Ring-opening of 3-β-D-ribofuranosyl-3,7,8,9-tetrahydropyrimido [1,2-i]purin-8-ol and preparation of 2-thio- and 2-aza-adenosine derivatives

Article abstract of DOI:10.1080/15257770802458162

The adduct 3-β-D-ribofuranosyl-3,7,8,9-tetrahydropyrimido[1,2-i]purin- 8-ol (2), obtained from adenosine and epichlorohydrin, underwent ring fission at basic conditions. The initial ring-opening took place at C2 of the pyrimidine unit resulting in 2-(5-amino-1-β-D-ribofuranosyl-imidazol-4-yl)-1,4,5,6- tetrahydropyrimidin-5-ol (3). Also the tetrahydropyrimidine ring of 3 could be opened resulting in 5-amino-1-(β-D-ribofuranosyl)-imidazole-4-(N-3-amino-2- hydroxyl-propyl)-carboxamide (4). In hot acid conditions, 2 was both deglycosylated and ring-opened yielding 2-(5-amino-imidazol-4-yl)-1,4,5,6- tetrahydropyrimidin-5-ol (7) as the final product. When reacting 3 with CS2 or HNO2 ring-closure took place and 3-β-D-ribofuranosyl-3,4,7,8,9- pentahydropyrimido[1,2-i]purin-8-ol-5-thione (5), and 3-β-D-ribofuranosyl- imidazo[4,5-e]-3,7,8,9-tetrahydropyrimido[1,2-c][1,2,3]triazine-8-ol (6), respectively, were obtained. Also, the pyrimidine ring of the epichlorohydrin adduct with adenine, 10-imino-5,6-dihydro-4H,10H-pyrimido[1,2,3-cd]purin-5-ol (10), underwent ring fission and the product was identified as 3-hydroxy-1,2,3,4-tetrahydroimidazo[1,5-a]pyrimidine-8-carboximidamide (11). Copyright Taylor & Francis Group, LLC.

Full text of DOI:10.1080/15257770802458162

Nucleosides, Nucleotides and Nucleic Acids, 27:1215–1226, 2008
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770802458162
RING-OPENING
OF 3-β-D-RIBOFURANOSYL-3,7,8,9-TETRAHYDROPYRIMIDO
[1,2-i]PURIN-8-OL AND PREPARATION OF 2-THIO- AND
2-AZA-ADENOSINE DERIVATIVES
Pernilla Sund and Leif Kronberg
˚
˚
Laboratory of Organic Chemistry, Abo Akademi University, Turku, Abo, Finland
The adduct 3-β-D-ribofuranosyl-3,7,8,9-tetrahydropyrimido[1,2-i]purin-8-ol (2), obtained from
2
adenosine and epichlorohydrin, underwent ring fission at basic conditions. The initial ring-opening
took place at C2 of the pyrimidine unit resulting in 2-(5-amino-1-β-D-ribofuranosyl-imidazol-4-yl)-
1,4,5,6-tetrahydropyrimidin-5-ol (3). Also the tetrahydropyrimidine ring of 3 could be opened re-
sulting in 5-amino-1-(β-D-ribofuranosyl)-imidazole-4-(N-3-amino-2-hydroxyl-propyl)-carboxamide
(4). In hot acid conditions, 2 was both deglycosylated and ring-opened yielding 2-(5-amino-
imidazol-4-yl)-1,4,5,6-tetrahydropyrimidin-5-ol (7) as the final product. When reacting 3 with
CS2 or HNO2 ring-closure took place and 3-β-D-ribofuranosyl-3,4,7,8,9-pentahydropyrimido[1,2-
i]purin-8-ol-5-thione (5), and 3-β-D-ribofuranosyl-imidazo[4,5-e]-3,7,8,9-tetrahydropyrimido[1,2-
c][1,2,3]triazine-8-ol (6), respectively, were obtained. Also, the pyrimidine ring of the epichloro-
hydrin adduct with adenine, 10-imino-5,6-dihydro-4H,10H-pyrimido[1,2,3-cd]purin-5-ol (10),
underwent ring fission and the product was identified as 3-hydroxy-1,2,3,4-tetrahydroimidazo[1,5-
a]pyrimidine-8-carboximidamide (11).
Keywords Adenosine; ring opening; adduct; epichlorohydrin
INTRODUCTION
The ring-opening of 1,N 6-etheno adenine derivatives has been studied
by Baker and Joseph,[1] Montgomery,[2] Tsou,[3] and others.[4] They used
the reaction for the preparation of 2-aza-1,N 6-etheno-adenosine from 1,N 6-
etheno-adenosine. Since then, adenine ring opening reaction has been
extensively studied and used for various synthetical purposes.[5,16] For
example, Koomen describes aminolytic ring opening,[6] and a way to use
Received 23 June 2008; accepted 20 August 2008.
Financial support for this work was obtained from Magnus Ehrnrooth Foundation, Finland.
˚
Address correspondence to Leif Kronberg, Laboratory of Organic Chemistry, Abo Akademi
˚
University, Biskopsgatan 8, 20500 Turku/Abo, Finland. E-mail: leif.kronberg@abo.fi
1215

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P. Sund and L. Kronberg
the fact that amine ring opening is reversible to avoid permanent ring
opening at basic conditions.[7] Adenine ring opening also occurs in the
biosynthesis of 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR),
the source of imidazole-moietes for purines and histidine in the cell. A
chemical simulation of the biochemical reaction path has been developed
by Ranganathan.[8] Ring opening is also one of the possible fates of the
1,N 6-ethenoadenine adduct in DNA.[9]
The ring-opening mechanism has the first step in common with the
Dimroth rearrangement, and was originally observed in 1888 by Rathke,
and in 1909 Dimroth provided a mechanistic explanation for the reaction.
To our knowledge, the ring-opening of 1,N 6-hydroxypropanoadenosi-
nes has not been studied previously. In the current work, we have explored
the ring-opening and utilized the reaction for the preparation of 2-thio and
2-azaadenosine derivatives.
RESULTS AND DISCUSSION
The preparation of 2 has earlier been described by Solomon[10] and
Sund and Kronberg.[11] In the current work, the epichlorohydrin reaction
was performed at pH 3.2 to avoid further attack by epichlorohydrin on 2.[11]
When 2 is held at pH 9 and 60◦C for a few days, ring opening occurs at
C2, giving compound 3. The symmetry of the formed tetrahydropyrimidine
1
ring was evident from the H NMR spectrum where the two axial H10
protons exhibited equivalent shifts. This was also found to be the case of
the equatorial protons. Also, the H2 signal has disappeared, and C6 (δ =
152.3 ppm) shows correlations to H10a and H10b.
When 2 is held at pH 12 and 60◦C for 24 hours, the initial ring-opening
is followed by a second ring-fission, which involves the hydroxypropano-
1
ring, giving 4. The H NMR spectrum of the imidazole derivative shows
the presence of an amide proton signal at δ = 7.46 ppm and separate
signals for H10 (δ = 3.25 and 3.21 ppm) and H12 (δ = 2.65 and 2.54
ppm), that is, the propano-moiety is no longer symmetric. Comparision
of the 13C NMR spectrum to that of a similar compound, 5-amino-1-(β-
D-2ꢁ-deoxyribofuranosyl)imidazole-[4-15N]-carboxamide), also supports this
interpretation.[19] The mechanism for the hydrolysis is thought to occur by
an attack of a hydroxide ion on C6 with subsequent ring-opening between
C6 and one of the tetrahydropyrimidine nitrogens.[12]
It was also found that treatment of 2 with 20% H3PO4 at 90◦C gives
the aglycon 7. Compound 7 could also be obtained by deglycosylation at
low pH of deoxy-3, the 2ꢁ-deoxyribose analogue of 3 (which was obtained
in the same way as 3). Acidic ring-opening of ethenoadenosine and 1-
methyladenosine has been previously described in the litterature.[13]

Adduct Ring Fission
1217
When 3 was treated with CS2 or HNO2, ring-closure took place and
2-thio- and 2-azaadenosine derivatives were obtained. In the product 5,
the symmetry of the propano-moiety was lost and a new carbon signal
at δ = 175.6 ppm was observed. The carbon signal was assigned to C2
based on a HMBC-correlation to H10a (δ = 4.97 ppm) in the propano-
moiety. The 13C NMR shift of C2 is consistent with that of a thioamide
(compared to 1,3-dimethylthiourea and 2-imidazolidinethione; SDBSWeb
at http://www.aist.go.jp/RIODB/SDBS/, National Institute of Advanced In-
dustrial Science and Technology). The broad peak in the 1H NMR spectrum
at δ = 9.16 ppm could be attributed to the N3-thioamide proton.
The product from ring-closure by HNO2 is compound 6. High-
resolution MS confirms the presence of six nitrogens in the structure. The
1H NMR spectrum shows separate signals for H10 (δ = 4.30 and 4.20 ppm)
and H12 (δ = 3.49 and 3.33 ppm).
In the reaction of epichlorohydrin and adenine at neutral conditions,
8 and 9 are formed.[14] In the current work, it was found that 10 can be
obtained by ring-closure of 8 and 9 at the same conditions. The product of
ring-opening of 10 at neutral conditions is 11. In the NMR spectra of the
compound, the carbon signal of C2 is missing and the signals of C4 and C8
shows correlations to protons in the propane moiety. However, there is also
the possibility that C8 is eliminated by ring opening, as this would also fit 2D
NMR data. This possibility could be excluded since the 13C NMR shifts of 11
is very similar to shifts reported for analogous compounds[15,16] and since
the UV spectrum of 11 have roughly the same features as the spectrum of
aglycon 7, indicating similar conjugation in both compounds.
CONCLUSIONS
The initial steps of both Dimroth rearrangement and all ring openings
presented in this paper (and even deaminations of adenosine analogs), is
an amidine hydrolysis. In the unmodified purine core of adenosine and
adenine, the amidine group at N7-C8-N9 is the target for the hydrolysis
and the final outcome is opening of the imidazole ring.[17] But when
one of the N1- or N3-nitrogens of the pyrimidine ring is alkylated, as
in the case of 2, the electrophilic amidine-group at N1-C2-N3 becomes
the preferred target for the hydrolysis and opening of the pyrimidine
ring becomes possible. Generally, the amidine hydrolysis is initiated by a
hydroxide attack on the protonated starting material. As it is known that
1-hydroxyisoguanine also undergoes ring opening, the electrophilic target
may also be a hydroxycarbamide function.[18]
In this work, several adenosine and adenine analogs were made by ring
opening of epichlorohydrin adducts 2 and 10. In the case of compound

1218
P. Sund and L. Kronberg
3, the ring can be reclosed by CS2 or HNO2, yielding 2-thio- and 2-
azaadenosine derivatives.
MATERIALS AND METHODS
(Caution: Epichlorohydrin has been found to be carcinogenic in mice
and rats and has been classified as a probable human carcinogen.)
Chemicals
Distilled water purified with a Millipore system (Simplicity 185, USA)
was used in the reactions and for all chromatography. The epichlorohydrin
was racemic. Adenosine (Adenine-9-β-D-ribofuranoside) was purchased
from Fluka AG (Switzerland). Deoxyadenosine (Adenine-9-β-D-2’-deoxyri-
bofuranoside) was purchased from Sigma-Aldrich Chemie (Germany).
Chromatographic Methods
Analytical HPLC was performed on an Agilent 1100 series liquid chro-
matograph equipped with a quaternary pump, a diode array detector (used
wavelength range: 190 to 400 nm) and the Chemstation software. The
column employed was a Hypersil BDS-C18, 5µm, 4.0 × 125 mm. It was
eluted isocratically for 2 minutes with 2% acetonitrile in 10 mM ammonium
acetate (pH 7) and then with a gradient from 2 to 20% over the course of
18 minutes at a flow rate of 0.5 mL/minute. The UV spectra were recorded
while the compounds eluted from the column.
Semipreparative HPLC was performed on a Varian 5000 liquid chro-
matograph equipped with a UV detector working at 254 nm or on the
Agilent 1100 system (see above) equipped with an Agilent fraction collector.
Spectroscopic and Spectrometric Methods
The mass spectrometry analyses were performed on an Agilent 1100
Series LC/MSD SL Trap system (Agilent Technologies, Finland), which was
equipped with an API electrospray interface and operated in the positive
ion mode. Nitrogen was used as nebulizer gas (40 psi) and as drying gas
(12 L/minute). The drying gas was heated to 350◦C. The capillary exit
offset had a value of 118.0 V and skim 1 was set at 40.0 V. In the scan
mode, the maximum ion accumulation time was 10 ms and the target
(ion) value was 20,000. Scanning from m/z 100 to 500 was applied for
recording of the full scan mass spectra. Collision induced dissociation
(CID) experiments coupled with multiple tandem mass spectrometry (MSn)
employed helium as collision gas. The fragmentation amplitude was 1.0 V.
An Agilent 254 nm wavelength UV detector was also coupled to the system,

Adduct Ring Fission
1219
and the LC conditions were identical to the analytical HPLC described
above, with the exception of the pump, which was of binary type. The
high-resolution mass spectrum for compound 6 was recorded on a LC/MSD
TOF mass spectrometer (Agilent G1969, USA). The system was operated in
the positive ion mode, and it was tuned and calibrated using the automated
functions, CheckTune and Calibrate, using Agilent TOF Electrospray Cal-
ibrant Solution (G1969–85001). The pressure of the nebulizer gas was 35
psig and the drying gas flow was 12 L/minute. The drying gas was heated to
300◦C. Scanning from m/z 100 to 1000 was applied for recording of the full
scan mass spectra. The fragmentor voltage was 150 V, skimmer voltage was
60 V and Oct RF was 250 V. The infusion rate was 10 µL/minute, and masses
were measured as an average of 11 measurements, with reference masses at
121.050873 and 922.009798. The high resolution mass spectra for all other
compounds were recorded on an Agilent 6220 Accurate-Mass TOF mass
spectrometer using ESI ionization in conjuction with HPLC, and operated
in positive ion mode. The Sciex software from Perkin-Elmer was utilized.
To prepare NMR samples, the solutions of the pure compounds were
evaporated to dryness, then residual water was removed by coevaporation
twice with 5 mL acetonitrile. DMSO-d6, 800 µL, was added and the solutions
were filtered trough a coarse-porosity sintered glass filter into NMR tubes.
The NMR spectra were recorded on a Bruker Avance 600 spectrometer
1
operating at 600.13 MHz for H and 150.90 MHz for 13C (Bruker Biospin
Corporation). The solvent was DMSO-d6, residual non-deuterated solvent
1
was used as internal reference for H NMR (at 2.50 ppm). For 13C NMR
1
the solvent signal was used as reference (at 39.5 ppm). The H NMR
1
signal assignments were based on chemical shifts and H-1H and 13C-1H
correlation data. Assignment of carbon signals was based on chemical shifts
and 13C-1H correlations.
Calculation of Yields
1
Product yields were determined in the following way: Quantitative H
NMR was performed, using residual undeuterated solvent as an internal
standard (the concentration of undeuterated solvent was determined for
the solvent lot used). HPLC standard solutions were prepared by diluting
aliquots from the NMR samples. The standards and the reaction mixtures
were analyzed by HPLC, and the peak areas in standards and reaction
mixtures were compared at 254 nm.
3-β-D-ribofuranosyl-3,7,8,9-tetrahydropyrimido[1,2-i]purin-8-ol (2)
Adenosine, (4 mmol, 1070 mg) was dissolved in 0.5 M potassium
phosphate buffer (50 ml) at pH 3.2 (pH was adjusted with phosphoric acid).
Epichlorohydrin, (3.2 ml, 35 mmol), was added and the mixture was stirred
and held at 37◦C for 2 days. Excess epichlorohydrin was removed by rotary

1220
P. Sund and L. Kronberg
evaporation, giving an oily and crystalline residue, which was suspended in
water (15 ml) and pH adjusted to 6.5 with 6 M sodium hydroxide solution.
The mixture was again rotary evaporated, and coevaporated three times
with dry methanol (20 ml). To separate phosphate salts, the residue was
washed three times with dry methanol (15 ml, each time vigorously shaken),
and the washings combined and evaporated to an oil. Dry acetone (20 ml)
was added (causing precipitation) and the mixture vigorously stirred until
the precipitate was evenly distributed. The mixture was centrifuged and
the supernatant was discarded (containing epichlorohydrin hydrolysates).
The precipitate was washed with acetone in this way three times. The white
precipitate was dissolved in dry methanol (3 ml), and purified on a short
silica column, eluted with a solvent gradient from acetone to methanol
to water, all eluents contained 10 mM TFA. The product still contained
some epichlorohydrin hydrolysis products, but was usable for the next step.
Yield 50% (of pure product, excluding contaminations). MS(ESI): m/z 324
(100%, M+H+).
2-(5-amino-1-β-D-ribofuranosyl-imidazol-4-yl)-1,4,5,6-tetrahydropyrimidin-
5-ol (3)
A solution of compound 2 (622 mg, 2 mmol) in water (15 ml) was held
at pH 9 and 60◦C for 2 days. The pH was adjusted with 6 M NaOH each
hour in the beginning of the reaction, then twice daily. The yield before
purification was 26%. The product was purified by repeated semipreparative
HPLC runs on the Varian LC, the column was a Thermo Hypersil Keystone
(BDS Hypersil C18), 250 × 10 mm, 5 µm particle size, the buffer was 10
mM ammonium acetate at pH 7 with an acetonitrile gradient. The flow rate
was 3 ml/minutes. UV spectrum: λmax 224, 225 (sh), 283 nm, λmin 249 nm,
1
ε = 5000 M−1cm−1. H NMR (DMSO-d6): δ = 7.52 (s, 1 H, H8); 5.54 (d, J
= 5.4 Hz, 1 H, H1ꢁ); 4.24 (t, J = 5.5 Hz, 1 H, H2’); 4.05 (t, J = 4.2 Hz, 1
H, H3’); 3.99 (m, 1 H, H11); 3.90 (m, J = 2.6 Hz, 1 H, H4’); 3.59 (qd, J =
11.5 and 2.6 Hz, 2 H, H5a’ and H5b’); 3.39 (br d, J = 12.3 Hz, 2 H, 2 ×
H10a); 3.21 (br d, J = 12.3 Hz, 2 H, 2 × H10b). 13C NMR (DMSO-d6): =
152.3 (C6); 141.8 (C4); 131.3 (C8); 107.4 (C5); 87.8 (C1’); 73.4 (C2’); 70.1
(C3’); 57.7 (C11); 85.7 (C4’); 60.9 (C5’); 44.81 and 44.82 (C10). MS(ESI):
m/z 314 (100%, M+H+). MS2 of m/z 314: 182 (100%, M+H+-ribosyl+H).
MS3 of m/z 314→182: 127 (100%), 110 (66%), 139 (33%). HRMS(ESI) m/z
314.1460 (M+H+, calcd. for C12H19N5O5+H+ 314.1459).
5-amino-1-(β-D-ribofuranosyl)-imidazole-4-(N-3-amino-2-hydroxyl-propyl)-
carboxamide (4)
A solution of unpurified compound 2 (196 mg, 0.6 mmol) in 0.085 M
phosphate buffer (9 ml) was held at pH 12 and 70◦C for 25 hours. The yield
before purification was 24%. The mixture was neutralized with phosphoric
acid and purified by repeated semipreparative HPLC runs on the Varian

Adduct Ring Fission
1221
LC with a Hypersil C18 column (250 × 10 mm). The buffer was 10 mM
phosphate at pH 7 with an acetonitrile gradient, and the flow rate was 3
ml/minutes. UV spectrum: λmax 268 nm, λmin 221 nm, ε = 8600 M−1cm−1.
1H NMR (DMSO-d6): δ = 7.46 (t, J = 6 Hz, 1 H, amide NH); 7.34 (s, 1 H,
H8); 5.48 (d, J = 5.6 Hz, 1 H, H1’); 4.26 (m, 1 H, H2’); 4.05 (m, 1 H, H3’);
3.89 (m, 1 H, H4’); 3.61 (d, J = 5.1 Hz, H11); 3.58 (s, H5a’ and H5b’); 3.25
(m, 1 H, H10a); 3.21 (m, 1 H, H10b); 2.65 (m, J = 10.5 Hz, 1 H, H12a); 2.54
(m, H12b). 13C NMR (DMSO-d6): δ = 164.8 (C6); 142.6 (C4); 128.7 (C8);
112.4 (C5); 87.6 (C1’); 85.4 (C4’); 73.0 (C2’); 70.1 (C3’); 69.1 (C11); 61.0
(C5’); 44.0 (C12); 41.5 (C10). MS(ESI): m/z 332 (100%, M+H+). MS2 of
m/z 332: 200 (100%, M+H+-ribosyl+H), 183 (34%), 314 (18%), 182 (16%).
MS3 of m/z 332→200: 183 (100%), 182 (61%). HRMS(ESI) m/z 332.1562
(M+H+, calcd. for C12H21N5O6+H+ 332.1565).
2-(5-amino-imidazol-4-yl)-1,4,5,6-tetrahydropyrimidin-5-ol (7)
Method A: A solution of 2 (50 mg, 0.28 mmol) in 20% H3PO4 (5.1 ml)
was held at 90◦C for one day, the yield of 7 before purification was 64%. Five
ml water was added, and pH was adjusted to 9.6 with 7 ml of 6 M NaOH. The
volume was reduced to 12 ml by rotary evaporation, the solution was allowed
to stand in a fridge overnight and filtered the following day. The filtrate was
evaporated to 5 ml and filtered again, and flashed on a silica column, eluted
with a solvent gradient from methanol to water, all eluents contained 0.01 M
pyridine. UV spectrum: λmax 202, 222, 283 nm, λmin 214, 247 nm, ε = 4900
M−1cm−1. MS(ESI): m/z 182 (100%, M+H+). MS2 of m/z 182: 127 (100%),
110 (86%), 139 (45%).
Method B (used for identification): The pH of a water solution of deoxy-3
(obtained by a method similar to that for 3) was adjusted to 1.8 with
H3PO4, and the solution was then held at 70◦C for 8 hours. The aglycon
was analyzed by LC-MS and UV-spectroscopy, and the compound was found
to be identical to that obtained by method A.
3-β-D-ribofuranosyl-3,4,7,8,9-pentahydropyrimido[1,2-i]purin-8-ol-5-
thione (5)
To a solution of purified compound 3 (10 mg, 0.032 mmol) in DMSO
(1 ml) was added K2CO3 (125 mg) and a solution of CS2 (100 µl) in DMSO.
After one day at ambient temperature, 17 mM phosphate buffer (15 ml) at
pH 4.7 was added and the solution was washed twice with dichloromethane
(5 ml) and rotary evaporated to 5 ml. The product was purified by repeated
semipreparative HPLC runs on the Agilent 1100 system with a Thermo
Hypersil BDS C18 column (250 × 10 mm). The buffer was 2 mM ammonium
acetate at pH 7 with an acetonitrile gradient, and the flow rate was 2
ml/minutes. The yield after purification was 33%. UV spectrum: λmax 208,
237, 296 nm, λmin 224, 258 nm, ε = 4000 M−1cm−1. 1H NMR (DMSO-d6): δ

1222
P. Sund and L. Kronberg
= 9.16 (br, 1 H, NH); 8.20 (s, 1 H, H8); 5.76 (d, J = 6.1 Hz, 1H, H1’); 5.47
(d, J = 2.5 Hz, 1 H, OH); 5.45 (d, J = 6.0 Hz, 1 H, OH); 5.24 (dd, J = 6.4
and 5.1, 1 H, OH5’); 5.17 (d, J = 3.4 Hz, 1 H, OH); 4.97 (dt, J = 14.3 and
3.2 Hz, 1 H, H10a); 4.50 (t, J = 5.5 Hz, 1 H, H2’); 4.32 (quin, J = 3.1 Hz,
1 H, H11); 4.11 (dd, J = 4.3 and 3.3 Hz, 1 H, H3’); 3.93 (q, J = 3.3 Hz, 1
H, H4’); 3.86 (d, J = 14.3 Hz, 1 H, H10b); 3.64 (dd, J = 12.1 and 3.5 Hz,
1 H, H5a’); 3.53 (dd, J = 12.2 and 3.2 Hz, 1 H, H5b’); 3.45 (dd, J = 12.8
and 1.7 Hz, 1H, H12a); 3.27 (dt, J = 13.2 and 2.9 Hz, 1 H, H12b). 13C NMR
(DMSO-d6): δ = 175.6 (C2); 148.3 (C6); 147.5 (C4); 139.2 (C8); 112.8 (C5);
86.9 (C1’); 53.1 (C10); 73.6 (C2’); 58.3 (C11); 70.5 (C3’); 85.8 (C4’); 61.5
(C5’); 44.3 (C12). MS(ESI): m/z 356 (100%, M+H+). MS2 of m/z 356: 224
(100%, M+H+-ribosyl+H). MS3 of m/z 356→224: 180, (100%), 170 (25%),
169 (18%), 151 (17%), 206 (16%), 152 (11%). HRMS(ESI) m/z 356.1027
(M+H+, calcd. for C13H17N5O5S+H+ 356.1023).
3-β-D-ribofuranosyl-imidazo[4,5-e]-3,7,8,9-tetrahydropyrimido[1,2-
c][1,2,3]triazine-8-ol (6)
To a solution of purified compound 3 (8 mg, 0.026 mmol) in water (7
ml), cooled with an ice bath, was added 12 M HCl (1.7 ml) and then a cooled
solution of KNO2 (170 mg) in water (1.5 ml). The reaction was held on the
ice bath for an hour, and then allowed to slowly reach ambient temperature.
The mixture was then neutralized with Na2HPO4 and NaOH, filtered and
purified by repeated semipreparative HPLC runs on the Agilent 1100 system
with a Thermo Hypersil BDS C18 column (250 × 10 mm). The buffer was 2
mM ammonium acetate at pH 7 with an acetonitrile gradient, and the flow
rate was 2 ml/minutes. The yield after purification was 31%. UV spectrum:
1
λmax 230, 258 (sh), 307 nm, λmin 211, 283 nm, ε = 16000 M−1cm−1. H
NMR (DMSO-d6): δ = 8.42 (s, 1 H, H8); 5.92 (dd, J = 5.5 Hz, 1 H, H1’);
4.49 (dt, J = 5.2 Hz, 1 H, H2’); 4.30 (d, J = 12.9 Hz, 1 H, H10a); 4.20 (1
H, H10b); 4.18 (1 H, H11); 4.13 (t, J = 4.2 Hz, 1 H, H3’); 3.96 (q, J = 4.0
Hz, 1 H, H4’); 3.66 (dd, J = 12.0 and 4.2 Hz, 1 H, H5a’); 3.55 (dt, J = 11.9
and 3.6 Hz, 1 H, H5b’); 3.49 (d, J = 16.2 Hz, 1 H, H12a); 3.33 (dt, J = 16.3
and 3.1 Hz, 1 H, H12b). 13C NMR (DMSO-d6): δ = 141.1 (C8); 138.6 (C4);
125.7 (C5); 88.2 (C1’); 85.8 (C4’); 74.4 (C2’); 70.1 (C3’); 61.1 (C5’); 60.2
(C11); 55.4 (C12); 49.8 (C10). MS(ESI): m/z 325 (100%, M+H+). MS2 of
m/z 325: 193 (100%, M+H+-ribosyl+H). MS3 of m/z 356→193: 147 (100%),
120 (59%), 93 (17%), 137 (10%). HRMS(ESI) m/z 325.1256 (M+H+, calcd.
for C12H16N6O5+H+ 325.1255).
General Procedure for Compounds 8, 9, and 10
To a solution of adenine (283.4 mg, 2.1 mmol) in 0.1 M phosphate
buffer (160 ml) at pH 7 was added epichlorohydrin (1 ml, 13 mmol). The
stirred reaction was allowed to proceed for one day in room temperature,

Adduct Ring Fission
1223
then the mixture was washed with toluene (20 ml), evaporated to 12 ml
and filtered. The filtrate was purified by repeated semipreparative HPLC
runs on the Varian 5000 chromatograph with a Hypersil C18 column (250
× 10 mm). The mobile phase was 5 mM phosphate buffer at pH 7 with
an acetonitrile gradient, and the flow rate was 3 ml/minutes. The fractions
were analyzed by LC-MS, and NMR spectra were recorded for those fractions
containing 8, 9, and 10.
1-(6-amino-3H-purin-3-yl)-3-chloropropan-2-ol (8)
UV spectrum: λmax 213, 276 nm, λmin 246 nm. H NMR (DMSO-d6): δ
1
= 8.21 (s, 1 H, H2); 8.03 (br, 1 H); 7.89 (br, 1 H); 7.76 (s, 1 H, H8); 4.54
(dd, J = 13.4 and 3.1 Hz, 1 H, H10a); 4.27 (m, 1 H, H11); 4.16 (dd, J =
13.4 and 8.8 Hz, 1 H, H10b); 3.73 (dd, J = 11.2 and 4.8 Hz, 1 H, H12a); 3.65
(dd, J = 11.2 and 5.4 Hz, 1 H, H12b). 13C NMR (DMSO-d6): δ = 155.1 (C6);
152.2 (C8); 149.7 (C4); 144.3 (C2); 120.3 (C5); 67.4 (H11); 52.9 (C10); 47.3
(C12). MS(ESI): m/z 228 (100%, M+H+), 230 (32%, M+H+).
1-(6-amino-9H-purin-9-yl)-3-chloropropan-2-ol (9)
UV spectrum: λmax 207, 262 nm, λmin 228 nm. 1H NMR (DMSO-d6): δ =
8.14 (s, 1 H, H2); 8.05 (s, 1 H, H8); 7.20 (br, 2 H, NH2); 5.68 (br, 1 H, OH);
4.31 (m, 1 H, H10a); 4.12 (m, 1 H, H11); 4.11 (m, 1 H, H10b); 3.66 (dd, J =
11.3 and 4.4 Hz, 1 H, H12a); 3.58 (dd, J = 11.3 and 4.8 Hz, 1 H, H12b). 13C
NMR (DMSO-d6): δ = 155.9 (C6); 152.3 (C2); 149.6 (C4); 141.5 (C8); 118.6
(C5); 68.4 (C11); 47.2 (C12); 46.5 (C10).MS(ESI): m/z 228 (100%, M+H+),
230 (32%, M+H+).
10-imino-5,6-dihydro-4H,10H-pyrimido[1,2,3-cd]purin-5-ol (10)
UV spectrum: λmax 209, 268 nm, λmin 241 nm. 1H NMR (DMSO-d6): δ =
8.35 (s, 1 H, H2); 8.30 (s, 1 H, H8); 4.47 (quin, J = 2.5 Hz, 1 H, H11); 4.34
(dt, J = 13.5 and 3.0 Hz, 1 H, H10a); 4.26 (d, J = 13.3 Hz, 1 H, H10b); 3.57
(dd, J = 13.9 and 1.7 Hz, 1 H, H12a); 3.44 (dt, J = 13.7 and 3.0 Hz, 1 H,
H12b). 13C NMR (DMSO-d6): δ = 148.2 (C4); 147.7 (C2); 143.6 (C8); 118.0
(C5); 57.6 (C11); 53.1 (C10); 45.5 (C12); C6 not observed. MS(ESI): m/z
192 (100%, M+H+). MS2 of m/z 192: 148 (100%), 107 (21%). HRMS(ESI)
m/z 192.0884 (M+H+, calcd. for C8H9N5O+H+ 192.0880).
3-hydroxy-1,2,3,4-tetrahydroimidazo[1,5-a]pyrimidine-8-
carboximidamide (11)
Fractions containing 8, 9 and 10 were pooled and held at 70◦C for
five days, then evaporated to 10 ml, filtered, and the filtrate was purified
by repeated semipreparative HPLC runs on the Varian 5000 cromatograph
with a Thermo Hypersil BDS C18 column (250 × 10 mm). The buffer was
1 mM ammonium acetate at pH 7 with an acetonitrile gradient, and the

1224
P. Sund and L. Kronberg
SCHEME 1 Structures and reactions of adenosine adducts. R = ribose. Purine numbering is retained
for all compounds with the exception of IUPAC names.
flow rate was 3 ml/minutes. Yield 6%. UV spectrum: λmax 207, 243 (sh), 297
1
nm, λmin 256 nm, ε = 1200 M−1cm−1. H NMR (DMSO-d6): δ = 7.36 (s,
1 H, H8); 4.13 (quin, J = 3.2 Hz, 1 H, H11); 3.99 (dd, J = 12.7 and 3.0
Hz, 1 H, H12a); 3.89 (dq, J = 12.7 and 3.8 Hz, 1 H, H12b); 3.31 (dd, J =
12.6 and 1.7 Hz, H10a); 3.24 (ddd, J = 12.8, 4.4 and 1.5 Hz, H10b). 13C NMR
(DMSO-d6): δ = 158.0 (C6); 141.3 (C4); 132.7 (C8); 105.1 (C5); 58.5 (C11);
47.6 (C12); 44.7 (C10). MS(ESI): m/z 182 (100%, M+H+). MS2 of m/z 182:
165 (100%). MS3 of m/z 182→165: 121 (100%), 147 (99%). HRMS(ESI)
m/z 182.1043 (M+H+, calcd. for C7H11N5O+H+ 182.1036).

Adduct Ring Fission
1225
SCHEME 2 Structures and reactions of adenine adducts.
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