Tuning the properties of cyclen based lanthanide complexes for phosphodiester hydrolysis; the role of basic cofactors

Article abstract of DOI:10.1039/b609923h

The synthesis of several cyclen based lanthanide [Eu(iii) and La(iii)] complexes is described; these metallo ribonuclease mimics are based on the use of alkyl amines as pendent arms, which give rise to fast hydrolysis within the physiological pH range of

Full text of DOI:10.1039/b609923h

View Article Online / Journal Homepage / Table of Contents for this issue
COMMUNICATION
www.rsc.org/chemcomm | ChemComm
Tuning the properties of cyclen based lanthanide complexes for
phosphodiester hydrolysis; the role of basic cofactors{
Ann-Marie Fanning, Sally E. Plush and Thorfinnur Gunnlaugsson*
Received (in Cambridge, UK) 12th July 2006, Accepted 4th August 2006
First published as an Advance Article on the web 22nd August 2006
DOI: 10.1039/b609923h
primary amines would be able to partake in general acid base
catalysis (i.e. function as cofactors) and potentially lower the pKa’s
of the metal bound water molecules. To the best of our knowledge
these are the first examples of such lanthanide complexes to
achieve such fast hydrolysis.
The synthesis of several cyclen based lanthanide [Eu(III) and
La(III)] complexes is described; these metallo ribonuclease
mimics are based on the use of alkyl amines as pendent arms,
which give rise to fast hydrolysis within the physiological pH
range of HPNP (an RNA model compound) that is highly
dependent on the length of the alkyl spacer.
The synthesis of 1–3 was achieved in three steps from the mono
Boc protected ethylene, propyl and butane diamine, which were
reacted with chloroacetyl chloride to give the appropriate
a-chloroamide derivatives 4–6, Scheme 1. The tetra-substituted
ligands 7–9 were formed by the reaction of cyclen with 4–6,
respectively, in MeCN, in the presence of Cs2CO3 and KI.
Deprotection of the four amines was achieved using a 15% (v/v)
aqueous HCl solution, giving ligands 1–3 as hygroscopic solids in
ca. 90%. The La(III) and Eu(III) complexes of 1–3 were prepared
by refluxing each of the ligands with 1.1 molar equivalents of the
relevant lanthanide triflate in dry MeOH for 16 h, followed by
precipitation using diethyl ether, filtration and washing. This gave
the desired lanthanide complexes La.1, Eu.1, La.2, Eu.2, La3 and
Eu.3 as pale yellow precipitates in 45–90% yields. All these
complexes were fully characterised by elemental analysis, HRMS,
IR and NMR spectroscopy (see ESI for full characterisation).{
Phosphoesters play major structural and mechanistic roles in
nature. They are the basis of energy formation and consumption,
the centre of phosphorylation processes and crucially, form the
backbone of both RNA and DNA. As protein synthesis is the
consequence of the translation of the genetic code, being able to
control the synthesis of non desirable proteins by directly targeting
nucleic acids through phosphodiester hydrolysis of RNA or DNA
is of great scientific and therapeutic value.1,2 In nature, nucleases
or ribonucleases achieve such hydrolysis in a fast and selective
manner, through the synergetic action of one or several cofactors
such as metal ion centres, basic amino acids and metal coordinated
water molecules.1,4 Hence, mimicking these features in a single
molecule, is of great current interest.1–7 We and others have
developed such ribonuclease mimics using macrocyclic lanthanide
cyclen (1,4,7,10-tetraazacyclododecane) complexes.7–9 By function-
alising these with hydrophobic amino acids and heterocycles as
cofactors we have demonstrated fast hydrolysis of phosphodiesters
such as HPNP (2-hydroxypropyl p-nitrophenyl phosphate)10 and
RNA oligomers. Even though this work has been extremely
successful maximum activity has only been achieved in a narrow
pH range; usually centred on pH y 8–10. This has been attributed
to: (1) the binding of the phosphodiester to the coordinated
lanthanide ion; and (2) the deprotonation of metal bound water
molecules (and hence, become nucleophilic) which had pKa’s
within this region.9 In separate studies we have also demonstrated
that the pKa’s of the aforementioned metal bound water molecules
could be significantly tuned by the structure of the pendent arms;
which can directly influence the hydrophobic environment around
the metal ion, which effects the pKa.11
1
The H NMR spectra (400 MHz, D2O) of the Eu(III) complexes
showed several resonances appearing between +25 and 213 ppm,
for the shifted axial and equatorial protons of the cyclen ring and
the acetamide protons of pendent arms, which is characteristic of
square antiprism geometry found in similar cyclen complexes in
solution.9,12 The HRMS (ESMS) also indicated the formation of
With the aim of achieving fast hydrolysis within the physiolo-
gical pH range, we set out to develop the new amino alkyl based
lanthanide complexes Ln1–Ln4. The design rationale behind Ln1–
Ln4 was that the alkyl spacers would give rise to a hydrophobic
environment around the lanthanide ions and at the same time the
School of Chemistry, Centre for Synthesis and Chemical Biology, Trinity
College Dublin, Dublin 2, Ireland. E-mail: gunnlaut@tcd.ie;
Fax: 00353 1671 2826; Tel: 00 353 1 608 3459
{ Electronic supplementary information (ESI) available: Synthesis of all
new ligands and complexes, 2nd order rate constant plot, potentiometric
titrations. See DOI: 10.1039/b609923h
Scheme 1 Synthesis of the cyclen ligands 1–3 and the corresponding
lanthanide complexes.
This journal is ß The Royal Society of Chemistry 2006
Chem. Commun., 2006, 3791–3793 | 3791

View Article Online
length of the spacer is crucial to its activity. This can be deduced
for two reasons; firstly, the alkyl spacers can give rise to a
hydrophobic environment around the metal ion;8 while secondly,
positioning the amines at an optimal distance from the metal
bound substrate. This enables the primary amines to either support
the binding of the substrate through hydrogen bonding or activate
the 29 hydroxyl group, improving its nucleophilicity. Moreover, we
predict that for the La(III) complexes, one of the metal bound
water molecules may possibly be deprotonated by these amines.
To confirm this, we investigated the pH dependence on the rate
of hydrolysis for La.1. Fig. 1 shows that the complex is very active
over a wide pH range. A ‘pseudo’ bell-shaped curve is observed
with a maximum rate constant centred on pH 7.4, and a shoulder
at ca. pH 8.3. These striking results indicate that the maximum
hydrolysis of HPNP does not occur within the pH range
associated with the pKa values expected for the two metal bound
water molecules of La.1 (see below). Having established this fast
hydrolysis for La.1, we measured the second order rate constant
for the hydrolysis of HPNP by La.1 at the rate maxima, pH 7.4.
Here the plot of k vs. [La.1] increased linearly (see ESI),{ indicating
that the reaction was first order in the complex concentration, with
a second order rate constant of 1.41 M21 s21. This is significantly
faster than those previously obtained for both tetra-amide cyclen
based La(III) complexes (0.016 M21 s21), and a dinuclear Zn(II)
complex (0.25 M21 s21) reported for the cleavage of HPNP.7,13
Furthermore, the latter of these was reported to be one of the
fastest second order rate constants determined for the hydrolysis of
HPNP by metal based ribonuclease mimics; clearly showing the
advantage of La.1 complex in promoting such transesterification.§
With the aim of gaining further insight into the role of the
amino moiety of the pendent arms, in the mechanism of
hydrolysis, potentiometric pH titrations were carried out on the
above La(III) and Eu(III) complexes of 1. For 1, five pKa’s were
determined (cf. Speciation diagram in Fig. 2) using the non-linear
least squares regression programme HYPERQUAD; pKa1 10.59
(¡0.01), pKa2 9.70 (¡0.04), pKa3 8.88 (¡0.01), pKa4 8.35 (¡0.05),
and pKa5 6.10 (¡0.09). The last three possible protonations of the
ligand occurred at very acidic pH and therefore could not be
accurately measured using a glass electrode. The first and the
second deprotonations, pKa1 and pKa2, respectively, may be
attributed to the protonation of the alkyl amines of the pendent
donor arms. The protonations pKa3, pKa4 and pKa5 are attributed
to the protonation of two of the cyclen amines and one further
alkyl amine in the pendent donor arms. For La.1, there are six
Scheme 2 Hydrolysis of HPNP by transesterification.10
the desired complexes with peaks found corresponding to the [M +
CF3SO3]+ ion and correct isotopic distribution patterns. The
hydration state (q), the number of metal bound water molecules,
was determined for the Eu.1, Eu.2 and Eu.3 complexes by
measuring their Eu(III) excited state lifetimes in H2O and D2O,
respectively.12 These indicated that each Eu(III) complex possessed
a single metal bound water molecule, giving an overall nine-
coordinate environment for these complexes. In comparison, the
La(III) complexes are usually found to have two metal bound
water molecules, due to their higher coordination requirement.9
The ability of the above complexes to promote phosphodiester
hydrolysis was evaluated using the RNA mimic substrate HPNP,
Scheme 2. The rate constant k for the hydrolysis of HPNP can be
obtained by monitoring spectrophotometrically the hydrolysis of
HPNP and the formation of p-nitrophenolate at 400 nm.{ We first
evaluated the ability of the La(III) and Eu(III) complexes to
hydrolyse HPNP at pH 7.4. The results from these measurements
clearly indicated that the length of the alkyl spacer had an effect on
the rate of hydrolysis. This can be seen in Table 1. For La.1, the
hydrolysis is, to the best of our knowledge, the fastest for such
stable coordination La(III) complexes, with a half-lifetime of 0.74 h.
It is almost four times faster than La.2 and twenty five times faster
than La.3. The same trend is also seen for the Eu(III) analogues.
Here, Eu.1 gives rise to extraordinary fast hydrolysis, which is
unprecedented for such Eu(III) coordination complexes, as in the
past, such Eu(III)-cyclen based complexes have been reported to be
either inactive, or very inefficient in promoting hydrolysis of
HPNP at this pH. Furthermore, these results clearly demonstrate
that the incorporation of ‘cofactors’ into our design, e.g. the
terminus amino functionalities and the length of the alkyl spacers,
play a crucial role in the promotion of hydrolysis.
With the aim of quantifying the role of the primary amines, we
also carried out hydrolysis of HPNP using analogues of these
molecules lacking the amino functions; and indeed these complexes
did not give rise to any measurable hydrolysis, clearly demonstrat-
ing their importance in our current design. It is also clear that the
Table 1 Results from the hydrolysis of HPNP at pH 7.4a
b
Complex
k/h21
t1/2/h
krel
La.1
Eu.1
La.2
Eu.2
La.3
Eu.3
a
0.933 (¡0.005)
0.365 (¡0.012)
0.170 (¡0.011)
0.076 (¡0.014)
0.032 (¡0.006)
0.021 (¡0.004)
0.74
1.89
4.08
9.02
21.73
33.01
7775 (¡42)
3000 (¡100)
1417 (¡92)
633 (¡116)
266 (¡50)
175 (¡33)
The rate constant (k) values were determined by fitting the data to
b
first order rate kinetics. krel, is the ratio between k and the rate
constant of the ‘uncatalysed’ hydrolysis of HPNP, kun 5 0.00012 h21
,
Fig. 1 The pH–rate profile for the hydrolysis of 0.14 mM HPNP at 37 uC
by La.1 (0.18 mM). The error bars are determined from the average of
2–3 measurements.
t1/2 5 5.87 6 103 h, at pH 7.4. Every rate constant that is quoted is
an average of 2–3 measurements, agreeing to within 15%.
3792 | Chem. Commun., 2006, 3791–3793
This journal is ß The Royal Society of Chemistry 2006

View Article Online
In summary, we have demonstrated that by simply incorporat-
ing primary amines into lanthanide cyclen complexes, the rate of
hydrolysis of phosphodiesters can be greatly increased and
maximum activity shifted towards the physiological pH range.
Moreover, such modifications greatly modulate the mechanism for
the hydrolysis, which can be switched from being dominantly
dependent on nucleophilic activation by metal bound hydroxide
molecules, to ligand based activation. We are currently investigat-
ing these and related systems in greater detail.
We thank Enterprise Ireland, IRCSET and TCD for financial
support and Dr John E. O’Brien for assisting with NMR.
Fig. 2 Speciation variation of ligand 1, showing the species present in
H2O at various pH in which [1]total 5 7.2 6 1023 M, [La(III)]total 5 7.0 6
1023 M, I 5 0.10 M (NEt4ClO4) at 25 uC. Speciation is shown relative to
the total concentration of ligand 1. yellow: 1H2; blue: 1H3; orange: 1H4;
light pink: 1H5; green: La.1; brown: La.1H; navy blue: La.1H2; red:
La.1H3; purple: La.1H4; pink: La.1OH2; black: La.1(OH2)2.
Notes and references
{ All of the experiments discussed in this paper were performed using either
an Agilent 8453 or a Cary 50 Scan spectrophotometer, both fitted to a
circulating temperature controlled water bath, and mechanically stirred. A
50 mM HEPES buffer solution was prepared in deionised water and the
pH of the solution was adjusted to the desired pH using 2 M NaOH or 2 M
HCl solutions. A 0.18 mM HPNP (Abs 5 1.22 at 300 nm, e 5
6777 M21 cm21) solution was subsequently prepared using this buffered
solution and 2.4 mL of this HPNP solution was then incubated in a UV cell
at 37 uC for 10 min. The addition of 0.18 mM EDTA had no effect on k.
§ As anticipated, the lanthanide complexes of 1 were thermodynamically
stable with logK of 13.64 (¡0.01) and 20.64 (¡0.01) for 1.La, and 1.Eu,
respectively, as established using HPERCHEM programme.
possible sites for protonation; two assigned to metal bound water
molecules [La.1OH2 and La.1(OH2)2] as well as four alkyl amines
(La.1H4, La.1H3, La.1H2 and La.1H). As expected the pKa values
decreased on increasing protonation; 8.13 (¡0.06), 8.17 (¡0.01),
6.80 (¡0.01) and 6.76 (¡0.05), were assigned to the alkyl amines.
The pKa values 9.21 (¡0.03) and 9.26 (¡0.03) were assigned to
the deprotonation of the two metal bound water molecules, which
are very close. However, the titration curve shows that this process
occurs from pH 8–10 (see ESI).{ Moreover, from the speciation
diagram in Fig. 2 it can be seen that the formation of La.1OH2
and La.1(OH2)2 occurs where the activity of La.1 is decreasing in
Fig. 1. This clearly indicates that these two species do not partake
directly in the hydrolysis, for instance through nuclephilic
activation of the 29 hydroxyl group of HPNP, as previously
demonstrated by us for related complexes where maximum activity
was achieved at alkaline pH. However, under physiological
conditions, one of these water molecules is most likely replaced
upon binding of the substrate to the La(III) centre (Lewis acid
activation). Such an activation mode is also possible for Eu.1.
However, here a single pKa 5 8.00 (¡0.06) was determined for the
deprotonation of the metal bound water. We demonstrated this by
measuring the hydration state in the presence of a phosphodiester
for Eu.1, and a q y 0 was determined. From Fig. 2, it can be seen
that within the pH window 6.5–8.5, which most closely overlaps
with that of the pH–rate profile in Fig. 1, the most active species is
La.1H2 (ca. 60% at pH 7.5) while lesser contributions are observed
from La.1H3 and La.1H4. This suggests that the amines can
strongly influence the reaction through two possible modes of
action, e.g. through hydrogen bonding and/or by a general acid–
base catalysis. Furthermore, they may be acting as a general base
by deprotonating the second sphere waters molecules, which
themselves become nucleophilic.14 For Eu.1 a similar species
distribution (cf. ESI){ was observed; clearly demonstrating the
important role of the primary amines in pendent donor arms in
the hydrolytic process. The amines may also participate directly in
the transesterification by functioning as nucleophiles (e.g. pendent
arm base nucleophilic activation). These results clearly show that
the trend observed in Table 1, e.g. the decrease in activity as a
function of the length of the alkyl spacer, is a measure of the role
the spacers plays in locating the primary amines around HPNP.
1 R. Breslow, in: Artificial Enzymes, Wiley-VCH, Weinheim, 2005.
2 S. K. Silverman, Org. Biomol. Chem., 2004, 2, 2701; J. R. Morrow and
O. Iranzo, Curr. Opin. Chem. Biol., 2004, 8, 192; S. J. Franklin, Curr.
Opin. Chem. Biol., 2001, 5, 201.
3 F. Mancin, P. Scrimin, P. Tecilla and U. Tonellato, Chem. Commun.,
2005, 2540; C. Liu, M. Wang, T. Zhang and H. Sun, Coord. Chem. Rev.,
2004, 248, 147; N. Trawick, A. T. Daniher and J. K. Bashkin, Chem.
Rev., 1998, 98, 939; J. K. Baskin, Curr. Opin. Chem. Biol., 1999, 3, 752.
4 S. Keiper and J. S. Vyle, Angew. Chem., Int. Ed., 2006, 45, 3306;
S. A. Kandadai, W. Chiuman and Y. Li, Chem. Commun., 2006, 2359;
A. S. Borovic, Acc. Chem. Res., 2005, 38, 54; D. M. Perreault and
E. V. Anslyn, Angew. Chem., Int. Ed. Engl., 1997, 36, 432; A. J. Kirby,
Angew. Chem., Int. Ed. Engl., 1996, 35, 707; D. E. Wilcox, Chem. Rev.,
1996, 96, 2435; J. Chin, Acc. Chem. Res., 1991, 24, 145.
5 G. Feng, J. C. Mareque-Rivas and N. H. Williams, Chem. Commun.,
2006, 1845; G. Feng, J. C. Mareque-Rivas, R. Torres, Martin de
Rosales and N. H. Williams, J. Am. Chem. Soc., 2005, 127, 13470.
6 R. T. Kovacic, J. T. Welch and S. J. Franklin, J. Am. Chem. Soc., 2003,
125, 6656; K. Worm, F. Chu, K. Matsumoto, J. Sumaoka, S. Tobey,
V. M. Lynch and E. Anslyn, Chem.–Eur. J., 2003, 9, 741.
7 P. S. Amin, J. R. Morrow, C. H. Lake and M. R. Churchill, Angew.
Chem., Int. Ed. Engl., 1994, 33, 773.
8 T. Gunnlaugsson, R. J. H. Davies, M. Nieuwenhuyzen, C. S. Stevenson,
R. Viguier and S. Mulready, Chem. Commun., 2002, 2136;
T. Gunnlaugsson, J. E. O’Brein and S. Mulready, Tetrahedron Lett.,
2002, 43, 8493.
9 T. Gunnlaugsson, R. J. H. Davies, P. E. Kruger, P. Jensen,
C. S. Stevenson, S. Mulready and A.-M. Fanning, Tetrahedron Lett.,
2005, 45, 3761.
10 R. Breslow and D.-L. Huang, Proc. Natl. Acad. Sci. U. S. A., 1991, 88,
4080.
11 T. Gunnlaugsson, D. F. Brougham, A.-M. Fanning,
M. Nieuwenhuyzen, J. E. O’Brien and R. Vigure, Org. Lett., 2004, 6,
4805.
12 A. Beeby, I. M. Clarkson, R. S. Dickins, S. Faulkner, D. Parker,
L. Royle, A. S. de Sousa, J. A. G. Williams and M. Woods, J. Chem.
Soc., Perkin Trans. 2, 1999, 493; T. Gunnlaugsson and J. P. Leonard,
Chem. Commun., 2005, 3114.
13 O. Iranzo, T. Elmer, J. P. Richard and J. R. Morrow, Inorg. Chem.,
2003, 42, 7737.
14 M. Botta, S. Aime, A. Barge, G. Bobba, R. S. Dickins, D. Parker and
E. Terreno, Chem.–Eur. J., 2003, 9, 2102.
This journal is ß The Royal Society of Chemistry 2006
Chem. Commun., 2006, 3791–3793 | 3793