New nucleoside analogs from 2-amino-9-(β-d-ribofuranosyl)purine

Article abstract of DOI:10.1039/b316413f

Four novel derivatives of 2-amino-9-(β-D-ribofuranosyl)purine (1) were synthesised and fully characterised. When 1 was reacted with chloroacetaldehyde (a), 2-chloropropanal (b), bromomalonaldehyde (c) and a mixture of chloroacetaldehyde + malonaldehyde (d), 3-(β-D-ribofuranosyl)-imidazo-[1, 2a]purine (2), 3-(β-D-ribofuranosyl)-5-methylimidazo-[1,2a]purine (3), 3-(β-D-ribofuranosyl)-5-formylimidazo-[1,2a]purine (4) and 9-(β-D-ribofuranosyl) 2-(3,5-diformyl-4-methyl-1,4-dihydro-1-pyridyl)purine (5) were formed, respectively. The products were isolated, purified by chromatography and characterised by MS, complete NMR assignment as well as fluorescence and UV spectroscopy. The yields of these reactions were moderate (14-20%). The fluorescence properties differed from those of the starting compound and the quantum yields were considerably lower.

Full text of DOI:10.1039/b316413f

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New nucleoside analogs from 2-amino-9-(ꢀ-D-ribofuranosyl)purine
a
a
a
b
a
Piritta Virta, Toni Holmström, Mattias U. Roslund, Peter Mattjus, Leif Kronberg and
a
Rainer Sjöholm
a
Åbo Akademi University, Department of Organic Chemistry, Biskopsgatan 8,
FIN-20500 Turku/Åbo, Finland. E-mail: piritta.virta@abo.fi; Fax: ϩ358 2 2144866;
Tel: ϩ358 2 2154502
Åbo Akademi University, Department of Biochemistry and Pharmacy, FIN-20520 Turku/Åbo,
b
Finland
Received 16th December 2003, Accepted 6th February 2004
First published as an Advance Article on the web 25th February 2004
Four novel derivatives of 2-amino-9-(β--ribofuranosyl)purine (1) were synthesised and fully characterised.
When 1 was reacted with chloroacetaldehyde (a), 2-chloropropanal (b), bromomalonaldehyde (c) and a mixture
of chloroacetaldehyde ϩ malonaldehyde (d), 3-(β--ribofuranosyl)-imidazo-[1,2a]purine (2), 3-(β--ribofuranosyl)-
5
2
-methylimidazo-[1,2a]purine (3), 3-(β--ribofuranosyl)-5-formylimidazo-[1,2a]purine (4) and 9-(β--ribofuranosyl)-
-(3,5-diformyl-4-methyl-1,4-dihydro-1-pyridyl)purine (5) were formed, respectively. The products were isolated,
purified by chromatography and characterised by MS, complete NMR assignment as well as fluorescence and UV
spectroscopy. The yields of these reactions were moderate (14–20%). The fluorescence properties differed from
those of the starting compound and the quantum yields were considerably lower.
Introduction
the reactivity of the unnatural nucleobase would be higher than
that of natural nucleobases, which are generally classified as
quite unreactive. The compound 1 was prepared in two steps
Haloacetaldehydes have been shown to react with nucleic acid
components yielding etheno derivatives of adenosine, guano-
sine and cytidine. The 1,N -ethenodeoxyadenosine derivatives,
due to their fluorescence, are useful compounds for e.g. the
studies of mutagenesis and enzymology. Fluorescence in
general is an extremely useful tool for the investigation of
biological material. The incorporation of fluorescent nucleo-
sides into oligonucleotides will greatly facilitate studies of the
structure–function of various RNAs, protein–RNA structures,
and DNA-RNA based diagnostic applications. That is why a
large variety of fluorescent molecules have been incorporated
into DNA/RNA and proteins e.g. 3-methyl-isoxanthopterin
and 2-aminopurine, 1,N -ethenoadenosine.
dihydropyridine derivatives of natural nucleosides and proteins
8
9
starting from guanosine. Other methods are also available.
1
6
In the current work, we report on the synthesis of four novel
derivatives of 2-amino-9-(β--ribofuranosyl)purine (1). They
are formed in the reaction of compound 1 with chloroacetalde-
hyde (a), 2-chloropropanal (b), bromomalonaldehyde (c)
and chloroacetaldehyde ϩ malonaldehyde (d), respectively
2
3
(
Scheme 1). We present in this contribution, the details of the
synthetic procedures, the complete structural analysis, which
4
1
13
15
includes MS, NMR analyses ( H-, C-, N- chemical shifts and
coupling constants), as well as fluorescence and UV spectral
properties. The fluorescence intensities and lifetimes compared
to those of 1 are discussed.
6
1f,5
Fluorescent
6
have also been previously reported.
Our research in the field of nucleoside chemistry originally7
aimed at synthesising modified fluorescent RNA bases.
However, our present investigations are also directed toward the
preparation of RNA base analogues, which might have other
interesting applications than only those associated with their
fluorescence properties. To our knowledge 2-amino-9-(β--
ribofuranosyl)purine (1) has not been used to produce different
etheno or dihydropyridine derivatives and it was expected that
Results and discussion
The products (2–5) were synthesised using the reagents shown
in Scheme 1. The reactions of 2-amino-9-(β--ribofurano-
syl)purine (1) with the aldehydes a–d were carried out in
aqueous solution at pH 4.5. The progress of the reactions was
monitored by HPLC analysis on a reversed phase (C 18)
column and the molecular weights of formed products were
Scheme 1
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 4
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 8 2 1 – 8 2 7
821

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obtained from LC-MS analyses of the reaction mixture. The
products were isolated by preparative reversed phase chromato-
graphy and were fully characterised by H-, C- and N NMR
spectroscopy, electrospray mass spectrometry, HRMS, fluor-
escence spectroscopy and UV absorption.
The yields of 2, 3, 4 and 5 were 14%, 17%, 20% and 16%
respectively. The NMR spectroscopic data, the mass spectro-
metric and UV properties are given in the Experimental section.
The fluorescence properties are presented in Tables 1 and 2 and
Fig. 2.
NMR parameters
In the reactions a–c the formation of two different regioisomers
was possible. Thus complete NMR spectroscopic analysis
was necessary for the determination of which isomer was
preferentially formed (Scheme 4).
1
13
15
Mechanisms
The formation of ethenoadenosine from adenosine on treat-
ment with chloroacetaldehyde has earlier been described in the
10
literature. The formation of compounds 2–4 from reactions
a–c can be expected to follow a similar mechanism. That is, the
reaction is initiated by attack of the exocyclic amino group of
the nucleobase on the carbonyl carbon of the different alde-
hydes used in a–c. Subsequently, the halogen atom is displaced
by intramolecular nucleophilic attack of the ring nitrogen.
Finally, the products are obtained through dehydration of the
cyclic intermediates (Scheme 2). LC-MS analysis of the reaction
mixture of aldehyde c and starting material 1, showed the
presence of a molecule with mass m/z 338 and according to
Scheme 4
1
3-(ꢀ-D-Ribofuranosyl)-imidazo-[1,2a]purine (2). In the
H
NMR spectrum of compound 2, two singlet and two doublet
resonance signals from the protons in the base unit were
observed. The signal at δ 8.79 ppm was assigned to H-2 based
on the observed long-range H–H (long-range COSY) and
HMBC correlation to the sugar unit. The other singlet at δ 9.56
ppm was assigned to H-9. The large downfield shift of H-9 can
be explained by the high electron density at C-9 (δ 126.67 ppm)
due to resonance of the non-aromatic double-bond system with
the non-bonding electrons on N-3 This resonance places a
partial negative charge on C-9 which repels the electron cloud
around H-9 causing a downfield shift of the proton. This
1
H NMR the compound was the carbinolamine intermediate
(
Fig. 1), which supports the described mechanism.
2
indicates that 2 has the linear (1,N -etheno) rather than the
angular (N ,3-etheno) tricyclic structure. The doublets at δ 7.95
J 1.5 Hz) and 7.68 ppm (J 1.5 Hz) were assigned to H-7 and
2
Scheme 2
(
H-6, respectively. Both doublets showed a strong H–H corre-
lation with each other. The linear tricyclic structure was
definitely assigned based on the 2D NOESY spectrum and the
1
15
H– N HMBC data. The signal at δ Ϫ191 ppm was assigned to
N-8 based on its chemical shift and its correlations to the
signals of H-6, H-7 and H-9. Correlations between the signals
of H-6, H-7 and the nitrogen signal at δ Ϫ155 ppm assigned the
signal to N-5 confirming that the etheno bridge was situated
between N-5 and N-8. The nitrogen signal at δ Ϫ216 ppm was
assigned to N-3 based on its chemical shift and its correlation
with the signals of H-1Ј and H-2Ј. The nitrogen signal at δ Ϫ142
ppm showed correlation to the signal from H-2 and was
assigned to N-1. The nitrogen signal at δ Ϫ161 ppm correlated
with the signal of H-9 and was assigned to N-4. No amino
Fig. 1 The carbinolamine intermediate observed by NMR and
LC-MS.
Malonaldehyde–acetaldehyde conjugates have previously
6
been reacted with 2Ј-deoxyadenosine, cytidine and proteins.
There have been different suggestions for the mechanism
11
involved. In the work of Gómez-Sánchez et. al. it was pre-
sented that condensation of aliphatic aldehydes with malon-
aldehyde is a general reaction giving first a 1 : 1 conjugate
and with an excess of a malonaldehyde, a 2 : 1 conjugate.
15
group nitrogen could be detected by N NMR spectroscopy.
13
In the C NMR spectrum seven signals from the base and
five signals from the ribosyl unit were observed. The signals of
carbons bonded to hydrogen atoms were assigned from the
one-bond C–H correlation spectra (HMQC or HETCOR).
Thus the signals at δ 126.67 ppm and 148.68 ppm were assigned
to C-9 and C-2, respectively. The latter signal also displayed
a long-range correlation to H-1Ј and H-2Ј in the HMBC
spectrum. The signal at δ 146.51 ppm displayed H–C long-
range correlation with the signals of H-6 and H-7 and was
assigned to C-4a. The carbon signal at δ 127.57 ppm was
assigned to C-9a based on its correlation with the signal of H-2
and its large trans-vicinal coupling constant to H-2 (J 13 Hz),
12
(
Scheme 3). In the present reaction the 2 : 1 conjugate would
then react with the amino group of compound 1, to form
product 5 (Scheme 3).
13
which has been observed previously for similar compounds.
The signal at δ 149.72 ppm displayed H–C long-range corre-
lation with the signals of H-2, H-9 and H-1Ј and was assigned
to C-3a. The signals of the ribosyl moiety were assigned using
correlation spectroscopy.
Scheme 3
8
22
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 8 2 1 – 8 2 7

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3
-(ꢀ-D-Ribofuranosyl)-5-methylimidazo-[1,2a]purine (3). In
Hz). The slightly broadened signal at δ 8.55 ppm was assigned
1
the H NMR spectrum of 3, besides the ribosyl protons four
signals from the base moiety were observed. The singlet at
δ 2.51 ppm was assigned to CH on the basis of the area integral
3H, doublet J 0.3 Hz) and the large upfield shift. Further, it
had NOESY correlations to H-9 (δ 9.29 ppm) and H-6 (δ 7.43
ppm, d, J 0.3 Hz). The proton giving a singlet at δ 8.76 ppm
displayed a NOESY interaction and a long-range correlation
with H-1Ј and H-2Ј in the ribose unit, and the signal was
assigned to H-2. All N signal shifts were determined from the
H– N HMBC spectrum as described for compound 2 and had
similar chemical shifts to those of compound 2. The C NMR
spectrum displayed eight signals that originated from the
modified base moiety. The proton binding carbons were
identified from C–H correlation spectra. The carbon signal for
the methyl group was observed at δ 8.89. The carbons C-6 (one
proton attached) and C-7 in the etheno bridge gave resonance
signals at δ 131.44 and 117.68 ppm, respectively. The methyl
group carbon and C-7 showed also long-range C–H couplings
to the H-9 in the base unit. The signals at δ 124.27 ppm and
to proton H-2Љ and the signal at 3.66 ppm (d, J 6.7 Hz) to H-4Љ.
The singlets at δ 9.57 and 9.56 ppm respectively, were assigned
to the formyl protons (C-5Љ) due to their long-range COSY
correlations to the H-4Љ and methyl group signals. The broaden-
ing of the δ 8.55 (H-2Љ) and the separate signals of the formyl
protons are most probably due to the presence of a mixture
3
(
15
of two diastereomers. All N signal shifts were determined
1
15
from the H– N HMBC spectrum. In addition to the ribosyl
15
13
carbons the C NMR spectrum displayed 12 signals from the
1
15
modified base moiety. The methine carbon signals at δ 145.60
and 149.02 ppm were assigned to C-8 and C-6, respectively,
from heteroatom correlations. The signal at δ 21.14 ppm was
assigned to the methyl group from the dihydropyridyl moiety
and the signal at δ 22.65 ppm was assigned to C-4Љ due to its
heteroatom correlation to H-4Љ. The signals at δ 140.81 and
140.76 ppm were assigned to C-2Љ according to heteroatom cor-
relations. H-2Љ displayed long-range H–C HMBC correlations
to C-2 at δ 150.07 ppm. The signal at δ 125.17 ppm was assigned
from long-range H–C HMBC correlations to both C-3Љ and the
signals detected at δ 190.92 and 190.87 ppm were assigned to
C-5Љ due to their heteroatom correlation to H-5Љ. The signals of
C-2Љ as well as those of the formyl carbons appeared as pairs of
peaks, most probably due to the presence of a mixture of two
diastereomers. This could, however, not be confirmed by HPLC
as only one peak was shown in the chromatogram. The carbon
signals at δ 132.05, 152.18 and 150.07 ppm were assigned to C-
13
1
48.38 ppm were assigned to be C-9 and C-2, respectively.
These assignments were supported by the 2D HMBC corre-
lation data. The carbon signals at δ 127.37 ppm, 146.07 ppm
and 148.83 ppm were assigned to C-9a, C-4a and C-3a, respect-
ively, based on the long-range H–C correlations in HMBC as
described for compound 2. The signals of the ribosyl moiety
were assigned using correlation spectroscopy.
5
, C-4 and C-2, respectively, from the long-range H–C corre-
lations in HMBC. All nitrogen shifts could be assigned from the
inverse detection experiment and the signals of the ribosyl
moiety were easily assigned using correlation spectroscopy.
3
-(ꢀ-D-Ribofuranosyl)-5-formylimidazo-[1,2a]purine (4). In
1
addition to the ribosyl protons the H NMR spectrum of
compound 4 also displayed four singlets. The singlet at δ 9.89
ppm was assigned to the formyl proton on the basis of its corre-
lation with the carbon signal at δ 178.13 ppm and its H–H
correlation to the proton signal at δ 8.67 ppm assigned to H-6
and the HMBC correlation to C-7 at δ 127.46 ppm identified
from the large geminal C–HCHO coupling (J 31 Hz). The large
downfield shift of H-9 (δ 10.10 ppm) can be explained by the
anisotropic effect of the formyl group. The proton signal at
δ 9.00 ppm was assigned to H-2 in the purine ring on the basis
of its COSY correlation to H-1Ј in the ribosyl moiety. An eight-
bond H–H correlation was also observed between the signals of
H-2 and H-6 suggesting a highly planar zig-zag arrangement of
the bonds and this supported the linear structure of the three-
Fluorescence properties
Unlike natural DNA bases, 2-amino-9-(β--ribofuranosyl)-
purine (1), is fluorescent at neutral pH and this native fluor-
escence has been demonstrated to be an extremely useful
probe of e.g. DNA conformational changes and DNA base
14
5c–d
2
flipping.
On the other hand, the linear 1,N -ethenoguano-
sine, which resembles the products 2–4, does not exhibit
1a
fluorescence at all. This indicates that the conjugated
2
systems in the products 2–4 and 1,N -ethenoguanosine are
different.
We were interested in comparing the fluorescence character-
istics of the products with those of 2-amino-9-(β--ribofurano-
syl)purine (1). It is well known that an etheno bridge between
15
ring system. All N signal shifts were determined from the
1
15
H– N HMBC spectra. Eight carbon signals from the modified
base moiety were detected. The methine carbons at δ 149.46 and
27.90 ppm were assigned to C-2 and C-9, respectively, C-2
6
N-1 and N of adenosine enhances the fluorescence intensity
1
considerably in comparison to unmodified adenosine and there-
fore it was of interest to investigate the fluorescence properties
of the similar products formed from 1. The results of the fluor-
escence measurements are shown in Tables 1 and 2 and Fig. 2.
All in all, the fluorescence properties of the products differed
quite a lot from those of compound 1. The quantum yields were
considerably lower for the products, but the fluorescence
lifetime of molecule 3 was equal and of molecule 4 it was more
than five ns longer than for the starting material. When
comparing the properties of the etheno derivatives 2–4 it was
obvious that a methyl group in the five membered-ring slightly
increased the fluorescence lifetime but did not affect the
quantum yield, while an aldehyde group increased both the life-
time and the quantum yield considerably. Product 3 had the
same emission maximum as 1, but all other compounds had
their emission maxima at a longer wavelength. The excitation
maximum for compound 3 was also the same as for 1, for com-
pound 2 at a slightly shorter wavelength (298 nm), for com-
pound 4 at a 31 nm shorter (276 nm) and for the product 5 at a
slightly longer (313 nm) wavelength. Therefore the Stokes shifts
for all other products than for 3 were also larger. For example,
the Stokes shift for compound 4 was 167 nm, but only 56 nm for
the reference molecule 1.
displayed a long-range H–C correlation to H-1Ј. The signals at
δ 148.77 and 122.46 ppm were assigned to the etheno bridge
carbons C-6 and C-7 based on the HMBC correlations to the
formyl group. In the coupled carbon spectrum the signal of C-7
was split into two doublets due to the coupling to the formyl
carbon (δ 178.13, J 31 Hz) and the geminal coupling to H-6
(
J 15 Hz). The high value of this coupling may be explained by
13
the electronegativity of the nitrogens and the carbonyl group.
The carbon signals at δ 129.03, 150.20 and 151.14 ppm were
assigned to C-9a, C-4a and C-3a, respectively, from the long-
range H–C correlations. All nitrogen shifts could be assigned
from the inverse detection experiment and the signals of the
ribosyl moiety were assigned using correlation spectroscopy.
9
-(ꢀ-D-Ribofuranosyl)-2-(3,5-diformyl-4-methyl-1,4-dihydro-
1
1
-pyridyl)purine (5). The H NMR spectrum of compound 5
showed four singlets and one doublet in addition to the ribosyl
moiety signals. The singlet at δ 8.81 ppm was assigned to H-8,
due to the long-range H–H correlation to H-1Ј. The other
proton (H-6) from the base moiety was detected at δ 9.08 ppm
(
long-range COSY correlation to H-8). The methyl group in the
dihydropyridyl ring displayed a doublet at δ 1.04 ppm (J 6.7
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 8 2 1 – 8 2 7
823

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a
Table 1 Fluorescence parameters of the molecules in water at 23 ЊC
Molecule
Excitationλmax(H O)/nm
Emissionλmax(H O)/nm
Quantum Yield
2
2
1
2
3
4
5
307 ± 0.5
298 ± 0.5
306 ± 0.9
276 ± 0.1
313 ± 0.1
363 ± 0.8
465 ± 0.9
363 ± 0.9
437 ± 0.1
442 ± 0.5
0.607 ± 0.110
0.073 ± 0.013
0.068 ± 0.021
0.168 ± 0.048
0.178 ± 0.013
a
The parameters are an average of at least three sets of experimental data. All experiments were performed using samples with an optical density
0.05
<
Table 2 Fluorescence decay parameters of the molecules in water at
the conjugation is more efficient in 4 than in the corresponding
adenosine adduct.
a
2
3 ЊC
For adenosine, the formation of a dihydropyridine ring-
system, has been reported to enhance fluorescence intensity
significantly. Instead in 1, the dihydropyridine ring quenched
the fluorescence. Both quantum yield and lifetime were lower
than for the starting material 1.
Molecule
Lifetime τ/ns
χ2
DW
6a
1
2
3
4
5
10.42 ± 0.21
7.94 ± 0.42
10.15 ± 0.47
16.07 ± 0.71
2.78 ± 0.06
1.120
0.903
1.081
0.906
1.068
1.60
2.18
0.99
1.96
1.83
Conclusions
a
The parameters were calculated using non-linear squared analysis,
and the goodness of fit was characterized using chi-squared (χ ) and the
Durbin–Watson parameter (DW)
2
Four 2-amino-9-(β--ribofuranosyl)purine (1) derivatives have
been synthesised. The compounds 2–5 were formed in the
reaction of compound 1 with chloroacetaldehyde (a), 2-chloro-
propanal (b), bromomalonaldehyde (c) and chloroacetaldehyde
ϩ malonaldehyde (d) (Scheme 1). The syntheses were straight-
forward but the yields were moderate and most of the starting
materials were recovered unchanged in the reaction mixture.
A 95% yield of etheno deoxyadenosine has been reported in
1a
the reaction of chloroacetaldehyde with deoxyadenosine.
However, with 2-amino-9-(β--ribofuranosyl)purine (1) the
yield was considerably lower, only 14%. For the other reactions
b–d, the yields were slightly higher than for the adenosine
6a,7,10c
reactions with the same reagents.
Consequently it seems
that the reactivity of 2-amino-9-(β--ribofuranosyl)purine (1)
towards these reagents, is much lower than that of adenosine.
The reactions were studied and tested in many different
reaction conditions applying different reaction times, temper-
atures, reactant concentrations and solvents but no increase
of the yields could be achieved. No definitive explanation to
the unreactivity of the starting material (1) can yet be given.
However, this topic is currently under investigation in our
laboratory.
The fluorescence properties of the products were determined
and compared with the starting material 2-amino-9-(β--
ribofuranosyl)purine. The fluorescence properties of the
products 2–5 differed considerably from those of compound 1
and it can be concluded that the “etheno”- or dihydropyridine
ring systems do not enhance the fluorescence characteristics
of 2-amino-9-(β--ribofuranosyl)purine in the same way they
do in the similar adenosine products. Therefore it must be
concluded that the conjugated double bond system of these
product molecules is critically different.
The NMR data were consistent with the structures. The
2
2
linear (1,N -etheno) rather than the angular (N , 3-etheno)
tricyclic structure was determined for compounds 2–4 from the
15
N NMR measurements and the NOESY spectra.
LC-MS analysis of the reaction mixtures revealed the
Fig. 2 Normalised emission (a) and excitation (b) spectra of the
molecules 1–5 in water at 23 ЊC.
presence of low concentrations of products with the same
masses as 2–4 indicating the formation of small amounts of the
angular derivatives (Scheme 4).
The fluorescence measurement results showed, that the
formation of a five membered ring to the 2-amino-9-(β--ribo-
furanosyl)purine changed the fluorescence properties dram-
atically and instead of enhancing the fluorescence the newly
introduced ring-structure quenched it. It can also be noted that
while in ethenoadenosine the fluorescence is decreased by the
Experimental
Chemicals
Chloroacetaldehyde diethyl acetal (99%) was obtained from
Acros Organics, 2-chloropropionaldehyde dimethyl acetal
(>96%) was obtained from Fluka AG and 1,1,3,3-tetra-
1
5
presence of an aldehyde group on the etheno bridge, the alde-
hyde group in 4 increased the quantum yield. This suggests that
8
24
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 8 2 1 – 8 2 7

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methoxypropane (99%) was obtained from Aldrich Chemical
Co. 2-Amino-9-(β--ribofuranosyl)purine was synthesised
according to known methods. The solvents for the synthesis
were of analytical grade and for HPLC of commercial HPLC
grade.
In these experiments, the excitation wavelength was set to 337
nm, and the emission wavelength to 430 nm. The slit width was
set to 5 nm. Analyses of the data were performed with the
software supplied by PTI (Time Master 1.2).
Chromatographic methods
Spectroscopic and spectrometric methods
The HPLC analyses were made on a Kontron Instruments
liquid chromatographic system consisting of a model 322
pump, a 440 diode array detector (UV), a Jasco FP-920
fluorescence detector, and a Kromasystem 2000 data handling
program. The chromatographic separations were performed
on an analytical 5 µm, 4 mm × 125 mm reversed phase C 18
column (Hypersil BDS-C18, Hewlett Packard/Agilent). The
column was eluted isocratically for 5 min with 0.01 M phos-
phate buffer (pH 7.1) and then with a gradient from 0 to 40%
acetonitrile over the course of 25 min at a flow rate of 1 mL
1
13
15
The H-, C- N- and 2D NMR spectra were recorded in
DMSO at 30 ЊC on a JEOL JNM A 500 Fourier transform
NMR spectrometer at 500.16, 125.78 and 50.69 MHz, respect-
1
13
ively. H and C shifts were referenced against DMSO-d
5
1
solvent signal 2.50 ppm and 39.51 ppm, respectively. The H
NMR signal assignments were based on chemical shifts from
the 2D H–H, H–C and H–N correlation spectroscopy data. The
assignment of carbon signals was based on the same techniques
and carbon–proton coupling constants. All nitrogen shifts were
from HMBC spectra and were referenced externally to 90%
Ϫ1
min .
The products were isolated from the reaction mixtures on
flash chromatography columns, packed with 40 µm C 18
reversed-phase silica gel, preparative column. The products
were eluted with an acetonitrile–water gradient.
nitromethane in CD NO2 (0.00 ppm). All inverse detected
HMBC- and HMQC experiments were recorded with an
3
inverse 5 mm probe with z-axis pulse-field gradient capability.
1
16
The H NMR spectra were analysed by PERCH software to
perform complete spectral analyses.
Syntheses
The mass spectrometric analyses were performed on an
Agilent 1100 Series LC/MSD Trap system equipped with an
electrospray source and operated in the positive mode. Nitrogen
was used as nebulizer gas (40 psi) and as drying gas (12 mL
Chloroacetaldehyde (a). A mixture of chloroacetaldehyde
diethyl acetal (5.0 mL, 32 mmol), 1 M HCl (15 mL) and ethanol
(
5.0 mL) was stirred for 2 hours at 70 ЊC. The solution was
Ϫ1
min ). The drying gas was heated to 350 ЊC. The capillary exit
stored at 20 ЊC and used for reactions without further
treatment.
offset had a value of 71.2 V and skim 1 was set at 29.9 V. The
maximum ion accumulation time was 50 ms and the target
value was 50000. Scanning from m/z 100–500 was applied for
the recording of the full mass spectrum.
The electron impact high-resolution mass spectra (EI) were
recorded on Fisons ZABSpec-oaTOF instrument. The spectra
were acquired using a direct insert probe scanning from 50 to
2
-Chloropropanal (b). A mixture of 2-chloropropionaldehyde
dimethyl acetal (5.0 mL, 34 mmol), 1.0 M HCl (15 mL) and
ethanol (5.0 mL) was stirred for 2 hours at 70 ЊC. The solution
was stored at 20 ЊC and used for reactions without further
treatment.
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
Bromomalonaldehyde (c). This reagent was prepared by the
method of Trofimenko.
19
8
000–10000 (at 10% peak height).
The UV spectra of the compounds were recorded with a
Shimadzu UV-160A spectrophotometer.
The fluorimetric properties of the compounds were studied
at 23 ЊC. Steady state fluorescence measurements were per-
2
-Amino-9-(ꢀ-D-ribofuranosyl)purine (1). The preparation of
the compound 1 was performed in two steps starting from
guanosine. In the first step 6-thioguanosine was prepared
according to a method by Kung and Jones. In the second step
-thioguanosine was reduced with Raney nickel according to a
8a
formed on
a
Photon Technology International (PTI)
6
Quantamaster 1 spectrofluorimeter operating in the T-format.
The emission wavelength scans were performed with the excit-
ation 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, to yield a product with a resistance of at
8b
method developed by Fox et al. to yield the starting material 1.
3
-(ꢀ-D-Ribofuranosyl)-imidazo-[1,2a]purine (2). Chloro-
acetaldehyde (8.5 mL, 11 mmol) was added to 1.0 g (3.7 mmol)
of 1 dissolved in 0.5 M KH PO buffer (15 mL, pH 4.5) and
ethanol (10 mL). The reaction mixture was stirred at 37 ЊC for
2 h. The pH was kept at 4.5 by addition of 0.5 M Na HPO4
pH 9.0) buffer solution. The reaction was stopped and the
reaction mixture was neutralised with NaHCO3 (aq.). The
mixture was filtered and the product was isolated from the solid
by preparative reversed phase chromatography. The fractions
containing the product were combined and evaporated to give
oil. The residue was precipitated from dry ethanol (151 mg,
2
4
2
(
2
Ϫ1
least 18.2 MΩcm .
The fluorescence quantum yield of the compounds was
estimated by comparison with the known quantum yield of a
17
standard. The quantum yield standard was quinine sulfate
(
Fluka AG, Switzerland) in 0.1 M H SO which is known to
2 4
17–18
have a quantum yield of 0.53 ± 0.023.
1
(
(
4%) of pure 2 as yellow powder. λ (H O)/nm 229 and 294
max 2
3 Ϫ1 Ϫ1
In order to avoid inner filter effects the optical density was
kept below 0.05 in all measurements. The quantum yield was
calculated according to eqn (1):
ε/dm mol cm 26600 and 5700); λmin(H O)/nm 204 and 261
2
3 Ϫ1 Ϫ1 ϩ
ε/dm mol cm 10000 and 2820); m/z (EI) 291.0966 (M ,
C H N O requires 291.0967), 159 (100%). δ (500.16 MHz;
12
13
5
4
H
a
DMSO) 3.61 (1 H, ddd , J
(1 H, ddd , J5
Ϫ11.9, J5Јb,5Ј-OH 4.8, H-5Јb), 3.73
3.6, H-5Јa), 4.00 (1 H, ddd , J
5Јb,5Јa
2
2
a
a
Q = Q (I/I ) (OD /OD) (n /n )
(1)
R
R
R
R
3.9, J4Ј,5Јb
Јa,5Ј-OH
4Ј,5Јa
a
3
.7, H-4Ј), 4.22 (1 H, ddd , J 4.1, J3Ј,3Ј-OH 4.8, H-3Ј), 4.65 (1 H,
3Ј,4Ј
a
where Q is the quantum yield, I is the integrated intensity OD
ddd , J2 3.3, J2Ј,2Ј-OH 5.3, H-2Ј), 5.11 (1 H, t, 5Ј-OH), 5.24 (1 H,
Ј,3Ј
17
is the optical density, and n is the refractive index. The
subscript R refers to the reference fluorophore of known
quantum yield.
d, 3Ј-OH), 5.56 (1 H, d, 2Ј-OH), 6.00 (1 H, d, J1Ј,2Ј 5.5, H-1Ј),
7.68 (1 H, d, J6,7 1.5, H-6), 7.95 (1 H, d, J7,6 1.5, H-7), 8.79 (1 H,
s, H-2), 9.56 (1 H, s, H-9). δ (125.78 MHz; DMSO) 61.20 (t,
C
1
1
1
The fluorescence decay parameters of the reaction products
JC,H 139, C-5Ј), 70.17 (d, J 151, C-3Ј), 73.13 (d, J 148,
C,H C,H
1 1
were determined using a PTI Timemaster instrument (N laser).
C-2Ј), 85.31 (d, J
148, C-4Ј), 87.15 (d, J 166, C-1Ј),
C,H
2
C,H
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 8 2 1 – 8 2 7
825

View Article Online
1
>1
1
1
1
9
2
10.65 (dd, J 196, JC,H 15, C-7), 126.67 (d, J 190, C-9),
9-(ꢀ-D-Ribofuranosyl)-2-(3,5-diformyl-4-methyl-1,4-dihydro-
C,H
C,H
>
1
1
>1
27.57 (dd, JC,H 13 and 2, C-9a), 133.90 (dd, J 188, JC,H
1-pyridyl)purine (5). 1,1,3,3-Tetramethoxypropane (3.74 g, 17
mmol) was hydrolysed with HCl (50 mL, 0.1 M) and the pH
was adjusted to 4.5. Chloroacetaldehyde diethyl acetal (0.75 g,
5.0 mmol) was added and the mixture stirred for 10 min. The
pH was adjusted again to 4.5. The starting material (1.0 g, 3.7
mmol) was dissolved in 35 mL of water. The solution was
added to the first made mixture of hydrolysed 1,1,3,3-
tetramethoxypropane and chloroacetaldehyde diethyl acetal
and the pH was adjusted to 4.5. The reaction mixture was
stirred at 37 ЊC for 45 h. The reaction was stopped and the
reaction mixture was filtered. The filtrate was evaporated and
C,H
>
1
1
, C-6), 146.51 (ddd, JC,H14; 7 and 4, C-4a), 148.68 (dd, J
14, JC,H 4, C-2), 149.72 (m, JC,H 7; 4 and 2, C-3a). δ (50.69
MHz; DMSO) Ϫ216 (N-3), Ϫ191 (N-8), Ϫ161 (N-4), Ϫ155 (N-
), Ϫ142 (N-1). The signals appear as broad triplets/quartets
C,H
>1
>1
N
a
5
(
JH,H ± 0.3 Hz).
3
-(ꢀ-D-Ribofuranosyl)-5-methylimidazo-[1,2a]purine (3). 2-
Chloropropanal (8.5 mL, 12 mmol) was added to the solution
of 1 (1.0 g, 3.7 mmol) in 0.5 M KH PO (25 mL, pH 4.5) buffer
and ethanol (10 mL). The reaction was allowed to proceed at
7 ЊC for 29 h. The pH was adjusted to 4.5. The reaction was
stopped and the mixture was neutralised with aqueous
NaHCO . The mixture was filtered and the product was
isolated from the solid by preparative reversed phase chromato-
graphy. The fractions containing the product were combined
and evaporated. The oily product was crystallised from dry
ethanol at ϩ8 ЊC to give 3 (195 mg, 17%) as a crystalline light
2
4
methanol added. The product crystallised to give (247 mg, 16%)
3
3
5
as orange crystals. λ (H O)/nm 235, 287, 313 and 369 (ε/dm
max 2
Ϫ1 Ϫ1
mol cm 17600, 16900, 22900 and 5860); λ (H O)/nm 213,
min
2
3
3
Ϫ1
Ϫ1
2
5
2
62, 294 and 349 (ε/dm mol cm 12000, 7780, 15700 and
170); m/z (EI): 401.1337 (M , C H N O requires 401.1335),
ϩ
18
19
5
6
54 (100%), 269 (7). δ (500.16 MHz; DMSO) 1.04 (3 H, d,
H
a
JCH3,4Љ 6.7, – CH ), 3.66 (1 H, d, H-4Љ), 3.67 (1 H, dd , J
Ϫ12.2, H-5Јb), 3.74 (1 H, dd , H-5aЈ), 4.04 (1 H, dd , J
J4Ј,5Јb 3.4, H-4Ј), 4.29 (1 H, dd , J 3.8, H-3Ј), 4.67 (1 H, dd ,
J2Ј,3Ј 5.0, H-2Ј), 6.10 (1 H, dd , J 5.4, H-1Ј), 8.55 (2 H, s, H-
3
5Јb,5Јa
a
a
3.8,
3
Ϫ1
4Ј,5Јa
yellow powder. λ (H O)/nm 231, 296 and 343 (ε/dm mol
max
2
a
a
Ϫ1
3
3Ј,4Ј
cm 35600, 6530 and 2880); λ (H O)/nm 265 and 320 (ε/dm
min
2
a
Ϫ1
Ϫ1
ϩ
1Ј,2Ј
mol
cm
1890 and 2500); m/z (EI) 305.1128 (M ,
2
9
1
1
8
Љ), 8.81 (1 H, s, H-8), 9.08 (1 H, s, H-6), 9.56 (1 H, s, H-5Љ) and
C H N O requires 305.1124), 173 (100%). δ (500.16 MHz;
13
15
5
4
H
1
.57 (1H, s, H-5Љ). δ (125.78 MHz; DMSO) 21.14 (dd, J
a
C
C,H
DMSO) 2.51 (3 H, JCH3,6 0.3, s, – CH ), 3.59 (1 H,ddd , J
3
5Јb, 5Јa
>1
1
1
28, JC,H 6, -CH ), 22.65 (dm, J 133, C-4Љ), 61.07 (t, J
a
3
C,H
C,H
Ϫ12.0, J5
Јb,5Ј-OH
5.8, H-5Јb), 3.71 (1 H, ddd , J
5.1, H-5Јa),
5Јa,5Ј-OH
a
1
1
42, C-5Ј), 70.07 (d, J 148, C-3Ј), 73.70 (d, J 150, C-2Ј),
5.70 (d, J 150, C-4Ј), 87.48 (d, J 166, C-1Ј), 125.17 (2C,
a
C,H
C,H
3
3
.98 (1 H, ddd , J4
4.00, J4Ј,5Јb 4.2, H-4Ј), 4.21 (1 H, ddd , J
Ј,5Јa 3Ј,4Ј
a
1
1
C,H
C,H
.7, J3
Ј,3Ј-OH
5.1, H-3Ј), 4.64 (1 H, ddd , J
H-2Ј), 5.10 (1 H, t, 5Ј-OH), 5.22 (1 H, d, 3Ј-OH), 5.56 (1 H, d,
Ј-OH), 5.98 (1 H, d, J1Ј,2Ј 5.6, H-1Ј), 7.44 (1 H, d, J6,CH3 0.3, H-
), 8.76 (1 H, s, H-2), 9.29 (1 H, s, H-9). δ (125.78 MHz;
2Ј,3Ј
4.7, J2Ј,2Ј-OH 5.9,
>1
>1
dm, JC,H 26, C-3Љ), 132.05 (dd, JC,H 12 and 6, C-5), 140.76
1
>1
1
and 140.81 (2C, dm, J 184,1 JC,H 5, C-2Љ), 145.60 (dd, J
C,H
C,H
2
6
>1
>1
2
1
1
16, JC,H 4, C-8), 149.02 (d, J 186, C-6), 150.07 (dt, JC,H
C,H
C
>1
2 and 3, C-2), 152.18 (ddd, JC,H 8; 3 and 3, C-4), 190.87 and
90.92 (2C, dt, JC,H 176, JC,H 7, C-5Љ). δ (50.69 MHz;
1
1
DMSO) 8.89 (q, J
129,-CH ), 61.23 (t, J
140, C-5Ј),
C,H
3
C,H
1
>1
1
1
N
7
0.20 (d, J
149, C-3Ј), 73.11 (d, J
148, C-2Ј), 85.30 (d,
C,H
>1
C,H
DMSO) Ϫ236 (N-1Љ), Ϫ208 (N-3), Ϫ164 (N-4), Ϫ136 (N-6),
1
1
JC,H 149, C-4Ј), 87.14 (d, J
4; 14 and 7, C-7), 124.27 (d, J 189, C-9), 127.37 (d, JC,H
2, C-9a), 131.44 (ddd, J
164, C-1Ј), 117.68 (ddd, JC,H
C,H
1
a
Ϫ135 (N-1). The signals appear as broad triplets/quartets (J
>1
H,H
1
1
(
1
(
C,H
>1
±
0.3 Hz).
1
186,
J
9 and 4, C-6), 146.07
C,H
C,H
1
>1
>1
dd, JC,H 13 and 4, C-4a), 148.38 (dd, J 214, JC,H 4, C-2),
C,H
>
1
48.83 (dd, JC,H 8 and 6, C-3a). δ (50.69 MHz; DMSO) Ϫ217
N
Acknowledgements
a
N-3), Ϫ190 (N-8), Ϫ161 (N-4), Ϫ156 (N-5), Ϫ141 (N-1). The
We are grateful to Mr Markku Reunanen for recording the
mass spectra. Financial support from the Finnish Graduate
School of Bioorganic and Medicinal Chemistry is gratefully
acknowledged (P. Virta). The work of Peter Mattjus was sup-
ported by the Academy of Finland, Sigrid Jusélius Foundation,
Magnus Ehrnrooth Foundation, Svenska Kulturfonden,
Medicinska understödsföreningen Liv och Hälsa r.f. and Åbo
Akademi.
signals appear as broad triplets/quartets (JH,H ± 0.3 Hz).
3
-(ꢀ-D-Ribofuranosyl)-5-formylimidazo-[1,2a]purine
Bromomalonaldehyde (1.7 g, 11 mmol) was added to the solu-
tion of 1 (0.78 g, 3.0 mmol) in 0.5 M KH PO (35 ml, pH 4.5)
(4).
2
4
buffer and ethanol (15 mL). The reaction was allowed to
proceed at 37 ЊC for 28 h and the pH was adjusted to 4.5. The
reaction was stopped and the mixture was filtered. The product
was isolated from the solid by preparative reversed phase
chromatography. Methanol was added to the syrup and evapor-
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2
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Ј-OH), 5.60 (1 H, s, 2Ј-OH), 6.07 (1 H, d, J1Ј,2Ј 5.2, H-1Ј), 8.67
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1
1
(
d, J
150, C-3Ј), 73.64 (d, J
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48, C-4Ј), 87.44 (d, J 166, C-1Ј), 122.46 (dd, JC,H 31 and
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5, C-7), 127.90 (d, J 193, C-9), 129.03 (dd, JC,H1 12 and 2,
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N
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