Fluorescent 7- and 8-methyl etheno derivatives of adenosine and 6-amino-9-ethylpurine: Syntheses and fluorescence properties

Article abstract of DOI:10.1081/NCN-120018625

Two fluorescent adenosine derivatives (5 and 7) (Sch. 1) and two 6-amino-9-ethyl-purine derivatives (6 and 8) (Sch. 1), were synthesised using 2-chloropropanal and 3-chloropropyne as reagents. The structures of the products were determined by spectroscopi

Full text of DOI:10.1081/NCN-120018625

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Fluorescent 7- and 8-Methyl Etheno Derivatives of
Adenosine and 6-Amino-9-ethylpurine: Syntheses and
Fluorescence Properties
Piritta Virta a c , Toni Holmström a , Tony Munter a , Thomas Nyholm b , Leif Kronberg a &
Rainer Sjöholm a
a Department of Organic Chemistry , Åbo Akademi University , Åbo, Finland
b Department of Biochemistry and Pharmacy , Åbo Akademi University , Åbo, Finland
c Department of Organic Chemistry , Åbo Akademi University , Biskopsgatan 8, Åbo,
FIN-20500, Finland
Published online: 24 Jun 2011.
To cite this article: Piritta Virta , Toni Holmström , Tony Munter , Thomas Nyholm , Leif Kronberg & Rainer Sjöholm (2003)
Fluorescent 7- and 8-Methyl Etheno Derivatives of Adenosine and 6-Amino-9-ethylpurine: Syntheses and Fluorescence
Properties, Nucleosides, Nucleotides and Nucleic Acids, 22:1, 85-98, DOI: 10.1081/NCN-120018625
To link to this article: http://dx.doi.org/10.1081/NCN-120018625
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
Vol. 22, No. 1, pp. 85–98, 2003
Fluorescent 7- and 8-Methyl Etheno Derivatives
of Adenosine and 6-Amino-9-ethylpurine:
Syntheses and Fluorescence Properties
1
1
2,#
Piritta Virta,1, Toni Holmstrom, Tony Munter, Thomas Nyholm,
*
¨
Leif Kronberg, and Rainer Sjoholm
1
1
¨
1Department of Organic Chemistry
˚
2
Department of Biochemistry and Pharmacy, Abo Akademi University,
˚
Abo, Finland
ABSTRACT
Two fluorescent adenosine derivatives (5 and 7) (Sch. 1) and two 6-amino-9-ethyl-
purine derivatives (6 and 8) (Sch. 1), were synthesised using 2-chloropropanal and
3-chloropropyne as reagents. The structures of the products were determined by
spectroscopic and spectrometric methods (1H-, 13C- and 2D NMR, MS, UV and
fluorescence spectrometry). Their fluorescence properties were determined and
found to be similar to those of ethenoadenosine. Also, the stabilites of 5 and 7
in aqueous solutions were determined and found to be higher than that of the
etheno derivative of adenosine.
Key Words: Nucleosides; Fluorescence.
˚
*Correspondence: Piritta Virta, Department of Organic Chemistry, Abo Akademi University,
˚
Biskopsgatan 8, FIN-20500, Abo, Finland; Fax: þ358 2 215 4866; E-mail: piritta.virta@abo.fi.
#Fluorescence measurements.
85
DOI: 10.1081/NCN-120018625
Copyright # 2003 by Marcel Dekker, Inc.
1525-7770 (Print); 1532-2335 (Online)
www.dekker.com

86
Virta et al.
Scheme 1.
INTRODUCTION
Flourescence based techniques are powerful tools for investigation of biological
material. The sensitivity of fluorescence spectroscopy is several orders of magnitude
better than that of UV detection and in some cases even superior to radioactivity
based techniques. Fluorescence can provide information on the structure, distance,
orientation, complexation and location of biomolecules. In addition, time-resolved
methods are used in measurements of molecular dynamics and kinetics. Fluorescent
oligonucleotides and nucleic acids are important tools in molecular biology, diagnos-
tics and structural studies.[1] In order to make oligonucleotides and nucleic acids
fluorescent several methods have been developed. Most of the methods are based
on the covalent attachment of a fluorescent dye molecule to the nucleoside either
at the base or the sugar moiety.[2]
Unlike radioisotopes, dyes may have a negative influence on the structure and
mobility of oligonucleotides and nucleic acids. Even small changes in the conforma-
tion of a nucleic acid can lead to reduced specificity of hybridisation with its target.
One way to minimise this problem is to use only slightly modified fluorescent nucleo-
side analogs as starting materials for the synthesis of oligonucleotides and nucleic
acids with fluorescent properties. Ideally, a fluorescent nucleoside analog should clo-
sely resemble the naturally occurring purine or pyrimidine base structure, especially
at hydrogen bonding sites critical to base pairing. It should also have reasonable
fluorescence under physiological conditions. That is, the developed fluorescent
structures should as far as possible chemically and physically resemble their natural
counterparts.[3]
The most attractive approach is the use of nucleosides which have been made
fluorescent by only slightly modifying their structure. However, in the literature there
are only a few examples of such compounds.[4] The ‘‘etheno-nucleosides’’, represent
fluorescent nucleosides synthesised from non-fluorescent starting materials. They are
formed when adenosine, cytidine or guanosine reacts with chloroacetaldehyde.[4a,5]
The etheno derivatives are commercially available as phosphoramidites. The major
drawback of the etheno derivatives is their gradual degradation upon long-term
storage in aqueous media.[6] However, we found that methyl substituted ethenoder-
ivatives posses more favourable stability properties than the unsubstituted ones.

Fluorescent Derivatives of Adenosine
87
The synthesis of methyletheno derivatives using 2-bromopropanal was reported 20
years ago by Krzyzosiak et al. However, the fluorescence properties and the stability
of the products were not studied. Complete 1H- and 13C NMR data were not
reported either.[5c] The reaction with 2-chloropropanal and adenosine has also been
performed previously but the authors did not determine the structure of the
product.[7] In this paper, we report the synthesis and complete characterisation of
two fluorescent adenosine derivatives (5 and 7) (Sch. 1) and the corresponding 6-
amino-9-ethylpurine derivatives (6 and 8) (Sch. 1). Their fluorescence properties
and stability data for compounds 5 and 7 are also given.
RESULTS AND DISCUSSION
The compounds were synthesised according to the reactions shown in Sch. 2.
The yields of the products (5–8) from 2-chloropropanal and 3-chloropropyne
reactions with 1 and 2 (Sch. 1) were about 10%. Most of the starting material was
recovered unchanged. It was found in both reactions, that besides the main product
the other isomer (7-methyl etheno for 7 and 8 and 8-methyl etheno for 5 and 6) was
formed in very small amounts. It has been shown that in the corresponding deriva-
tives of 2-aminopyrimidine a Dimroth rearrangement takes place leading to isomer-
isation.[5d] However, the methyletheno derivatives of adenine (5 and 6) were found
Scheme 2.

88
Virta et al.
no to undergo isomerisation to 7 and 8 upon storage for two months in aqueous
solutions at room temperature.
The reactions were first performed with 2 as the starting material and after its
successful reaction, the same reaction was performed with 1.
The reactions were studied and tested in many different reaction conditions
applying different reaction times, temperatures (e.g., room temperature, 50ꢀC,
100ꢀC) and solvents (e.g., water, methanol, ethanol, DMF, 2-methoxyethanol,
acetate buffer pH 4.5). The best method for the synthesis of 5 and 6, was to use water
(pH 4-5) as solvent, a temperature of 90ꢀC and 1d reaction time. The best method for
the synthesis of 7 and 8 was to use methanol as solvent, a temperature of 60ꢀC and
10 d reaction time. The same product was also obtained using chloroacetone as
reagent (water solution pH 4-5), but the yield was not any higher. The details of
the procedures are described in the Experimental Section.
The progress of the reactions was monitored by HPLC analysis on a reversed
phase (C 18) column. The products were isolated by preparative reversed phase chro-
matography and characterised by UV absorption (etheno derivatives of adenosine
have a characteristic UV absorption spectrum[8a]),1H-,13C-, and 2D NMR spect-
roscopy, electrospray mass spectrometry and fluorescence spectroscopy. The
fluorescence spectroscopic data are presented in Fig. 1 and Tables 1 and 2. The
NMR spectroscopic and the mass spectrometric data are given in the Experimental
Section.
The formation of these products may be rationalised by the mechanisms
discussed.
Figure 1. Normalised excitation (I) and emission (II) spectra of the 5–8 in water at 23ꢀC.
3 and 4 were used as reference molecules.

Fluorescent Derivatives of Adenosine
Table 1. Fluorescence parameters of the molecules in water at 23ꢀCa
89
Sample
Excitation max (nm)
Emission max (nm)
Quantum yield f
3
4
5
7
6
8
314 ꢁ 0.5
313 ꢁ 1.0
322 ꢁ 0.1
316 ꢁ 0.3
322 ꢁ 0.1
316 ꢁ 0.5
422 ꢁ 0.8
422 ꢁ 0.5
432 ꢁ 0.7
428 ꢁ 0.5
433 ꢁ 0.8
427 ꢁ 0.8
0.540 ꢁ 0.028
0.553 ꢁ 0.041
0.319 ꢁ 0.020
0.438 ꢁ 0.024
0.329 ꢁ 0.013
0.474 ꢁ 0.035
aThe parameters are the average of at least three sets of experimental data. All experiments
were performed using samples with an O.D < 0.05.
Mechanisms
The formation of etheno adenosine from adenosine on treatment with chloroa-
cetaldehyde has been described in the literature.[5a,5b,8] The formation of 5 and 6
from 2-chloropropanal follows a similar mechanism.[5c]
The reaction is initiated by attack of the exocyclic amino group of the nucleo-
base on the carbonyl carbon of 2-chloropropanal. Subsequently, the chlorine is
displaced by intramolecular nucleophilic attack of the ring nitrogen. Finally, the
product is obtained through dehydration of the cyclic intermediate (Sch. 3).
Based on the structures of the reaction products a mechanism for the formation
of compounds 7 and 8 from 3-chloropropyne is suggested in Sch. 4.
A reaction involving an allylic rearrangement is probable and the first step
results in an allene derivative, which then forms the product by cyclisation.
Fluorescence Properties
The fluorescence properties for the four products (5–8) were measured and com-
pared with commercially available ethenoadenosine (3) (Sch. 1) and ethendeoxyo-
Table 2. Fluorescence decay parameters of the molecules in water at 23ꢀCa
Molecule
Lifetimes (ns) t
w2
DW
3
4
5
7
6
8
23.77 ꢁ 0.17
23.17 ꢁ 0.16
17.03 ꢁ 0.08
20.34 ꢁ 0.17
16.84 ꢁ 0.08
21.27 ꢁ 0.01
1.01
1.06
0.944
0.758
0.954
0.895
1.98
1.59
1.78
1.62
2.67
2.09
aThe parameters were calculated using non-linear squared analysis, and the goodness of fit was
characterised using chi-squared (w2), the Durbin Watson parameter (DW), and by evaluating
the randomness of the residual pattern.

90
Virta et al.
Scheme 3.
Scheme 4.
adenosine (4) (Sch. 1) (Fig. 1, Tables 1 and 2). The fluorescence excitation maxima of
3 and 4 were observed at 314 and 313 nm, respectively and fluorescence emission
maxima at 422 nm (Table 1). The maxima were the same as those reported on the
ethenoadenosine.[8a] The methyl-etheno derivatives have fluorescence excitation
maxima at 322 (5 and 6) and 316 nm (7 and 8) and fluorescence emission maxima
at 432 (5), 433 (6), 428 (7) and 427 nm (8) (Table 1). This shows that the Stokes shift
in methyl-etheno derivatives (5–8) is slightly larger than in the etheno derivatives (3
and 4). That is, Stokes shifts are from 10 to 12 nm in 5 to 8 and, 8 and 9 nm in 3 and 4.
The methyl-etheno derivatives (5 to 8) have lower fluorescence quantum yields
(f 0.319–0.474) and shorter lifetimes (16.84–21.27 ns) than ethenoadenosine and
ethenodeoxyadenosine (f 0.540–0.553 and 23.17–23.77 ns) (Tables 1 and 2). The
results show that the type of sugar moiety does not have much effect on the fluores-
cence lifetimes (Table 2). On the other hand, molecules with an ethyl group instead
of the sugar group (6 and 8) have higher fluorescence quantum yield (Table 1).
Stability Tests of 5 and 7
The stability of 5 and 7 was determined and compared with the stability of ethe-
noadenosine (3). It was found that 5 and 7 were most stable at basic (pH 9.0) con-
ditions than the unsubstituted etheno derivatives. At these conditions 7 was found to
be the most stable compound. At neutral conditions (pH 7.4), all tested compounds
were quite stable, but again 7 was the most stable one. At acidic conditions (pH 4.6)
all compounds were more stable than at basic conditions after 8 d, but less stable
than at neutral conditions after 32 d. It can be concluded that both 5 and 7 have

Fluorescent Derivatives of Adenosine
91
Figure 2. Relative amounts of the tested compounds 3, 5, and 7 after 8 d and 32 d. The Y-
axis shows the relative area of the peak observed in the UV-chromatogram at the wavelength
254 nm, and the X-axis shows the time.
comparable or higher stability than 3 and that 7 is the most stable one at neutral or
alkaline conditions (Fig. 2).
It is known that the etheno derivatives of adenosine are not ‘‘fully’’ stable
in water solutions. The major degradation products have been identified as bi-
imidazoles.[9]
NMR Parameters of 5 and 7
1
The H NMR spectrum of 5 displayed besides the signals from the protons of
the ribose moiety, one-proton singlets at d ¼ 8.56 (H-2) and 9.06 ppm (H-5) and a
1
one-proton doublet at d ¼ 7.31 ppm (H-8). The H NMR spectrum of 7 displayed
besides the ribose moiety signals one-proton singlets at d ¼ 8.57 (H-2) and
9.20 ppm (H-5) and a one-proton quartet at d ¼ 7.80 ppm (H-7). The three-proton
doublets for the methyl group appeared at d ¼ 2.57 ppm in 5 and at d 2.38 ppm in
1
1
7. The H NMR spectra of the two isomers differed in many respects. In the H
NMR spectrum of 5 the signal of H-5 was shifted upfield (in comparison to 7), pos-
sibly due to the inductive effect of the methyl group at C-7. Moreover, the signals
of the protons of the methyl group were shifted downfield, probably due to steric
compression.
The 13C NMR spectrum of 5 showed besides the signals from purine and ribose
moieties, carbon signals at d ¼ 120.16 (C-7) and 130.19 ppm (C-8). In the spectrum of
7, the same carbons were observed at 108.41 and 142.17 ppm, respectively. The steric

92
Virta et al.
1
compression observed in the H NMR spectrum is also observed in the relative 13C
NMR shifts of the methyl carbons of the two isomers (d ¼ 8.81 ppm for 5 and
d ¼ 14.12 ppm for 7).
In the H-H correlation spectra both isomers displayed connection between the
methyl protons and H-5. Conclusive evidence for the structures was obtained from
the NOESY spectra. A strong methyl – H-5 interaction was observed in the spectrum
of 5 while this interaction was absent in the spectrum of 7.
CONCLUSIONS
In conclusion, two adenosine and two 3-amino-9-ethylpurine derivatives have
been synthesised. The compounds 5 and 6 were formed in the reaction of 1 and 2
with 2-chloropropanal, and 7 and 8 were formed in the reaction of 1 and 2 with
3-chloropropyne. The syntheses were straightforward but the yields were quite poor
and most of the starting materials were recovered unchanged in the reaction mixture.
The products have been identified and their structures determined. The fluorescent
properties and relative stabilities of the products were determined and compared
with commercially available ethenoadenosine (3) and ethenodeoxyadenosine (4).
The fluorescence properties (quantum yields and lifetimes) are somewhat lower for
the methyl-etheno derivatives than for the corresponding etheno derivatives (3 and
4), but otherwise quite similar to those of 3 and 4. The results show that the
methyl-etheno derivatives are more stable at basic conditions and that 5 is more
stable at acidic conditions than the unsubstituted ethenoadenosine (3).
EXPERIMENTAL
Chemicals
Adenosine (1), 2-chloropropionaldehyde dimethyl acetal and 1,5-diazabicy-
clo[4.3.0]non-5-ene (DBN) were obtained from Fluka AG (Buchs, Switzerland).
6-Amino-9-ethylpurine (2) was synthesised according to a known method.[10] 3-
Chloropropyne (propargyl chloride) was obtained from Aldrich Chemical Co. (Mil-
waukee, Wisconsin, USA). The solvents were of analytical grade. The solvents for
HPLC were of commercial HPLC grade.
Spectroscopic and Spectrometric Methods
1
The H-,13C- and 2D NMR spectra were recorded in DMSO or CDCl3 at 30ꢀC
on a JEOL JNM A 500 Fourier transform NMR spectrometer (JEOL, Tokyo,
1
Japan) at 500.16 and 125.78 MHz, respectively. The H NMR signal assignments
were based on chemical shifts and H-H and C-H correlation data. The assignment
of carbon signals was based on chemical shifts, C-H correlations, and carbon-proton
coupling constants.
The electrospray ionisation mass spectra (EI-MS) and the high resolution
mass spectra (HRMS) were recorded on Fisons ZABSpec-oaTOF instrument

Fluorescent Derivatives of Adenosine
93
(Manchester, U.K.). Ionisation was carried out using nitrogen as both the nebulizing
and bath gas. A potential of 8.0 kV was applied to the ESI needle. The temperature
of the pepperpot counter electrode was 90ꢀC. The isolated component was intro-
duced by loop injection at a flow rate of 20 mL=min (80=20=1 H2O=CH3CN=acetic
acid). Poly(ethyleneglycol) (PEG) was used as the standard for the exact mass
determination. The mass spectrometer was working at a resolution of 7000. The
UV spectra of the isolated compounds were recorded with a diode-array detector
as the peaks eluted from the HPLC column.
The fluorimetric properties of the compounds were studied at 23ꢀC. Steady state
fluorescence measurements were performed on a Photon Technology International
(PTI) Quantamaster 1 spectrofluorimeter operating in the T-format. The emission
wavelength scans were performed with the excitation wavelength set at 320 nm. Exci-
tation 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 resistivity
of 18.2 MOcm.
The fluorescence quantum yield of the compounds was estimated by comparison
with the known quantum yield of a standard.[11] The quantum yield standard was
quinine sulfate (Fluka AG, Switzerland) in 0.1 M H2SO4 which is known to have
a quantum yield of 0.53 ꢁ 0.023.[11,12]
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,
2
Q ¼ QRðI=IRÞðODR=ODÞðn2=nR
Þ
where Q is the quantum yield, I is the integrated intensity, OD is the optical density,
and n is the refractive index.[11] The subscript R refers to the reference fluorophore of
known quantum yield.
The fluorescence decay parameters of the reaction products were determined
using a PTI Timemaster instrument (N2 laser). 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. Analysis of the data were performed with the software supplied by
PTI (Time Master 1.2).
Chromatographic Methods
The HPLC analyses were made on a Kontron Instruments liquid chromato-
graphic system consisting of a model 322 pump, a 440 diode array detector (UV)
(Kontron Instruments S. P. A., Milan, Italy), a Jasco FP-920 fluorescence detector
(Jasco Corporation, Tokyo, Japan), and a Kromasystem 2000 data handling pro-
gram (Kontron Instruments S. P. A., Milan, Italy). The chromatographic separa-
tions were performed on an analytical 5 mm, 4 mm ꢂ 125 mm reversed phase C 18
column (Hypersil BDS-C18, Hewlett Packard, Espoo=Esbo, Finland). The column
was eluted isocratically for 5 min with 0.01 M phosphate buffer (pH 7.1) and then
with a gradient from 0 to 50% acetonitrile over the course of 25 min at a flow rate
of 1 mL=min.

94
Virta et al.
The products were isolated from the reaction mixtures by chromatography on a
manually packed 40 mm, 4 cm ꢂ 8 cm C 18 preparative column (Bondesil, Analyti-
chem International, Harbor City, California, USA). The column was eluted with
100 ml of water and then with 100 mL of 2.5%, 5%, 7.5%, 10%, 12.5%, 15%,
17.5%, 25% and 40% acetonitrile solutions in water.
The Stability Determinations
The stability determinations were done at 37ꢀC at three different pH conditions
(4.6, 7.4 and 9.0), using 0.1 M phosphate buffer as the solvent. The degradation of
methyl-etheno products of adenosine (5 and 7) and ethenoadenosine (3) were mon-
itored by HPLC using the analytical RP C 18 column.
Preparation of 7-Methyl-3-(b-D-ribofuranosyl) imidazo[2,1-i ]purine (5).
2-Chloropropionaldehyde dimethyl acetal (6.3 mL, 48 mmol) was hydrolysed with
0.1 M HCl (150 mL) at 90ꢀC for 30 min. The pH was adjusted to 4–5 with 4 M
sodiumhydroxide and adenosine (1) (6.5 g, 24 mmol) was added to solution. The
reaction was allowed to proceed for 1 d at 90ꢀC. The solution was cooled down
to room temperature and then evaporated to dryness at 40ꢀC in a rotary eva-
porator. The product was isolated from the reaction mixture by preparative
reversed phase chromatography. The fractions containing the product were com-
bined and evaporated to dryness. The product was obtained as a white solid.
The yield was ca. 10%. The purified product had the following spectral charac-
teristics: UVmax 230 nm, 268 nm, 278 nm and 301 nm, UVmin 250 nm and 289 nm
(HPLC eluent, 26% acetonitrile in 0.01 M phosphate buffer, pH 7.1). The fluor-
escence excitation and emission maxima were observed at 322 nm and 432 nm,
respectively.
In the positive ion electrospray mass spectra (EI-MS), the following ions were
observed [IP 70 eV; m=z (% r. a.) ]: 305 (10, Mþ), 173 (100, Mþ -Rib). High resolu-
tion mass spectrometry (HRMS) gave the protonated molecular formula as
C13H15N5O4 (MHþ 305.1124 gmolꢃ1, calculated mass 305.1124 gmolꢃ1). The fluor-
escence characteristic data of 5 are presented in Tables 1 and 2 and Fig. 2.
1H NMR (500.16 MHz, DMSO) : d ¼ 9.06 (s, 1H, H-5), 8.56 (s, 1H, H-2), 7.31
(d, 1H, H-8, J ¼ 1.0 Hz), 6.07 (d, 1H, H-10, J ¼ 5.6 Hz), 5.55 (d, 1H, 20-OH,
J ¼ 5.7 Hz), 5.26 (d, 1H, 30-OH, J ¼ 5.0 Hz), 5.10 (t, 1H, 50-OH, J ¼ 5.5 Hz), 4.61
(t, 1H, H-20, J ¼ 5.3 Hz), 4.21 (q, 1H, H-30, J ¼ 4.1 Hz), 4.00 (q, 1H, H-40, J ¼ 4.0 Hz),
3.72 (dd, 1H, H-50, J ¼ 11.9 and 4.6 Hz), 3.61 (ddd, 1H, H-500, J ¼ 11.9; 5.5 and
4.3 Hz), 2.57 (d, 3H, -CH3, J ¼ 1.0 Hz).
>1
13C NMR (125.78 MHz, DMSO) : d ¼ 140.14 (ddd, C-10a,
J
4 Hz), 138.02 (ddd, C-3a,
11; 5 and
C,H
1
1 Hz), 139.81 (dd, C-2, JC,H 214 Hz,
>1
J
214 Hz,
>1
J
12;
C,H
C,H
>1
>1
1
6 and 3 Hz), 135.42 (d, C-5,
>1
J
4 Hz), 122.92 (d, C-10b,
J 1 Hz), 130.19 (dq, C-8, JC,H
C,H
11 Hz), 120.16 (dq, C-7,
C,H
>1
>1
187 Hz,
J
J
J
16
C,H
C,H
C,H
1
1
and 7 Hz), 87.80 (d, C-10, JC,H 166 Hz), 85.56 (d, C-40, JC,H 149 Hz), 74.11 (d, C-
1
1
1
20, JC,H 147 Hz), 70.31 (d, C-30, JC,H 148 Hz), 61.32 (t, C-50, JC,H 140 Hz), 8.81
1
(q, -CH3, JC,H 129 Hz).

Fluorescent Derivatives of Adenosine
95
Preparation of 3-Ethyl-3H-7-methyl imidazole[2,1-i]purine (6). Compound 6
was prepared as described for 5. 2-Chloropropionaldehyde dimethylacetal (2.4 mL,
18 mmol), 0.1 M HCl (60 mL) and 6-amino-9-ethylpurine (2) (1.5 g, 9.1 mmol). The
product was obtained as a white solid. The yield was ca. 10%. The purified product
had the following spectral characteristics: UVmax 232 nm, 270 nm, 280 nm and
301 nm, UVmin 252 nm and 289 nm (HPLC eluent, 32% acetonitrile in 0.01 M phos-
phate buffer, pH 7.1). Fluorescence excitation and emission maxima were observed
at 322 nm and 433 nm, respectively.
In EI-MS the following ions were observed [IP 70 eV; m=z (% r. a.) ]: 201 (100,
Mþ), 186 (4, Mþ-Me), 172 (20, Mþ-Et). HRMS gave the protonated molecular for-
mula as C10H11N5 (MHþ 201.1016 gmolꢃ1, calculated mass 201.1014 gmolꢃ1).
1H NMR (500.16 MHz, DMSO) : d ¼ 9.00 (s, 1H, H-5), 8.31 (s, 1H, H-2), 7.28
(d, 1H, H-8, J ¼ 1.2 Hz), 4.37 (q, 2H, -CH2CH3 J ¼ 7.3 Hz), 2.56 (d, 3H, -CH3,
J ¼ 0.9 Hz), 1.48 (t, 3H, -CH2CH3, J ¼ 7.3 Hz).
1
>1
13C NMR (125.78 MHz, DMSO) : d ¼ 140.55 (dt, C-2, JC,H 211 Hz,
J
C,H
>1
>1
4 Hz), 140.40 (ddd, C-10a,
and 3 Hz), 134.89 (dd, C-5, JC,H 213 Hz,
J
12; 5 and 1 Hz), 138.17 (ddd, C-3a,
J
J
12; 6
C,H
C,H
1
>1
1
1 Hz), 129.93 (dq, C-8, JC,H
C,H
>1
>1
>1
187 Hz,
and 7 Hz), 38.72 (tq, -CH2CH3, JC,H 141 Hz,
J
5 Hz), 122.53 (d, C-10b,
1
J
11 Hz), 119.77 (dq, C-7, J
>1
5 Hz), 15.49 (qt, -CH2CH3,
15
C,H
C,H
C,H
J
C,H
1JC,H 128 Hz,
J
3 Hz), 8.79 (q,-CH3).
C,H
>1
Preparation of 8-Methyl-3-(b-D-ribofuranosyl) imidazo[2,1-i ]purine (7). Adeno-
sine (1) (6.5 g, 24 mmol) was dissolved in methanol and 3-chloropropyne (14 mL,
195 mmol) and DBN (6.0 mL, 49 mmol) were added to the solution. The mixture
was stirred 10 days at 60 ꢀC. The solution was cooled down to room temperature
and then evaporated to dryness at 40ꢀC in a rotary evaporator. The product was
isolated from the reaction mixture by preparative reversed phase chromatography.
The fractions containing product were combined and evaporated to dryness. The
product was obtained as a white solid. The yield was ca. 10%.
The purified product had the following spectral characteristics: UVmax 230 nm,
268 nm, 278 nm and 301 nm, UVmin 250 nm and 289 nm (HPLC eluent, 26% aceto-
nitrile in 0.01 M phosphate buffer, pH 7.1). Fluorescence excitation and emission
maxima were observed at 316 nm and 428 nm, respectively.
In EI-MS the following ions were observed [IP 70 eV; m=z (% r. a.)]: 305 (7, Mþ),
173(21, Mþ, -Rib).). HRMS gave the protonated molecular formula as C13H15N5O4
(MHþ 305.1122 gmolꢃ1, calculated mass 305.1124 gmolꢃ1).
The fluorescence characteristic data of 7 are presented in Tables 12 and Figure 2.
1H NMR (500.16 MHz, DMSO) : d ¼ 9.20 (s, 1H, H-5), 8.57 (s, 1H, H-2), 7.80
(q, 1H, H-7, J ¼ 1.0 Hz), 6.07 (d, H-1’, J ¼ 5.6 Hz), 5.60 (br, 1H, 2’-OH), 5.30 (br,
1H, 30-OH), 5.13 (br, 1H, 50-OH), 4.63 (t, 1H, H-20, J ¼ 5.3 Hz), 4.24 (dd, 1H, H-
30, J ¼ 3.8 and 4.6 Hz), 4.03 (q, 1H, H-40, J ¼ 3.9 Hz), 3.74 (dd, 1H, H-50, J ¼ 12.1
and 3.8 Hz), 3.63 (dd, 1H, H-500, J ¼ 12.1 and 4.1 Hz), 2.38 (d, 3H, -CH3, J ¼ 1.0 Hz)
>1
13C NMR (125.78 MHz, DMSO) : d ¼ 142.17 (dq, C-8,
J
JC,H 6 Hz), 139.83 (dd, C-2, JC,H 214 Hz, 4 Hz), 138.43 (ddd,
9 and 7 Hz),
C,H
>1
1
140.06 (t, C-10a,
1
C-3a, JC,H 13; 5 and 3 Hz), 136.35 (d, C-5, JC,H 214 Hz,
1
>1
J
1 Hz), 122.48 (d,
C,H
5 Hz), 87.78 (d C-10,
>1
1
12 Hz), 108.41 (dq, C-7, JC,H 195 Hz,
>1
C-10b,
J
J
C,H
C,H

96
Virta et al.
1
1
1JC,H 166 Hz), 85.56 (d C-40, JC,H 148 Hz), 74.11 (d C-20, JC,H 148 Hz), 70.31 (d
1
1
1
C-30, JC,H 149 Hz), 61.31 (t C-50, JC,H 141 Hz), 14.12 (q -CH3, JC,H 127 Hz).
Preparation of 3-Ethyl-3H-8-methyl imidazo[2,1-i]purine (8). Compound 8 was
prepared as described for 7. 6-amino-9-ethylpurine (2) (1.5 g, 9.1 mmol), 3-chloro-
propyne (5.3 mL, 74 mmol) and DBN (2.3 mL, 49 mmol). The product was obtained
as a white solid. The yield was ca. 10%.
The purified product had the following spectral characteristics: UVmax 232 nm,
270 nm, 280 nm and 301 nm, UVmin 252 nm and 289 nm (HPLC eluent, 32% aceto-
nitrile in 0.01 M phosphate buffer). Fluorescence excitation and emission maxima
were observed at 316 and 427 nm, respectively.
In EI-MS the following ions were observed [IP 70 eV; m=z (% r. a.) ]: 201 (100,
Mþ), 186 (13, Mþ-Me), 173 (35, Mþ-Et). HRMS gave the protonated molecular for-
mula as C10H11N5 (MHþ 201.1015 gmolꢃ1, calculated mass 201.1014 gmolꢃ1).
1H NMR (500.16 MHz, CDCl3) : d ¼ 8.71 (s, 1H, H-5), 7.93 (s, 1H, H-2), 7.42 (q,
1H, H-7, J ¼ 1.0 Hz), 4.33 (q, 2H, -CH2CH3, J= 7.3 Hz), 2.48 (d, 3H, -CH3,
J ¼ 1.0 Hz), 1.57 (t, 3H, -CH2CH3, J ¼ 7.3 Hz).
>1
13C NMR (125.78 MHz, CDCl3) : d ¼ 143.74 (dq, C-8,
J
9 and 7 Hz),
C,H
>1
1
141.28 (t, C-10a,
(C-3a), 134.44 (dd, C-5, JC,H 210 Hz,
J
6 Hz), 139.79 (dt, C-2, JC,H 209 Hz ,>1JC,H 4 Hz), 139.05
C,H
1
>1
>1
J
1 Hz), 123.39 (d, C-10b,
5 Hz), 39.38 (tq, -CH2CH3, JC,H
J
C,H
C,H
1
11 Hz), 107.14 (dq, C-7, JC,H 192 Hz,
>1
1
J
C,H
>1
1
5 Hz), 15.75 (qt, -CH2CH3, JC,H 128 Hz,
>1
141 Hz,
1
J
CH3, JC,H 127 Hz).
J
3 Hz), 14.40 (q, -
C,H
C,H
ACKNOWLEDGMENTS
This project was financed by TEKES (The National Technology Agency). We
are grateful to Mr. Markku Reunanen for the mass spectra.
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Received June 11, 2002
Accepted November 11, 2002