Synthesis of [1,3, NH2-15N3] (5′S)-8,5′-cyclo-2′-deoxyguanosine

Article abstract of DOI:10.1002/jlcr.3051

To facilitate NMR studies and low-level detection in biological samples by mass spectrometry, [1,3, NH₂-¹⁵N₃] (5′S)-8,5′-cyclo-2′-deoxyguanosine was synthesized from imidazole-4,5-dicarboxylic acid in 21 steps. The three <

Full text of DOI:10.1002/jlcr.3051

Research Article
Received 4 December 2012,
Revised 6 February 2013,
Accepted 15 March 2013
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.3051
Synthesis of [1,3, NH2-15N3] (50S)-8,50-cyclo-20-
deoxyguanosine{
Chanchal K. Malik, Rajat S. Das, and Ashis K. Basu
†
†
*
To facilitate NMR studies and low-level detection in biological samples by mass spectrometry, [1,3, NH2-15N3] (50S)-8,50-cyclo-
20-deoxyguanosine was synthesized from imidazole-4,5-dicarboxylic acid in 21 steps. The three 15N isotopes were
introduced during the chemo-enzymatic preparation of [1,3, NH2-15N3]-20-deoxyguanosine using an established procedure.
15
The N-labeled 20-deoxyguanosine was converted to a 50-phenylthio derivative, which allowed the 8-50 covalent bond
formation via photochemical homolytic cleavage of the C–SPh bond. SeO2 oxidation of C-50 followed by sodium
borohydride reduction and deprotection gave the desired product in good yield. The isotopic purity of the [1,3, NH2-15N3]
(50S)-8,50-cyclo-20-deoxyguanosine was in excess of 99.94 atom% based on liquid chromatography–mass spectrometry
measurements.
Keywords: cyclo-dG; g-radiation damage; tandem DNA lesion; 15N-S-cdG
Because the commercially available 15N-labeled 20-deoxyguanosine
is very expensive, we started the synthesis from imidazole-4,5-
dicarboxylic acid and generated the S-cdG in 21 steps. Most of the
steps gave good yield making preparation of the final product in
Introduction
The tandem DNA lesions, 8,50-cyclo-20-deoxyadenosine (cdA)
and 8,50-cyclo-20-deoxyguanosine (cdG) diastereomers (Figure 1),
are formed by g-radiation and reactive oxygen species.1 These
either large or small scale feasible by this approach.
lesions have been detected in DNA derived from various
cells and organisms.2,3 An interesting characteristic of these
Results and discussion
DNA damages is that they are repaired by nucleotide excision
repair and not by base excision repair. Their likely role in
The synthesis of [1,3, NH2-15N3]-20-deoxyguanosine ([1,3,
NH2-15N3]-20-dG) was based on a published procedure by Jones
neurologic diseases in xeroderma pigmentosum patients
with defects in nucleotide excision repair has been suggested.4
S-cdA accumulates in the genomic DNA of Cockayne
syndrome B-deficient mice, suggesting that the cyclopurine
deoxynucleosides may accumulate in Cockayne syndrome
patients.5 Adverse biological effects of these DNA lesions have
been implicated in that both S-cdA and S-cdG are mutagenic in
Escherichia coli,6,7 whereas S-cdA is also a strong block of gene
expression in human cells.8 To explore the structural basis of
their biological effects, the structures of DNA duplexes
containing an S-cdG lesion placed opposite dC, dT, or dA were
obtained by a combination of solution NMR spectroscopy and
molecular dynamics calculations.9,10 These studies showed that
the S-cdG deoxyribose is in the O40-exo (west) pseudorotation,
whereas typically the ‘south’ (C20-endo) and the ‘north’ (C30-endo)
pseudorotation are observed in B-DNA and A-DNA, respectively.
However, the Watson–Crick base pairing was conserved in the
S-cdGꢀdC pair, whereas the S-cdGꢀdT pair adopts a wobble pairing.
But no hydrogen bonding was observed for the S-cdGꢀdA pair,
which differs in conformation from the dGꢀdA mismatch pair. The
biological and chemical effects of these DNA lesions are being
actively pursued by many research groups.
and coworkers,12 which starts with imidazole-4,5-dicarboxylic
acid (1) (Scheme 1). Two 15N isotopes were introduced by
diazocoupling and amidation using Na15NO2 and 15NH4Cl,
respectively, of the two carboxyl groups to yield the key
compound [NH2, CONH2-15N2]-5-amino-4-imidazolecarboxamide
(3) in six steps. Ring closure of compound 3 with sodium ethyl
xanthate gave the hypoxanthine derivative 4 with two 15N labels,
which was converted to [1,3-15N2]-2-(methylthio)hypoxanthine
(5) with iodomethane. We have evaluated several enzymes
for the subsequent transglycosylation step13,14 to attach the
2-deoxyribose from 20-deoxyguanosine to the 15N containing
2-(methylthio)hypoxanthine base and found that purine
nucleotide phosphorylase (PNP; Aldrich, St. Louis, MO, USA) gave
the best (70%) yield. However, ~10% glycosylation occurred at
the N7 position, and this isomer was separated from the desired
Department of Chemistry, University of Connecticut, Storrs, CT 06269, USA
*Correspondence to: Ashis K. Basu, Department of Chemistry, University of
Connecticut, Storrs, CT, USA.
E-mail: ashis.basu@uconn.edu
† These two authors contributed equally to this work.
Detection and quantification of low-level DNA damages in
biological samples can be efficiently accomplished by liquid
chromatography–mass spectrometry with the availability of the
DNA lesion standards containing stable isotopes such as 15N.11 The
15N-labels are also of value for NMR studies of the DNA lesion
structures. Here, we report the synthesis of [1,3, NH2-15N3]-S-cdG.
{ Supporting information may be found in the online version of this article.
List of abbreviations: S-cdG and R-cdG, (50S)- and (50R)-8,50-cyclo-20-
deoxyguanosine, respectively; S-cdA and R-cdA, (50S)- and (50R)-8,50-cyclo-20-
deoxyadenosine, respectively.
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C. K. Malik et al.
30 position and the isobutyryl group at the exocyclic amine were
deprotected to give the desired product [1,3, NH2-15N3]-S-cdG 12.
Although we began the synthesis in millimole scale, most of the
steps starting from compound 4 were carried out successfully in
hundred-micromole to one-millimole scale. This approach allows
one to synthesize 30–35 mg of the final product, 12, from a 6-g
starting material, 1.
The 1H-NMR of 12 (Figure 2) showed the typical 15N–1H coupling
constants of ~90 Hz for both 15NH2 and 15NH at 10.54 and 6.45 ppm,
respectively. By contrast, both these signals appear as singlets for
the unlabeled S-cdG (data not shown). It is also noteworthy that
proton-decoupled 15N signals of [1,3, NH2-15N3]-dG at 166.12 (N3),
147.65 (N1), and 73.45 (NH2) were only slightly shifted to 165.26,
147.54, and 73.14, respectively, in [1,3, NH2-15N3]-S-cdG (Figure 3).
The isotopic purity of the [1,3, NH2-15N3]-S-cdG was in excess of
99.94 atom% based on liquid chromatography–mass spectrometry
(LC-MS) measurements (for details, see Supporting Information).
Figure 1. Structures of (50S)-8,50-cyclo-20-deoxyguanosine (S-cdG) and (50S)-8,50-
cyclo-20-deoxyadenosine (S-cdA).
N9 glycosylated product 6 by reversed-phase chromatography.
The methylthio group at the 2 position was oxidized to
methylsulfoxyl group, which was slowly replaced by an amino
group using 15NH4Cl in 14 days to give [1,3, NH2-15N3]-20-dG (7).
The synthesis of cyclopurine deoxynucleosides is usually
accomplished by a method developed by Matsuda and coworkers,
which relies on the phenylthio group as a surrogate for methylene Experimental
radical that generates a new covalent bond between C-50 of 20-
Bulk solvents were purchased either from Sigma Aldrich (St. Louis, MO) or
deoxyribose and C8 of the purine base.15 Accordingly, our initial
goal was to synthesize a protected labeled dG with a phenylthio
group at the 50 carbon. The exocyclic amine group of 15N3-labeled
dG 7 was protected with isobutyryl group, and the 50-hydroxyl
group was replaced with a phenylthio group using diphenyl
disulfide to generate compound 9 (Scheme 2). Ultraviolet (UV)
irradiation of 9 at 254 nm in argon-purged acetonitrile provided
the 8,50-cyclo-20,50-dideoxyguanosine derivative via the 50-
methylene radical. The released phenylthiyl radical during the UV
irradiation was trapped with triethyl phosphate. The 30-hydroxyl
group of this cyclopurine 20-deoxynucleoside derivative was
protected with a tert-butyldimethylsilyl (TBDMS) group to afford
compound 10, which was refluxed in 1,4-dioxane with selenium
oxide to oxidize the 50-CH2 group. Subsequent reduction by
sodium borohydride generated the S-cdG derivative 11 with
Fisher Scientific (Clifton, NJ). Deuterated solvents were purchased from
Cambridge Isotope Laboratories (Andover, MA). The water used for
this study was obtained from a Barnstead Nanopure System (Thermo
Scientific, Dubuque, IA). All reagents used were of highest purity available.
The silica gel used for flash chromatography (40–63 mm) and gravitational
column (63–200 mm) were purchased from Sorbent Technology (Norcross,
GA). Reversed-phase preparative chromatography was performed on a
Combiflash Rf medium pressure liquid chromatography (MPLC) system
(Teledyne Isco, NE) using C-18 columns (20–40 mm).
4,5-Imidazoledicarboxylic acid, 4-bromoaniline, 1-ethyl-3-(3-dimethy-
laminopropyl) carbodiimide (EDCI), hydroxybenzotriazole (HOBt) 1,8-
diazabicyclo[5.4.0]undec-7-ene (DBU), N-bromosuccinimide (NBS) 10% Pd,
1% Pt, methyl iodide, K2HPO4, and oxone were obtained from Acros
Organics, Geel, Belgium. NaSCSOEt was from Fisher Scientific.
Deoxyguanosine was from Berry & Associates, Dexter, MI. The 15NH4Cl
and Na15NO2 were from Cambridge Isotope Laboratories, Andover, MA.
high stereoselectivity.16 The TBDMS group of the latter at the PNP was purchased from Sigma Aldrich.
Scheme 1. Synthesis of [1,3, NH2-15N3]-2’-deoxyguanosine from imidazole-4,5-dicarboxylic acid.
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C. K. Malik et al.
Scheme 2. Conversion of 15N3-labeled 2’-deoxyguanosine to 15N3-labeled (5’S)-8,5’-cyclo-2’-deoxyguanosine.
1
Figure 2. H-NMR of (50S)-[1,2,3-15N3]-8,50-cyclo-20-deoxyguanosine on a 500-MHz Bruker Advance spectrometer.
2 days. The solvent was evaporated, and the reaction mixture was
HPLC analysis
neutralized with Na2CO3. The compound was extracted with CH2Cl2
(3 ꢁ 200 mL), and the combined organic layers were washed with 0.1 M
NH4HCO3. The solvent was dried over Na2SO4 and removed under
vacuum to afford the compound diethyl 1H-imidazole-4,5-dicarboxylate
(UV lmax 257 nm) (6.9 g, 85%).
The HPLC analyses were performed on a reversed-phase column (Synergi
4-mm Hydro-RP 80 Å LC column 250 ꢁ 4.6 mm, Phenomenex, Torrance,
CA) using a Waters 1525 HPLC system (Milford, MA) equipped with a
diode-array detector (model 2996). A linear gradient elution program
with 100% water to 15% acetonitrile with a flow rate of 1 mL/min in
20 min was used.
Diethyl 2-bromo-1H-imidazole-4,5-dicarboxylate
Diethyl 1H-imidazole-4,5-dicarboxylate
Diethyl 1H-imidazole-4,5-dicarboxylate (6.9 g, 32.53 mmol) and freshly
Imidazole-4,5-dicarboxylic acid (6 g, 38.5 mmol) was combined with crystallized NBS (8.71 g, 48.9 mmol) were combined in a round-bottom
ethanol (350 mL) and concentrated H2SO4 (35 mL) in a 500-mL round- flask under Ar, and dry acetonitrile (90 mL) was added. The reaction
bottom flask, and the reaction mixture was refluxed under nitrogen for mixture was stirred in the dark for 30 h, after which the solvent was
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C. K. Malik et al.
Figure 3. Proton-decoupled N-NMR spectrum of (50S)-[1,2,3-15N3]-8,50-cyclo-20-deoxyguanosine.
15
evaporated. The residue was diluted with EtOAc and washed with brine dried under vacuum over P2O5 for 2 days to afford pure [5, CONH2-15N2]-
and saturated Na2SO3 solution. The organic layer was concentrated and 5-((4-bromophenyl)azo)-2-bromo-4-imidazolecarboxamide (3.7g, 90%). UV
purified by column chromatography to afford the diethyl 2-bromo-1H- lmax 275 nm and HRMS (ESI); [M+ H]+ calcd C10H8Br2N135N2O: 375.9982,
imidazole-4,5-dicarboxylate (UV lmax 286 nm) (8.96 g, 95%).
observed 375.9009.
[5-15N]-5-((4-Bromophenyl)azo)-2-bromo-4-imidazole-
carboxylic acid
[NH2,CONH2-15N2]-5-Amino-4-imidazolecarboxamide (3)
To
a
mixture of [5, CONH2-15N2]-5-((4-bromophenyl)azo)-2-bromo-4-
imidazolecarboxamide (2g, 5.35 mmol), 10% Pd-C (1.15g) and 1% Pt
(90 mg) under N2-atmosphere NH3 (7N) in MeOH (440 mL) was added.
The reaction mixture was purged with H2 gas for 2h and then allowed to stir
for an additional 7h under H2-atmosphere. The reaction mixture was
filtered and evaporated. The residue was dissolved in water (2mL) and
washed with ether (2ꢁ 5 mL). The aqueous layer was purified on a
reversed-phase MPLC column eluted with water. Formic acid was added
to the fraction containing the product. Evaporation of the solvent afforded
compound 3 (465 mg, 50%). UV lmax 230nm (shoulder), 267nm. HRMS (ESI)
: m/z calcd [M+ H]+ C4H7N215N2O: 129.0561, observed 129.0463.
A
mixture of diethyl 2-bromo-1H-imidazole-4,5-dicarboxylate (1 g,
3.45 mmol) and Na2CO3 (960 mg, 9.057 mmol) in 34-mL water was heated
at 100ꢂC for 36 h, then cooled to 0ꢂC. A cold solution of Na15NO2
(228 mg, 3.26 mmol) in 4-mL water was added dropwise to a cold
solution of 4-bromoaniline (536 mg, 3.14 mmol) in 3.1 mL of 10%
HCl. After 25 min, an ice-cold solution of Na2CO3 (440 mg, 4.15 mmol) in
5-mL water was slowly added. This cold mixture was added to cold
aqueous solution of the diethyl 2-bromo-1H-imidazole-4,5-dicarboxylate
and Na2CO3. A yellow color appeared within 5 min, which gradually
turned red, and a thick precipitate was formed. The reaction mixture
was allowed to stir for an additional 2 h, and the precipitate was filtered
and dissolved in Na2CO3 (400 mg,) in 25-mL water. This aqueous Na2CO3
solution was filtered, and the remaining black residue was discarded. The
[1,3-15N2]-2-Mercaptohypoxanthine (4)
aqueous solution was washed with CH2Cl2 (3 ꢁ 100) and acidified with To a mixture of compound 3 (150 mg, 0.862 mmol) and sodium ethyl
concentrated HCl to afford a yellow precipitate. The precipitate was xanthate (200 mg, 1.39 mmol), anhydrous N,N'-dimethylformamide
collected and washed with cold water to provide compound 2 (1 g, (DMF) (5 mL) was added under N2 atmosphere and stirred the reaction
80%). UV lmax 235 nm; HRMS (ESI):: m/z calcd [M + H]+ C10H7Br2N315NO2: mixture for 30 min at room temperature and refluxed for 5 h. The solution
375.8916, observed 375.8902.
was allowed to cool, CH3CN (15 mL) was added, and a white precipitate
appeared that was collected by filtration. The filtrate was evaporated
and purified by reversed-phase MPLC to afford product 4 (110 mg, 75%)
UV lmax 280 nm.
[5, CONH2-15N2]-5-((4-Bromophenyl)azo)-2-bromo-4-
imidazolecarboxamide
[1,3-15N2]-2-(Methylthio)hypoxanthine (5)
In a 1-L round-bottom flask, compound 2 (4.12 g, 11.02 mmol) and
15NH4Cl (780 mg, 14.4 mmol) were dissolved in 600-mL anhydrous CH3CN
under N2 at ꢃ15ꢂC. The reaction mixture was stirred for 2 h. Separately,
EDCI (2.38 g, 12.42 mmol) was dissolved in 195-mL CH3CN in a 250-mL
round-bottom flask at ꢃ15ꢂC under N2 and stirred for 2 h. The cold EDCI
was added dropwise to the compound 2 solution. During the EDCI
addition, 2.4-mL DBU (16 mmol) was also added, and the reaction
mixture was allowed to stir at ꢃ15ꢂC. Aliquots of water (3 ꢁ 10) were
added after 4, 6, and 8 h, and stirring was continued at ꢃ10ꢂC for 36 h.
The solvent was then evaporated, and the solid residue was suspended
in 200-mL water for overnight at 4ꢂC. The solution was filtered, and the
precipitate was collected. The orange cake was dissolved in 1.5-L warm
aqueous Na2CO3 (26 g) solution and filtered. The solid residue was
In a round-bottom flask, compound
4 (100 mg, 0.588 mmol) was
dissolved in water (2 mL), and MeI (0.2 mL) was added. The reaction
mixture was stirred vigorously for 2 days. A white precipitate appeared
that was filtered to afford compound 5. After partial evaporation of the
filtrate, another crop of the compound was obtained. The compound
was purified by reversed-phase MPLC. Yield of compound 5 was 82 mg
(76%). UV lmax 262 nm; HRMS (ESI): m/z calcd [M + H]+ C6H7N215N2O5:
185.0281, observed 185.0218.
[1,3-15N2]-2-(Methylthio)-20-deoxyinosine (6)
discarded. The aqueous Na2CO3 solution was washed with CH2Cl2 In a 20-mL glass vial, compound 5 (200 mg, 1.087 mmol) (combined from
(3 ꢁ 200 mL) and acidified with concentrated HCl. The precipitate was multiple synthesis of this base) and 20-deoxyguanosine (300 mg,
filtered and washed with cold water (2 ꢁ 25 mL). The compound was 1.8 mmol) were suspended in aqueous K2HPO4 (10 mL, 0.02 M), and 1 N
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C. K. Malik et al.
KOH was added dropwise to maintain the pH of the solution at 8.4. PNP (0.58 mL, 3.3 mmol) were dissolved in 450-mL dry acetonitrile by
(100 mL, 9.4 units) was added to the reaction mixture, and the vial was
heated for 2 days at 45ꢂC. It was then filtered, and the solid residue was sealed under argon and irradiated with 254-nm UV light for 20 h. The
was extracted with 2% NH4OH. The aqueous part was concentrated reaction was monitored by TLC (85/15 CHCl3/MeOH, v/v). The solution
sonication, placed in a 2-L quartz reactor, and purged with Ar. The reactor
and purified by reversed-phase column chromatography to afford was evaporated to dryness, and the resulting brownish yellow solid
compound 6 (228 mg, 70%). UV lmax 263 nm. HRMS (ESI): m/z calcd was purified on a 30-g C-18 column with a step gradient of acetonitrile
[M + Na]+ C11H14N215N2O4SNa: 323.0574, observed 323.0954.
(0–30%) in water. The product was isolated as a white solid (47.4 mg,
45%). ESI-MS (positive mode): m/z calcd [M + H]+ C14H18N215N3O4: 323.1,
observed 323.0 Da.
[1,3, NH2-15N3]-20-Deoxyguanosine (7)
[1,3,NH2-15N3]-N2-Isobutyryl-30-O-(tert-butyldimethylsilyl)-
In a 20-mL vial, compound 6 (340 mg, 1.133 mmol) was suspended in
63-mL water at 0ꢂC, and chilled oxone (398 mg, 2.62 mmol) in 9.7-mL
water was added dropwise over 30 min. The reaction mixture was
stirred for 2 h. Subsequently, Na2SO3 (25 mg, 0.198 mmol) was added
and stirred for an additional 10–15 min, and the pH was adjusted to
6.7 by adding NaHCO3. The solution was concentrated and purified
by reversed-phase MPLC to afford the compound [1,3-15N2]-2-
(methylsulfoxyl)-20-deoxyinosine (UV lmax 258 nm) (304 mg, 85%). It
was added to a mixture of 15NH4Cl (175 mg, 3.24 mmol) and KHCO3
(206 mg, 2.06 mmol) in anhydrous dimethyl sulfoxide (DMSO) (3.3 mL)
in a round-bottom flask, which was sealed with a septum and kept
for 14 days at 78ꢂC. Subsequently, the mixture was evaporated to
dryness and diluted with hot water and purified by reversed-phase
MPLC to afford compound 7 (207 mg, 80%) UV lmax 254 nm; 1H-NMR
(400 MHz, DMSO) d 2.19 (1H, J = 4, 6, 14 Hz), 2.47–2.50 (1H, m), 3.51–3.57
(2H, m), 3.79–3.82 (1H, m), 4.33–4.34 (1H, m), 4.95 (1H, s), 5.26 (1H, s), 6.12
(1H, t, J = 8 Hz), 6.45 (2H, d, J = 92 Hz), 7.92 (1H, s), 10.61 (1H, d, J = 92 Hz);
15N-NMR (DMSO) (50 MHz, DMSO-d6) 73.45, 147.65, 166.12. HRMS (ESI):
m/z calcd [M+ H]+ C10H14N215N3O4: 271.0957, observed 271.0921.
8,50-cyclo-20,50-dideoxyguanosine (10)
[1,3,NH2-15N3]-N2-Isobutyryl-8,50-cyclo-20,50-dideoxyguanosine (47.4 mg,
0.147 mmol) and imidazole (40 mg, 0.589 mmol) were dried and
dissolved in 2-mL dry DMF. TBDMS-Cl (44.3 mg, 0.294 mmol) was added
to the reaction mixture while stirring under nitrogen. Stirring continued
at ambient temperature for 20 h when TLC monitoring (93/7 CHCl3/
MeOH, v/v) showed the reaction to be complete. The solvent was dried
under nitrogen, and the resulting semi-solid was purified on a silica gel
column with a step gradient of MEOH (0–4%) in CH2Cl2. The product
was isolated as a white solid (45 mg, 70%). ESI-MS (positive mode): m/z
calcd [M + H]+ C20H31N215N3O4Si: 436.2, observed 436.1 Da.
(50S)-[1,3,NH2-15N3]-N2-Isobutyryl-30-O-(tert-
butyldimethylsilyl)-8,50-cyclo-20-deoxyguanosine (11)
[1,3,NH2-15N3]-N2-Isobutyryl-30-O-(tert-butyldimethylsilyl)-8,50-cyclo-20,50-
dideoxyguanosine 10 (45 mg, 0.103 mmol) was dissolved in 20-mL dry
1,4 dioxane. SeO2 (57.1 mg, 0.515 mmol) was added, and the mixture
was refluxed for 20 h when TLC (93/7 CHCl3/MeOH, v/v) showed that
the reaction was complete. The hot solution was passed through a celite
pad and washed with 20-mL 10% MEOH in chloroform. The filtrate
was dried under reduced pressure to produce a brownish white powder
of [1,2,3-15N3]-N2-isobutyryl-30-O-(tert-butyldimethylsilyl)-50-oxo-8,50-cyclo-
20-deoxyguanosine. This product was suspended in 5-mL MEOH, and
NaBH4 (8mg, 0.21 mmol) was added. This reaction mixture was stirred at
room temperature for an hour and checked for completion by TLC (93/7
CHCl3/MeOH, v/v). The excess borohydride was carefully neutralized by
dropwise addition of 1 N HCl in MEOH. The solution was passed through
a celite pad and evaporated to dryness. The resulting yellow solid was
purified by silica gel column chromatography with a step gradient of MEOH
(0–6%) in CH2Cl2. Compound 11 was isolated as a white solid (16.8mg,
36%), and as reported,16 its 1H-NMR exhibited a d5.27 doublet for
[1,3, NH2-15N3]-N2-Isobutyryl-20-deoxyguanosine (8)
[1,3, NH2-15N3]-20-deoxyguanosine 7 (292 mg, 1.1 mmol) was evaporated
three times with anhydrous pyridine. The residue was suspended in
10 mL of dry pyridine under argon. Chlorotrimethylsilane (1.195 g,
1.40 mL) was added dropwise to the stirred solution. After 15 min,
isobutyric anhydride (1.74 g, 1.82 mL) was added, and stirring was
continued for 3 h. The reaction mixture was cooled in an ice bath to
~5ꢂC, and 2-mL water was added to quench the excess reagent. After
5 min, 2 mL of 29% aqueous NH4OH was added. After stirring for
15 min, the reaction mixture was evaporated to dryness. The residue
was suspended in 10-mL water and washed with 10-mL 1:1 EtOAc/ether.
The organic layer was discarded, and the aqueous layer containing 8
was dried. The solid residue was purified by chromatography on a 4-g
C-18 column with a step gradient of 3–30% acetonitrile/water. The
product was isolated as a white solid (226 mg, 61%). ESI-MS (positive
mode): m/z calcd [M + H]+ C14H20N215N3O5: 341.1, observed 341.1 Da.
the H-50 proton (J4 5ꢃ ~ 7 Hz) indicating the 50S stereo-configuration.
0
ESI-MS (positive mode): m/z calcd [M + H]+ C20H32N215N3O5Si: 453.2,
observed 453.2 Da.
[1,3, NH2-15N3]-N2-Isobutyryl-50-phenylthio-20,50-
dideoxyguanosine (9)
(50S)-[1,3,NH2-15N3]-N2-Isobutyryl-8,50-cyclo-20-
deoxyguanosine
[1,3, NH2-15N3]-N2-Isobutyryl-20-deoxyguanosine 8 (171 mg, 0.503 mmol)
and diphenyl disulfide (435 mg, 2 mmol) were dissolved in 10-mL dry
DMF under argon. PBu3 (0.492 mL, 2 mmol) was dropwise added, and
the reaction mixture was stirred at room temperature. The reaction was
found to be complete after 6 h by thin layer chromatography (TLC)
monitoring (90/10 CH2Cl2/MeOH, v/v). The reaction mixture was
quenched with 10-mL water and evaporated to a glassy syrupy
residue. It was purified on a 30-g C-18 column with a step gradient
of acetonitrile (0–40%) in water as the mobile phase. The product
was isolated as a white solid (141 mg, 65%). ESI-MS (positive mode):
m/z calcd [M + H]+ C20H24N215N3O4S: 433.1, observed 433.1 Da.
(50S)-[1,3,NH2-15N3]-N2-Isobutyryl-30-O-(tert-butyldimethylsilyl)-8,50-cyclo-20-
deoxyguanosine 11 (16.6mg, 0.037 mmol) was dissolved in 2-mL dry THF. A
solution of tetrabutylammonium fluoride (TBAF) (74mL, 0.074 mmol) was
added, and the mixture was stirred for 6 h. The solvent was removed under
reduced pressure. The resulting gummy residue was purified by silica gel
column chromatography with a step gradient of MEOH (0–10%) in CH2Cl2.
The product was isolated as a white solid (11.8mg, 95%). ESI-MS
(positive mode): m/z calcd [M + H]+ C14H18N125N3O5: 339.1, observed 339.1Da.
(50S)-[1,3,NH2-15N3]-8,50-Cyclo-20-deoxyguanosine (12)
(50S)-[1,3,NH2-15N3]-N2-Isobutyryl-8,50-cyclo-20-deoxyguanosine (8.2 mg,
0.024 mmol) was dissolved in 1.5-mL MEOH, and 0.5 mL of 29% aqueous
ammonia was added to the solution. The mixture was stirred overnight
at room temperature, and the reaction was monitored by HPLC using a
[1,3, NH2-15N3]-N2-Isobutyryl-8,50-cyclo-20,50-
dideoxyguanosine
Previously crushed [1,3, NH2-15N3]-N2-isobutyryl-50-phenylthio-20,50- C-18 column (0–20% acetonitrile in water, 20 min). The solution was
dideoxyguanosine
9 (141 mg, 0.327 mmol) and triethyl phosphite dried down to 400 mL and filtered through 0.2-mm nylon membrane.
J. Label Compd. Radiopharm 2013
Copyright © 2013 John Wiley & Sons, Ltd.
www.jlcr.org

C. K. Malik et al.
The product was isolated as a white solid (6.1 mg, 95%). UV lmax 258 nm;
ESI-MS (positive mode): m/z calcd [M + H]+ C10H12N215N3O4: 269.1,
observed 269.1 Da. [M + H]+. 15N-NMR (50 MHz, DMSO-d6): d 165.26,
147.54 and 73.14.
[5] G. Kirkali, N. C. de Souza-Pinto, P. Jaruga, V. A. Bohr, M. Dizdaroglu,
DNA Repair (Amst) 2009, 8, 274–278.
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5945–5954.
[7] V. P. Jasti, R. S. Das, B. A. Hilton, S. Weerasooriya, Y. Zou, A. K. Basu,
Biochemistry 2011, 50, 3862–3865.
Acknowledgement
[8] P. J. Brooks, D. S. Wise, D. A. Berry, J. V. Kosmoski, M. J. Smerdon,
R. L. Somers, H. Mackie, A. Y. Spoonde, E. J. Ackerman, K. Coleman,
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This work was supported by the National Institute of
Environmental Health Sciences (NIEHS grant ES013324).
[10] H. Huang, R. S. Das, A. K. Basu, M. P. Stone, Chem. Res. Toxicol. 2012,
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Conflict of Interest
The authors did not report any conflict of interest.
2012, 25, 2007–2035.
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