Synthesis, characterisation and theoretical calculations of 2,6-diaminopurine etheno derivatives

Article abstract of DOI:10.1039/b505508c

Four derivatives of 2,6-diaminopurine (1) were synthesised and characterised. When 1 was reacted with chloroacetaldehyde, 5-aminoimidazo[2,1-i] purine (2), 9-aminoimidazo[2,1-b]purine (3), 9-aminoimidazo[1,2-a]purine (4) and diimidazo[2,1-&:2′,1′-i]purine (5) were formed. The purified products (3-5) were fully characterised by MS, complete NMR assignments as well as fluorescence and UV spectroscopy. The purified, isolated yields of these products (3-5) varied from 2.5 to 30%. The relative stability of different tautomers was investigated by theoretical calculations. Fluorescence characteristics are also discussed and compared to the starting material 1 and a reference molecule 2-aminopurine. The Royal Society of Chemistry 2005.

Full text of DOI:10.1039/b505508c

View Article Online / Journal Homepage / Table of Contents for this issue
A R T I C L E
Synthesis, characterisation and theoretical calculations of
,6-diaminopurine etheno derivatives†
2
a
b
a
c
b
Piritta Virta,* Andreas Koch,‡ Mattias U. Roslund,§ Peter Mattjus,¶ Erich Kleinpeter,‡
a
a
d
Leif Kronberg, Rainer Sj o¨ holm and Karel D. Klika*
a
˚
˚
Department of Organic Chemistry, Abo Akademi University, FI-20500 Turku/Abo, Finland.
E-mail: piritta.virta@abo.fi; Fax: +358 2 2154866; Tel: +358 2 2154502
Department of Chemistry, University of Potsdam, P. O. Box 69 15 53, D-14415 Potsdam,
Germany
Department of Biochemistry and Pharmacy, Abo Akademi University, FI-20520 Turku/Abo,
Finland
Department of Chemistry, University of Turku, FI-20014 Turku, Finland.
E-mail: klikakd@yahoo.co.uk; Fax: +358 2 3336700; Tel: +358 2 3336826
b
c
d
˚
˚
Received 20th April 2005, Accepted 1st June 2005
First published as an Advance Article on the web 6th July 2005
Four derivatives of 2,6-diaminopurine (1) were synthesised and characterised. When 1 was reacted with
chloroacetaldehyde, 5-aminoimidazo[2,1-i]purine (2), 9-aminoimidazo[2,1-b]purine (3), 9-aminoimidazo[1,2-a]purine
ꢀ
ꢀ
(
4) and diimidazo[2,1-b:2 ,1 -i]purine (5) were formed. The purified products (3–5) were fully characterised by MS,
complete NMR assignments as well as fluorescence and UV spectroscopy. The purified, isolated yields of these
products (3–5) varied from 2.5 to 30%. The relative stability of different tautomers was investigated by theoretical
calculations. Fluorescence characteristics are also discussed and compared to the starting material 1 and a reference
molecule 2-aminopurine.
Introduction
Our research in the field of nucleoside chemistry originally
aimed at synthesising modified fluorescent RNA/DNA bases.
5
The reaction of genotoxic chemicals with DNA and RNA,
particularly reactions with the base moieties, to form adducts ac-
counts for the detrimental and toxic effects of these compounds.
The DNA is a continuous target for endogenous and exogenous
damage and some of the most commonly encountered DNA
lesions are the exocyclic DNA adducts. In particular, etheno
adducts, with an additional five-membered ring fused to the
base moiety, have gained much attention since they can result
from reactions with a variety of chemicals (e.g. chloroacetalde-
hyde, chloropropanal, bromomalonaldehyde). These etheno
compounds formed can be used as models to investigate what
may be happening detrimentally on the biomolecular scale (e.g.
fluorescence markers), as some etheno derivatives of purine are
However, our present investigations are also directed toward the
preparation of nucleoside base analogues, which might also have
interesting applications other than those associated with their
5,6
fluorescence properties. To our knowledge 2,6-diaminopurine
(
1) has not previously been used to produce different etheno
derivatives.
Owing to the similarity of the expected products and
many tautomers (possibly even dimers through hydrogen
7
bonding), theoretical calculations of carbon and nitrogen NMR
chemical shifts and the relative stability of the different tau-
tomeric forms of the products were also included into this
work.
In the current work, we report on the synthesis of four
derivatives of 2,6-diaminopurine (1). They are formed in the
reaction of compound 1 with chloroacetaldehyde (Scheme 1).
In this contribution we present the details of the synthetic
procedures, the complete structural analysis of the products,
1
known to be fluorescent.
As mentioned above, haloacetaldehydes have been shown to
react with nucleic acid components yielding etheno derivatives
2
6
of adenosine, guanosine and cytidine. The 1,N -ethenodeoxy-
adenosine derivatives, due to their fluorescence, are proven to be
1
13
15
which includes MS, and NMR analyses ( H, C, N NMR
data), as well as fluorescence and UV spectroscopic properties
for isolated compounds 3–5 and NMR analyses for compound
3
4
useful compounds, e.g. studies of mutagenesis and enzymology.
2
. The fluorescence intensities and lifetimes of compounds 3, 4
†
Electronic supplementary information (ESI) available: modelling
details for all tautomers of compounds 1–5. See http://dx.doi.org/
and 5 are compared to those of 1 and 2-aminopurine (reference)
and are discussed. Also, the theoretical calculations of the NMR
chemical shifts are compared to the experimental ones and the
relative stabilities of the different tautomers in the gas phase for
all compounds are discussed.
1
‡
§
0.1039/b505508c
Theoretical calculations.
NMR measurements.
¶
Fluorescence measurements.
◦
Scheme 1 Reagents and conditions: (a) chloroacetaldehyde (ClCH
2
CHO), pH 4–5, 80 C, ca. 1 d.
2
9 2 4
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 9 2 4 – 2 9 2 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 5

View Article Online
Results and discussion
the etheno bridge protons. This left the identification of the
structure of 2 by default.
Owing to the lack of a sugar moiety attached to N-3 (cf. ref. 5),
many tautomeric forms of the products were possible. The only
The products (2–5) were synthesised according to Scheme 1.
The reaction of 2,6-diaminopurine (1) with chloroacetaldehyde
was carried out in water/DMF solution at pH 4–5 and
the progress of the reaction was monitored by HPLC. The
products 3–5 were isolated by Ca-enriched silica gel (for
acid labile compounds) followed by reversed-phase column
product for which distinct tautomers could be observed directly
◦
(
18.5 C in DMSO) was compound 4 where two tautomers
8
were present. The major tautomer was identified as 1H-9-
aminoimidazo[1,2-a]purine after modifying a magnitude-mode
HMBC NMR pulse sequence. The assignment of the major
tautomer was based on the sign of the proton–carbon coupling
constants [between H-1 to C-3a ( J positive), between H-1 to C-
( J negative) and between H-1 to C-9a ( J negative)]. For
chromatography and deprotonation with NaHCO , after which
the compounds were fully characterised by H, C and
3
1
13 15
N
NMR spectroscopy, electrospray ionisation mass spectrometry,
fluorescence spectroscopy and UV absorption. Compound 2
was only observed and characterised in one of the fractions
containing a mixture of all three-ring products 2–4. Owing to
the ease of protonation and the many possible tautomers of
the products, their absolute characterisation was demanding
and during this project one standard magnitude-mode HMBC
NMR pulse sequence was also modified in order to identify
the major tautomer of compound 4. The major tautomer was
assigned using different 2D techniques, like hetero NOESY
3
2
2
8
2
the other products, distinct tautomers were not seen under
similar conditions. For the compounds 3 and 5, additional
indirect evidence for the identity of the tautomers present was
2
forthcoming by the correlations of H-2 to two sp -hybridised
nitrogens. This could be interpreted as evidence for either the
H-7 or H-8 amino tautomers in the case of 3 (i.e. disregarding
imino tautomers for which there was a lack of evidence in all
cases based on the chemical shifts of the NH groups) or the
2
(
PHOESY) and high resolution HMBC for the determination of
H-11 tautomer in the case of 5. However, in the former case (3)
the chemical shifts of −162 and −191 ppm for N-7 and N-8
heteronuclear coupling constants. Dry DMSO-d was used for
NMR measurements as these compounds were not soluble in
other solvents better suited for low-temperature NMR to slow
6
(
an appropriate value with an exocyclic double bond at position
7
a), respectively, clearly indicate the predominance of either the
8
down the fast exchange of the NH-protons.
H-1 or the H-3 amino tautomer, or possibly both, together
with the presence of the H-7 and/or H-8 amino tautomer(s)
based on the comparison of the calculated and experimental
chemical shifts. It is not possible to be more precise than this
due to an insufficient number of constraints. In the case of 5, the
situation is less complex as only three tautomers are possible.
Again, the chemical shift of −153 ppm for N-11 discounts
the predominance of the H-11 tautomer. Comparison of the
calculated and experimental chemical shifts revealed that the
tautomeric composition is dominated by H-1 and H-3 together
with a minor contribution of H-11. Compound 2 was clearly
dominated by only either the H-1 or H-3 tautomers or a mixture
of both, but no clear indication was provided as to which was
the dominate one.
The isolated yields of 3, 4 and 5 were 9, 2.5 and 30% respec-
tively. The NMR spectroscopic data, the mass spectrometric
and UV properties are given in the Experimental section. The
fluorescence properties are presented in Tables 1 and 2 and Fig. 2.
Structure determination and modelling
In the reaction, the formation of different regioisomers was
possible. Thus, complete NMR spectroscopic analysis was
necessary for the determination of which isomers were formed
and isolated. Owing to the great similarity of the structures,
theoretical calculations of the carbon and nitrogen NMR
chemical shifts were also performed as this has been found to be
a reliable method for structural determinations, even in the case
of distinguishing between tautomeric or other kinds of extremely
To obtain information of the different tautomers present,
theoretical tautomeric equilibrium calculations for all products
in the gas phase were performed, with additional calculations
considering DMSO solvation employing the polar-continuum
model. The results at the different levels of theory (HF/6-
311G**, B3LYP/6-311G**, MP2/6-31G**) are presented in
the ESI† though it is known that it is notoriously difficult to
accurately replicate tautomeric energies, even with inclusion of
6b,9
similar structures.
The formed products were easily protonated and NMR shifts
changed considerably upon protonation (e.g. even the chemical
shifts of the protons adjacent to the site of protonation can be
6,10
deshielded by as much as 1 ppm ). Also, different states of
protonation had different NMR shifts. Owing to this it was very
important to perform the NMR measurements for all of the
molecules under similar conditions, after deprotonation with
11
the solvent (hence the multiple calculations). These aberrations
can have a variety of origins – correct representation of the
solvent, the formation of H-bonds with the molecules and, for
these kinds of compounds, the possibility for dimer formation
(Fig. 1) – thus moving to higher levels of theory does not
NaHCO
3
. Structural determinations and signal assignments
were accomplished by the application of a comprehensive
1
13
8
1
13
1
15
set of 2D FG H-{ C}-PHOESY, H-{ C}- and H-{ N)-
1
13
1
15
12
HSQC and H-{ C}- and H-{ N)-HMBC experiments. Un-
fortunately, homo-NOEs were never observed between labile
protons and non-labile protons, leaving structural analysis (in
particular) to rely mainly on long-range J hetero-correlations.
Other methodologies, when necessary due to ambiguity or
lack of correlations, were used on occasion. For example, the
assignment of the etheno- and exchangeable-protons was based
on the relative magnitude of JH,C, JH,C, JH,C, JH,N and JH,N
and determination of the sign of JH,C to distinguish between 2-
and 3-bond couplings.
necessarily improve matters.
3
2
1
3
2
,
n
8
Compound 5 was readily discernible by NMR from the
presence of the additional etheno bridge, further confirmed by
8
MS analysis. Compound 4 has previously been identified by us,
thus leaving only the distinction between 2 and 3. Compound 3
was identified based on the correlation from the amino protons
Fig. 1 An example of possible dimer formation for product 2.
For compound 4, two favourable tautomers in the gas phase
with an energy difference of 1.40 kcal mol were found:
tautomers 4_1 (3H) and 4_8a in Scheme 2. However, at no
time was there any evidence for imine tautomers for any
of the NH
2
group to C-9a, a highly distinctive carbon which
−
1
is outstandingly shielded. This was further confirmed by the
2
correlations from the NH
2
-protons to an sp -hybridised nitrogen
which was not one of the two nitrogens that were correlated to
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 9 2 4 – 2 9 2 9
2 9 2 5

View Article Online
addition to the known 1H and 3H tautomers must be involved
in a fast exchange equilibrium. This third tautomer could well be
structure 3_3 (7H tautomer), but with too many unknowns (the
number of participating tautomers and mole fractions thereof) it
is not possible to describe the system definitively. Although 3_3
always remained the most stable species, significant alterations
in the relative energies of 3_1 and 3_2 (3H tautomer) occurred,
particularly with higher levels and upon inclusion of the solvent.
For compound 2, the two most stable tautomers were found to
−
1
differ in energy by 0.83 kcal mol (2_1 and 2_2, Scheme 2) and
these two tautomers were always close in energy regardless of the
level or upon inclusion of the solvent (in fact, both the B3LYP
and MP2 methods reversed their preference upon inclusion of
the solvent, i.e. 2_1 became favoured).
For the tetracyclic product 5, only one major tautomer in the
gas phase was found, tautomer 5_2, and this did not change
upon inclusion of the solvent. For both these compounds (2
and 5) it was not possible to evaluate the experimental chemical
shifts in terms of the calculated chemical shifts to discern the
state of the system and thus it must be concluded that both
systems are comprised of multiple tautomers in fast exchange.
Thus, the clear preference of two tautomers (in the case of 2) and
one tautomer (for 5) provided by the energy calculations is at
6b,9
odds with the chemical shifts which are known to be reliable.
Fluorescence
Unlike natural DNA bases, 2-aminopurine is fluorescent at neu-
tral pH and this native fluorescence has been demonstrated to be
an extremely useful probe of, for example, DNA conformational
1
4
Scheme 2
changes and DNA base flipping. On the other hand, the linear
2
1
,N -ethenoguanosine, which resembles the product 4, does not
2d
of the compounds, and generally it has been shown that
exhibit fluorescence at all.
purines are a mixture of 1H and 3H tautomers with the 1H
We were interested in comparing the fluorescence characteris-
tics of the isolated products 3–5 with those of 2,6-diaminopurine
(1), and 2-aminopurine as references. It is well known that an
11,13
tautomer dominating.
(Indeed, this was found to be the
case for the calculations on 1 where little variance amongst
the levels of theory was evident.) Experimentally, it was the
6
etheno bridge between N-1 and N of adenosine enhances the
1
H tautomer that was found to be the most stable with the
fluorescence intensity considerably in comparison to unmodified
adenosine and therefore it was of interest to investigate the
fluorescence properties of the etheno products formed from
compound 1. The results of the fluorescence measurements are
shown in Tables 1 and 2 and Fig. 2.
All in all, the fluorescence properties of the products differed
quite significantly from those of the reference compound
2-aminopurine but not much from the starting material 1. The
quantum yields of the products were considerably lower and
the fluorescence lifetimes were also much shorter than for 2-
aminopurine. The presence of etheno ring(s) did not have drastic
effect on quantum yields or fluorescence lifetimes compared to
the starting material 1. For compound 3, two lifetimes were
minor tautomer presumed to be the 3H tautomer (the 4H could
not be excluded). But comparison of the chemical shifts does
in fact correlate to these two structures (i.e. the 1H and 3H
tautomers) and thus confirms our earlier findings. Moreover,
the 4H tautomer can now be categorically discounted based on
8
1
3
15
the calculated C and N chemical shifts.
Also for compound 3, two major tautomers were found with
−
1
an energy difference of 1.98 kcal mol (3_1 and 3_3, Scheme 2).
However, no sense could be made of the calculated chemical
shifts in comparison to the experimental values (as individual
tautomers or as combinations of two tautomers in fast exchange)
leading to the conclusion that at least one other tautomer in
◦
Fig. 2 Normalised excitation (left) and emission (right) spectra of the compounds 3–5 in water at 25 C.
2
9 2 6
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 9 2 4 – 2 9 2 9

View Article Online
◦
a
Table 1 Fluorescence parameters of the compounds in water at 25 C
Compound
Absorbance, kmax(H
2
O)/nm
Excitation, kmax(H
2
O)/nm
Emission, kmax(H
2
O)/nm
Quantum yield
1
280.3 ± 0.5
303.7 ± 2.6
278.3 ± 1.2
282.7 ± 1.2
283.3 ± 0.5
295.7 ± 0.9
312.0 ± 0.8
353.3 ± 3.3
334.7 ± 3.7
294.0 ± 0.0
349.7 ± 0.9
366.0 ± 1.0
418.3 ± 1.7
419.5 ± 2.5
327.0 ± 0.0
0.02 ± 0.01
0.50 ± 0.03
0.02 ± 0.00
0.01 ± 0.01
0.03 ± 0.00
2
3
4
5
-Aminopurine
a
The parameters are an average of at least three sets of experimental data. All experiments were performed using samples with an optical density <
0
.1.
Table 2 Fluorescence decay parameters of the compounds in water at
action times, temperatures, reactant concentrations and solvents
but no increase of the yields could be achieved.
◦
a
2
5 C
One reason for the moderate yields and difficult purification
could be the protonation of the starting material and products.
Protonation of the products was first noticed when NMR proton
shifts of the samples changed after normal silica column purifi-
cation. Therefore all of the products had to be deprotonated
2
Compound
Lifetime, s/ns
v
DW
1
2
1.73 ± 0.01
10.85 ± 0.31
1.04 ± 0.05
1.10 ± 0.12
0.10 ± 0.05
1.07 ± 0.05
1.95 ± 0.00
1.98 ± 0.00
1.85 ± 0.10
-Aminopurine
3
5
.81 ± 0.37
with NaHCO
powder was added and the mixture stirred for 5 min before
the NaHCO was filtered off) before characterisation, and for
3
(to the MeOH solution of the product, NaHCO
3
4
5
2.85 ± 0.37
1.06 ± 0.05
1.76 ± 0.02
1.19 ± 0.02
1.44 ± 0.27
1.93 ± 0.05
3
a
purification 0.1% Ca-enriched silica was used.
The parameters were calculated using non-linear squared analysis, and
the goodness of fit was characterised using chi-squared (v ) and the
2
The fluorescence properties of the products were determined
and compared with those of the starting material 1 and 2-
aminopurine. The fluorescence properties of the products 3–5
did not differ considerably from those of compound 1 and were
much lower than for 2-aminopurine. It can be concluded that
the ‘etheno’ ring(s) system does not enhance the fluorescence
characteristics of 2,6-diaminopurine products in the same way
Durbin–Watson parameter (DW).
found, one of which was three times longer than the lifetime
for the starting material 2,6-diaminopurine. These two lifetimes
could be explained, for example, by two different tautomers of
the compound 3. When comparing the lifetimes and quantum
yields of the etheno derivatives 3–5 it can be concluded that they
are quite similar.
The emission maxima for compounds 3 and 4 were at longer
wavelength, and for compound 5 at shorter wavelength, than the
emission maximum for the starting material 1. The excitation
maxima for compounds 3 and 4 were also at longer wavelength,
and for compound 5 close to the excitation maximum of the
starting material. Therefore the Stokes shifts for products 3 and
1
as they do in similar adenosine products. Therefore it must
be concluded that the conjugated double bond system and
substituent effects of these product molecules are critically
different compared to 2-aminopurine or ethenoadenosine.
Theoretical tautomeric equilibrium calculations for all prod-
ucts in the gas phase were performed in order to obtain
information on the different tautomers present. It has to be
noted that tautomers in the gas phase can differ from those
present in DMSO as DMSO can form H-bonds with the
molecules, and also for these kinds of compounds there is also
4
were also slightly larger, and for product 5 slightly smaller than
for the starting material 2,6-diaminopurine (1). For example, the
Stokes shift for compound 4 was 85 nm, and for compound 5 it
was 33 nm in comparison to the Stokes shift of 54 nm for the
starting material.
The fluorescence measurement results showed that the
formation of additional five-membered ring(s) to the 2,6-
diaminopurine structure did not change the fluorescence prop-
erties dramatically and therefore the formation of a more
conjugated double bond system did not enhance the fluorescence
properties for these molecules. This could be explained by the
fact that all of the products contain many tautomers in rapid
exchange. Different tautomers have been shown to have different
12a
the possibility for dimer formation.
Regarding the product distribution it may be concluded that
the NH
reactive than the NH
,6-diaminopurine 1. It might be then assumed that the four-ring
2
group at position 2 in the purine ring system is more
2
group at position 6 in the starting material
2
product 5 is formed from product 3 as the reaction proceeds.
Experimental
Chemicals
2,6-Diaminopurine (1, 98%) was obtained from Adrich.
Chloroacetaldehyde diethyl acetal (99%) was obtained from
Acros Organics. The solvents for the synthesis were of an-
alytical grade and for HPLC of commercial HPLC grade.
2-Aminopurine (98%) was obtained from MP Biomedicals
13,15
fluorescent charasteristics.
Comparison of the data to those of 2-aminopurine shows
that these molecules 3–5 are not fluorescent enough to be used
as fluorescent probes.
(formerly ICN Biochemicals, Inc.).
Conclusions
Spectroscopic and spectrometric methods
The isolated yield for compound 4 (2.5%) was low and for
compounds 3 (9%) and 5 (30%) moderate. Part of the starting
material (1) was recovered unchanged from the reaction mixture.
Yields of the products 3 and 4 in the reaction mixture were higher
1
13
15
The H-, C- N- and 2D NMR spectra were recorded in 100%
◦
DMSO-d
6
at 18.5 and 25 C. The NMR experiments were
performed at 14.1 T using an NMR spectrometer equipped
(
ca. 15–20%), but isolation of the pure products was difficult. The
with a z-axis field gradient 5 mm inverse broadband probe
1
13
yield of the product 5 increased as the reaction time increased
to ca. 24 h, but essentially not after that. The separation of this
tetracyclic product from the other products and starting material
on Ca-enriched silica column, using dichloromethane/methanol
as eluent, was good. The reactions were studied and tested
under many different reaction conditions applying different re-
operating at 600.13, 150.92 and 60.81 MHz for H, C and
5
1
N, respectively. Spectral widths for the 2D experiments were
optimised from the 1D spectra and acquired with an appropriate
1
13
level of resolution. Both H and C spectra were referenced
to the solvent signal (2.50 and 39.51 ppm, respectively). All
nitrogen shifts for the products were from HMBC spectra
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 9 2 4 – 2 9 2 9
2 9 2 7

View Article Online
and referenced externally to 90% nitromethane in CD
3
NO
2
(UV), a Jasco FP-920 fluorescence detector (Jasco), and a
ChemStation data handling program. The chromatographic
separations were performed on an analytical 5 lm, 4.6 mm ×
125 mm Zorbax SB-CN column (Agilent Technologies). The
column was eluted isocratically for 5 min with 0.01 M acetate
buffer (pH 7.1) and then with a gradient from 1 to 30%
acetonitrile over the course of 15 min at a flow rate of
1
(
0.00 ppm). The H NMR signal assignments were based on
1
1
1
13
chemical shifts and correlations from 2D H– H, H– C and
1
15
H– N correlation spectra. The assignment of carbon signals
was based on the same techniques and carbon–proton coupling
constants.
The electron impact high-resolution mass spectra (EI) were
recorded on a Fisons ZABSpec-oaTOF instrument. The spectra
were acquired using a direct insert probe scanning from 50 to
−
1
1.0 mL min .
Compounds 3–5 were isolated from the reaction mixtures
by flash column chromatography, initially with a Ca-enriched
silica gel 60 (Fluka) column and afterwards with a 40 lm
C-18 reversed-phase column. The products were eluted over
silica using a dichloromethane/methanol gradient containing
2% triethylamine and over reversed-phase silica using a wa-
ter/acetonitrile gradient.
1
500 amu and using electrons energised to 70 eV. Accurate mass
measurements were performed using a peak matching technique
with PFK as a reference substance at a resolution of 8000–10 000
(
at 10% peak height).
The UV spectra of the compounds were recorded with a
Shimadzu UV-160A spectrophotometer. The absorbance mea-
surements (Table 1) for the compounds were obtained using a
Varian Cary 50 Conc UV-visible spectrophotometer.
Synthesis
The fluorimetric properties of the compounds were studied at
◦
5
-Aminoimidazo[2,1-i]purine (2), 9-aminoimidazo[2,1-b]purine
2
5 C. Steady-state fluorescence measurements were performed
ꢀ
ꢀ
(
3), 9-aminoimidazo[1,2-a]purine (4) and diimidazo[2,1-b:2 ,1 -
on a Photon Technology International (PTI) Quantamaster
spectrofluorimeter operating in the T-format. The emission
i]purine (5). Chloroacetaldehyde diethyl acetal (1.0 mL,
1
◦
6
.7 mmol) was added to 0.1 M HCl (5.0 mL) and stirred at 80 C
wavelength scans were performed with the excitation wavelength
set at 320 nm. Excitation wavelength scans were made with
the emission monochromator set at 430 nm. In the steady-state
measurements, the slit widths were kept at 5 nm. The water used
in the experiments was purified by reverse osmosis followed by
passage through a Millipore UF Plus water purification system
for ca. 1 h. The clear solution containing the chloroacetaldehyde
was then added to a solution of 2,6-diaminopurine (0.50 g,
3
.3 mmol) in DMF (ca. 10 mL). The pH was adjusted to 4–5 and
◦
the reaction mixture was stirred at 80 C for ca. 24 h. The pH was
maintained at 4–5 by the appropriate addition of either 1.0 M
NaOH or 1.0 M HCl. Upon completion of the reaction, the
−
1
to yield a product with a resistance of at least 18.2 MX cm .
The fluorescence quantum yield of the compounds was
estimated by comparison with the known quantum yield of a
solution was neutralised with NaHCO (aq.). The mixture was
3
evaporated to dryness and crude purified using a Ca-enriched
silica column and then further purified using a reversed-phase
C-18 column to yield pure 3 (52 mg, 9%), 4 (10 mg, 2,5%) and 5
16
standard. The quantum yield standard was quinine sulfate
(
Fluka AG, Switzerland) in 0.1 M H
2
6,17
SO which is known to
4
1
(
200 mg, 30%) as white powders.
have a quantum yield of 0.53 ± 0.02.
In order to avoid inner filter effects the optical density was
kept below 0.1 in all measurements. The quantum yield was
calculated according to eqn. (1),
5
-Aminoimidazo[2,1-i]purine (2).
H
d (600.13 MHz; DMSO)
7
.36 (2 H, s, NH
2
), 7.38 (1 H, s, J8,7 = 1.6 Hz, H-8), 7.90 (1
H, d, J7,8 = 1.6 Hz, H-7), 8.02 (1 H, s, H-2), H-1/H-3 not
observed; d (150.92 MHz; DMSO) 104.3 (C-7), 108.9 (C-10b),
31.8 (C-8), 140.5 (C-5), 141.6 (C-2), 149.3 (C-10a), 151.8 (C-3a);
(60.81 MHz; DMSO) −307 (NH ), −220 (N-1/N-3), −209
2
2
Q = Q
R
(I/I
R
)(OD
R
/OD)(n /n
R
)
(1)
C
1
where Q is the quantum yield, I is the integrated intensity OD is
16
d
N
2
the optical density, and n is the refractive index. The subscript
R refers to the reference fluorophore of known quantum yield.
The fluorescence decay parameters of the reaction products
(
N-6), −163 (N-9), −148 (N-1/N-3).
9
-Aminoimidazo[2,1-b]purine (3).
H
d (600.13 MHz; DMSO)
were determined using a PTI Timemaster instrument (N
In these experiments, the excitation wavelength was set to
37 nm, and the emission wavelength to 430 nm. The slit width
2
laser).
6
(
3
(
.13 (2 H, s, NH ), 7.00 (1 H, d, J6,5 = 1.5 Hz, H-6), 7.34
2
1 H, s, H-2), 7.39 (1 H, d, J5,6 = 1.5 Hz, H-5), H-1/H-
not observed; d (150.92 MHz; DMSO) 104.5 (C-5), 115.6
C-9a), 127.3 (C-6), 144.0 (C-3a), 145.7 (C-2), 148.5 (C-7a),
3
C
was set to 5 nm. Analyses of the data were performed with the
software supplied by PTI (Time Master 1.2).
1
51.9 (C-9); d
N
(60.81 MHz; DMSO) −308 (NH
2
), −212 (N-4),
2
−
191 (N-8), −162 (N-7), −151, −143 (sp -hybridised N-1/N-
Modelling
3
3
), sp -hybridised N-3/N-1 not observed. m/z (EI) 174.0654
+
Geometry optimisations were performed using the Gaussian
(M , C
7
H
6
N
6
requires 174.0654). UV: kmax(H
2
O)/nm 220 and
O)/nm 252
1
8
3
−1
9
3
8 program package and either ab initio (at either the HF/6-
276 (e/dm mol−1 cm 2260 and 1150); kmin(H
2
19
20
3
−1
−1
11G** level of theory or the MP2/6-31G** level of theory )
(e/dm mol cm 770).
or density functional theory methods (at the B3LYP/6-311G**
21
9
-Aminoimidazo[1,2-a]purine (4).
level of theory ). Additional calculations were also made
including the effect of the solvent (SCIPCM method, dielectric
constant = 46.7 for DMSO). The calculation of the energies and
chemical shifts were both performed at the same level of theory
in each case. The isotropic magnetic shieldings (r) so obtained
were computed as chemical shifts (d) based on their difference
Major tautomer. d (600.13 MHz; DMSO) 7.38 (1 H, s, H-6),
H
7
.41 (2 H, s, NH
H, s, H-1); d (150.92 MHz; DMSO) 109.4 (C-7), 115.5 (C-9a),
31.2 (C-6), 135.1 (C-2), 141.2 (C-3a), 142.2 (C-4a), 144.7 (C-9);
2
), 7.81 (1 H, s, H-2), 7.92 (1 H, s, H-7), 12.68 (1
C
1
d
N
(60.81 MHz; DMSO) −306 (NH
2
), −223 (N-1), −202 (N-8),
1
3
15
from the shieldings of TMS (for C) or nitromethane (for N)
and then calibrated based on the results for 1. This has been
found to be a reliable method for the calculation of chemical
−
150 (N-5), −138 (N-3).
Minor tautomer. d
H
(600.13 MHz; DMSO) 7.19 (2 H, s, NH
2
),
7
.44 (1 H, s, H-6), 7.94 (1 H, s, H-7), 7.97 (1 H, s, H-2), 13.20* (1
H, s, H-3); d (150.92 MHz; DMSO) 109.8 (C-7), 115.5 (C-9a),
31.2 (C-6), 137.9 (C-2), 138.7 (C-4a), 143.9 (C-9), 149.4 (C-3a);
(60.81 MHz; DMSO) −309 (NH ), −231 (N-3)*, −226 (N-8),
180 (N-5). m/z (EI) 174.0648 (M , C requires 174.0654).
O)/nm 214 and 277 (e/dm mol cm 2180 and
6b,9
shifts for these nuclei. Various platforms were used for the
C
calculations, e.g. an SGI Octane workstation or Linux cluster.
1
d
N
2
+
Chromatographic methods
−
7
H
6
3
N
6
−1
−1
HPLC analyses were carried out using an Agilent Technologies
UV: kmax(H
1010); kmin(H
the minor tautomer to be the N-3.
2
3
−1
−1
1
100 series liquid chromatographic system consisting of an
2
O)/nm 247 (e/dm mol cm 590). *Assuming
autosampler, degasser, quaternary pump, diode array detector
2
9 2 8
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 9 2 4 – 2 9 2 9

View Article Online
ꢀ
ꢀ
Diimidazo[2,1-b:2 ,1 -i]purine (5).
d
H
(600.13 MHz; DMSO)
P. Virta, T. Holmstr o¨ m, M. U. Roslund, P. Mattjus, L. Kronberg and
R. Sj
o¨ holm, Org. Biomol. Chem., 2004, 2(6), 821–827.
7
1
.15 (1 H, d, J6,5 = 1.5 Hz, H-6), 7.23 (1 H, d, J10,9 = 1.4 Hz, H-
6
(a) J. M a¨ ki, K. D. Klika, R. Sj o¨ holm and L. Kronberg, J. Chem.
Soc., Perkin Trans. 1, 2001, 1216–1219; (b) J. M a¨ ki, P. T a¨ htinen, L.
Kronberg and K. D. Klika, J. Phys. Org. Chem., 2005, 18, 240–249.
R. J. Sijbesma and E. W. Meijer, Chem. Commun., 2003, 5–16.
M. U. Roslund, P. Virta and K. D. Klika, Org. Lett., 2004, 6(16),
2673–2676.
9 P. T a¨ htinen, A. Bagno, K. D. Klika and K. Pihlaja, J. Am. Chem.
Soc., 2003, 125, 4609–4618; K. D. Klika, J. Imrich, I. Danihel, S.
B o¨ hm, P. Kristian, S. Hamul’akov a´ , K. Pihlaja, A. Koch and E.
Kleinpeter, Magn. Reson. Chem., 2005, 43, 380–388; E. Kleinpeter
and A. Koch, J. Phys. Org. Chem., 2001, 14, 566–576.
0), 7.30 (1 H, s, H-2), 7.70 (1 H, d, J5,6 = 1.5 Hz, H-5), 7.81 (1 H,
d, J9,10 = 1.4 Hz, H-9), H-1/H-3 not observed; d
C
(150.92 MHz;
DMSO) 109.1 (C-9), 109.4 (C-5), 116.0 (C-12b), 126.4 (C-6),
29.8 (C-10), 136.2 (C-7a), 137.6 (C-3a), 141.4 (C-12a), 144.9
7
8
1
(
(
C-2); d
N
(60.81 MHz; DMSO) −215 (N-4), −213 (N-8), −164
2
N-7), −153 (N-11), −150, −137 (sp -hybridised N-1/N-3),
3
+
sp -hybridised N-3/N-1 not observed. m/z (EI) 198.0658 (M ,
requires 198.0654). UV: kmax(H O)/nm 234, 283 and
95 (e/dm mol cm 7840, 3680 and 2690); kmin(H O)/nm 252
C
2
(
9
H
6
N
6
2
3
−1
−1
−1
2
3
−1
e/dm mol cm 1840).
1
1
0 J. Arpalahti and K. D. Klika, Eur. J. Inorg. Chem., 1999, 1199–1201.
1 A. Laxer, D. T. Major, H. E. Gottlieb and B. Fischer, J. Org. Chem.,
2
001, 66, 5463–5481.
Acknowledgements
1
1
1
2 (a) R. Marek, personal communication; (b) I. Alkorta and J. Elguero,
Struct. Chem., 2003, 14(4), 377–389.
We are grateful to Mr Markku Reunanen for recording the
mass spectra. Professor J. Peter Slotte is acknowledged for
access to the fluorescence facility, and Dr Thomas Nyholm
is acknowledged for assistance. Financial support from the
Graduate School of Bioorganic and Medicinal Chemistry (P. V.),
Academy of Finland, Sigrid Jus e´ lius Foundation, Magnus
Ehrnrooth Foundation, Medicinska underst o¨ dsf o¨ reningen Liv
och H a¨ lsa, K. Albin Johansson Foundation (P. M.) is gratefully
acknowledged.
3 S. K. Mishra, M. K. Shukla and P. C. Mishra, Spectrochim. Acta,
Part A, 2000, 56, 1355–1384.
4 J. Fujimoto, Z. Nuesca, M. Mazurek and L. C. Sowers, Nucleic Acids
Res., 1996, 24(4), 754–759; B. Holz, S. Klimasauskas, S. Serva and
E. Weinhold, Nucleic Acids Res., 1998, 26(4), 1076–1083.
15 R. W. Wilson and P. R. Callis, Photochem. Photobiol., 1980, 31, 323–
27.
3
1
1
1
6 Principles of Fluorescence Spectroscopy, J. R. Lakowicz, ed., Kluwer
Academic/Plenum Publishers, New York, 1999, 2nd edn.
7 M. J. Adams, J. G. Highfield and G. F. Kirkbright, Anal. Chem.,
1
977, 49(12), 1850–1852.
8 Gaussian 98 (Revision A.12), M. J. Frisch, G. W. Trucks, H. B.
Schlegel, G. E. Scuseria, M. A. Robb, J. R. Cheeseman, V. G.
Zakrzewski, J. A. Montgomery, Jr., R. E. Stratmann, J. C. Burant, S.
Dapprich, J. M. Millam, A. D. Daniels, K. N. Kudin, M. C. Strain,
O. Farkas, J. Tomasi, V. Barone, M. Cossi, R. Cammi, B. Mennucci,
C. Pomelli, C. Adamo, S. Clifford, J. Ochterski, G. A. Petersson, P. Y.
Ayala, Q. Cui, K. Morokuma, N. Rega, P. Salvador, J. J. Dannenberg,
D. K. Malick, A. D. Rabuck, K. Raghavachari, J. B. Foresman, J.
Cioslowski, J. V. Ortiz, A. G. Baboul, B. B. Stefanov, G. Liu, A.
Liashenko, P. Piskorz, I. Komaromi, R. Gomperts, R. L. Martin,
D. J. Fox, T. Keith, M. A. Al-Laham, C. Y. Peng, A. Nanayakkara,
M. Challacombe, P. M. W. Gill, B. Johnson, W. Chen, M. W. Wong,
J. L. Andr e´ s, C. Gonzalez, M. Head-Gordon, E. S. Replogle and J. A.
Pople, Gaussian, Inc., Pittsburgh PA, 2002.
References
1
J. R. Barrio, J. A. Secrist III and N. J. Leonard, Biochem. Biophys.
Res. Commun., 1972, 46(2), 597–604; S. C. Srivastava, S. K. Raza and
R. Misra, Nucleic Acid Res., 1994, 22, 1296–1304.
(a) S. Mikkola, N. Koissi, K. Ketom a¨ ki, S. Rauvala, K. Neuvonen
and H. L o¨ nnberg, Eur. J. Org. Chem., 2000, 2315–2323; (b) R.
Khazanchi, P.-L. Yu and F. Johnson, J. Org. Chem., 1993, 58(9),
552–2556; (c) J. T. Kusmierek, D. E. Jensen, S. J. Spengler, R.
Stolarski and B. Singer, J. Org. Chem., 1987, 52(12), 2374–2378;
d) P. D. Sattsangi, N. J. Leonard and C. R. Frihart, J. Org. Chem.,
977, 42(20), 3292–3296; (e) P. D. Sattsangi, J. R. Barrio and N. J.
Leonard, J. Am. Chem. Soc., 1980, 102, 770–774.
G. A. Pandya and M. Moriya, Biochemistry, 1996, 35, 11 487–11 492.
J. A. Secrist, J. R. Barrio, N. J. Leonard and G. Weber, Biochemistry,
2
2
(
1
3
4
19 W. J. Hehre, L. Random, P. V. R. Schleyer and J. A. Pople, Ab Initio
Molecular Orbital Theory, John Wiley & Sons Inc., New York, 1986.
20 C. Møller and M. S. Plesset, Phys. Rev., 1934, 46, 618–622.
21 A. D. Becke, J. Chem. Phys., 1993, 98, 5648–5652.
1
972, 11(19), 3499–3506.
P. Virta, T. Holmstr o¨ m, T. Munter, T. Nyholm, L. Kronberg and
R. Sj o¨ holm, Nucleosides Nucleotides Nucl. Acids, 2003, 22(1), 85–97;
5
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 9 2 4 – 2 9 2 9
2 9 2 9