Macrocyclic amines as catalysts of the hydrolysis of the triphosphate bridge of the mRNA 5′-cap structure

Article abstract of DOI:10.1039/b306268f

The reactions of a 5′-cap model compound P ¹-(7-methylguanosine) P³-guanosine 5′,5′-triphosphate, m⁷GpppG, were studied in the presence of three different macrocyclic amines (2-4) under neutral conditions. The only products observed in the absence of the macrocycles resulted from the base-catalysed imidazole ring-opening and the acid-catalysed cleavage of the N⁷-methylguanosine base, whereas in the presence of these catalysts hydrolysis of the triphosphate bridge predominated. The latter reaction yielded guanosine 5′-monophosphate, guanosine 5′-diphosphate, 7-methylguanosine 5′-monophosphate and 7-methylguanosine 5′-diphosphate as the initial products, indicating that both of the phosphoric anhydride bonds were cleaved. The overall catalytic activity of all three macrocycles was comparable. The hydrolysis to guanosine 5′-diphosphate and 7-methylguanosine 5′-monophosphate was slightly more favoured than the cleavage to yield guanosine 5′-monophosphate and 7-methylguanosine diphosphate. All the macrocycles also enhanced the subsequent hydrolysis of the nucleoside diphosphates, 2 being more efficient than 3 and 4.

Full text of DOI:10.1039/b306268f

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Macrocyclic amines as catalysts of the hydrolysis of the
triphosphate bridge of the mRNA 5Ј-cap structure
Zhibo Zhang, Harri Lönnberg and Satu Mikkola*
University of Turku, Department of Chemistry, FIN-20014 Turku, Finland
Received 3rd June 2003, Accepted 19th August 2003
First published as an Advance Article on the web 1st September 2003
The reactions of a 5Ј-cap model compound P1-(7-methylguanosine) P3-guanosine 5Ј,5Ј-triphosphate, m7GpppG,
were studied in the presence of three different macrocyclic amines (2–4) under neutral conditions. The only products
observed in the absence of the macrocycles resulted from the base-catalysed imidazole ring-opening and the
acid-catalysed cleavage of the N 7-methylguanosine base, whereas in the presence of these catalysts hydrolysis
of the triphosphate bridge predominated. The latter reaction yielded guanosine 5Ј-monophosphate, guanosine
5Ј-diphosphate, 7-methylguanosine 5Ј-monophosphate and 7-methylguanosine 5Ј-diphosphate as the initial products,
indicating that both of the phosphoric anhydride bonds were cleaved. The overall catalytic activity of all three
macrocycles was comparable. The hydrolysis to guanosine 5Ј-diphosphate and 7-methylguanosine 5Ј-monophosphate
was slightly more favoured than the cleavage to yield guanosine 5Ј-monophosphate and 7-methylguanosine
diphosphate. All the macrocycles also enhanced the subsequent hydrolysis of the nucleoside diphosphates, 2 being
more efficient than 3 and 4.
opening of the imidazole ring of the N 7-methylguanine base,
Introduction
are not accelerated.26 Recent results suggest that the 5Ј-cap
The 5Ј-terminus of RNA polymerase II synthesised mRNA
moiety is a promising target for artificial nucleases. It has
molecules contains a unique dinucleoside 5Ј,5Ј-triphosphate
been shown that 5Ј-capped oligonucleotides undergo selective
cleavage of the triphosphate bridge when treated with a
moiety where one of the nucleosides is N 7-methylguanosine
(Scheme 1). This so called 5Ј-cap structure is required as a recog-
complementary oligonucleotide conjugate bearing an N-(2-
nition site of various enzymes that are involved in splicing,1–5
mercaptopropionyl)glycine–Cu2ϩ complex at the 3Ј-terminus.20
transport6,7 and translation8,9 of mRNA. The 5Ј-cap structure
In addition, a 15-meric oligonucleotide conjugated with a
also protects the mRNA against intracellular exonucleases,1,10
Eu3ϩ–THED chelate has been shown to cleave the cap structure
and viral RNA polymerases use 5Ј-capped mRNA sequences
of an intracellular ICAM-1 RNA, significantly inhibiting the
from the host cell as primers for the viral RNA synthesis.11
expression of the respective protein.13
Interactions with enzymes, and, hence, also the consequent
Studies with metal ion-based catalysts have shed light on the
processes, are sensitive to the correct 5Ј-terminal struc-
catalysis mechanisms of the cleavage of the cap structure,
though all details of the catalysis mechanism are not yet
ture.3,5,6,9,10,12 The 5Ј-cap structure is therefore a potential target
site for artificial nucleases, chemical agents that sequence-select-
known. It is generally believed that a hydroxo ligand of a
ively eliminate intracellular mRNA molecules. The principle
phosphate-bound metal ion catalyst acts as a nucleophile that
of such catalysts is simple: an oligonucleotide recognizes a
attacks a phosphate group, while the coordinated metal ion
sequence of the mRNA target by hybridization, and a catalyt-
electrostatically enhances the nucleophilic attack.22–26 Altern-
ically active function attached enhances the cleavage of the
atively, a functional group on the ligand may serve as the
5Ј-terminal cap structure.13 Alternatively, the catalysts may be
nucleophile.21,25 Metal ion catalysts may, however, have a more
designed to cleave internucleosidic phosphodiester bonds,14–18
complex role in the reaction, a metal ion aquo ligand possibly
and several oligonucleotide conjugates bearing either a metal
serving as a general acid-catalyst.26
ion complex or an organic catalyst have been shown to cleave
The rate-enhancement of hydrolysis by metal ions is rather
their target RNAs selectively.
significant. A Cu2ϩ–bipyridine complex in 2 mM concentration
at pH 8 and 60 ЊC enhances the hydrolysis of a dinucleoside
triphosphate at least by a factor of 20000,26 and various
lanthanide ion complexes25 and dinuclear Cu2ϩ complexes22,23
appear to be even better catalysts reducing the half-life of the
hydrolysis of the triphosphate to a few hours under neutral
conditions and 37 ЊC. While efficient catalysis by metal ions has
been observed, no reports on rate-enhancement by organic
molecules have been published, even though they would be able
to provide catalysis by the mechanisms discussed above. An
organic cleaving agent, particularly if intracellular cleavage is
attempted, might have advantages over the metal ion-based
ones. For example, the selectivity and efficiency of metal ion-
based catalysts may be reduced by coordination of the metal
ion with other ligands available inside the cell. Even though no
data on the cleavage of 5Ј-cap models have been reported, it is
known that protonated forms of some cyclic polyamines
modestly enhance the cleavage of ATP.27–33 Under neutral con-
ditions hexaazadioxa (1)27,29 and heptaaza (2)31 macrocycles are
the most efficient catalysts studied. The mechanisms suggested
Scheme 1
The cleavage of the 5Ј-cap has been studied by using dinucleo-
side triphosphates as model compounds,19–26 and it has been
shown that Cu2ϩ and lanthanide ion complexes, for example,
efficiently enhance the hydrolysis of the triphosphate bridge. In
contrast, the other potential reactions, viz. the hydrolysis of the
N-glycosidic bond of the N 7-methylguanosine moiety and the
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 4 0 4 – 3 4 0 9
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 3
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Scheme 2
for the macrocycle promoted hydrolysis of ATP are basically
Results and discussion
similar to those of metal ion promoted hydrolysis of dinucleo-
side triphosphates. The reaction with 1 and 2 proceeds via a
nucleophilic attack of ring nitrogen on the phosphate group
resulting in a transient formation of a phosphoramidate
intermediate.27–29,31 In cases where a phosphoramidate inter-
mediate has not been observed, the macrocycle has been pro-
posed to enhance the nucleophilic attack of a water molecule
on the phosphate electrostatically, by hydrogen bonding or as a
general acid catalyst.30,32–34
The reaction of m7GpppG (5) in the presence of macrocyclic
amines 2–4 was studied in 0.1 M buffer solutions at 60 ЊC. The
concentration of m7GpppG was kept below 50 µM to maintain
pseudo first-order conditions. Samples taken from reaction
solutions were analysed by capillary zone electrophoresis (CZE)
using a 0.15 M boric acid buffer as the background electrolyte.
First-order rate constants of the disappearance of m7GpppG
were calculated on the basis of the integrated signal areas in
electropherograms. The concentrations of the more stable
hydrolysis products, GMP and GDP, were determined using
calibration curves obtained with commercially available
compounds. The concentration of 5 in standard solutions was
determined spectrophotometrically using the log ε value
reported previously.35 Samples of known concentrations were
analysed using the CZE conditions mentioned above. The
signal areas in the electropherograms were normalised by
dividing the signal area by the migration time. Rate constants
of the triphosphate hydrolysis were calculated on the basis of
the increase of the concentration of the hydrolysis products as a
function of time.
Under all the conditions studied, the reactions of m7GpppG
resulted in a complex product mixture. Of the hydrolysis
products, 7-methylguanosine 5Ј-phosphate (m7GMP; 6),
guanosine 5Ј-phosphate (GMP; 7), guanosine 5Ј-diphosphate
(GDP; 8), and 7-methylguanosine 5Ј-diphosphate (m7GDP; 9)
were identified by co-injecting with authentic samples. How-
ever, in addition to 6–9, several other products were formed.
These unknown products were difficult to identify, since several
minor peaks were observed which could not be fully separated
by any HPLC-method tested. Therefore, a sufficiently accurate
HPLC-MS analysis or isolation of the products by chromato-
graphy were not possible. Comparison of the product mixtures
obtained in the presence and in the absence of the amine
catalysts shows, however, that the unknown products result
from the imidazole ring-opening of the N 7-methylguanine
base. This reaction takes place via a nucleophilic attack of
a hydroxide ion on the C8 of the base, and it is known to result
The present paper studies the effect of macrocyclic amines 2–
4 on the hydrolysis of a 5Ј-cap model compound, P1-(7-methyl-
guanosine) P3-guanosine 5Ј,5Ј-triphosphate (5). We show that
the amines significantly enhance the hydrolysis of the triphos-
phate bridge to yield nucleoside monophosphate and diphos-
phate products (6–9 in Scheme 2). To our knowledge, this is the
first report on the hydrolysis of a 5Ј-cap structure promoted by
purely organic catalysts.
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Fig. 1 The observed rate constants of disappearance of 5 (kobs) as a
function of pH in the presence of macrocyclic amine 4 at 60ЊC.
the 7-methylguanosine moiety37,38 gradually becomes the pre-
dominant reaction, which is clearly shown by the formation
of m7Guo as the predominant product under slightly acidic
conditions. Since the macrocycle promoted hydrolysis is
observed only over a narrow pH-range, the competing reactions
predominating under more acidic and basic conditions, the
effect of pH on the hydrolysis could not be studied in detail.
As evidence of 5 reacting via the hydrolysis of the triphos-
phate bridge under neutral conditions, hydrolysis products 6–9
were formed to a significant extent in the presence of the
macrocyclic amines. In the absence of the catalysts these
products were not observed at all. The proportion of hydrolysis
products among the product mixture was determined by
calculating the concentrations of 5 and the non-methylated
hydrolysis products 7 and 8. The results of this analysis show
that after 75% of 5 has reacted, the hydrolysis products con-
stitute 70–80% of all products formed. Further evidence in
favour of macrocycle catalysis was obtained by studying the
effect of the amine concentration on the kobs values. As is shown
in Fig. 2, the rate constants increase as the concentrations of
the amine catalysts increase. The hydrolysis products became
clearly more predominant in the product mixture as the amine
concentration increased.
Scheme 3
in a complex product mixture (Scheme 3a).36 Products 6 and 9
undergo the ring-opening reaction slightly faster than
m7GpppG,36 which further increases the complexity of the
product mixture. In addition, pH-independent depurination of
the 7-methylguanosine moiety yields an apurinic dimer which,
owing to rapid anomerisation of the sugar moiety, may exist in
two different forms (Scheme 3b).37,38
Fig. 1. shows the pH-dependence of the rate constant of the
overall disappearance of m7GpppG (kobs) in the presence of
macrocyclic amine 4 at 10 mM concentration. The kobs values
shown consist of contributions of the imidazole ring-opening
(Scheme 3a), depurination of the N 7-methylguanosine moiety
(Scheme 3b) and hydrolysis of the triphosphate bridge. The
proportions of the three different reactions vary depending on
pH. The macrocycle-promoted hydrolysis is most prominent in
neutral solutions. Under these conditions hydrolysis products
6–9 were formed to a significant extent in the presence of
the macrocyclic amines, but not at all in the absence of these
catalysts. At pH > 7, the base-catalysed imidazole ring-
opening36,39,40 gradually becomes the only reaction, and above
pH 8 the hydrolysis products 6–9 are not observed at all. Con-
sequently, the rate increase on increasing pH refers entirely
to the base-catalysed imidazole ring-opening. The macrocycle-
promoted hydrolysis also becomes less significant at pH < 7.
The macrocycle-dependent phosphate bridge hydrolysis is
decelerated and, hence, the pH-independent depurination of
Fig. 2 The effect of concentration of 2–4 on the kobs values of the
cleavage of 5 at pH 7.2 and 60 ЊC. Notation: 2 – filled triangles, 3 – open
triangles and 4 – filled squares.
The macrocycle-promoted hydrolysis was studied in more
detail at pH 7.2. The rate constants determined in the absence
of azacrowns 2–4 and in their 10 mM solutions are collected in
Table 1. The rate constants, kmc, of the macrocycle dependent
reactions were obtained by subtracting from the observed rate
constant (kobs) the rate constant in the absence of the polyamine
catalyst (kuncat). In other words, azacrowns 2–4 are assumed
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Table 1 Rate constants of the reaction of a 5Ј-cap model m7GpppG
(5) and of nucleoside diphosphates 8 and 9 in the presence and absence
of macrocyclic amines 2–4 at pH 7.2 and 60 ЊC.
(Scheme 2). The cleavage between the α and β phosphates is
slightly favoured in both cases: after 75% of 5 has reacted, the
GDP : GMP concentration ratios were approximately 2 : 1 and
3 : 1 with amines 3 and 4, respectively. In the presence of 2 the
situation is a bit more complicated, since the catalyst also effi-
ciently promoted the hydrolysis of GDP to GMP, and the only
product observed to any significant extent was GMP. Most
probably, however, 2 also enhances the hydrolysis of 5 between
α and β phosphates as well as between β and χ phosphates, but
the proportion of the two reaction routes cannot be determined
on the basis of the data available.
m7GpppG (5)
GDP (8)
m7GDP (9)
kuncat/10Ϫ6 sϪ1
0.97 0.03
3.97 0.14
4.81 0.19
3.83 0.17
3.0
3.8
2.9
3.89 0.09
kobs(2)/10Ϫ6 sϪ1
kobs(4)/10Ϫ6 sϪ1
kobs(4)/10Ϫ6 sϪ1
kmc(2)/10Ϫ6 sϪ1 a
kmc(3)/10Ϫ6 sϪ1 a
kmc(4)/10Ϫ6 sϪ1 a
36.8
2.50 0.06
1.94 0.09
1.2
28.5
13.6
6.5
0.8
0.5
0.4
24.7
9.8
3.6
As was mentioned in the introduction, several mechanistic
alternatives have been proposed for the macrocycle-promoted
hydrolysis of ATP. These include nucleophilic catalysis by
unprotonated ring nitrogens, facilitation of nucleophilic attack
on phosphorus by electrostatic interaction or hydrogen bonding
of the phosphate oxygens with the protonated ring nitrogens
and general acid/base catalysed attack of a water molecule. The
same mechanisms may also operate in the present case. The
species distribution curves in Fig. 3, based on reported pKa
values,41,42 show that the predominant species of the hepta-
amine 2 and hexamine 4 at pH 7.2 at 60 ЊC is a trication. Such
a structure, containing both protonated and unprotonated
amines, could, in principle, catalyse the hydrolysis of the tri-
phosphate bridge by any of the mechanisms proposed. An MS
analysis of a sample from the reaction solution was carried out
to detect a possible phosphoramidate intermediate, but without
success. This cannot, however, be taken as conclusive evidence
against the mechanism where the attacking nucleophile is ring
nitrogen since the intermediate may be too short-lived to
accumulate. On using ATP as a substrate, the phoshoramidate
was only observed with the most efficient polyamine catalysts,
1 and 2. Even though relatively high concentrations of the
reactants were used, the decomposition of the intermediate
generally was faster than its formation.28 The fact that the
catalytic efficiency of azacrowns 2–4 is highest when approx-
a kobs Ϫ kuncat
.
to accelerate only the hydrolysis of the triphosphate bridge,
leaving the imidazole ring-opening of the N 7-methylguanine
base and depurination of the N 7-methylguanosine moiety of
5 unaffected. This assumption appears to be valid, since the rate
constants of the uncatalysed reaction (kuncat) in Table 1 corre-
spond to 20–30% of the observed kobs values, which is well
consistent with the proportion of hydrolysis products
mentioned above. This is also consistent with previous
observations that simple acyclic amines, such as triethylamine,
do not promote the ring-opening reaction.26,36
The data in Table 1 show that azacrowns 2–4 at a 10 mM
concentration increase the rate constants of the disappearance
of m7GpppG by a factor of four. The catalytic activity of all
three amines is comparable, 3 being a slightly better catalyst
than 2 or 4. In the absence of 2–4, the hydrolysis of m7GpppG
is very slow in comparison to the imidazole ring-opening. No
hydrolysis products (6–9) were observed during 10 days, sug-
gesting that the rate constant for the macrocycle-independent
hydrolysis must be smaller than 5 × 10Ϫ8 Ϫ1. Evidently the
s
reaction is even slower, since the rate constant of the uncata-
lysed hydrolysis of the triphosphate bridge of P1,P3-bis(adenos-
ine) 5Ј,5Ј-triphosphate (10) has been estimated to be less than
5 × 10Ϫ9 sϪ1 at 60 ЊC.26 Accordingly, macrocyclic amines 2–4 at
a 10 mM concentration appear to enhance the hydrolysis of
m7GpppG by a factor of a few hundred.
The hydrolysis of the triphosphate bridge of 5, in principle,
results in the formation of four different products (Scheme 2).
Cleavage of a bond between α and β phosphates would result
in the formation of m7GMP and GDP, while cleavage between
β and χ phosphates would yield m7GDP and GMP. The pre-
dominant hydrolysis products observed however varied depend-
ing on the catalyst employed. This results partly from the fact
that the ring-opening reaction of the N 7-methylated products
is faster than that of 5. Furthermore, in some cases the
macrocyclic amines enhance the cleavage of the products as
well. The effect of 2–4 on the hydrolysis products was studied
by independent experiments, and the rate constants obtained
with nucleoside diphosphates 8 and 9 are included in Table 1.
With 3 and 4 as the catalyst of the cleavage of 5, the pre-
dominant products observed were GMP (7) and GDP (8), the
amounts of m7GMP (6) and m7GDP (9) were clearly smaller,
particularly for longer reaction times. Both amines enhance the
hydrolysis of m7GDP and 3 also slightly enhanced the dis-
appearance of m7GMP. There is no doubt that 3 and 4 promote
the cleavage between both α and β, and β and γ phosphates
Fig. 3 a. Species distribution of macrocyclic amine 2 at 60 ЊC. pKa
values taken from ref. 41 have been extrapolated using the temperature
dependence of pKa values of 4 reported in ref. 42. b. Species
distribution of macrocyclic amine 4 at 60 ЊC. pKa values at 60 ЊC have
been extrapolated using the temperature dependence reported in ref. 42.
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imately half of the nitrogen atoms are protonated could well be
accounted for by a mechanism involving an attack of unproton-
ated ring nitrogen on phosphorus assisted by interaction of the
phosphate oxygens with the positively charged ammonium ion
centers on the ring.
amino]ethanesulfonic acid, pKa = 9.3 at 25 ЊC) buffer. The pH
of the reaction solutions was checked with a pH-meter at 25 ЊC.
The ionic strength was adjusted to 0.1 M with NaNO3.
Kinetic measurements
Polyamines 2–4 show interesting selectivity that cannot be
explained by the present data. Even though 2–4 hydrolyse
m7GpppG approximately equally efficiently, their abilities to
hydrolyse m7GDP and GDP differ considerably, 2 being the
most and 4 the least effective catalyst. Comparison of the pres-
ent data with those reported for ATP shows that the effect of
cyclic polyamines on the hydrolysis of the cap structure cannot
be predicted by their influence on the hydrolytic stability of
ATP. For example, even though 2 and 4 are equally efficient
catalysts of the hydrolysis of m7GpppG, only 2 significantly
hydrolyses ATP.31 Another indication of the dissimilarity of
the two systems is that 1, which is a good catalyst of ATP
hydrolysis,27–29 does not enhance the hydrolysis of a 5Ј-cap
model 10.26 In contrast, a 2Cu2ϩ–1 complex efficiently catalyses
the hydrolysis of 10,26 whereas complex formation does not
affect the catalysis of ATP hydrolysis.29
Reactions were carried out in Eppendorf tubes immersed in a
water bath, the temperature of which was thermostated at
60 ЊC. The reactions were initiated by adding substrate stock
solution (a few microliters) to give a concentration of 50 µM.
The total reaction volume was 1.5 ml. Aliquots (12) of 100 µl
were withdrawn at appropriate intervals to cover approximately
two half-lives of the reaction. The aliquots were immediately
cooled down over an ice-bath, and stored in the freezer until
analysed.
Analysis of aliquots
Aliquots were analysed by capillary zone electrophoresis
(CZE). Capillary electrophoretic analysis was carried out in a
fused silica capillary (75 µm id, 110 cm) with 0.15 M boric acid,
pH 8.5 as the run buffer. A voltage of 30 kV was applied. The
compounds were detected at 254 nm. Under these conditions
the migration time of the starting material was about 60 min.
Experimental
Materials
Acknowledgements
Guanosine 5Ј-diphosphate and guanosine 5Ј-monophosphate
were products of Sigma and they were used as received.
1,4,7,10,13,16-Hexaazacycloicosane trisulfate (Sigma) was
transferred to the free base eluting through Dowex (OHϪ) resin.
N 7-Methylguanosine, N 7-methylguanosine 5Ј-monophosphate
and N 7-methylguanosine 5Ј-diphosphate were prepared using
methyl iodide as methylating reagent as described before.43
m7GpppG was synthesised according to a literature method36,44
starting from guanosine 5Ј-phosphorimidazolide and 7-methyl-
ated guanosine diphosphate. The coupling reaction was carried
out in the presence of ZnCl2 in anhydrous DMF. The com-
The authors wish to thank Dr. Attila Jancso for producing the
species distribution curves.
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1
products were characterized by H NMR and 13C NMR and
1
mass spectra. 1,4,7,10,13,16,19-Heptaazacycloeicosane (2): H
NMR (D2O, 400 MHz): 3.582 (s, 28H). 13C NMR (D2O, 400
MHz): 43.789. Mass: 302.1 (M ϩ 1). 1,4,7,10,13,17-Hexaaza-
1
cycloeicosane (3): H NMR (D2O, 400 Hz): 1.4994 (m, 4H),
2.454 (m, 9H), 2.5063 (m, 15H); 13C NMR (D2O, 400 Hz):
48.168, 48.115, 47.963, 47.819, 46.460, 46.187; HRMS:
287.292320 (found), 287.292300 (calculated for C12N6H30).
Preparation of reaction solutions
The pH of the reaction solutions was adjusted with 0.1 M acetic
acid, MES (morpholino ethanesulfonic acid; pKa 6.2 at 25 ЊC),
MOBS (4-[N-morpholine] butanesulfonic acid; pKa =7.6 at
25 ЊC), HEPES (2-[4-(2-hydroxyethyl)-1-piperazino]ethane-
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