Modeling of prebiotic catalysis with adenylated polymeric templates: Crystal structure studies and kinetic characterization of template-assisted phosphate ester hydrolysis

Article abstract of DOI:10.1002/1521-3765(20021115)8:22<5184::AID-CHEM5184>3.0.CO;2-2

We have synthesized and characterized novel, copper-metalated, polymeric templates that contain adenine nucleobases. These promote hydrolysis of nonnatural and natural phosphate ester substrates in a highly efficient and catalytic fashion. The crystal structure of the copper-containing adenylated monomer reveals the formation of a polymeric array, through coordination to both N1 and N7 atoms. Possible implications of these studies for prebiotic catalysis, involving synergism between adenine and copper ions, are also discussed.

Full text of DOI:10.1002/1521-3765(20021115)8:22<5184::AID-CHEM5184>3.0.CO;2-2

FULL PAPER
Modeling of Prebiotic Catalysis with Adenylated Polymeric Templates:
Crystal Structure Studies and Kinetic Characterization of Template-Assisted
Phosphate Ester Hydrolysis
[
a]
[b]
[a]
S. G. Srivatsan, Masood Parvez, and Sandeep Verma*
This paper is dedicated to Prof. Dr. Fritz Eckstein on the occasion of his 70th birthday
Abstract: We have synthesized and characterized novel, copper-metalated, poly-
meric templates that contain adenine nucleobases. These promote hydrolysis of non-
natural and natural phosphate ester substrates in a highly efficient and catalytic
fashion. The crystal structure of the copper-containing adenylated monomer reveals
the formation of a polymeric array, through coordination to both N1 and N7 atoms.
Possible implications of these studies for prebiotic catalysis, involving synergism
between adenine and copper ions, are also discussed.
Keywords: adenine
heterogeneous catalysis ¥ prebiotic
¥ polymerization
¥
copper
¥
Introduction
ysis in the primordial era. In this context, Bernal pointed
towards the possible assistance of mineral surfaces for bio-
logical polymerization reactions.[ Towards this end, adsorp-
tion of adenine and other nucleotides on graphite, zeolites,
feldspars, and montmorillonite, and a favorable assistance
6]
Ahypothetical scenario of a prebiotic RNAworld, in which
RNAserved as a genetic template and possessed catalytic
features necessary for self-replication, has long been postu-
lated.[ However, due to the lack of a credible mechanism for
prebiotic RNAsynthesis and RN A× s inherent instability, it
seems likely that life processes originated with a nucleic-acid-
like polymer possessing the desired templating and catalytic
features.[ Although artificial selection has discovered nucleic
1]
II
from Cu for optimal binding to the mineral surfaces have
already been documented.[
7]
In general, mimicry of artificial nucleases employs ligands
of synthetic or natural origin, in conjunction with an
2]
appropriate metal ion, to accomplish phosphate ester hydrol-
[3]
[8, 9]
acid enzymes with predetermined catalytic activities,
a
ysis.
Reaction catalysis is mostly homogenous; however,
plausible mechanism for de novo nucleic-acid template
synthesis still remains elusive. In this context, numerous
chemical and biochemical studies under simulated prebiotic
conditions have appeared, thanks to pioneering efforts by
Orgel, Miller, Ferris, and others.[
some reports have also documented the use of these reagents
in a heterogeneous environment.[ Most of the reported
examples use small, monomeric ligands that are often not
suited to mimic the intricate three-dimensional architectures
of biomolecules, such as protein enzymes. We have recently
described the activity of nucleobase-containing polymeric
frameworks, which conform to classical Michaelis ± Menten
10]
4]
Studies pertaining to prebiotic catalysis and the chemical
origin of life have attracted considerable attention over the
years.[ One theme of these investigations relates to the
preponderance of homogenous versus heterogeneous catal-
5]
[11]
kinetics, for the hydrolysis of activated phosphate esters.
Interestingly, one such template also displayed a remarkable
enzyme-like activity for oxygen insertion in an aromatic CÀH
bond,[ a reaction catalyzed by hydroxylase/oxidase protein
enzymes and required for the biosynthesis of the essential
amino acid tyrosine and catecholamine neurotransmitters.[
As a bio-inspired approach, we report the construction of two
new adenylated polymeric templates and their catalytic
assistance of 2',3'-cyclic adenosine monophosphate (2',3'-
cAMP) cleavage. Thorough kinetic analyses have been
performed to confirm the true catalytic nature of metalated
polymeric adenylated templates, and preliminary attempts
have been made to understand the underlying mechanism, by
employing activated phosphate esters.
12]
[
a] Dr. S. Verma, S. G. Srivatsan
Department of Chemistry
13]
Indian Institute of Technology-Kanpur
Kanpur-208016 (UP) (India)
Fax : (‡91)512-597436
E-mail: sverma@iitk.ac.in
[
b] Dr. M. Parvez
Department of Chemistry
The University of Calgary, 2500 University Drive NW
Calgary, Alberta (Canada) T2N 1N4
Supporting information for this article is available on the WWW under
http://www.chemeurj.org/ or from the author.
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5
184±5191
Results and Discussion
Syntheses and characterization:
9-(4-Vinylbenzyl)adenine (9-
VBA) was synthesized by treat-
ment of 4-vinylbenzyl chloride
with adenine, in the pres-
ence of potassium carbonate
(
Scheme 1). The vinyl group
was introduced in order to ach-
ieve more effective polymeriza-
tion than obtained with the
allylated monomer used by us
in previous studies,[
11]
which
resulted in a better product
yield. 9-VBAwas co-polymer-
ized either with 1,4-divinylben-
zene or with ethyleneglycol di-
methacrylate in the presence of
azobis(isobutyronitrile) (AIBN),
followed by metalation with
CuCl in methanol. The extent
2
of nucleobase incorporation in
templates
1 (DVB) and 2
(
EGDMA) was determined by
elemental analysis, while the
amount of copper in the meta-
lated templates was estimated
by atomic absorption spectrosco-
Scheme 1.
py (AAS). The coordination around copper in the polymeric
matrix was partially characterized by EPR spectroscopy.
Templates 1 and 2 were found to display isotropic symmetry
with giso ˆ 2.112 and 2.120, respectively (Figure 1). Both the
templates 1 and 2 were hydrophobic and completely insoluble
in common solvents. To make the polymer compatible with
hydrolysis in aqueous buffer, it was necessary to pre-wet it
with the minimum possible volume of methanol. Thus, catalysis
of phosphate ester cleavage was heterogeneous in nature.
others, have used such complexes to generate novel coordi-
nation architectures.[ We have determined the X-ray crystal
structure of the 9VBA-Cu complex in order to ascertain the
nature of coordination around the copper atom. Green, plate-
like 9VBA-Cu crystals were grown, with N,N-dimethyl
formamide (DMF) as a solvent and hexane as a precipitant.
The ORTEP diagram (Figure 2) of the unit cell shows two
pentacoordinate copper atoms. Coordination occurs through
the ring nitrogen atoms of modified adenine, two chlorine
atoms, and an oxygen atom of DMF. The geometry around
both copper atoms is distorted trigonal bipyramidal, with
nitrogens N19 and N21 occupying the axial positions around
Cu11, and N11 and N29 around Cu12. The equatorial
positions are taken up by Cl11, Cl12, and O11, and by Cl21,
Cl22, and O21, respectively. The axial angles N19-Cu11-N21
and N11-Cu12-N29 are close to linear, with angles of 170.3(3)8
and 168.6(3)8, respectively. Distortions of the two copper
centers from perfect trigonal bipryamidal geometry are
evident from the equatorial angles Cl1-Cu11-O (95.2(17)8
and 155.8(18)8), Cl2-Cu12-O (94.9(18)8 and 158.0(18)8), and
Cl-Cu-Cl (108.9(8)8 and 107.1(8)8). The rest of the angles
subtended at copper centers are close to right angles, as
expected for trigonal bipyramidal geometry. The distortion
may be due to the steric bulk of the vinylbenzyl substituent.
Important bond lengths and bond angles are listed in Table 1.
Simultaneous coordination to N1 and N7 has been observed
14]
X-ray structural studies: Metal ± nucleobase coordination
complexes have received considerable attention over the past
several decades. Several strategies, pioneered by Lippert and
II
[15]
before in Pt -metalated modified purine crystal structures.
In addition, coordination to other nitrogen atoms has also
been observed for various copper-metalated adenine crystal
structures.[
Figure 1. Solid-state EPR spectra for templates 1 and 2 at room temper-
ature.
16]
Chem. Eur. J. 2002, 8, No. 22
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S. Verma et al.
FULL PAPER
Figure 3. POVRAY diagram showing the polymeric structure of the
9VBA-Cu complex. Hydrogen atoms are omitted for clarity.
Figure 2. POVRAY-rendered ORTEP diagram showing the asymmetric
unit of 9VBA-Cu complex. Hydrogen atoms are excluded for clarity.
8
Table 1. Important bond lengths [ä] and angles [ ] for 9VBA-Cu
complex.
plates.[7] This modification was introduced to mimic adenine
adsorbed on mineral surfaces, and it ensured that any
resulting catalytic assistance was heterogeneous in nature.
Analogously to mineral surfaces, the polymeric matrix
provided a constellation of adenine residues and a synthetic
scaffold for nucleobase/metal-ion coordination. All of the
kinetic studies were performed with the metalated polymeric
template to exploit their reusability[ and are reported in
subsequent sections.
[
a]
Cu11ÀO11
Cu11ÀN19
2.000(5)
2.026(6)
2.037(6)
2.274(2)
2.572(2)
N11ÀCu12
Cu12ÀO21
Cu12ÀN29
Cu12ÀCl22
Cu12ÀCl21
2.027(6)
2.014(5)
2.015(6)
2.270(2)
2.576(2)
#
1
Cu11
ÀN21
Cu11ÀCl12
Cu11ÀCl11
O11-Cu11-N19
O11-Cu11-N21
87.8(2)
87.3(2)
O21-Cu12-N29
O21-Cu12-N11
N29-Cu12-N11
O21-Cu12-Cl22
N29-Cu12-Cl22
N11-Cu12-Cl22
O21-Cu12-Cl21
N29-Cu12-Cl21
N11-Cu12-Cl21
Cl22-Cu12-Cl21
88.1(2)
87.6(2)
#
1
11]
N19-Cu11-N21#1
O11-Cu11-Cl12
N19-Cu11-Cl12
170.3(3)
155.83(18)
92.44(18)
88.63(19)
95.20(17)
94.40(19)
94.3(2)
168.6(3)
157.96(18)
91.69(19)
88.38(19)
94.86(18)
95.34(18)
95.5(2)
Efforts have been made to understand the coordination
around copper in metalated templates, as compared with
copper in the complex, by employing spectroscopic techni-
ques. The solid-state EPR spectrum of the monomeric copper
complex displayed an axial symmetry with g ˆ 2.160 and g ˆ
#
1
N21 -Cu11-Cl12
O11-Cu11-Cl11
N19-Cu11-Cl11
#
1
N21 -Cu11-Cl11
Cl12-Cu11-Cl11
108.86(8)
107.09(8)
k
?
2
.044 (Figure 4), while both the polymeric templates dis-
[
a] Symmetry transformations used to generate equivalent atoms: #1: x À 1,
played isotropic symmetry (Figure 1). Diffuse reflectance
UV/visible spectra were recorded for the complex as well as
for the templates 1 and 2. The polymeric templates were
found to follow similar UV/Vis patterns, with a d ± d transition
at 795 nm, ligand-to-metal charge transfer (LMTC) at 420 nm,
and a p ± p* transition at 282 nm. Though there was no change
in LMTC and p ± p* transitions for the copper± adenine
complex, the d ± d transition was blue-shifted (717 nm). This
observation suggests a subtle difference between the coordi-
nation environments of copper at the monomer and at the
polymer level, which is not quite clear in the latter case.
y, z; #2: x‡1, y, z.
Anotable feature of the 9-(4-vinylbenzyl)adenine ± copper
complex is that it extends into a coordinated polymeric array
assisted by metal centers (Figure 3). Such an extended
framework bears a striking resemblance to the periodicity of
single-strand nucleic acids. In this case, modified adenine
monomers appear to be tethered noncovalently through the
participation of copper ions, and this assembly extends in the
solid-state solely through coordination to copper ions, which
act in a fashion analogous to the phosphodiester bridges in
natural nucleic acids.
It is highly plausible that heterocyclic nucleobases, con-
taining various metal ion coordination sites, could have been
assembled in situ with the aid of metal ions, in the absence of
N-glycosyl substituents and the phosphodiester backbone.
Therefore, such nucleobase assemblies could have served the
role of protonucleic acids to accomplish initial, primordial
templating and catalytic reactions.
Kinetic investigation of phosphate ester hydrolysis: Kinetic
parameters have been derived for the hydrolysis of activated
phosphate esters, such as p-nitrophenyl phosphate (pNPP),
bis(p-nitrophenyl) phosphate (bNPP) and 2-hydroxypropyl-p-
nitrophenyl phosphate (hNPP), under catalysis by templates 1
and 2. pNPP is an example of a phosphate monoester, bNPP is
a phosphodiester, while hNPP is a phosphate diester that also
serves as an RNAmodel, owing to the presence of an internal
hydroxyl group. Hydrolytic reactions catalyzed by copper-
adenylated templates were monitored at 400 nm as a function
Furthermore, modified adenine has been cross-polymerized
and post-synthetically metalated to yield metalated tem-
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Adenylated Polymeric Templates
5184±5191
Figure 5. Michaelis ± Menten saturation kinetics for bNPP hydrolysis
catalyzed by template 2.
Figure 4. Solid-state EPR spectrum of the 9VBA-Cu complex.
and 2 were subjected to thorough kinetic analysis in order to
determine kinetic parameters for the hydrolysis of activated
substrates (Table 3, Figure 6), by means of Lineweaver± Burk
plots (1/V against 1/[S]). For pNPP hydrolysis catalyzed by
of release of p-nitrophenolate anion with respect to time
4
À1
À1
(
1.65 Â 10 m cm ).
Significant rate acceleration was observed for the hydrol-
ysis of activated phosphate esters when catalyzed by tem-
plates 1 and 2, relative to uncatalyzed reaction. Pseudo-first-
order rate constants (kobs) for template-assisted hydrolysis
were determined from plots of ln [A /(A À A )] versus time
Table 3. Kinetic parameters for templates 1 and 2.[a]
À1
À1
À1
Substrate K
m
[mm] Vmax [mm min ] kcat [min ] (kcat/K
m
)/kuncat [m ]
1[b] pNPP
bNPP
1.18
0.62
2.95
1.42
1.33
1.12
4.01 Â 10
9.79 Â 10
1.71 Â 10
6.23 Â 10
1.72 Â 10
8.70 Â 10
À5
À5
À3
À5
À4
À4
5.51 Â 10 9.49 Â 10
À5
4
1
1
t
À4
8
5
4
8
5
(
Table 2). For pNPP hydrolysis, k was found to be 3.52 Â
1.34 Â 10 2.77 Â 10
obs
À3
À4
À4
À1
hNPP
2.34 Â 10 4.01 Â 10
10
and 5.11 Â 10 min for templates 1 and 2, correspond-
2 3
2[c]
pNPP
bNPP
hNPP
6.29 Â 10 9.00 Â 10
À5
ing to rate enhancements of 7.15 Â 10 - and 1.04 Â 10 -fold,
À4
1.75 Â 10 1.69 Â 10
À4
8.79 Â 10 3.96 Â 10
Table 2. Pseudo-first-order rate constants for templates 1 and 2.[a]
[a] All hydrolytic reactions were performed in duplicate in 3 mL of 0.01 mm
N-ethylmorpholine buffer in 10% aqueous methanol (pH 8.0, 308C). The
reference cell contained substrate without polymer to correct for back-
Substrate[b]
k
obs [min
À1
]
k
rel
[c]
Template
À1
À4
À4
À3
2
5
3
ground hydrolysis. Template concentrations were 1 mgmL , correspond-
1
pNPP
bNPP
hNPP
3.52 Â 10
1.23 Â 10
2.57 Â 10
5.11 Â 10
1.68 Â 10
3.54 Â 10
7.15 Â 10
1.58 Â 10
1.30 Â 10
1.04 Â 10
2.15 Â 10
1.79 Â 10
ing to 0.73 and 0.99 mm of copper if the polymeric templates 1 and 2 were
completely soluble in the buffer. [b] [S] concentration ranges of pNPP,
bNPP, and hNPP for 1 were 1.0 ± 5.0mm, 0.50 ± 4.0mm, and 0.20 ± 1.0mm,
respectively. [c] [S] concentration ranges of pNPP, bNPP, and hNPP for 2
were 1.50 ± 3.0mm, 0.25 ± 3.0mm, and 0.80 ± 1.60mm, respectively.
À4
À4
À3
3
5
3
2
pNPP
bNPP
hNPP
[
a] All hydrolytic reactions were performed in duplicate in 3 mL of 0.01 mm
N-ethylmorpholine buffer in 10% aqueous methanol (pH 8.0, 308C). The
reference cell contained substrate without polymer to correct for back-
ground hydrolysis. The concentration of templates 1 and 2–pre-wetted
with methanol (15 mL)–was 1 mgmL . [b] Concentration of pNPP, bNPP,
and hNPP was 5.0 mm, 5.0 mm, and 3.0 mm, respectively. [c] krel is with
template 1, K , Vmax, and kcat were found to be 1.18mm,
m
À5
À1
À5
À1
4
.01 Â 10 mm min , and 5.51 Â 10 min , while with 2 the
À5 À1
À1
parameters were 1.42mm, 6.23 Â 10 mm min , and 6.29 Â
À5 À1
10 min , respectively. Similarly, K , V , and k values
m max cat
respect to the uncatalyzed reaction rate.
for the hydrolysis of bNPP by 1 and 2 were found to be
À5
À1
À4
À1
0
1
.62mm, 9.79 Â 10 mm min , and 1.34 Â 10 min for 1 and
À4 À1 À4 À1
.33mm, 1.72 Â 10 mm min , and 1.75 Â 10 min for 2.
respectively, relative to the uncatalyzed reaction.[17] The
These parameters were also determined for the hydrolysis of
values of kobs for the hydrolysis of bNPP by 1 and 2 were
RNAmodel substrate hNPP with catalysis by templates 1 and
À4
À4
À1
À3
À1
À3
À1
À1
found to be 1.23 Â 10 and 1.68 Â 10 min , respectively,
2 to give 2.95mm, 1.71 Â 10 mm min , and 2.34 Â 10 min
À4 À1 À4
with relative rate enhancements relative to the uncatalyzed
for 1, and 1.12mm, 8.70 Â 10 mm min , and 8.79 Â 10 min
for 2.
5
5
[17]
reaction of 1.58 Â 10 - and 2.15 Â 10 -fold, respectively.
Similarly, the k values for the hydrolysis of hNPP with
The catalytic proficiency ([kcat/K ]/kuncat), which is a meas-
m
obs
catalysis by templates 1 and 2 were determined to be 2.57 Â
ure of an enzyme×s ability to lower the activation energy of the
reaction, was also calculated for these templates for the
hydrolysis of activated substrates (Table 3).[ These values
indicate that both templates have high affinities for the
À3
À3
À1
10
and 3.54 Â 10 min , respectively, corresponding to rate
19]
enhancements relative to the uncatalyzed reaction of 1.30 Â
3
3
[18]
1
0 - and 1.79 Â 10 -fold.
The higher rate enhancement
À3
observed with template 2 than with 1 may be attributed to
higher copper loading in template 2.
substrates pNPP, bNPP, and hNPP, at concentrations of 10 ,
À7
À4
10 , and 10 m, respectively.
Both of the templates followed Michaelis ± Menten satu-
ration kinetics, as was confirmed by plotting substrate
concentration versus initial velocity (Figure 5). Templates 1
As in our previous studies with allyl polymers, the hydro-
lytic activity of templates 1 and 2 was found to be pH
dependent, with hydrolytic rates peaking at nearly pH 8.3
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S. Verma et al.
FULL PAPER
Figure 7. HPLC traces for the cleavage of 2',3'-cAMP at pH 8.0 and 308C.
1
: 2',3'-cAMP; 2: cleavage of 2',3'-cAMP by template 2; 3: cleavage of 2',3'-
cAMP by template 1; 4: recycled template 1. Retention times: 3'-AMP
.91 min; 2',3'-cAMP 8.33 min; 2'AMP 11.95 min.
6
First-order rate constants (k ) for templates 1 and 2 were
obs
À7 À1
À6
2
.43 Â 10 and 4.06 Â 10 s , respectively, which corre-
sponded to enhancements of ꢀ1600- and 270-fold relative
to the uncatalyzed reaction.[
20b]
Furthermore, the template-
assisted hydrolytic reaction led to preferential formation of 3'-
AMP as the major regioisomer, in 70% yield.
All cleavage reactions were performed under heteroge-
neous conditions, since the polymers are insoluble in aqueous
buffer. Consequently, attempts were made to recycle the
polymeric template for subsequent cleavage reactions. In a
simple procedure, after one complete cleavage reaction, the
polymer was filtered, washed with aqueous buffer, methanol,
and acetone, and air-dried. In one such recycling experiment,
template 1 was reused for 2',3'-cAMP hydrolysis and,
interestingly, the extent of cleavage for the recycled template
remain unchanged when compared to the fresh catalyst
(Figure 7). The amounts of copper in 1 and 2 after one cycle
of hydrolytic cleavage of 2',3'-cAMP were found to be 37.0 and
42.0 mg per gram of polymer, respectively. Atomic absorption
spectroscopy estimated similar copper contents for fresh and
recycled polymer, suggesting a lack of change in copper-ion
content during hydrolytic reactions. This observation was
further reinforced by arrest experiments as detailed below.
The reusability of the polymeric templates indicates that
such constructs act as rugged, insoluble scaffolds for holding
multiple adenine residues and copper ions. The rate enhance-
Figure 6. Lineweaver ± Burk plots for hydrolysis of pNPP (top), bNPP
(
middle), and hNPP (bottom) by templates 1 (^) and 2 (*).
(
data not shown). While solvent studies confirmed preferen-
tial involvement of metal ± hydroxy intermediates in phos-
phate ester hydrolysis, a linear dependence of assisted hydro-
lysis rate with respect to temperature revealed the high thermal
stability and activity of these templates (data not shown).
Cleavage of 2',3'-cAMP: We have employed 2',3'-cAMP to
evaluate the catalytic potential of our templates toward
natural, unactivated substrate (Scheme 2). Adenylated tem-
plates 1 and 2 catalyzed the hydrolysis of 2',3'-cAMP with
appreciable rate enhancement and regioselectivity. Hydro-
lytic reactions were performed in N-ethylmorpholine buffer
ments obtained with our polymers are comparable to those
given in recent reports,[
20a,c]
except for dinuclear and trinuclear
(
pH 8.0) at 308C, and the appearance of product was
homogeneous copper complexes, for which relatively higher
[20a]
monitored by C-18 reversed-phase HPLC (Figure 7).
rate accelerations have been achieved.[
20b,c]
However, the rare
reusability feature of these templates clearly suggests a
rugged and catalytic nature of our systems.
31P NMR spectroscopy: Efforts were made to establish a
working mechanism for these templates by study of assisted
hydrolysis of hNPP. This reaction could yield either a mono-
phosphorylated product, through an intermolecular nucleo-
philic attack, or a cyclic phosphate, through an intramolecular
attack. It has already been shown for certain artificial systems
Scheme 2.
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Adenylated Polymeric Templates
5184±5191
that metal-ion-promoted hNPP hydrolysis proceeds through
an intramolecular pathway to yield a cyclic phosphate.[
Exclusive, time-dependent formation of a cyclic phosphate
product as a result of template-mediated hNPP hydrolysis was
detected in this study, thus suggesting the activation of the
internal hydroxyl group by metalated templates for trans-
esterification (Figure 8). However, in the absence of an
Rate inhibition by vanadate ions: Phosphatases are inhibited
by vanadate ions, as they mimic the transition state of
phosphate ester hydrolysis.[ We have studied the inhibitory
effect of vanadate ion on phosphate ester hydrolysis catalyzed
by our templates, and Lineweaver± Burk plots were conse-
quently derived for bNPP hydrolysis catalyzed by 2 in the
presence or absence of ammonium vanadate (0.10mm) at
pH 8.0 (Figure 10). The values of K and V in the presence
21]
22]
m
max
Figure 10. Lineweaver± Burk plots for bNPP hydrolysis by template 2: (*)
in absence of inhibitor; (^) in the presence of inhibitor.
Figure 8. 31P NMR spectra for the hydrolysis of hNPP by template 2.
of inhibitor were found to be 0.69mm and 8.99 Â
Chemical shifts for hNPP and for the cyclic phosphate product are
À5
À1
1
0 mm min , respectively, and in its absence 1.33mm and
[
21d]
À4.32 ppm and 18.58 ppm, respectively.
The chemical shifts are with
À4
À1
1
.72 Â 10 mm min . The kinetic data obtained suggest an
respect to 85% H PO as external reference.
3
4
uncompetitive mode of inhibition by vanadate ions, with K ˆ
i
0
.11mm. Aprecise explanation for this uncompetitive inhib-
ition is not available at present.
internal nucleophile for 2',3'-cAMP, an intermolecular attack
by metal-bound hydroxyl seems a probable mechanism. A
Fenton-type, free-radical-mediated cleavage of activated non-
natural phosphate esters has already been ruled out for such
polymeric templates.[ However, it is worth mentioning that
these templates might display an alternative mechanism of
action while interacting with natural substrates.
Conclusion
11a]
Curiously, the crystal structure of metalated vinylbenzyl
adenine monomer has the form of an extended strand-like
structure, which bears a resemblance to single-strand nucleic
acids. Copper ions act as tethers (or the backbone), in the
absence of sugar residues and phosphodiester linkage, to
stabilize the metalated assembly.
Arrest experiments: Implications for a crucial role of
coordinated copper ions was obtained from ™reaction arrest∫
experiments. In this assay, 2 was incubated with bis(p-nitro-
phenyl) phosphate (bNPP) as a model phosphodiester
substrate for 6 h, followed by its removal by filtration. This
resulted in the complete abrogation of bNPP hydrolysis when
observed over a time period of 75 h (Figure 9), thus indicating
The adenylated polymeric templates described here are
insoluble due to cross-linking, and act heterogeneously to
accomplish non-natural and natural phosphate ester hydrol-
ysis. Their prolonged and sustained reactivity, as demonstrat-
ed by recycling experiments and reaction arrest assay, indicate
that they can retain metal-ion-dependent catalytic properties
for long durations, without altered reaction rates. Hence, we
propose that these insoluble templates, containing multiple
adenine residues, mimic metal-coordinated adenine aggre-
gates or primordial adenine-based oligomers adsorbed on
II
mineral surfaces. Favorable Cu interactions observed in this
study also allude to synergistic interactions between nucleo-
bases and metal ions in the prebiotic era for catalyzing
fundamental biochemical reactions.[ Interestingly, a modi-
fied adenosine moiety has previously been examined for its
role in catalyzing ester hydrolysis.[
23]
24]
Figure 9. Reaction arrest experiment for bNPP hydrolysis by template 2.
In short, the metalated adenine-containing polymers de-
scribed in this report combine a scaffold for substrate binding
and a nucleobase matrix for copper-ion coordination, to
produce a catalytically active, nucleic-acid-like polymeric
template with a biological function. It is proposed that studies
with such model constructs might provide further insight into
intricate involvement of coordinated copper ions, and not the
leached out ions, for the catalytic activity of the polymeric
templates. As control reactions, nonmetalated template or
copper salt alone did not support 2',3'-cAMP hydrolysis under
the reaction conditions.
Chem. Eur. J. 2002, 8, No. 22
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5189

S. Verma et al.
FULL PAPER
primitive organization of nucleic acid constituents and into
their cooperative interaction with metal ions for prebiotic
catalysis. Another intriguing possibility is that such metal ±
nucleobase assemblies could have functioned as short, pre-
biotic templates for recognition of other bases and amino
acids, for subsequent primordial oligomerization reactions.
We are currently evaluating these templates for nucleic acid
modification, while template-assisted biosynthetic reactions
have also been envisaged.
anisotropically, while hydrogen atoms were included at geometrically
idealized positions. The final cycle of full-matrix, least-squares refinement
by use of SHELXL97[ converged with unweighted and weighted agree-
ment factors, R ˆ 0.091 and wR ˆ 0.262 (all data), respectively, and
goodness of fit, S ˆ 0.998.
26]
CCDC 181927 contains the supplementary crystallographic data for this
paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/
conts/retrieving.html (or from the Cambridge Crystallographic Data
Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: (‡44)1223-
3
36033; or deposit@ccdc.cam.uk).
Cross-polymers of 9VBA with DVB and EGDMA: AIBN-initiated free-
radical polymerization was employed for the synthesis of nucleobase cross-
polymers. 9-VBA(1.0 g, 4.0 mmol, 1 equiv), DVB (0.23 mL, 0.8 mmol,
0
.2 equiv) or EGDMA(0.15 mL, 0.8 mmol, 0.2 equiv), and AIBN (15 mg)
were taken up in dry DMSO (8.0 mL) and the reaction mixture was purged
with oxygen-free N gas for 45 min. Polymerization was performed at 808C
Experimental Section
2
Instrumentation: HPLC assay was performed on a Perkin ± Elmer 785 A
for 18 h in the presence of DVB and 6 h in the presence of EGDMA. A
white gel was formed in both cases, and this was filtered, washed with hot
DMSO (5 Â 20 mL), methanol (5 Â 20 mL), and acetone (3 Â 20 mL), and
dried under vacuum. The amounts of nucleobase cross-polymer obtained
with DVB and with EGDMAwere 0.85 g and 1.05 g, respectively.
1
31
UV/Vis detector. H and P NMR spectra were recorded on a JEOL-JNM
LAMBDA 400 model operating at 400 and 161.7 MHz, respectively. EPR
spectra were recorded on a Varian 109E Line Century Series X-band
spectrometer. The amount of copper in the metalated nucleobase polymer
was determined by AAS spectrometry (GBC), at FEAT Laboratory, IIT-
Kanpur. Elemental analyses and mass spectra were recorded at the
Regional Sophisticated Instrumentation Centre, Lucknow (India). Reac-
tion kinetics were examined on a Shimadzu UV-160 UV/Vis spectropho-
tometer.
Copper-metalated cross-polymer of 9VBA and DVB (template 1):
Unmetalated template 1 (0.75 g) and CuCl ¥ 2H O (0.51 g) were taken
2 2
up in methanol (25 mL). The reaction mixture was allowed to stir at room
temperature for 24 h. Metalated polymer was isolated by filtration, washed
with methanol (4 Â 25 mL) and acetone (3 Â 10 mL), powdered, and dried
under vacuum. Template 1 was obtained as a green, amorphous powder
Chemicals and Reagents: 4-Vinylbenzyl chloride, ethyleneglycol dimeth-
acrylate (EGDMA), and 1,4-divinylbenzene (DVB) were purchased from
Fluka (Switzerland). 2',3'-cAMP was purchased form Sigma. Azobis(iso-
butyronitrile) (AIBN; Ajax Chemicals, India) was recrystallized from
(
0.84 g) and was found to be insoluble in common solvents.
Copper-metalated cross-polymer of 9VBA and EGDMA (template 2):
Unmetalated template 2 (0.80 g) and CuCl ¥ 2H O (0.543 g) were taken up
2
2
2
methanol. Copper chloride ¥ 2H O (Merck, India), ammonium vanadate
in methanol (25 mL). The reaction mixture was allowed to stir at room
temperature for 24 h. Metalated polymer was isolated by filtration, washed
with methanol (4 Â 25 mL) and acetone (3 Â 10 mL), powdered, and dried
under vacuum. Template 2 was obtained as a green, amorphous powder
(
S.D. Fine Chemicals, India) and pNPP (SRL, Mumbai) were used as
supplied. Potassium carbonate was purchased from Lancaster (England).
The sodium salt of bNPP (Fluka, Switzerland) was prepared from the
commercial sample and used for kinetic assays. N-Ethylmorpholine,
methanol (spectroscopic grade), and ethyleneglycol were from S.D. Fine
Chemicals (India) and were distilled prior to use. 2-Hydroxypropyl-p-
nitrophenyl phosphate (hNPP) was synthesized by the literature proce-
dure.[ Triply distilled water was used in all assays, and HPLC grade
solvents were used for HPLC analysis. All solvents were distilled or dried
when necessary by general procedures.
(
0.99 g) and was found to be insoluble in common solvents.
Characterization of templates 1 and 2: Powder EPR spectra at room
temperature indicated the presence of isotropic symmetry for templates 1
and 2, with giso ˆ 2.112 and 2.120, respectively. Elemental analyses found
(%): Template 1: C 61.11, H 9.85, N 17.01; Template 2: C 53.29, H 13.52, N
16.26. It is thus determined from elemental analyses that every gram of
templates 1 and 2 contains 610 mg (2.43 mmol) and 583 mg (2.32 mmol) of
monomer, respectively. Copper in the polymeric matrices was estimated by
AAS and was found to be 46.3 and 63.1 mg per gram of polymer for 1 and 2,
respectively.
25]
9
3
-(4-Vinylbenzyl)adenine (9-VBA): 4-Vinylbenzyl chloride (0.5 mL,
.5 mmol, 0.9 equiv) and K CO (0.61 g, 4.4 mmol, 1.2 equiv) were stirred
2
3
in anhydrous DMSO (15 mL) at 308C for 5 h, under a nitrogen atmosphere.
The solvent was evaporated under reduced pressure, and silica gel column
chromatography afforded the pure compound [R
methanol 9:1)] in 71% yield. M.p. 2028C; H NMR (400 MHz, [D
f
ˆ 0.6 (ethyl acetate/
General assay for the hydrolysis of pNPP, bNPP, and hNPP by 1 and 2
(spectrophotometric method): All hydrolytic reactions were performed in
duplicate in centrifuge tubes thermostatted at 308C. The assay mixture
contained 3 mL of substrate solution of appropriate concentration,
prepared in 0.01m N-ethylmorpholine buffer (pH 8.0) in 10% aqueous
1
6
]DMSO,
), 5.8
3
2
(
58C, TMS): d ˆ 5.2 (d, J(H,H) ˆ 11 Hz, 1H; CÀH), 5.4 (s, 2H; CH
2
3
3
d, J(H,H) ˆ 18 Hz, 1H; CÀH), 6.7 (dd, J(H,H) ˆ 18, 11 Hz, 1H; CÀH),
2
2
7
8
4
1
.3 (d, J(H,H) ˆ 8 Hz, 2H; ArCÀH), 7.4 (d, J(H,H) ˆ 8 Hz, 2H; ArCÀH),
1
3
À1
.1 (s, 1H; ArCÀH), 8.2 (s, 1H; ArCÀH), 7.2 ppm (br; NH
5.8, 114.5, 118.6, 126.3, 127.8, 136, 136.5, 136.6, 140.7, 149.4, 152.5,
); C NMR: d ˆ
methanol. The amount of polymer was 1 mgmL of buffered substrate
2
solution, and the concentration of the catalyst corresponded to the amount
of copper present in the polymeric matrix. The polymers were pre-wetted
with methanol (15 mL). Initial velocities were determined as a function of
time-dependent release of p-nitrophenolate anion (e400 ˆ 1.65 Â
‡
‡
55.9 ppm; MS [EI]: m/z (%): 251 (10) [M ], 250 (54) [M À H].
VBA-Cu complex: 9VBA(0.10 g, 0.4 mmol, 1.0 equiv) was dissolved in
9
methanol/chloroform (4:1; 5.0 mL), and CuCl
0
2
2
¥ 2H
.5 equiv) was added to this solution. The reaction mixture was stirred for
h at room temperature. Abrownish-green precipitate was formed, and
2
O (0.035 g, 0.20 mmol,
4
À1
À1
1
0 m cm ). Michaelis ± Menten parameters were calculated from corre-
sponding Lineweaver ± Burk plots. Pseudo-first order rate constants were
derived from ln [A /(A )] against time plots, where A and A are the
1
1
À A
t
1
t
this was isolated by filtration and washed with methanol (25 mL),
chloroform (25 mL), and acetone (25 mL). The complex was dried under
vacuum. Crystals suitable for X-ray studies were grown in N,N-dimethyl-
formamide by diffusion. Agreen plate crystal was coated with Para-
tone 8277 oil (Exxon) and mounted on a glass fiber. All measurements
were made on a Nonius Kappa CCD diffractometer with graphite
monochromated Mo-Ka radiation.
absorbances at infinite time and at time t. All hydrolytic reactions were
performed over at least four half-lives of each substrate.
31P NMR experiment for hydrolysis of hNPP by 1 and 2: Hydrolytic
reactions were performed in 1.0 mL N-ethylmorpholine buffer in 10%
aqueous methanol (0.01m, pH 8.0, 308C). The concentration of the
À1
substrate, hNPP, was 5 mm and the polymer weight was 3 mgmL for 1
3
1
and 2. P NMR spectra were recorded in solutions of reaction mixture and
O (2:1) at appropriate time intervals, and the reported chemical shifts
are with respect to 85% H PO (40 mm) standard, in the same buffer.
Crystal data for the 9VBA-Cu complex: C40
H
54Cl
4
Cu
2
N
14
O
4
: M
r
ˆ 1063.85,
≈
D
2
triclinic, space group P1, a ˆ 13.255(7), b ˆ 13.680(5), c ˆ 14.500(8) ä, a ˆ
3
À3
3
4
6
7.16(2), b ˆ 89.80(2), g ˆ 88.46(3)8, Vˆ 2422.1(2) ä , 1calcd ˆ 1.459 Mgm
,
À1
Tˆ 173(2) K, Z ˆ 2, m(MoKa) ˆ 1.15mm , 20508 reflections measured,
Cleavage of 2',3'-cAMP: Cleavage of cyclic phosphate catalyzed by
templates 1 and 2 was monitored by HPLC. In a typical procedure, 2',3'-
cAMP solution (1.0 mm, 1.0 mL) in N-ethylmorpholine buffer (0.01m in
10% aqueous methanol, pH 8.0), was stirred in an Eppendorf tube at 308C.
1
0
0984 unique (Rint ˆ 0.13), final R1 ˆ 0.091, wR2 ˆ 0.212 [I > 2s(I)], R1 ˆ
.223 and wR2 ˆ 0.262 (all data). The structure was solved with SIR92 and
expanded by Fourier techniques. Non-hydrogen atoms were refined
5190
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Chem. Eur. J. 2002, 8, No. 22

Adenylated Polymeric Templates
5184±5191
À1
À1
Polymer weights for 1 and 2 were 5 mgmL and 3 mgmL of buffer,
respectively. The reaction mixture was centrifuged and filtered to remove
any particulates. Samples (20 mL) were analyzed on a C-18 RP-HPLC
column (100 mm  4.6 mm, Brownlee). An isocratic gradient of 95% A
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2
PO
4
(10 mm); B ˆ methanol/water (3:2)] was used for
elution and the eluents were monitored at 260 nm. First-order rate
constants were obtained as the slopes of plots of ln (A /A ) against time,
where A and A are the integrations of the area of the HPLC peak for 2',3'-
cAMP at time ˆ 0 and t, respectively.
0
t
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Received: April 2, 2002 [F3985]
Chem. Eur. J. 2002, 8, No. 22
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0947-6539/02/0822-5191 $ 20.00+.50/0
5191