Semisynthesis of 6-chloropurine-2′-deoxyriboside 5′- dimethoxytrityl 3′-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite and its use in the synthesis of fluorescently labeled oligonucleotides

Article abstract of DOI:10.1080/15257770.2010.530332

An efficient enzymatic synthesis of 6-chloropurine-2′-deoxyriboside from the reaction of 6-chloropurine with 2′-deoxycytidine catalyzed by nucleoside-2′-deoxyribosyltransferase (E.C. 2.4.2.6) followed by chemical conversion into the 5′-dimethoxytrityl 3′-

Full text of DOI:10.1080/15257770.2010.530332

Nucleosides, Nucleotides and Nucleic Acids, 29:831–840, 2010
Copyright ꢀC Taylor and Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770.2010.530332
SEMISYNTHESIS OF 6-CHLOROPURINE-2ꢀ-DEOXYRIBOSIDE
5ꢀ-DIMETHOXYTRITYL 3ꢀ-(2-CYANOETHYL-N,N-DIISOPROPYLAMINO)
PHOSPHORAMIDITE AND ITS USE IN THE SYNTHESIS
OF FLUORESCENTLY LABELED OLIGONUCLEOTIDES
Md. Jashim Uddin,1 Michael I. Schulte,1 Leena Maddukuri,1 Joel Harp,2
and Lawrence J. Marnett1
1A. B. Hancock Jr. Memorial Laboratory for Cancer Research, Departments of Biochemistry,
Chemistry, and Pharmacology, Vanderbilt Institute of Chemical Biology, Vanderbilt
University School of Medicine, Nashville, Tennessee, USA
2Center for Structural Biology, Vanderbilt University, Nashville, Tennessee, USA
An efficient enzymatic synthesis of 6-chloropurine-2ꢁ-deoxyriboside from the reaction of 6-
2
chloropurine with 2ꢁ-deoxycytidine catalyzed by nucleoside-2ꢁ-deoxyribosyltransferase (E.C. 2.4.2.6)
followed by chemical conversion into the 5ꢁ-dimethoxytrityl 3ꢁ-(2-cyanoethyl-N,N-diisopropylamino)
phosphoramidite derivative is described. The phosphoramidite derivative was incorporated site-
specifically into an oligonucleotide and used for the introduction of a tethered tetramethylrhodamine-
cadaverine conjugate. The availability of an efficient route to 6-chloropurine-2ꢁ-deoxyriboside 5ꢁ-
dimethoxytrityl 3ꢁ-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite enables the facile synthesis
of oligonucleotides containing a range of functional groups tethered to deoxyadenosine residues.
Keywords
Semisynthesis; nucleoside deoxyribosyltransferase; 6-chloropurine-2ꢁ-
deoxyriboside; fluorescent oligonucleotide
INTRODUCTION
Oligonucleotides containing tethered functional groups (e.g., fluo-
rescent dyes) are useful for polymerase chain reaction (PCR) analy-
sis, probing of whole cells, and characterization of genetic polymor-
phisms inter alia.[1–3] 6-Chloropurine-2ꢁ-deoxyriboside 5ꢁ-dimethoxytrityl
3ꢁ-(2-cyanoethyl-N,N -diisopropylamino) phosphoramidite (5) (Scheme 1)
Received 3 February 2010; accepted 5 October 2010.
The authors are grateful to Dr. Carol A. Rouzer for a critical reading of this manuscript and to the
National Institutes of Health for financial support of this work (CA87819).
Address correspondence to Lawrence J. Marnett, A. B. Hancock Jr. Memorial Laboratory for
Cancer Research, Departments of Biochemistry, Chemistry, and Pharmacology, Vanderbilt Institute
of Chemical Biology, Vanderbilt University School of Medicine, Nashville, TN 37232-0146, USA. E-mail:
larry.marnett@vanderbilt.edu
831

832
Md. J. Uddin et al.
SCHEME 1 Semisynthesis of 5.
is an important precursor that provides access to oligonucleotides ad-
ducted through deoxyadenosine residues.[4,5] Synthesis of the precursor,
6-chloropurine-2ꢁ-deoxyriboside (3) is complicated due to the involvement
of unstable intermediates and the requirement for multistep procedures.[6]
In addition, chemical synthesis requires removal of protecting groups un-
der conditions that affect the nucleoside scaffold. Some success has been
achieved in the synthesis of this type of compound using microbiological[7] or
enzymatic[8] methodologies. Both approaches offer distinct advantages over
multistep chemical procedures. These methodologies involve the transfer of
a sugar residue from a donor to an acceptor base and require the presence of
nucleoside phosphorylases or nucleoside deoxyribosyltransferases either as
purified enzymes or within the bacterial cells used for synthesis. This report
describes a semisynthetic procedure for the preparation of 5 using purified
nucleoside 2ꢁ-deoxyribosyltransferase (E.C. 2.4.2.6).[9] The phosphora-
midite 5 was used in the synthesis of 5ꢁ-TATCATGTCTGPuClTTCCCGGT-3ꢁ
(6), which was conjugated with 5-TAMRA cadaverine to give 5ꢁ TATCAT-
GTCTGPuTAMRAATTCCCGGT-3ꢁ (7).
RESULTS AND DISCUSSION
To develop an efficient, alternative route for the synthesis of 6-
chloropurine-2ꢁ-deoxyriboside, we investigated a number of biosynthetic
approaches. Whole cell approaches employing Bacillus stearothermophilus
as a source of transglycosylase for the reaction of 2ꢁ-deoxyinosine and
6-chloropurine in the presence of xanthine oxidase were unsuccessful.[10]
We next tried purified nucleoside 2ꢁ-deoxyribosyltransferase (E.C. 2.4.2.6)

Synthesis of Fluorescently Labeled Oligonucleotides
833
to catalyze the transglycosylation between 2ꢁ-deoxycytidine (1) and 6-
chloropurine (2) in aqueous morpholinoethanesulfonic acid (MES) at pH
6.4.[11] Nuclear magnetic resonance (NMR) analysis and mass spectrometry
revealed the presence of some product but purification was difficult. Repeti-
tion of this reaction with the omission of MES followed by a silica gel column
chromatographic purification afforded 3 in 40% yield. Compound 3 was con-
verted successively into the 5ꢁ-dimethoxytrityl derivative (4) followed by the
3ꢁ-(2-cyanoethyl-N,N -diisopropylamino) phosphoramidite derivative (5) in
good yields (Scheme 1). Phosphoramidite 5 was synthesized as a mixture of
two diastereoisomers and one of them was isolated from the diastereomeric
mixture by flash chromatography.
All of the compounds were characterized by one-dimensional and
two-dimensional NMR and by mass spectrometry. Furthermore, the ab-
solute stereochemistry of compound 3 was unambiguously confirmed
by a single crystal x-ray analysis, which showed that compound 3 is a
purine—N9—glycoside, rather than an N7—glycoside. The conformation
of the hydroxymethyl group with respect to the sugar ring is gauche—trans.
The relative configuration of the sugar and base at the glycosidic linkage is
β (Figure 1).
The 1H NMR spectrum of each of these compounds contains two charac-
teristic purine protons that can be used for differentiation of other aromatic
protons in the molecule. For compound 5, these characteristic signals, iden-
tified as two separate singlets at around 8. 7 and 8.8 ppm, were assigned to
the protons at position-2 and position-8 of the purine ring, respectively. The
other aromatic protons in the molecule are trityl protons that are assigned
as three different multiplets located at 7.28–7.30, 7.15–7.20, and 6.74–6.79
ppm. Assignments were confirmed by a 1H-1H COSY experiment, which re-
vealed the correlations of the neighboring protons in each aromatic system.
A single phosphorous atom was detected in the separated diastereomer of 5
at 8.31 ppm in a 31P-NMR experiment.
The oligonucleotide 5ꢁ-TATCATGTCTGPuClTTCCCGGT-3ꢁ (6) was pre-
pared by automated DNA synthesis using 5 for incorporation of the 6-Cl-
purine residue. After removal of the protecting groups by hydrolysis with
concentrated ammonium hydroxide, the product was desalted by gel fi-
tration chromatography. Nucleophilic displacement of the chloro group
of the purine nucleus of 6 with 5-TAMRA cadaverine in the presence of
N,N -diisopropylethylamine gave the expected fluorescent oligonucleotide 5ꢁ
TATCATGTCTGPuTAMRAATTCCCGGT-3ꢁ (7) (Scheme 2). Oligonucleotide
7 was purified by HPLC. The HPLC retention time of compound 7 (16
minutes) was much longer than that of the starting oligonucleotide 6
(8.9 minutes) in the reverse-phase chromatography. Fluorescent oligonu-
cleotide, 7, was characterized by electrospray mass spectrometry and
exhibited a molecular ion at 6381.8 (calcd., 6381.4). The steady-state
fluorescence excitation and emission of 7 were determined to be λexc
540 nm, λemit = 572 nm (Figure 2).
=

834
Md. J. Uddin et al.
SCHEME 2 Synthesis of fluorescent oligonucleotide, 7.
Semisynthesis of 5 by a combination of enzymatic and chemical synthesis
from 2 provides significant advantages over chemical synthesis. Compound
3 is produced in moderate yield in a single step by enzymatic synthesis
and can be protected and activated for DNA synthesis by standard chem-
istry. This enables the site-specific incorporation of 6-chloropurine residues
into oligonucleotides for post-synthetic coupling of amine-containing flu-
orophores or other pharmacophores. This enables introduction of a wide
range of functionality into the oligonucleotide scaffold.
EXPERIMENTAL
Materials and Methods
Reagents and Enzymes
Silica gel column chromatography was performed using Sorbent silica
˚
gel, standard grade, porosity 60A, particle size 32–63 µm (230 × 450 mesh),

Synthesis of Fluorescently Labeled Oligonucleotides
835
FIGURE 1 Single crystal x-ray structure of 3.
surface area 500–600 m2/g, bulk density 0.4 g/mL, pH range 6.5–7.5. High
performance liquid chromatography (HPLC) grade solvents were obtained
from Fisher (Pittsburgh, PA, USA). Reagent grade chemicals and deuterated
solvents were obtained from Aldrich (Milwaukee, WI, USA) and were used as
received. Nucleoside 2ꢁ-deoxyribosyltransferase (EC 2.4.2.6, transferase) was
a generous gift from S. Short (GlaxoSmithKline, Research Triangle Park,
NC, USA).
Instrumental Analysis
1H NMR spectra were obtained on a Bruker AV-I console operating at
1
400.13 MHz. H COSY experiments were executed using a 9.4 T Oxford
magnet equipped with a Bruker AV-I console operating at 400.13 MHz.
Experimental conditions included 2048 × 512 data matrix, 13 ppm sweep
width, recycle delay of 1.5 seconds, and 4 scans per increment. The data
were processed using a squared sine-bell window function, symmetrized,

836
Md. J. Uddin et al.
FIGURE 2 (A) HPLC chromatogram of the oligonucleotide, 6 (retention time = 8.9 minutes) with
detection at 260 nm; (B) HPLC chromatogram of oligonucleotide, 7 (retention time = 16 minutes)
with detection at 540 nm; (C) fluorescence excitation (λ
540 nm) spectra of compound 7 in Hank’s
max
Balanced Salt solution pH 7.4; (D) fluorescence emission (λ
572 nm) spectra of compound 7 in
max
Hank’s Balanced Salt solution pH 7.4.
and displayed in magnitude mode. 13C direct detection, heteronuclear single
quantum coherence (HSQC), and heteronuclear multiple bond correlation
HMBC (HMBC) NMR experiments were conducted using an 11.7 T Oxford
magnet equipped with a Bruker DRX console operating at 500.13 MHz.
Multiplicity-edited HSQC spectra were acquired using a 2048 × 256 data
matrix, a J (C-H) value of 145 Hz resulting in a multiplicity selection delay
of 34 ms, a recycle delay of 1.5 seconds, and 16 scans per increment along
with GARP decoupling on 13C during the acquisition time (150 ms). The
data were processed using a p/2 shifted squared sine window function and
displayed with CH/CH3 signals phased positive and CH2 signals phased
negative. J 1(C-H) filtered HMBC experiments were acquired using a 2048
× 256 data matrix, a J (C-H) value of 9 Hz for detection of long range
couplings resulting in an evolution delay of 55ms, a J 1(C-H) filter delay
of 145 Hz (34 ms) for the suppression of one-bond couplings, a recycle
delay of 1.5 seconds and 128 scans per increment. The HMBC data were
processed using a p/2 shifted squared sine window function and displayed

Synthesis of Fluorescently Labeled Oligonucleotides
837
in magnitude mode. Mass spectrometry was performed on a quantum triple
quadrupole instrument in ESI positive or negative ion mode operated in-line
with a ThermoElectron Surveyor pump and autosampler.
Crystallography
A single crystal of 6-chloropurine-2ꢁ-deoxyriboside was used for diffrac-
tion data collection at 293 K on an Oxford Diffraction XCalibur PX2 Ultra
with an Onyx CCD detector with CuKα radiation. Diffraction data were in-
dexed, integrated, and scaled using Chrysalis software.[12] The structure was
solved using SIR2002[13] and refined with SHELXL.[14] The ortep images
were prepared using XP.[14]
Fluorescence Spectroscopy
The steady-state fluorescence excitation and emission were determined
with a Spex 1681 Fluorolog spectrofluorometer, equipped with a 450 W
xenon arc lamp. The excitation and emission monochromator slit widths
were 1–2 mm.
HPLC Analysis
The purification of fluorescent oligonucleotide was performed on a
Waters 2996 HPLC system with a photodiode array detector using a Phe-
nomenex C18 column (25 × 0.46 cm, 3 mL/min) with 0.1 M aqueous
ammonium formate and acetonitrile. HPLC gradients: (A) initially 1% ace-
tonitrile, 15 minutes linear gradient to 10% acetonitrile, 5 minutes linear gra-
dient to 20% acetonitrile, 5 minutes isocratic at 20% acetontrile, 3 minutes
linear gradient to 100% acetonitrile, 4 minutes isocratic in 100% acetonitrile
followed by 3 minutes linear gradient to initial conditions. (B) Initially 1%
acetonitrile, 5 minutes linear gradient to 5% acetonitrile, 25 minutes linear
gradient to 8% acetonitrile, 3 minutes linear gradient to 99% acetonitrile,
isocratic at 99% acetonitrile for 6 minutes, and 3 minutes linear gradient to
the initial conditions, isocratic at 1% acetonitrile for 3 minutes. (C) Initially
1% acetonitrile, 5 minutes linear gradient to 5% acetonitrile, 20 minutes
linear gradient to 5.7% acetonitrile, 3 minutes linear gradient to 99% ace-
tonitrile, isocratic at 99% acetonitrile for 5 minutes, and then a 3 minute
linear gradient to the initial conditions, isocratic at 1% acetonitrile for 2
minutes.
Chemistry
Synthesis of 6-Chloropurine-2ꢀ-deoxyriboside (3)
2ꢁ-Deoxycytidine (1) (1.25 g, 5.5 mmol), 2 (0.85 g, 5.5 mmol), and deion-
ized water (100 mL) were mixed in a 250 mL bottle and capped. The bottle
was agitated in a shaker at 37◦C for approximately 1 hour or until all of the
2 was dissolved. Purified E. coli nucleoside 2ꢁ-deoxyribosyltransferase (E.C.

838
Md. J. Uddin et al.
2.4.2.6) was added (300 µg in 2 µL 100 mM sodium phosphate buffer,
pH 6.0) with continued agitation at 37◦C for 4 hours. The reaction was
cooled to room temperature, and water was removed by rotary evaporation.
The resulting residue was purified by flash chromatography using 35:7:1
CHCl3:MeOH:NH4OH resulting in a white solid. The product was recrystal-
lized using 6:1 EtOAc:MeOH to give colorless crystals of 3 (0.59 g, 40%).
1H NMR (400 MHz, D2O) 2.62–2.65 (m, 1H), 2.86–2.88 (m, 1H), 3.81–3.84
(m, 2H), 4.17 (q, 1H), 4.67–4.70 (m, 1H), 6.52 (t, 1H), 8.64 (s, 1H), 8.68 (s,
1H); MS (ESI) m/z (M+H)+ calcd 271.05; found 271.06
Synthesis of 5ꢀ-Dimethoxytrityl 6-chloropurine-2ꢀ-deoxyriboside (4)
To a stirred solution of 3 (174 mg, 0.64 mmol) in dry pyridine
(2.5 mL) was added diiospropylethylamine (168 mg, 0.96 mmol) followed
by dimethoxytrityl chloride (304 mg, 0.9 mmol). The reaction mixture
was stirred at room temperature for 5 hours. Then MeOH (1 mL) was
added with further stirring for 10 minutes. Water (18 mL) was added
and the organic compounds were extracted with CH2Cl2 and washed with
10% K2CO3. The organic layer was dried with Na2SO4 and evaporated in
vacuo, and the residue was purified by flash chromatography using 4:95:1
CHCl3:MeOH:diisopropylethylamine to give the product (4) as a brown
amorphous solid (280 mg, 77%). 1H NMR (400 MHz, DMSO d6) 2.36–2.40
(m, 1H), 2.85–2.89 (m, 1H), 3.12–3.21 (m, 2H), 4.00–4.04 (m, 1H), 4.49–4.52
(m, 1H), 5.40 (d, 1H, OH), 6.47 (t, 1H), 6.50- 6.78 (m, 4H), 7.13–7.21 (m,
7H), 7.27–7.30 (m, 2H), 8.69 (s, 1H), 8.79 (s, 1H); MS (ESI) m/z (M+H)+
calcd 573.18; found 573.20.
Synthesis of 6-Chloropurine-2ꢀ-deoxyriboside 5ꢀ-dimethoxytrityl
3ꢀ-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite (5)
To a stirred solution of 4 (40 mg, 0.07 mmol) in dry CH2Cl2 (0.5 mL) was
added diisopropylethylamine (13 mg, 0.1 mmol) followed by β-cyanoethyl-
N,N -diisopropylamino-3-chlorophosphoramidite (20 mg, 0.084 mmol). The
reaction mixture was stirred at room temperature for 1 hour. Then EtOAc
(0.5 mL) and water (1 mL) were added, and the organic compounds were
extracted and washed with saturated NaHCO3 and water. The organic layer
was dried with Na2SO4 and evaporated in vacuo, and the residue was purified
by flash chromatography using 49:50:1 EtOAc:n-Hex:triethylamine to give
the product 5 as a colorless gummy mass (86% total). A diastereomerically
pure single isomer was obtained with a silica column using a gradient elution
of EtOAc:n-Hex:TEA (10:89:1 to 30:69:1). Single isomer—1H NMR (400
MHz, DMSO d6) 1.12 (s, 3H), 1.13 (s, 3H), 1.14 (s, 3H), 1.15 (s, 3H),
2.54–2.56 (m, 1H), 2.67 (t, 2H), 3.12–3.14 (m, 1H), 3.22–3.28 (m, 2H),
3.57–3.58 (m, 2H), 3.65–3.68 (m, 2H), 3.71 (s, 3H), 3.72 (s, 3H), 4.18–4.21
(m, 1H), 4.82–4.86 (m, 1H), 6.50–6.52 (m, 1H), 6.74–6.79 (m, 4H), 7.15–7.20

Synthesis of Fluorescently Labeled Oligonucleotides
839
(m, 7H), 7.28–7.30 (m, 2H), 8.67 (s, 1H), 8.82 (s, 1H); MS (ESI) m/z (M+H)+
calcd 773.29; found 773.31.
Synthesis of 5ꢀ-TATCATGTCTGPuClATTCCCGGT-3ꢀ (6)
The 21-mer oligonucleotide 6 was synthesized from the 3ꢁ to 5ꢁ end on
0.2 µmol scale using standard automated DNA synthesis using an ABI 395
DNA synthesizer at Midland Certified Reagents. A 0.1 M solution of phospho-
ramidite 5 in dry acetonitrile was used for standard automated solid-phase
synthesis. After synthesis, the oligonucleotide was cleaved from the solid
support by incubation at 55◦C with concentrated ammonium hydroxide, re-
sulting in complete deprotection of the oligonucleotide. The product was
desalted by gel filtration chromatography and the oligonucleotide was pu-
rified using reverse-phase HPLC (C18; acetonitrile:water; 10−50% gradient
over 50 minutes; monitoring at 254 nm). The purified oligonucleotide ex-
hibited one peak on HPLC. Electrospray mass spectrometry of the oligonu-
cleotide confirmed the formation of 5ꢁ-TATCATGTCTGPuClATTCCCGGT-
3ꢁ (6), ESMS m/z [M-H]− calcd 6381.4, found 6381.8.
Synthesis of 5ꢀ-TATCATGTCTGPuTAMRAATTCCCGGT-3ꢀ(7)
Oligonucleotide 6 (1 mg) was mixed with diisopropylethylamine
(50 µL), DMSO (100 µL), and TAMRA cadaverine (1.4 mg,) in a plastic
test tube. The reaction mixture was stirred at 75◦C for 3 days. HPLC analy-
sis showed complete disappearance of 6. Water was added and the sample
was lyophilized to give the crude product, which was purified by sequential
chromatography on the three reversed-phase HPLC systems listed above to
afford the oligonucleotide 7 (61%). ESMS m/z [(M-5H)/5z]− calcd 1371.0,
found 1371.2.
TABLE 1 Crystallographic parameters
Cell parameters
Values
˚
20.658 A
a
b
c
˚
8.156 A
˚
7.047 A
β
97.637
1176.79
C2
3
˚
V (A )
Space group
µ (mm−3
)
2.97
Reflections
Parameters
2474
166
Goodness of Fit (all data)
R1 (all data)
1.026
0.045

840
Md. J. Uddin et al.
Crystal Data
A crystal structure determination of compound 3 was performed, where
diffraction data were collected from a single crystal at room temperature
(293 K). The crystal was monoclinic with space group C2. Unit cell parame-
ters are shown in Table 1. The structure was phased by direct methods and
atom assignments verified as expected. Structure refinement with riding hy-
drogens and anisotropic temperature factors resulted in a final R1 of 0.045
and a Goodness of Fit (S) of 1.026. The absolute structure was confirmed rea-
sonably well with a Flack-x parameter of 0.084 (esd 0.0227). A displacement
thermal ellipsoid plot is shown in Figure 1.
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