Cyclen based lanthanide ion ribonuclease mimics: The effect of pyridine cofactors upon phosphodiester HPNP hydrolysis

Article abstract of DOI:10.1016/j.tetlet.2005.03.150

The cyclen based pyridine complexes 1Ln-3Ln (Ln = La(III) and Eu(III)) were synthesised as metallo-ribonuclease mimics and their ability to hydrolytically cleave the phosphodiester of HPNP at 37°C was investigated using UV-vis spectroscopy, whereas the bi

Full text of DOI:10.1016/j.tetlet.2005.03.150

Tetrahedron
Letters
Tetrahedron Letters 46 (2005) 3761–3766
Cyclen based lanthanide ion ribonuclease mimics: the effect
of pyridine cofactors upon phosphodiester HPNP hydrolysis
Thorfinnur Gunnlaugsson,a,* R. Jeremy H. Davies,b Paul E. Kruger,a Paul Jensen,a
Thomas McCabe,a Sinead Mulready,a John E. OꢀBrien,a Clarke S. Stevensonb and
Ann-Marie Fanninga
aDepartment of Chemistry, Centre for Synthesis and Chemical Biology, Trinity College Dublin, Dublin 2, Ireland
bSchool of Biology and Biochemistry, Queen’s University of Belfast, Belfast BT9 7BL, Northern Ireland
Received 22 November 2004; revised 14 March 2005; accepted 21 March 2005
Available online 9 April 2005
Abstract—The cyclen based pyridine complexes 1Ln–3Ln (Ln = La(III) and Eu(III)) were synthesised as metallo-ribonuclease mim-
ics and their ability to hydrolytically cleave the phosphodiester of HPNP at 37 ꢀC was investigated using UV–vis spectroscopy,
whereas the binding of the substrate was evaluated using 31P NMR and Eu(III)-luminescent measurements. In contrast 2La gave
rise to fast pH dependent hydrolysis of HPNP, with maximum efficiency at ca. pH 8.2, and with a half-lifetime of ꢀ1 h, the 1Ln
and 3Ln complexes were found to be inactive, emphasizing the importance of the nature of the pyridine isomer as a cofactor in
the hydrolytic process.
ꢁ 2005 Elsevier Ltd. All rights reserved.
Phosphoesters are structural motifs found in biological
systems such as DNA and RNA. Nature has developed
ribonucleases that hydrolytically cleave such esters in a
controllable manner often thought to involve the syner-
gic action of more than one metal ion.1,2 Moreover, the
active sites of such enzymes have basic amino acid
residues, which in addition to metal coordination, act
as cofactors that participate in general acid–base cataly-
sis, helping to stabilise the transition state and assist
leaving group departure. Unlike DNA, RNA has the
20-hydroxyl group available as an internal nucleophile,
making RNA much less stable in comparison to DNA.
It is postulated that metal ions participate in the hydro-
lysis, either directly or indirectly, through Lewis-acid
activation (via coordination to the phosphate anion),
stabilisation of the transition state, or through the pro-
vision of nucleophiles in the form of metal-bound waters
or hydroxyl-containing molecules.1,2 We3 and others4
have developed synthetic nuclease molecules that can
mimic the behaviour of these metallo-enzymes. Such
mimics are potentially important for use in biotechno-
logy as they can be utilised in the manipulation of genes,
as structural probes, and in medicine as novel therapeu-
tics for blocking gene transcription.1,2,5 We have already
focused our efforts on synthesising lanthanide ion-based
ribonuclease mimics by incorporating dipeptides such as
GlyGly and GlyAla into 1,4,7,10-tetraazacyclododecane
(cyclen),3 which gave large enhancements in the rate of
hydrolysis of RNA phosphodiester mimics such as
HPNP (2-hydroxypropyl-p-nitrophenylphosphate) 7.
Herein, we describe the synthesis and structural analysis
of three new Ln(III) cyclen complexes 1Ln–3Ln
(Ln = La(III) and Eu(III)) where we have incorporated
the three pyridine isomers onto the macrocyclic struc-
ture as part of the tetra-amide pendent arms. The main
objectives were to investigate the role of the pyridine
moieties (which can give rise to a hydrophobic cavity)3,6
in conjunction with the lanthanide ion centre in the
hydrolysis and to demonstrate the potential role of these
molecules as ribonuclease mimics or artificial enzymes
using non-ꢁnaturalꢀ metal ions.
The synthesis of 1–3 is shown in Scheme 1, commencing
with the a-chloroamides 4–6. The precursor 5 was
formed in 98% yield by reacting chloroacetyl chloride
with 3-aminopyridine in acetone at 0 ꢀC, in the absence
of any base. However, the synthesis of 4 and 6 was only
successful in DCM at 0 ꢀC using triethylamine as base.
Keywords: Ribonuclease mimics; Lanthanides; Eu(III); La(III); Hydro-
lysis; HPNP; Cyclen.
*
Corresponding author. Tel.: +353 1 608 3459; fax: +353 1 671
2826; e-mail: gunnlaut@tcd.ie
0040-4039/$ - see front matter ꢁ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2005.03.150

3762
T. Gunnlaugsson et al. / Tetrahedron Letters 46 (2005) 3761–3766
N
H
H
H
H
N
N
N
N
H
N
NH
O
Cl
H
N
N
O
N
N
N
O
N
Cs2CO3, KI
MeOH or DMF
O
N
4- 2-position (49%)
5- 3-position (98%)
6- 4-position (48%)
N
H
N
O
3+
N
HN
1- 2-position (74%)
2- 3-position (86%)
3- 4-position (41%)
N
NH
N
O
Ln
O
H
N
N
N
Ln O , H O
2
3
2
O
O
N
N
N
H
or Ln(CF SO ) ,
3
3 3
N
MeOH
HN
1Ln- 2-position; Ln = La (61%) and Eu (64%)
2Ln- 3-position; Ln = La (64%) and Eu (74%)
3Ln- 4-position; Ln = La (62%) and Eu (70%)
N
Scheme 1. Synthesis of 1–3 and the corresponding lanthanide ion complexes 1Ln–3Ln.
The next step involved the incorporation of these arms
into the macrocycle. For 1 and 3 the desired products
were obtained using 5.5 equiv of 4 and 6, respectively,
in the presence of cyclen, Cs2CO3 and KI in DMF
(80 ꢀC for 64 h). The products were purified by precipi-
tation from dry CHCl3 yielding only the desired tetra-
substituted cyclen derivatives 1 and 3 in 74% and 86%
yields, respectively. However, the synthesis of 2 under
these conditions was not successful. It was isolated as
a pale orange powder in 41% yield, by reacting 5
(5.5 equiv) and cyclen in refluxing MeOH in the pres-
ence of Cs2CO3 and KI for five days. This yielded both
the tri- and tetra- (2) substituted cyclen derivatives,
which were separated by gradient elution on alumina
rial and the axial protons of the cyclen ring and the pen-
dent arms were substantially shifted due to the presence
of the paramagnetic Eu(III) ion. For 2Eu these appeared
(in D2O, 400 MHz) at 16.35, 8.08, 7.18, 4.61, 3.16, 1.59,
1.03, ꢁ1.31, ꢁ4.21 and ꢁ10.36 ppm, respectively. We
also determined the hydration state (q), the number of
metal-bound water molecules for the europium com-
plexes, as this would confirm the coordination number
of the complexes in solution. For all of these, a q-value
of ca. 1 was determined, Table 1. We propose that this
geometry would give rise to a concave structural motif
as previously discussed.3 We were able to grow single
crystals of 2La, 2Eu and 2Gd as colourless needles, by
slow evaporation of water solutions. The structure of
2La is shown in Figure 1. It is known that such
La(III)–cyclen complexes usually adopt a 10 coordina-
tion geometry.6,8,9 This was found to be the case for
2La, which clearly showed the concave nature of the
complex, with the La(III) ion centrally located, coordi-
nating to the four nitrogen atoms of the cyclen ring
and four oxygen atoms of the carboxylic amides, giving
a disordered geometry. Furthermore, the complex had
the ninth coordination site occupied by a metal-bound
water molecule, and in the tenth coordination site was
a triflate anion.8 We predict that in water this second
1
[CH2Cl2:MeOHÆNH3 (1–20%)]. The H NMR spectra
of 1–3 showed the presence of C4 symmetry for all these
complexes, for example, for 2 (400 MHz, DMSO-d6) the
methylene protons on the acetamide arms and the cyclen
ring protons appeared as singlets at 3.20 and 2.72 ppm,
respectively, with the pyridine protons appearing at
8.51, 8.16 and 7.03 ppm, while the amide proton ap-
peared at 10.39 ppm. Similar results were seen for 1
and 3. The corresponding La(III) and the Eu(III) com-
plexes of 1 and 3 were formed in water from either
La2O3 or Eu2O3, respectively, under reflux. After filtr-
ation, the solvent was removed under reduced pressure,
giving oils that were redissolved in the minimum amount
of MeOH followed by trituration using CHCl3. This
yielded 1Ln and 3Ln (Ln = La and Eu) in ca. 60% yields.
This method was, however, unsuccessful for 2La and
2Eu (as well as the Gd(III) complex 2Gd which was also
prepared) which were formed by reacting 2 with
La(CF3SO3)3 and Eu(CF3SO3)3 in refluxing MeOH
followed by precipitation from CHCl3, giving 2La and
2Eu in 75 and 65% yields, respectively.7
Table 1. Determination of the hydration state of the Eu(III) com-
plexesa,ꢂ
Complex
k (msꢁ1
H2O
)
s (ms)
H2O
k (msꢁ1
D2O
)
s (ms)
D2O
q ( 0.5)
1Eu
2Eu
3Eu
1.74
2.60
2.56
0.36
0.38
0.39
0.60
1.07
1.41
1.66
0.94
0.71
1.09
1.18
0.90
a Determined using qEu = 1.2[(1/sH O ꢁ 1/sD O) ꢁ 0.25 ꢁ 0.075x] (x = 4
2
2
for 1–3).10
The 1H NMR spectra of all the Eu(III) complexes
showed, as expected, that the resonances for the equato-
ꢂ We were unable to determine the q-value for the La(III) complexes
using this method.6,8,9

T. Gunnlaugsson et al. / Tetrahedron Letters 46 (2005) 3761–3766
3763
Figure 2. X-ray crystal structure of 2Eu (D(kkkk)) showing the nine
coordinate environment of the Eu(III) complex. Hydrogen atoms and
triflate anions have been removed for clarity.
Figure 1. X-ray crystal structure of 2La showing the 10 coordinate
environment of the La(III) complex. Hydrogen atoms, solvent and
non-coordinated triflate anions have been removed for clarity.
coordination site is occupied by the solvent, due to fast
H2O exchange, giving rise to the diaqua species. As
shown later, the presence of these metal-bound water
molecules is vital to the activity of the ribonuclease
mimic and towards phosphodiester hydrolysis.1–4,10 In
comparison to these results, the X-ray crystal structure
of 2Eu, Figure 2, showed a typical monocapped square
antiprism geometry, with the enantiomeric conforma-
tion D(kkkk) where the water molecule was in the axial
position and the overall coordination number of Eu(III)
was nine. The X-ray crystal structure of the 2Gd
complex was isostructural, supporting the results of
the solution studies which showed that these complexes
have a single bound water molecule.
OH
O
P
O
O
+
+
-O
H+
NO2
O
O
O
O
P
-O
9
8
NO2
7
Scheme 2. The hydrolysis of HPNP.
no measurable HPNP hydrolysis was observed over
48 h, indicating that the rate enhancement was indeed
due to the hydrolysis of the substrate by these
complexes.
In order to investigate the ability of these complexes to
promote phosphodiester hydrolysis, we used the RNA
mimic compound HPNP, 7, Scheme 2. The rate constant
of hydrolysis of HPNP can be followed by observing the
appearance of p-nitrophenolate 9 at 400 nm, upon
hydrolysis (HPNP absorbs at 300 nm), Scheme 2.ꢃ The
resulting absorption changes at 400 nm were fitted to
give the rate constants kobs. Under these conditions,
2La was found to promote hydrolysis of 7 with a kobs
of 0.189 ( 0.003) hꢁ1 and a s1/2 ꢀ 3.7 h. This is a rate
enhancement of 1600.§ In direct contrast to these results,
neither 1La nor 3La gave rise to any significant hydroly-
sis at this pH. We also evaluated the hydrolysis of 2Eu
under identical conditions, which gave kobs = 0.078
( 0.0028) hꢁ1 and s1/2 = 8.9 h. When these measure-
ments were repeated in the absence of either complexes
To investigate the hydrolytic ability of 2La and 2Eu
further, we evaluated the hydrolysis of 7 as a function
of pH. These results, where kobs is shown as a function
of pH, are shown in Figure 3. For both complexes, the
hydrolysis was too slow to evaluate in acidic media
below pH ꢀ 6. The overall results demonstrate that for
both complexes some hydrolysis is observed at around
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
ꢃ All initial kinetic experiments were carried out at 37 ꢀC, over several
half-lifetimes, using 4.32 · 10ꢁ7 mol of HPNP (giving 0.18 mM) and
50 mM HEPES buffer to maintain constant pH. A solution contain-
ing 4.32 · 10ꢁ7 mol of the corresponding lanthanide complex was
added and the reaction monitored over 16 h, with constant stirring.
Measurements were recorded with either an Agilent 8453 or a Cary 50
Scan spectrophotometer, both fitted to a circulating temperature
controlled water bath and mechanically stirred. Errors are within
10%.
pH
9.5
0
6.5
7.5
8.5
Figure 3. The changes in the rate constant of hydrolysis of HPNP
for 2La (blue open circles) and 2Eu (pink circles) as a function of pH.
The error bars are determined from two set of measurements.
§ kucat = 0.00012 hꢁ1 14
.

3764
T. Gunnlaugsson et al. / Tetrahedron Letters 46 (2005) 3761–3766
pH 7.4, and that for 2Eu the hydrolysis becomes pH
independent above pH 7.5, Figure 3. However, for
2La it is clear that the hydrolysis is significantly pH
dependent in alkaline media, as a ꢁbell-shapedꢀ curve is
observed. Here, 2La has highest activity at ca. pH 8.2,
kobs = 0.71 ( 0.014) hꢁ1 and s1/2 = ꢀ1 h, which is a rate
enhancement of ꢀ4.8 · 103. We attribute this ꢁbell-
shapeꢀ nature to the presence of two metal-bound water
molecules in 2La versus one for 2Eu, as the main differ-
ence is the higher coordination environment of the
La(III) complex. This consequently affects the nature
of the cavity in 2La, Figure 1 versus Figure 2, and we
would expect that such a structural difference would also
exist in solution though we have no direct evidence for
that at present. These results clearly demonstrate, since
only 2Ln is able to promote significant hydrolysis of
HPNP, that minor structural modifications have an
enormous effect on the hydrolytic activity of the com-
plex as neither 1La nor 3La gave rise to such enhanced
hydrolysis at pH ꢀ 8. This could possibly be due to the
different affinities of these complexes for the substrate,
caused either by their ability to bind the substrate, or
any resulting intermediates/leaving groups, through
hydrogen bonding and/or base–acid catalysis.11 More-
over, the resonance stabilisation of any deprotonated
amides can also have an effect here, and we are currently
investigating that possibility in greater detail. The fact
that the hydrolysis is slower above pH 8.5 suggests that
the second water molecule becomes deprotonated, giv-
ing the dihydroxy species that prevents the binding of
the substrate to the metal centre. Hence, the observed
enhancements in kobs between pH 7.5 and 8.5 are not
due to background hydrolysis (or a buffer effect), but
to the synergic action of Lewis-acid activation of the
substrate and nucleophilic activation of the 2-hydroxy
nucleophile.
nucleophile activator. This would give rise to subsequent
deprotonation of the 2-hydroxypropyl group in HPNP,
after coordination of HPNP through the phosphate an-
ion to the metal ion, and tandem expulsion of the second
water molecule. The resulting activated nucleophile
could then attack the metal-bound/coordinated phos-
phodiester, which would produce a five-coordinate phos-
phorane intermediate, which would break down to give a
cyclic metal bound phosphate and 9. Finally, the cyclic
phosphate would be released by ligand exchange with
water. For the Eu(III) complex, only one water molecule
is present in the complex and if the second water mole-
cule is necessary for hydrolysis to take place, then the
rate constant should be independent of pH. This was in-
deed found to be the case as shown in Figure 3.
To test if the substrate was binding to the metal com-
plexes, we carried out 31P NMR binding studies in
H2O using 2Eu and 2La, and barium diethylphosphate
½ðCH3CH2OÞ POꢁ2 ꢂ (DEP), which lacks the 20-hydroxyl
2
group. For 2Eu the phosphorus resonance was shifted
upon coordination, demonstrating fast exchange on
the NMR timescale. For 2La, the changes were, how-
ever, found to be in slow exchange, with a signal that
became saturated after addition of ca. 1 equiv of DEP.
We also evaluated the changes in the q-value for the
Eu(III) complex in the presence of HPNP and DEP.
Whereas the q-value was affected for DEP, it remained
constant, for example, ꢀ1, for HPNP, suggesting that
the water was not displaced by the phosphodiester. We
also observed the changes in the Eu(III) emission of
2Eu as a function of HPNP and DEP. For the former,
the emission remained constant, whereas for DEP it in-
creased in intensity. This suggests that HPNP was not
binding, whereas DEP did. We are evaluating these
effects in greater detail.
We propose a possible mechanism for 2La where both
the metal-bound water molecules participate in the
hydrolytic process, Figure 4.3,12 We propose that depro-
tonation of one of these water molecules would give rise
to a metal-bound hydroxy group that could function as a
In summary, we have synthesised three new cyclen-
based pyridine isomers and their corresponding La(III)
and Eu(III) complexes. Of these only the 3-isomer
(2Ln) gives rise to hydrolysis of HPNP, demonstrating
that the pyridine isomer plays a pivotal role in
2+
+
3+
OH2
OH
OH
OH
-H+
-H+
H+
La
La
La
H+
OH2
OH2
O
O
+H2O
HO
-H2O
2+
P
+H2
-H2O
O
O
O
P
O
O
+
NO2
O
OH2
OH
HO
La
La
O
O
O
O
P
P
O
O
O
O
O
NO2
NO2
Figure 4. The proposed mechanism for the hydrolysis of HPNP by the La(III) complex 2La (see text for further details).

T. Gunnlaugsson et al. / Tetrahedron Letters 46 (2005) 3761–3766
3765
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N-Pyridin-3-yl-2-[2,7,10-tris-(pyridin-3-ylcarbamoylmeth-
yl)-1,4,7,10-tetraazacyclododec-1-yl]-acetamide 2: Cal-
culated for C36H44N12O4ÆH2OÆCHCl3: C, 52.53; H,
5.60; N, 19.86. Found: C, 52.23; H, 5.21; N, 19.87; Calcd
for C36H451N12O4: [M+H] m/z (ES+): 709.3687. Found:
709.3653; H (DMSO-d6, 400 MHz), d 10.39 (br s, 4H,
NH), 8.51 (s, 4H, CCHN), 8.16 (t, J = 6.0 Hz, 8H,
NCHCH), 7.03 (s, 4H, NCHCHCH), 3.20 (s, 8H,
CH2), 2.72 (16H, s, NCH2CH2N); 13C (DMSO-d6,
100 MHz), d 172.2, 143.0, 140.1, 163.0, 127.3, 124.1,
56.8, 52.3, 51.6; m/z (ES+): 709.2 (M+H)+, 731.2
(M+Na)+; IR mmax (cmꢁ1) 3385, 3218, 3170, 3066,
2969, 2828, 1696, 1548, 1483, 1305, 1204, 1106, 950,
805, 706. 2La: Calcd for C36H44N12O4La: [(M)3+ peak]
m/z (ES+): 847.2672. Found: 847.2654; 1H (D2O,
400 MHz), d 8.46, 8.11, 7.82, 7.1, 3.8, 3.6, 2.9, 2.7, 2.4;
m/z (ES+): 423.02 (M)2+, 497.99 (M+Trif)2+; IR mmax
(cmꢁ1) 3463, 3284, 1651, 1486, 1282, 1030, 963, 639.
2Eu: Calcd for C36H44N12O4Eu: [(M)3+ peak] m/z
(ES+): 861.2821. Found: 861.2827; 1H (D2O,
400 MHz), d 16.35, 8.08, 7.18, 4.61, 3.16, 1.59, 1.03,
ꢁ1.31, ꢁ4.21, ꢁ10.36; m/z (ES+): 430.17 (M)2+, 505.21
(M+Trif)2+, 1159.37 (M+2Trif); IR mmax (cmꢁ1) 3463,
3284, 1651, 1486, 1282, 1168, 1030, 963, 639.
Acknowledgements
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¨
We thank TCD, QUB and Enterprise Ireland for finan-
cial support.
jugate Chem. 2002, 13, 945–951; (b) Sakamoto, S.;
Tamura, T.; Fukukawa, T.; Komatsu, Y.; Ohtsuka, E.;
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6. Parker, D.; Dickins, R. S.; Puschmann, H.; Cossland, C.;
Howard, J. A. K. Chem. Rev. 2002, 102, 1977–
2020.
7. We have previously developed lanthanide cyclen com-
plexes as luminescent sensors and switches: (a) Gunn-
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We have not quantified this hydrolysis.

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