH°
8.1
20.5
7.7
3.8
7.2
21.5
8.1
20.8
6.7
21.4
7.7
21.3
7.9
21.3
0
100
200
300
400
500
[b]
298
concentration / µM
[a] determined by spectrophotometric titration (Supporting Information). [b]
energy difference between tautomers in kJ mol-1, calculated on the
semiempirical AM1 level.
Figure 8. Cleavage of RNA substrate 13 by 2-aminoperimidine derivatives 24
and 27–31 as a function of catalyst concentration. Conditions: 150 nM RNA 13,
3.13 – 500 µM cleaver, 50 mM TRIS-HCl, pH 7 or 8, 37 °C, 20 h. Data points,
determined at least in duplicate, are connected by lines for the sake of clarity.
70
60
50
40
30
20
10
0
28, pH 8
28, pH 7
29, pH 8
29, pH 7
27, pH 8
27, pH 7
100
4
80
60
40
20
0
24
25
26
0
100
200
300
400
500
0
100
200
300
400
500
concentration / µM
concentration / µM
Figure 7. Cleavage of RNA substrate 13 by tris(2-aminobenzimidazole) 4 and
2-aminoperimidines 24, 25, and 26 as a function of catalyst concentration.
Conditions: 150 nM RNA 13, 3.13–500 µM cleaver, 50 mM TRIS-HCl, pH 8,
37 °C, 20 h. Data points, determined at least in duplicate, are connected by
lines for the sake of clarity.
Figure 9. pH-dependend cleavage of RNA substrate 13 by aminoperimidine
derivatives 27, 28, and 29. Conditions: 150 nM RNA 13, 6.25 – 500 µM
catalyst, 50 mM TRIS-HCl pH 7 or 8, 37 °C, 20 h. Data points are connected
by lines for the sake of clarity.
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10.1002/ejoc.202100950
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coming close to the pKa of the catalyst is demonstrated in Figure
9. Methylperimidine 28 (pKa = 8.1) cleaves faster at pH 8
calculations were performed. The comparison is shown in Figure
11. The values for ΔrH° obtained by AM1 have a similar order
298
whereas ester 29 (pKa
=
6.7) works better at pH 7.
of magnitude as ΔrG° calculated by the other methods and
298
Bromoperimidine 27 (pKa = 7.2), a weak catalyst already at pH 7, show overall similar qualitative trends. Due to the semi-empirical
does not work at all under more basic conditions (Figure 9).
Upon titration with NaOH solution under air, 2-
aminoperimidine 24 turns brown in the strongly basic pH region
by irreversible oxidation.[31,32] The same effect occurred with
most other aminoperimidine derivatives. Although we have not
seen problems in the RNA cleavage experiments, oxygen
sensitivity may complicate or even prevent the synthesis of
oligonucleotide conjugates. A notable exception is ester 29,
protected by its electron withdrawing substituent.
nature of AM1, zero point vibrational energies and thermal
corrections are already included in the definition of self-
consistent field energies, so that we did not attempt to
disentangle different contributions and omitted furthermore
entropical corrections in directly comparing here ΔrH° values
from semi-empirical AM1 with ΔrG°values from ab initio methods.
For the relevant compounds 6–12, 19, 22, 24, 25 and 27–31 the
mean absolute error (MAE) between AM1 and the hybrid density
functional B3LYP is 7.7ꢀkJꢀmol-1 which is similar to the MAE
between AM1 and second-order Møller–Plesset perturbation
theory with the resolution of identity approximation RI-MP2
(6.9ꢀkJꢀmol-1) and not much larger than the MAE between B3LYP
The cleavage pattern induced in the enantiomeric TAR RNA
[14]
15
by compound 24 (Figure 10a) and by imidazole buffer
(Figure 10b) reveals a striking difference. Probing of the stable
stem-loop structure with imidazole shows, as expected,
and RI-MP2 (5.9ꢀkJꢀmol-1). For the relevant compounds ΔrG°
298
cleavage restricted to the bulge and the loop regions. In contrast, by RI-MP2 is always larger than ΔrG°298 by B3LYP. ΔrG°298 based
24 induces hydrolysis in all possible positions. This requires full
denaturation of the RNA stem-loop and suggests binding of 15
to polycationic aggregates formed by 24. At 20 µM, however, the
amount of cleavage induced by 24 drops drastically and a
pattern similar to Figure 10b occurs. In contrast, the cleavage
pattern of compounds 6 and 18 represents the bulge and loop
structure of RNA 15 even at the highest concentration of 10 mM.
on high level coupled cluster theory with the resolution of identity
approximation RI-CCSD(T) is between the values of B3LYP and
RI-MP2. As very accurate energy differences seem not to be
important in the classification whether the compound has
cleaving abilities or not, AM1 is a fast method to get a first
estimate.
Figure 10. Cleavage pattern of TAR RNA 15 induced by different agents and
analyzed with an ALFexpress II sequencer detecting the fluorescence of Cy5-
labeled fragments. a) 150 nM RNA 15, 100 µM 2-aminoperimidine 24, 50 mM
TRIS-HCl pH 8, 37 °C, 20 h. b) 150 nM RNA 15, 2 M imidazole pH 7, 40 mM
NaCl, 500 µM EDTA, 37 °C, 20 h. The non-denaturing imidazole buffer
cleaves RNA 15 preferentially in the single-stranded bulge and loop regions
(indicated in grey, see Figure 3).
Direct evidence for aggregates formed from aminoperimidine
24 and oligonucleotides is provided by fluorescence correlation
spectroscopy (FCS).[14] Dye labeled DNA 16 (19 nM) when
diluted with oligo 17 (131 nM; 150 nM total oligonucleotide
concentration as in the cleavage experiments) in buffer free of
heterocyclic guanidines shows high mobility consistent with a
monomeric state. At concentrations of 250 μM and more, 24
massively raises the diffusion time of 16 indicating the presence
of large aggregates. According to Figure 10a, however,
aggregation is not absent even at lower concentrations.
Aminoimidazole 6, in contrast, has no impact on substrate
mobility up to 10 mM. Some effects are found for compounds 10
and 18, but only in the absence of cosolvent whereas compound
19 due to its more lipophilic structure causes considerable
aggregation above 2 mM.
Figure 11. Energy difference between the relevant tautomers of the various
compounds calculated by different theoretical methods (RI-CCSD(T) only for
6–12, 23–27 and those where it is zero due to symmetry). Data points are
connected by lines for the sake of clarity.
Conclusions
The aim of the present study is to identify heterocyclic amidines
and guanidines as building blocks of improved artificial
ribonucleases related to conjugate 5 - and to understand why
some are better catalysts than others. Parameters we consider
relevant are the pKa values, the steric demand and the
hydrophobicity of the compounds: The weakly basic candidates
To test how reliable the semi empirical description of the
energy difference by AM1 is, more advanced quantum chemical
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It is difficult in general to ascribe changes in reactivity in a
strict sense to changes of a single parameter. Thus, a final
evaluation of catalysts is done best in form of conjugates with
oligonucleotides such as 5. Nanomolar absolute concentrations
then avoid aggregation phenomena whereas high local
concentrations of catalyst can lead to fast and specific cleavage
of complementary RNA strands. Not every compound is
sufficiently stable to synthesize such conjugates, but 6, 19 and
29 seem to be good starting points for developing the next
generation of synthetic RNA cleavers.
Experimental Section
Materials. Commercially available guanidines and amidines
were purchased from Acros (8), Aldrich (7), Alfa Aesar (6, 10),
Fluka (9, hydrobromide of 24), and Maybridge (12). The
remaining compounds were prepared as described in the
literature (11, 18, 19, 23, 25–27) and in the Supporting
Information (18–22 and 28–31). 21, 22, and 28–31 are new
compounds.
Figure 12. Classification of the cleavage behaviour (“-“ marks compounds that
do not cleave, “+” marks compounds that are active cleavers and “(+)” marks
compounds that show only little cleavage at pH 7) of various compounds in
dependence of their experimental pKa and computed ΔrH°
values as
298
obtained on the AM1 level (for a variant of this figure with ΔrG° from RI-MP2
298
Cleavage experiments and aggregation studies. RNA
handling and FCS measurements were carried out as described
before.[14] Cleavage experiments and fragment analysis were
done similar to the described procedures.[14] However, to prevent
precipitation of RNA fragments in the presence of heterocycles
during incubation or in the gel pockets, some changes were
made to the loading buffer and the gel composition (see below).
RNA cleavage assay. 150 nM Cy5-labelled RNA 13, 14, or 15
was incubated in a final volume of 10 μL with the indicated
cleaver concentration (0.031-10 mM) in a 50 mM Tris-HCl buffer
at pH 6.0, 7.0 or 8.0 (checked with a glass electrode and
adjusted if required). All cleavage reactions were performed at
37 °C for 20 h.
see Supporting Information).
such as 8, 9, and 11 all fail in the cleavage assay. In addition,
we have found a weak correlation between catalytic activity and
the energy difference between amino and imino tautomers
(Figure 12). This parameter prompted us to investigate 2-
aminoperimidine 24. At first glance, 24 is the most effective
catalyst of this study, a powerful RNA cleaver even in the
micromolar concentration range. In contrast, much less cleavage
occurs in the presence of 2-aminobenzimidazole 10, the active
component of our previous artificial ribonucleases. A direct
comparison, however, is complicated by the fact that RNA
cleavage is not catalyzed by a single guanidine. Instead, a
concerted interaction of two or more subunits is required. When
monomeric building blocks are tested, this cooperativity can
result from high initial concentrations or by local enrichment
caused by aggregation phenomena. The latter is obviously the
case with compound 24. We assume that in general RNA
hydrolysis by monomeric guanidine analogs occurs in non-ideal
solutions. Compounds with large, flat and hydrophobic ring
systems, therefore, may become active at lower concentrations
than small and hydrophilic molecules. On the other hand, when
linked together by appropriate frameworks, derivatives of 2-
aminobenzimidazole such as 4 and 5 turn into powerful catalysts.
Against this background, the cleavage activity of aminoimidazole
6 is quite remarkable - a compound with minimal aggregation
potential. It is superior to compound 10, in spite of a less
favorable value of ΔrH°298. The difference may be attributed to
the steric hindrance caused by the benzene ring of 10. When
ΔrH°298 is reduced to zero by the symmetry of imidazoimidazole
18, a further increase in reactivity is seen. Although this does not
prove the relevance of proton shuttling as depicted in Figure 4, it
may be wise to take tautomeric equilibria into account. The effort
to predict them by AM1 calculations is insignificant.
Polyacrylamide gel electrophoresis. The oligonucleotide
fragments were separated by denaturing PAGE (16 % monomer,
8
M urea) on a DNA sequencing device (ALFexpress,
Amersham Biosciences). Prior to electrophoresis, 15 µL of
loading buffer (8 M urea, 20 mM EDTA and 0.2 % crocein
orange in DEPC-treated H2O) were added to each sample and
10 μL of the sample were loaded on the gel. Following running
conditions were chosen: 1500 V (maximum), 60 mA (maximum),
25 W (constant), 60 °C, 2 s sampling interval and 400 min
running time. For analysis of the electropherograms, the
AlleleLinks 1.01 software package (Amersham Biosciences,
Uppsala, Sweden) was used. The peak areas under the curves
were added up, and the percentage of degraded RNA was
calculated. Multiple cleavage reactions were disregarded in this
system. All data were averaged over a minimum of three
experiments.
Computational Details
The structures of the different tautomeric forms of the molecules
in this study were analyzed with different theoretical methods.
This article is protected by copyright. All rights reserved.

10.1002/ejoc.202100950
European Journal of Organic Chemistry
FULL PAPER
Either with AM1[33] or by density functional theory
Acknowledgements
(B3LYP[34,35]/def2-TZVPP[36,37]
)
or with post-Hartree–Fock
methods (RI-MP2[38-40]/def2-TZVPP[36,37] or RI-CCSD(T)/cc-
pVTZ[41-44]). In all calculations solvent effects were disregarded.
In the case of AM1 the structures were energy minimized
using Gaussian[45]. The convergence criterion for the energy
change between two successive cycles was 1 µEh. Additionally
the maximal change in the density matrix was smaller than 10-6ꢀ
and the root mean square (RMS) of its elements was smaller
than 10-8. The structure was varied till the maximum force in
This work is dedicated to the memory of Prof. Klaus Hafner, to
whom RB is extremely grateful for a welcoming, friendly and
open atmosphere, numerous stimulating discussions as well as
support and advice whenever needed during his time at TU
Darmstadt. We also thank the Center for Scientific Computing
(CSC) Frankfurt for computer time.
Keywords: Ab initio calculations • Catalysis of phosphoryl
transfer • Post-Hartree-Fock methods • RNA cleavage • Semi-
empirical calculations
-1
internal coordinates was below 15ꢀµEhꢀa0 and 15ꢀµEhꢀrad-1 and
-1
the RMS deviation of the force was below 10ꢀµEhꢀa0 and
10ꢀµEhꢀrad-1.
For B3LYP (with the VWN5 local correlation functional) the
structural optimizer of Gaussian[45] was used together with
Turbomole[46-48] that provided the energy and gradients. An m4-
grid[49,50] was used for numerical integration. The energy was
minimized till the change between two successive SCF cycles
was below 0.01ꢀµEh. For the energy minimization of the
structures the same convergence criteria for the force were used
as in the AM1 case (see previous paragraph).
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10.1002/ejoc.202100950
European Journal of Organic Chemistry
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Heterocyclic amidines and guanidines
have been tested as RNA cleaving
catalysts, which led to the identifica-
tion of potent candidates. Besides pKa
values and steric effects, the energy
difference between tautomeric forms
seems to be one of the relevant
parameters, accessible by computa-
tional methods on different levels of
sophistication. The fast semi-empirical
AM1-method was found sufficient to
provide qualitative to semi-quantitative
estimates of corresponding trends.
Author(s), Corresponding Author(s)*
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RNA Hydrolysis by Heterocyclic
Amidines and Guanidines:
Parameters Affecting Reactivity
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Title
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