Photo-cleavable nucleotides for primer free enzyme mediated DNA synthesis

Article abstract of DOI:10.1039/c6ob01371f

The synthesis, characterization and potential application of eight 3′-O modified 2′-deoxyribonucleoside triphosphates (dNTPs) are discussed. These nucleotide analogues are modified by capping the 3′-OH with a photolabile protecting group which can temporarily cease DNA strand growth and can smoothly reinitiate the growth by the photodecomposition of the protecting group and setting the 3′-OH of dNTPs free to propagate. The synthesis of 3′-O-(2-nitrobenzyl)-2′-deoxyribonucleoside triphosphates (NB-dNTPs) and 3′-O-(4,5-dimethoxy-2-nitrobenzyl)-2′-deoxyribonucleoside triphosphates (DMNB-dNTPs) is discussed in detail with structural confirmation using NMR. The UV-cleaving studies are monitored and quantified using LCMS and ¹H NMR spectral traces. The synthesised nucleotides are employed for terminating and reinitiating template-less DNA synthesis, using primer independent Terminal Deoxynucleotidyl Transferase (TdT) enzyme. The use of this photolabile nucleotide in one step stop-start DNA synthesis is a novel strategy towards the precise assembly of dNTPs with the potential to reinforce present technologies.

Full text of DOI:10.1039/c6ob01371f

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Organic and Biomolecular Chemistry
ARTICLE
Received 00th January
Photo-cleavable Nucleotides for Primer Free Enzyme mediated
20xx,
Accepted 00th January
DNA synthesis.
20xx
DOI:
Anu Stella Mathews, a, b Haikang Yang, a, b Carlo Montemagno* a, b
10.1039/x0xx00000x
Abstract
www.rsc.org/
Synthesis, chatacterization and a potential application of eight 3′ modified 2′-deoxy ribonucleoside
triphosphates (dNTPs) is discussed. These nucleotide analogues are modified by capping the 3′-OH by a
photolabile protecting group which can temporarily cease DNA strand growth and can smoothly reinitiate the
growth by the photo decomposition of protecting group and setting the 3′-OH of dNTPs free to propagate. The
synthesis of 3′-O-(2-nitrobenzyl)-2′-deoxy ribonucleoside triphosphates (NB-dNTPs) and 3′-O-(4,5-dimethoxy-
2-nitrobenzyl)- 2′-deoxy ribonucleoside triphosphates (DMNB-dNTPs) is discussed in details with structural
1
confirmation using NMR. The UV-cleaving studies is monitored and quantified using LCMS and H NMR
spectral traces. The synthesised nucleotides are employed for terminating and reinitiating, templateless DNA
synthesis, using primer indepentent Terminal Deoxynucleotidyl Transferase (TdT) enzyme. The use of this
photolabile nucleotides as one step stop-start DNA synthesis is a novel strategy towards the presice assemply
of dNTPs with the potential to reinforce present technologies .
Table of contents
Presented will be the synthesis and characterization of eight 3′ modified 2′-deoxyribonucleoside triphosphates
for template less enzyme mediated photo-triggered “stop-start” DNA synthesis.
Introduction
inheritance called DNA. Modification of DNA through its basic
building blocks 2′ deoxyribonucleoside triphosphates (dNTPs)
Researches, from Friedrich Miescher’s discovery of
phosphorous-rich chemical in the nuclei of leukocytes, which he
named nuclein, in 18691 to the completion of gold-standard
genome sequence in 2003,2 have set the stage for understanding
and exploring the complex macromolecular carriers of
paves the way for wide applications in modern molecular
biology, clinical medicine and health care. dNTPs modified at
the 3′ hydroxyl position can act as enzyme mediated DNA
synthesis terminators, finding immense applications such as
DNA synthesis,3 DNA and RNA 3′ end labelling,4 mechanistic
probes, antimetabolites and antiviral agents.5 Thus, 3′ OH
modification of dNTPs with photolabile moieties promises a
versatile way of terminating or labelling sequence ends with the
further possibility of breaking bonds smoothly without the need
of any reagent and reinitiating any sort of reaction, leading to the
controlled presentation, patterning and release of bioactive sites.6
a.Ingenuity Lab, 11421 Saskatchewan Drive, Edmonton, Alberta T6G 3M9,
Canada
b.Department of Chemical and Materials Engineering, University of Alberta,
Edmonton, Alberta T6G 2V4, Canada
*Phone: +1 780-641-1617. E-mail: montemag@ualberta.ca.
Electronic
Supplementary
Information
(ESI)
available:
See
DOI: 10.1039/x0xx00000x
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We report the synthesis of eight 3′-O modified dNTPs and dNTPs. The purity of all the compounds was >90%. Molecular
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DOI: 10.1039/C6OB01371F
their UV response studies. 2-Nitrobenzyl and 4,5-dimethoxy-2- mass obtained from negative ion mode of LCMS showed
nitrobenzyl groups are used as the photo labile protecting groups [M+3H] (M is the calculated mass) due to the presence of
due to its high photo cleavable efficiency at near-UV light triphosphate ions.
irradiation.7 This study is the first of its kind which gives a better
understanding of the photo decomposition nature of all the four
dNTPs with respect to the base as well as the photolabile moiety
attached. Though the termination of DNA synthesis by 3′-O
modified dNTPs have been reported earlier,8,9 template-less
precise assembly of nucleotides with terminal deoxynucleotidyl
transferase (TdT) enzyme is a novel sequencing strategy. Present
work also demonstrates the successful application of synthesized
dNTP analogues as DNA sequence terminators during enzymatic
synthesis using TdT, which is a template-independent DNA
polymerase that catalyses the sequential addition of dNTPs to
oligonucleotides.
Results and discussion
The synthesis of eight 3′-O modified dNTPs were done by
selective protection strategy.10 Scheme 1 shows the general
synthetic route adapted to make 3′-O-(2-nitrobenzyl)-2′
deoxyribonucleoside triphosphates (NB-dNTPs) and 3′-O-(4,5-
dimethoxy-2-nitrobenzyl)-2′-deoxyribonucleoside triphosphates
(DMNB-dNTPs)
Scheme 1: General synthetic scheme of NB-dNTPs and DMNB-
dNTPs.
The synthesis was started using 5′-O caged deoxyribo purine
/ pyrimidine compounds. The primary and secondary amines in
the base were protected in order to avoid unwanted side reactions
which can decrease the yield. Site specific introduction of
photolabile group to the 3′- hydroxyl position of the well-sealed
starting material in the first step followed by the base and 5′-O
Table 1: Structure and molecular mass of 3′-O-modified dNTPs
confirmed using 1H NMR, HPLC and LCMS analysis.
deprotection procedures were carried out in dark inert
Biochemical and chemical researches, employing various
atmosphere. Each intermediate was purified using flash column
photo-active groups and irradiation protocols, show that the
1
chromatography and structure was confirmed using H NMR.
photoreactions carried out in the body tend to use photo source
with comparable long wavelength (>300 nm) to minimize
The final triphosphorylation procedure was modified to improve
purity and yield. The final products were purified by ion
exchange chromatography. The structural confirmation of the
modified dNTPs was done using NMR spectra and the purity was
assessed using HPLC and LCMS techniques. Table 1 shows the
structure, molecular mass and HPLC traces of all the eight
damage to tissue.11 Therefore, we choose nitrobenzyl derivatives
as the caging moiety of 3′OH of dNTPs. Photo-decomposition
reactions were carried out by irradiation using 365 nm UV source (1.4
mW/cm2 and 10 mW/cm2) with a working distance of 5 cm. The
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concentration and volume of all samples in water were fixed to 1 during deprotection to get an insight into the background activities
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DOI: 10.1039/C6OB01371F
mg/mL and 1 mL respectively.
that can arise from the contamination by the deprotected materials.
1H NMR monitored the structural changes of the dNTPs at regular
intervals of time. Base and sugar structures of the modified dNTPs
exhibited no changes in 1H NMR after irradiation while the 2H peaks
of 3′ O-CH2-nitrobenzyl, around 5.02-4.90 ppm, showed integration
decrease. The aromatic region corresponding to nitrobenzyl group and
O-CH3 of DMNB-dNTPs in NMR spectra was also altered while no
new peaks were formed proving the absence of formation of new
bonds resulting from contamination reactions with deprotected group
and dNTPs. Figure 2 shows the photo decomposition of NB-dTTP at
0, 5 and 10 minutes, of 1 mL of 1 mg/mL solutions of samples in D2O,
with 365 nm 10 mW /cm2. The H NMR spectra showed that all the
1
peaks of pyrimidine and sugar in thymidine remained intact, subjected
to small peak shift as a result of change in hydrogen interaction, while
the 3
′ O-CH2 peak gradually disappeared showing complete
decomposition.
Figure 1: 3D Mass traces of 3′-O-caged-dNTPs before and after
UV irradiation.
The photo removability of the protecting groups was first verified
using the mass changes in LCMS spectra. Figure 1 shows the 3D mass
traces quantified using LCMS for four model dNTPs when irradiated.
365 nm 10 mW/cm2 light source was used for complete photo
cleavage of 1 mL of 1 mg/mL solutions of samples in water. A
subtraction of mass ~ 135 and 195 was determined after irradiation
proving the exact detachment of the nitrobenzyl and
dimethoxynitrobenzyl groups respectively from 3′-O position leaving
the base and sugar structures of dNTPs intact and 3′-OH available for
further reactions.
Figure 3: Decomposition - irradiation time histograms of NB-
dTTP using 1.4 mW/cm2 and 10 mW/cm2power at 365 nm.
Prolonged UV exposure at 365 nm can damage biomolecules.12
To decrease the exposure time, we have used two different strategies
i.e. 1. increase the power of UV source and 2. change the protecting
group. Integration changes of the 3′ O-CH2 peak in comparison with
the 1H of 1′H of the sugar ring, fixed as 1, in 1H NMR spectral analysis
of the dNTPs in D2O were calculated as degree of decomposition. To
quantify the photo lability of NB-dNTPs and DMNB-dNTPs the
decomposition percentage was plotted as a function of time (Figure 3
and 4). Figure 3 shows the decomposition degree as a function of
power of UV light. The photo decompositions were carried out with
all 8 dNPTs with two different power (1.4 mW/cm2 and 10 mW/cm2)
for 60 minutes under fixed conditions of concentration and solvent.
Faster dissociation kinetics was obtained at elevated power of light.
More than 50% of deprotection was observed within 5 minutes of
irradiation for 10 mW/cm2 power source while only 5% cleavage was
observed for 1.4 mW/cm2 source. The complete deprotection
occurred within 10 minutes for 10 mW /cm2 and within 55 minutes
Figure 2: 1H NMR spectra of NB-dTTPs at 0, 5 and 10 minutes
of UV irradiation
When dealing with the dNTP molecules which have several
functional groups, it is important to study the structural changes
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for 1.4 mW/cm2 power source. Major concern was the distortion of the base is well isolated from the aci-nitro intermediate forming
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DOI: 10.1039/C6OB01371F
dNTP molecule by the use of high power UV. This was ruled out as photo-tautomerization occurring in the 3′-O position of the sugar ring
the 1H NMR spectra shows the dNTP structure to be very well stable due to the absence of any pi electrons on its way, we attribute the
after UV exposure till completion reaction. The result was confirmed difference in the dissociating rate to the spatial orientation of the bulky
by the Mass obtained by LCMS analysis.
imidazole fused pyrimidine on adenosine and guanine base containing
dATPs and dGTPs.
Figure 4: Decomposition - irradiation time plots of selected 3′-
O-caged-dNTPs at 365 nm 10 mW/cm2.
Figure 5: Peak Integration - irradiation time plots of NB-dCTP,
NB-dGTP, DMNB-dCTP and DMNB-dGTP in D2O at 365 nm
10 mW/cm2.
The impact of the structure of protecting group on the time of
decomposition is portrayed in figure 4. It was observed when the 4,5-
dimethoxy-2-nitrobenzyl moiety was replaced by 2-nitrobenzyl
photo-liable group the rate of decomposition was enhanced. 50%
photodecomposition was noted at 3 minutes for NB-dNTPs and at 5
minutes for DMNB-dNTPs. All the NB-dNTPs were completely
cleaved in 10 minutes of irradiation with 365 nm 10 mW/cm2 light
while it took 15 minutes for the complete deprotection of DMNB-
dNTPs. The presence of electron donating –OCH3 groups in the 4, 5
position of the nitrobenzyl ring have significantly reduced the rate of
photo decomposition showing that photo lability of the nucleotides
can be tailored according to the substitution in the nitrobenzyl ring.
The photodecomposition of 2-nitrobenzyl compounds occurs via
electronic excitation generating aci-nitro tautomers.13,14 The rate of
formation, stability and dissociation of this intermediates play a
significant role in the rate of this photo reactions. Higher power source
can accelerate the formation of the aci-nitro transient resulting in the
increased rate of photo decomposition. The effect of -OCH3
substitution can be attributed to the difference in the photo
tautomerisation mechanism15 leading to the aci-nitro
intermediate imparted by the electron donating methoxy
substitution of nitro benzene. The substitution can either slow
down the rate of tautomerization or can stabilize the aci-nitro
intermediate therefore decreasing the dissociation rate of the
intermediate to form final product.
The behaviour of the dNTP analogues in one step spot-start DNA
synthesis was also evaluated. Studies show that 3′-O modification of
dNTPs possess great challenge for incorporation by natural
polymerase, especially when the 3′-O cage is a bulky one as nitro
benzyl moiety.16 Keeping this in mind we selected terminal
deoxynucleotidyl transferase (TdT) enzyme to catalyse the DNA
symthesis. TdT belongs to a class of DNA polymerase with
relatively simple mechanism for catalysis due to the template
free polydeoxynucleotide direction for DNA synthesis. Together
with the efficiency at catalysing the growth of DNA from a DNA
initiator, TdT can also incorporate multiple unnatural fluorescent
nucleotides.17 Enzymatic, one step off-on, DNA extension was
performed by incubating 92.5 ng/L of a blunt-end 750 base-pair
DNA strand (750bp-DNA) with TdT at 20 U/L in presence of
cobalt containing buffer and 1mM of mono 3′-O protected dNTP
at 37 oC for 1 hour. Half the volume of samples was withdrawn
after 1 hour and subjected to UV light at 365 nm 10 mW/cm2 for
15 minutes and was again treated with TdT and unprotected
mono dNTP and incubated at 37 oC for another 1 hour. Figure 6
Shows the agarose gel electrophoresis image of termination and
reinitiation of the DNA sequencing before and after UV
irradiation. The caged dNTPs terminated the addition of more
nucleotides after initial addition of the first NB-dNTP / DMNB-
dNTP as indicated by bands 3, 4, 5, and 6. The original activity
was found to be restored after photolysis as indicated by bands
8, 9, 10 and 11. The result also shows a pronounced difference
between purine and pyrimidine triphosphates which can be
attributed to the difference photodecomposition depending
on the purine, pyrimidine stereochemistry14 and binding
Figure 5 plots the decrease in 3′-O-CH2 peak integration of all four
NB-dNTPs, showing the effect of base structure can on photo
decomposition rate. Purine based adenosine and guanine are
decomposed slightly faster than pyrimidine based cytosine and
thymine. The stability of the azi-nitro intermediate imparted by the
base can contribute to the difference in decomposition rates.7 Since
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1. Synthesis
triphosphate
of
3′-O-(2-nitrobenzyl)-2′-deoxyadenosine-5′-
pattern depending on TdT enzyme base preference while
adding different dNTPs to a DNA strand.18
View Article Online
(NB-dATP)
and
3D′O-OI:-1(04.,150-3d9i/mC6eOthBo0x13y7-21F-
nitrobenzyl)-2′-deoxyadenosine-5′-triphosphate (DMNB-dATP).
Scheme 2: Synthesis of NB-dATP and DMNB-dATP.
1.3b. Synthesis of 9-[β-D-5’-O-(tert-butyldimethylsilyl)-3’-O-(4,5-
dimethoxy-2-nitrobenzyl)-2’-deoxyribofuranosyl]-6-
chloropurine.
Figure 6: Gel electrophoresis showing the incorporation of NB-
dNTPs by TdT to 750bp-DNA causing the temperarory
termination of sequencing and the reinitiation after photo-
cleaving the 3′-O-2′-nitrobenzyl group and incorporating
nonprotected dNTP. (1. Standard Ladder, 2. Commercial dNTP
mix, 3. NB-dATP, 4. NB-dCTP,5. NB-dGTP,6. NB-dTTP, and
samples after UV irradiation and reintiation with 7. Commercial
dNTP mix, 8. dATP, 9. dCTP, 10. dGTP, and 11. dTTP). (Figure
16 in ESI shows the gel electrophoresis data for DMNB-dNTPs)
To a stirred solution of 9-[β-D-5’-O-(tert-butyldimethylsilyl)-2’-
deoxyribofuranosyl]-6-chloropurine 1.210(1.73 g, 4.48 mmol) in
DCM (135 mL) were added tetrabutylammonium bromide (0.722 g,
2.24 mmol), 4,5-dimethoxy-2-nitrobenzyl bromide (3.09 g, 11.2
mmol) and 40% aq. NaOH (65 mL). The reaction mixture was stirred
at room temperature for 1 h, diluted with EtOAc (300 mL) and then
the organic layer was separated. The aqueous layer was extracted with
EtOAc (2x125 mL). The combined organic layers were washed with
sat. NaHCO3 (80 mL), brine (80 mL) and dried over Na2SO4. After
concentration of the filtrate, the residue was purified by flash column
chromatography over silica gel using EtOAc/hexanes (1:2) to give
Conclusions
Photoselective template independent enzymatic DNA
synthesis was adapted and developed to give a novel strategy for
precise stacking of single nucleotides. Photo release of 2-
nitrobenzyl and 4,5-dimethoxy-2-nitrobenzyl groups from
dNTPs was quantified as a function of UV source power, caging
moiety and base. The rate of photo decomposition was perceived
to be dependent on the aci-nitro transients, as observed form the
altered release of photo labile group, in accordance with the
power of irradiation and substitution on the nitrobenzyl group.
Purine and pyrimidine base structure was found to affect the
photodecomposition. The study was well-applied for termination
and reinitiation of DNA synthesis with the merit of enzyme
specificity and non-chemical reaction. Ours is the first example
of “stop-start” DNA synthesis allowing the efficient use of the
unique template independent nature of TdT enzyme in a
controlled manner, which opens a new direction of research and
potentially new applications for photo labile nucleotides.
1
compound 1.3b was obtained as a yellow solid (2.22 g, 85%). H
NMR (600 MHz, CDCl3) δ 8.74 (s, 1H, 2-H), 8.51 (s, 1H, 8-H), 7.73
(s, 1H, one of C6H2), 7.26 (1H, one of C6H2), 6.59 (dd, J = 6.0, 7.8
Hz, 1H, 1′-H), 5.03-4.94 (m, 2H, OCH2C6H2), 4.49-4.45 (m, 1H, 3’-
H), 4.38-4.33 (m, 1H, 4′-H), 4.04 (s, 3H, OCH3), 3.97 (s, 3H, OCH3),
3.94 (dd, J = 3.6, 11.4 Hz, 1H, one of 5′-H), 3.86 (dd, J = 3.0, 11.4
Hz, 1H, one of 5′-H), 2.82-2.68 (m, 2H, 2′-H), 0.90 (s, 9H, C(CH3)3),
0.11 (s, 3H, one of SiCH3), 0.10 (s, 3H, one of SiCH3).
1.4b. Synthesis of 3′-O-(4,5-dimethoxy-2-nitrobenzyl)-2′-
deoxyadenosine.
To a stirred solution of 9-[β-D-5′-O-(tert-butyldimethylsilyl)-3′-
O-(4,5- dimethoxy -2- nitro benzyl) -2′- deoxyribo furanosyl]-6-
chloropurine 1.3b (2.22 g, 3.83 mmol) in THF (40 mL) was added 1.0
M tetrabutylammonium fluoride (TBAF) in THF solution (4.20 mL,
4.20 mmol) dropwise over 5 min. The reaction mixture was stirred at
room temperature for 1.5 h. After concentration, the residue was
dissolved in a mixture of dioxane (20 mL) and 7 N NH3 in MeOH (40
mL). The solution was stirred in a sealed flask at 85-90 °C for another
18 h. After evaporation, the residue was purified by flash column
chromatography over silica gel using MeOH/DCM (1:20) to
compound 1.4b as a pale yellow solid (0.953 g, 56%). 1H NMR (600
MHz, DMSO-d6) δ 8.34 (s, 1H, 2-H), 8.13 (s, 1H, 8-H), 7.69 (s, 1H,
Experimental
Synthesis of NB-dNTPs and DMNB-dNTPs: Experimental
procedures were done according to previous literature,10 with some
modifications. The repeated reactions are explained in Electronic
Supplementary Information (ESI). The 1H NMR, 31P NMR, LCMS of
all 3’-O-modified dNTPs and 13C NMR and HRMS of novel
compounds DMNB-dNTPs are given in ESI. The modified synthetic
procedures are explained below.
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one of C6H2), 7.32 (brs, 3H, 6-NH2 and one of C6H4), 6.35 (dd, J =
6.0, 8.4 Hz, 1H, 1′-H), 5.34 (t, J = 5.4 Hz, 1H, 5′-OH), 4.96-4.88 (m,
2H, OCH2C6H2), 4.38-4.35 (m, 1H, 3′-H), 4.16-4.12 (m, 1H, 4′-H),
2.5b. Synthesis of 4-O-allyl-5′-O-(4,4′-dimethoxytrityl)-3′-O-(4,5-
dimethoxy-2-nitrobenzyl)-2′-deoxyuridine.DOI: 10.1039/C6OB01371F
View Article Online
To
a vigorously stirring mixture of 4-O-allyl-5′-O-(4,4′-
3.94 (s, 3H, OCH3), 3.88 (s, 3H, OCH3), 3.66-3.53 (m, 2H, 5′-H), dimethoxytrityl)-2′-deoxyuridine 2.410 (1.91 g, 3.35 mmol), Bu4NOH
2.90-2.84 (m, 1H, one of 2′-H), 2.58-2.52 (m, 1H, 2′-H).
(tetrabutylammonium hydroxide) (1.5 mL, 55-60% in water) and NaI
(50.0 mg, 0.335 mmol) in DCM/water (10 mL/10 mL) was added 1.0
M NaOH solution (10 mL, 10 mmol). The mixture was stirred for 10
min at room temperature, a solution of 4,5-dimethoxy-2-nitrobenzyl
DMNB-dATP. Synthesis of 3′-O-(4,5-dimethoxy-2-nitrobenzyl)-
2′-deoxyadenosine-5’-triphosphate.
To
a suspension of 3′-O-(4,5-dimethoxy-2-nitrobenzyl)-2′- bromide (1.84 g, 6.70 mmol) in 10 mL of DCM was added over 5
deoxyadenosine (134 mg, 0.300 mmol) in anhydrous pyridine (1.0 min and the resulting reaction mixture was stirred for another 7 h at
mL) and DMF (1.0 mL) was added a solution of 2-chloro-4H-1,3,2- room temperature. The reaction mixture was diluted with DCM (150
benzodioxaphosphorin-4-one (85.0 mg, 0.405 mmol) in anhydrous mL) and then washed with brine (20 mL). The organic layer was dried
dioxane (1.0 mL) over 1 min at room temperature, leading to a clear over anhydrous Na2SO4. After evaporation, the crude product was
solution. After 20 min, a mixture of tributylammonium pyrophosphate then purified by flash column chromatography over silica gel using
(380 mg, 0.690 mmol) in anhydrous DMF (1.5 mL) and tributylamine hexanes/ethyl acetate (3:1~1:1) to give compound 2.5b as a pale
(0.350 mL, 1.47 mmol) was added over 20 seconds. After 20 min, a yellow solid (2.31 g, 90%). 1H NMR (600 MHz, CDCl3) δ 8.05 (d, J
solution of iodine (105 mg, 0.410 mmol) and water (0.16 mL) in = 7.2 Hz, 1H, 6-H), 7.72 (s, 1H, one of C6H2), 7.39-7.36 (m, 2H, two
pyridine (8 mL) was added over 5 min. After 20 min, the reaction was aromatic protons of DMTr), 7.30-7.20 (m, 7H, seven aromatic protons
quenched by addition of 5% Na2SO3 until excess iodine disappeared. of DMTr), 7.19 (s, 1H, one of C6H2), 6.84-6.79 (m, 4H, four ortho
After 10 min, 2.0 mL of 1.0 M triethyl ammonium bicarbonate protons to CH3O of DMTr), 6.37 (t, J = 6.6 Hz, 1H, 1′-H), 6.07-5.99
solution (TEAB) was added. After stirring at room temperature for 1 (m, 1H, CH2=CHCH2), 5.70 (d, J = 7.2 Hz, 1H, 5-H), 5.38 (dd, J =
h, the solvents were removed in vacuo. Water (20 mL) was added, and 1.2, 18.0 Hz, 1H, one of CH2=CHCH2), 5.28 (dd, J = 1.2, 10.2 Hz,
the mixture was filtered. The filtrate was then purified by anion 1H, one of CH2=CHCH2), 4.93-4.82 (m, 4H, CH2=CHCH2 and
exchange chromatography on DEAE-Sephadex A-25 using a gradient OCH2C6H2), 4.33-4.26 (m, 2H, 3′-H and 4′-H), 3.96 (s, 6H, two
of TEAB (pH 8, 0.1-1.0 M) to give compound DMNB-dATP as a CH3O), 3.78 (s, 6H, two CH3O), 3.51 (dd, J = 3.6, 10.8 Hz, 1H, one
pale yellow gum of TEA salt (144 mg, 44%) after lyophilization. 1H of 5′-H), 3.42 (dd, J = 3.6, 10.8 Hz, 1H, one of 5′-H), 2.82-2.76 (m,
NMR (600 MHz, D2O) δ 8.60 (brs, 1H, 2-H), 8.25 (brs, 1H, 8-H), 7.75 1H, one of 2′-H), 2.22-2.16 (m, 1H, one of 2′-H).
(s, 1H, one of C6H2), 7.35 (s, 1H, one of C6H2), 6.52-6.47 (m, 1H, 1′-
H), 5.04-4.94 (m, 2H, OCH2C6H2), 4.73-4.69 (m, 1H, 3′-H), 4.57-4.52
(m, 1H, 4′-H), 4.29-4.15 (m, 2H, 5′-H), 4.00 (s, 3H, OCH3), 3.94 (s,
3H, OCH3), 2.85-2.73 (m, 2H, 2′-H); 13C NMR (150.8 MHz, D2O)
165.8, 156.4, 155.2, 154.5, 152.8, 151.4, 146.8, 138.7, 128.8, 110.4,
107.6, 83.9, 83.8, 80.5, 67.9, 58.5, 56.1, 55.8, 36.6. 31P NMR (242.7
MHz, D2O) δ -10.0 (brs, 2P), -21.7 (brs, 1P); HRMS (ES-) calcd for
2.6b. Synthesis of 3′-O-(4,5-dimethoxy-2-nitrobenzyl)-2′-
deoxycytidine
To an ice cold solution of 4-O-allyl-5′-O-(4,4′-dimethoxytrityl)-
3′-O-(4,5-dimethoxy-2-nitrobenzyl)-2′-deoxyuridine 2.5b (2.30 g,
3.00 mmol) in DCM (45 mL) was added trifluoroacetic acid (2.70 mL,
35.0 mmol) was added slowly over 5 min. The resulting red solution
was stirred for 15 min at room temperature and then quenched with
saturated aqueous NaHCO3 solution until pH = 8-9. The mixture was
diluted with DCM (150 mL). The organic layer was separated, washed
with saturated NaHCO3 solution (2x20 mL) and dried over anhydrous
Na2SO4, and then concentrated. The residue was dissolved in 7 N NH3
-
C19H24N6O16P3 [(M+3H)]: 685.0467, found: 685.0459; HPLC:
89.8%.
2. Synthesis
triphosphate
of
3′-O-(2-nitrobenzyl)-2′-deoxycytidine-5′-
and 3′-O-(4,5-dimethoxy-2-
(NB-dCTP)
o
in methanol (55 mL) and stirred in a sealed tube for 20 h at 55 C.
nitrobenzyl)-dCTP (DMNB-dCTP)
After evaporation, the crude product was then purified by flash
column chromatography over silica gel using MeOH/DCM (1:10) to
afford compound 2.6b as a pale yellow solid (0.51 g, 40%). 1H NMR
(600 MHz, DMSO-d6) δ 7.75 (d, J = 7.8 Hz, 1H, 6-H), 7.67 (s, 1H,
one of C6H2), 7.26 (s, 1H, one of C6H2), 7.15 and 7.09 (s, 2H, NH2),
6.17 (dd, J = 6.0, 8.4 Hz, 1H, 1′-H), 5.71 (d, J = 7.8 Hz, 1H, 5-H),
5.04 (t, J = 5.4 Hz, 1H, OH), 4.86 (s, 2H, OCH2C6H2), 4.19-4.16 (m,
1H, 3′-H), 4.03-4.00 (m, 1H, 4′-H), 3.90 (s, 3H, one of CH3O), 3.84
(s, 3H, one of CH3O), 3.58-3.53 (m, 2H, 5′-H), 2.36-2.31 (m, 1H, one
of 2′-H), 2.06-1.99 (m, 1H, one of 2′-H).
DMNB-dCTP. Synthesis of 3′-O-(4,5-dimethoxy-2-nitrobenzyl)-
2′-deoxycytidine-5′-triphosphate.
Using the same preparation procedure of DMNB-dATP, 3′-O-
(4,5-dimethoxy-2-nitrobenzyl)-dCTP was obtained as a pale yellow
gum of TEA salt (90 mg, 28%). 1H NMR (600 Hz, D2O) δ 8.26 (d, J
= 7.2 Hz, 1H, 6-H), 7.73 (s, 1H, one of C6H2), 7.21 (s, 1H, one of
Scheme 3: Synthesis of NB-dCTP and DMNB-dCTP
6 | Organic & Biomolecular Chemistry, 2016, 00, 1-3
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C6H2), 6.36 (d, J = 7.8 Hz, 1H, 5-H), 6.28 (dd, J = 6.0, 8.4 Hz, 1H, 1′-
H), 4.97-4.87 (m, 2H, OCH2C6H2), 4.60-4.57 (m, 1H, 3′-H), 4.54-4.51
(m, 1H, 4′-H), 4.32-4.21 (m, 2H, 5′-H), 3.98 (s, 3H, one of CH3O),
[(dimethylamino)methylene]-3′-O-(4,5-dimethoxy-2V-inewitrAortibcleenOznylinl)e
-2′-deoxyguanosine
DOI: 10.1039/C6OB01371F
To a solution of 6-Chloro-2-[(dimethylaminomethylene)amino]-
3.93 (s, 3H, one of CH3O), 2.74-2.69 (m, 1H, one of 2′-H), 2.39-2.32 9-[β-D-5′-O-(tert–butyl dimethyl silyl) - 3′ - O - ( 4,5-dimethoxy -2-
(m, 1H, one of 2′-H); 13C NMR (150.8 MHz, D2O)  159.0, 152.8,
nitrobenzyl)-2′-deoxyribofuranosyl]purine 3.4b (1.23 g, 1.89 mmol)
in anhydrous DMF (12.0 mL) were added cesium acetate (1.09 g, 5.68
mmol), 1,4-diazabicyclo[2.2.2]octane (DABCO) (0.212 g, 1.89
mmol) and triethylamine (0.790 mL, 5.68 mmol) under argon and
stirred overnight at room temperature with exclusion of air and light.
Ac2O (5.4 mL) was added to the above reaction mixture and stirred
for another 30 min. The reaction mixture was then quenched with
water (20 mL) and extracted with ethyl acetate (3x50 mL). The
organic layer was dried over Na2SO4. After evaporation, the crude
product was purified by flash column chromatography over silica gel
using MeOH/DCM (1:20) to afford compound 3.5b as a pale yellow
solid (0.52 g, 44%). 1H NMR (600 MHz, CDCl3) δ 8.65 (s, 1H, NH),
8.61 (s, 1H, CHN(CH3)2), 7.90 (s, 1H, 8-H), 7.73 (s, 1H, one of C6H2),
7.25 (s, 1H, one of C6H2), 6.39 (dd, J = 5.4, 8.4 Hz, 1H, 1′-H), 5.05-
4.92 (m, 2H, OCH2C6H2), 4.45-4.42 (m, 1H, 3′-H), 4.29-4.26 (m, 1H,
4′-H), 4.02 (s, 3H, one of CH3O), 3.97 (s, 3H, one of CH3O), 3.87-
3.79 (m, 2H, 5′-H), 3.18 (s, 3H, one of N(CH3)2), 3.10 (s, 3H, one of
N(CH3)2), 2.70-2.65 (m, 1H, one of 2′-H), 2.60-2.54 (m, 1H, one of
2′-H), 0.91 (s, 9H, C(CH3)3), 0.10 (s, 6H, two of SiCH3).
148.0, 146.7, 144.2, 138.6, 128.9, 110.1, 107.5, 95.0, 86.8, 84.1, 80.6,
67.6, 58.4, 56.0, 55.7, 37.3. 31P NMR (242.7 MHz, D2O) δ -10.6 (brs,
-
2P), -23.0 (brs, 1P); HRMS (ES-) calcd for C18H24N4O17P3 [(M+3H)]:
661.0355, found: 661.0356; HPLC: 93.3%.
3. Synthesis of 3′ -O- (2-nitrobenzyl) -2′- deoxyguanosine -5′-
triphosphate
(NB-dGTP)
and
3′-O-(4,5-dimethoxy-2-
nitrobenzyl)- 2′-deoxyguanosine-5′-triphosphate (DMNB-dGTP)
3.6b. Synthesis of N2-[(dimethylamino)methylene]-3′-O-(4,5-
dimethoxy-2-nitrobenzyl)-2′-deoxyguanosine.
To an ice cold solution of 5′-O-(t-butyldimethylsilyl)-N2-
[(dimethylamino)methylene]-3′-O-(4,5-dimethoxy-2-nitrobenzyl)-2′-
deoxyguanosine 3.5b (0.51 g, 0.70 mmol) in THF (6.0 mL) was added
1.0 M TBAF in THF solution (1.6 mL, 1.6 mmol) over 1 min. The
reaction mixture was allowed to warm to room temperature and stirred
for 2.5 h with exclusion of air and light, diluted with EtOAc (100 mL),
washed with water (15 mL), brine (15 mL) and dried over anhydrous
Na2SO4. After concentration of the filtrate, the residue was purified
by flash column chromatography over silica gel using MeOH/DCM
Scheme 4: Synthesis of NB-dGTP and DMNB-dGTP
3.4b. Synthesis of 6-Chloro-2-[(dimethylaminomethylene) amino]
-9- [ β-D -5′ -O- (tert-butyldimethylsilyl) -3′- O- (4,5-dimethoxy -
2-nitrobenzyl)-2′-deoxyribofuranosyl] purine.
To
an
ice
cold
solution
of
6-chloro-2-
1
(1: 20) to afford 3.6b (0.350 g, 85%) as yellow solid. H NMR (600
[(dimethylaminomethylene)amino]-9- [β-D-5′-O-(tert-butyldimethyl
silyl) -2′-deoxyribofur anosyl] purine 3.310 (2.50 g, 5.50 mmol) in
anhydrous THF (25 mL) was added 95% NaH powder (0.306 g, 12.1
mmol) in portions. After stirring for 50 min at room temperature, a
solution of 4,5-dimethoxy-2-nitrobenzyl bromide (3.04 g, 11.0 mmol)
in THF (5.0 mL) was added, and then the reaction mixture was stirred
for another 2.5 h at room temperature with exclusion of air and light.
After concentration, the resulting residue was dissolved in ethyl
acetate (250 mL), washed with saturated aqueous NaHCO3 (20 mL),
brine (20 mL) and dried over anhydrous Na2SO4. After concentration,
the crude product was purified by flash column chromatography over
silica gel using EtOAc/hexanes (1:1-2:1) to afford compound 3.4b as
a pale yellow foam (1.25 g, 35%). 1H NMR (600 MHz, CDCl3) δ 8.74
(s, 1H, CHN(CH3)2), 8.28 (s, 1H, 8-H), 7.73 (s, 1H, one of C6H2), 7.27
(s, 1H, one of C6H2), 6.66 (dd, J = 6.0, 8.4 Hz, 1H, 1′-H), 4.98-4.92
(m, 2H, OCH2C6H2), 4.42-4.36 (m, 1H, 3′-H), 4.32-4.27 (m, 1H, 4′-
H), 4.05 (s, 3H, one of CH3O), 3.97 (s, 3H, one of CH3O), 3.93-3.84
(m, 2H, 5′-H), 3.18 and 3.17 (s, 6H, N(CH3)2), 2.78-2.73 (m, 1H, one
of 2′-H), 2.50-2.44 (m, 1H, one of 2′-H), 0.92 (s, 9H, C(CH3)3), 0.13
(s, 3H, one of SiCH3), 0.12 (s, 3H, one of SiCH3).
MHz, DMSO-d6) δ 11.3 (s, 1H, NH), 8.56 (s, 1H, CHN(CH3)2), 8.04
(s, 1H, 8-H), 7.67 (s, 1H, one of C6H2), 7.29 (s, 1H, one of C6H2), 6.25
(dd, J = 6.0, 8.4 Hz, 1H, 1′-H), 5.04 (t, J = 6.0 Hz, 1H, OH), 4.94-4.86
(m, 2H, OCH2C6H2), 4.35-4.31 (m, 1H, 3′-H), 4.10-4.06 (m, 1H, 4′-
H), 3.92 (s, 3H, one of CH3O), 3.85 (s, 3H, one of CH3O), 3.60-3.52
(m, 2H, 5′-H), 3.12 (s, 3H, one of N(CH3)2), 3.01 (s, 3H, one of
N(CH3)2), 2.76-2.60 (m, 1H, one of 2′-H), 2.54-2.48 (m, 1H, one of
2′-H overlapped with DMSO signal).
DMNB-dGTP. Synthesis of 3′-O-(4,5-dimethoxy-2-nitrobenzyl)-
2′-deoxyguanosine-5′-triphosphate.
Using the same preparation procedure of DMNB-dATP,
compound
3′-O-(4,5-dimethoxy-2-nitrobenzyl)-dGTP
was
1
obtained as a pale yellow gum of TEA salt (118 mg, 35%). H NMR
(600 MHz, D2O) δ 8.11 (s, 1H, 8-H), 7.71 (s, 1H, one of C6H2), 7.23
(s, 1H, one of C6H2), 6.29-6.25 (m, 1H, 1′-H), 5.02-4.89 (m, 2H,
OCH2C6H2), 4.69-4.64 (m, 1H, 3′-H), 4.53-4.47 (m, 1H, 4′-H), 4.25-
4.13 (m, 2H, 5′-H), 3.99 (s, 3H, one of CH3O), 3.93 (s, 3H, one of
CH3O), 2.87-2.71 (m, 2H, 2′-H); 13C NMR (150.8 MHz, D2O) 
158.1, 153.4, 152.9, 146.8, 146.7, 138.6, 138.4, 129.1, 129.0, 110.2,
107.5, 83.9, 83.6, 80.6, 67.8, 58.5, 56.1, 56.8, 36.2. 31P NMR (242.7
3.5b.
Synthesis
of
5′-O-(t-butyldimethylsilyl)-N2-
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MHz, D2O) δ -1.31 (brs, 1P), -11.7 (brs, 1P), -22.9 (brs, 1P); HRMS concentration, the resulting crude product was purified by flash
View Article Online
-
(ES-) calcd for C19H24N6O17P3 [(M+3H) -]: 700.0576, found: column chromatography over silica gel usingD1O0-I:2150%.10e3t9h/yCl6aOcBe0ta1t3e71inF
700.0581; HPLC: 76.2%.
hexanes to afford compound 4.3b as a pale yellow foam (0.60 g, 33%
over two steps). 1H NMR (600 MHz, CDCl3) δ 7.94-7.90 (m, 2H, two
of C6H5), 7.80 (s, 1H, one of C6H2), 7.65-7.61 (m, 2H, one of C6H5
and 6-H), 7.51-7.46 (m, 2H, two of C6H5), 7.20 (s, 1H, one of C6H2),
6.38 (dd, J = 5.4, 9.0 Hz, 1H, 1′-H), 4.92(s, 2H, OCH2C6H2), 4.31-
4.29 (m, 1H, 3′-H), 4.26-4.24 (m, 1H, 4′-H), 3.98-3.90 (m, 1H, one of
5’-H), 3.95 (s, 3H, one of CH3O), 3.94 (s, 3H, one of CH3O), 3.87-
3.84 (m, 1H, one of 5′-H), 2.59-2.54 (m, 1H, one of 2′-H), 2.14-2.08
(m, 1H, one of 2′-H), 1.97 (s, 3H, 5-CH3), 0.95 (s, 9H, C(CH3)3), 0.15
(s, 3H, one of SiCH3), 0.14 (s, 3H, one of SiCH3).
4. Synthesis of 3'-O-(2-nitrobenzyl)- thymidine-5′-triphosphate.
(NB-dTTP)
and
3'-O-(4,5-dimethoxy-2-nitrobenzyl)
thymidine-5′-triphosphate (DMNB-dTTP).
4.5b. Synthesis of 5′-O-(tert-butyldimethylsilyl)-3′-O-(4,5-
dimethoxy-2-nitrobenzyl) thymidine.
To a mixture of 3-N-benzoyl-5′-O-(tert-butyldimethylsilyl)-3′-O-
(4,5-dimethoxy-2-nitrobenzyl) thymidine 4.3b (0.600 g, 0.920
mmol)) in ethanol (10 mL) was added 30% ammonium hydroxide
solution (1.50 mL). The reaction mixture was stirred for 3 h at room
temperature with exclusion of light, and then subjected to evaporation.
The residue was extracted with DCM (3x50 mL). The organic layers
were combined and washed with brine (20 mL) and dried over
anhydrous Na2SO4. After concentration, the residue was purified by
flash column chromatography over silica gel using ethyl
acetate/hexanes (1:2) to afford compound 4.5b as a pale yellow solid
(0.46 g, 91%). 1H NMR (600 MHz, CDCl3) δ 8.18 (s, 1H, NH), 7.72
(s, 1H, one of C6H2), 7.51 (s, 1H, 6-H), 7.25 (s, 1H, one of C6H2), 6.38
(dd, J = 5.4, 9.0 Hz, 1H, 1′-H), 4.98-4.90 (m, 2H, OCH2C6H2), 4.31-
4.28 (m, 1H, 3′-H), 4.25-4.22 (m, 1H, 4′-H), 4.02 (s, 3H, one of
CH3O), 3.96 (s, 3H, one of CH3O), 3.95-3.91 (m, 1H, one of 5′-H),
3.85-3.82 (m, 1H, one of 5′-H), 2.55-2.50 (m, 1H, one of 2′-H), 2.08-
2.03 (m, 1H, one of 2′-H), 1.94 (s, 3H, 5-CH3), 0.94 (s, 9H, C(CH3)3),
0.13 (s, 6H, two of SiCH3).
Scheme 5: Synthesis of NB-dTTP and DMNB-dTTP
4.2. Synthesis of 3-N-benzoyl-5′-O-(tert-butyldimethylsilyl)
thymidine.
To a cooled (0 oC) solution of 5′-O-(tert-butyldimethyl)-
thymidine 4.1 (1.00 g, 2.81 mmol) in DCM (15 mL) was added
triethylamine (0.470 mL, 3.37 mmol) followed by benzoyl chloride
(0.36 mL, 3.09 mmol). The reaction mixture was stirred at 0 oC for 6
h, and then warmed to room temperature overnight, diluted with DCM
(50 mL), washed with water (20 mL). The organic layer was dried
over anhydrous Na2SO4. Evaporation of the filtrate gave compound
4.2 as a crude product (1.33 g, quantitative yield) without further
purification, directly used in the next step. 1H NMR (600 MHz,
CDCl3) δ 7.93 (d, J = 7.8 Hz, 2H, two of C6H4), 7.64 (d, J = 7.8 Hz,
1H, one of C6H4), 7.61 (s, 1H, 6-H), 7.52-7.47 (m, 2H, two of C6H4),
4.6b. Synthesis of 3′-O-(4,5-dimethoxy-2-nitrobenzyl) thymidine.
To a cooled (0 oC) solution of 5′-O-(tert-butyldimethylsilyl)-3′-O-
(4,5-dimethoxy-2-nitrobenzyl) thymidine 4.5b (0.460 g, 0.835 mmol)
in THF (8 mL) was added 1.0 M TBAF in THF solution (1.67 mL,
1.67 mmol) over 2 min. The solution was allowed to warm to room
temperature and continue stirring for 2 h with exclusion of air and
light. The mixture was poured into cold water (30 mL), and the
resulting mixture was extracted with ethyl acetate (3x40 mL). The
organic layers were combined, washed with brine (20 mL) and dried
6.35 (dd, J = 6.0, 8.4 Hz, 1H, 1′-H), 4.51-4.48 (m, 1H, 3′-H), 4.05-
4.02 (m, 1H, 4′-H), 3.94-3.92 (m, 1H, one of 5′-H), 3.88-3.85 (m, 1H,
one of 5′-H), 2.39-2.35 (m, 1H, one of 2’-H), 2.20-2.15 (m, 1H, one
of 2′-H), 1.97 (s, 3H, 5-CH3), 1.83 (d, J = 3.6 Hz, 1H, OH), 0.95 (s,
9H, C(CH3)3), 0.15 (s, 3H, one of SiCH3), 0.14 (s, 3H, one of SiCH3).
over anhydrous Na2SO4 After concentration, the residue was purified
.
by flash column chromatography over silica gel using 3-5% MeOH in
DCM to afford compound 4.6b as a pale yellow solid (0.35 g, 96%).
1H NMR (600 MHz, CDCl3) δ 8.26 (s, 1H, NH), 7.71 (s, 1H, one of
C6H2), 7.41 (s, 1H, 6-H), 7.20 (s, 1H, one of C6H2), 6.22 (dd, J = 6.0,
8.4 Hz, 1H, 1′-H), 4.98-4.90 (m, 2H, OCH2C6H2), 4.42-4.39 (m, 1H,
3′-H), 4.24-4.21 (m, 1H, 4′-H), 4.01 (s, 3H, one of CH3O), 4.01-3.96
(m, 1H, one of 5′-H), 3.96 (s, 3H, one of CH3O), 3.88-3.83 (m, 1H,
one of 5′-H), 2.52-2.47 (m, 1H, one of 2′-H), 2.42-2.35 (m, 1H, one
of 2′-H), 1.93 (s, 3H, 5-CH3).
4.3b. Synthesis of 3-N-benzoyl-5′-O-(tert-butyldimethylsilyl)-3′-
O-(4,5-dimethoxy -2-nitrobenzyl) thymidine.
To an ice cold mixture of 3-N-benzoyl-5′-O-(tert-
butyldimethylsilyl) thymidine 4.2 (crude 1.33 g, 2.81mmol) in DCM
(10 mL), aqueous Bu NOH (60%, 0.9 mL), NaI (84 mg, 0.56 mmol),
4
water (10 mL) and aqueous NaOH solution (1.0 M, 10 mL) was added
a solution of 2-nitrobenzyl bromide (0.630 g, 2.92 mmol) in DCM (10
mL) dropwise over 10 min. The reaction mixture was stirred at 0oC
for 2 h and then stirred at room temperature for 6 h with exclusion of
light. The reaction mixture was diluted with water (20 mL) and then
extracted with DCM (3x50 mL). The organic layer was washed with
brine (30 mL) and dried over anhydrous Na2SO4. After filtration and
DMNB-dTTP. Synthesis of 3′-O-(4,5-dimethoxy-2-nitrobenzyl)
thymidine-5′-triphosphate.
Using the same preparation procedure of DMNB-dATP,
8 | Organic & Biomolecular Chemistry, 2016, 00, 1-3
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ARTICLE
compound 3′-O-(4,5-dimethoxy-2-nitrobenzyl)-dTTP was obtained 11 V. Ramamurthy, K.S. Schanze, OrganicViewMArtoiclleecOunllianer
as a pale yellow gum of TEA salt (100 mg, 31%). 1H NMR (600 MHz,
D2O) δ 7.81 (s, 1H, one of C6H2), 7.29 (s, 1H, one of C6H2), 6.37 (dd,
J = 5.4, 9.6 Hz, 1′-H), 5.03-4.93 (m, 2H, OCH2C6H2), 4.64-4.61 (m,
1H, 3′-H), 4.47-4.43 (m, 1H, 4′-H), 4.29-4.21 (m, 2H, 5′-H), 4.01 (s,
3H, one of CH3O), 3.96 (s, 3H, one of CH3O), 2.59-2.54 (m, 1H, one
of 2′-H), 2.41-2.34 (m, 1H, one of 2′-H), 1.94 (s, 3H, 5-CH3); 13C
NMR (150.8 MHz, D2O)  166.1, 152.9, 151.3, 147.0, 139.0, 137.0,
129.1, 111.6, 110.5, 107.8, 85.1, 83.3, 80.6, 67.8, 58.3, 56.1, 55.9,
36.0, 11.6. 31PNMR (242.7 MHz, D2O) δ -10.1 (brs, 1P), -10.6 (brs,
1P), -22.7 (brs, 1P); HRMS (ES-) calcd for C19H25N3O18P3- [(M+3H)-
]: 676.0351, found: 676.0339; HPLC: 95.5%.
Photochemistry, Marcel Dekker, Inc: NDewOI:Y10o.r1k0,391/9C969O.B01371F
12 J. W. Walker, J. A. McCray, G. P. Hess, Biochemistry, 1986,
25, 1799
13 M. Schworer and J. Wirz, Helv. Chim. Acta, 2001, 84, 1414-
1425.
14 P. Klꢀn, T. ꢁolomek, C. G. Bochet, A.Blanc, R. Givens, M.
Rubina, V.. Popik, A. Kostikov, J. Wirz. Chem. Rev. 2013,
113, 119−191
15 C. G. Bochet. J. Chem., Perkin Trans. 2002, 1, 125-142
16 G. Lu, K. Burgess, Bioorg. Med. Lett, 2006, 16, 3902.
17 F.J. Bollum, The Enzyme- 3rd edition (P. D. Boyer, ed) Vol 10
145, Academic Press, New York.
,
18 V. Pandey and M.J. Modak, J. Bio. Chem. 1988, 263 (8), 3744.
Photodecomposition Studies: The photo decay of each dNTP was
done with 1 mg/mLsolution of compound in water / D2O. 1 mL of the
solution was irradiated with 365 nm UV light and samples were
withdrawn at regular intervals for 1H NMR and LCMS analysis.
Stop-Start DNA synthesis: For a standard reaction, 5 L of DNA
(92.5 ng/L, 750bp) was mixed with 5L of 5XTdT Buffer, CoCl2
solution (100mM, 0.5 L), dNTP (10 mM, 1 L) and TdT (20 un/L)
in a micro centrifuge tube and the final volume was made up to 50ml
by adding milliQ water. The mixture was then incubated at 37oC for
60 minutes. After completion of DNA end caging, 25 L was loaded
into 2% Agarose gel and remaining 25 L of the mixture was taken
out and subjected to UV irradiation (365 nm, 10 mW/cm2) for 15
minutes. After UV irradiation the mixture was again treated with
dNTP (10 mM, 1 L) and TdT (15 un) and incubated for another 60
minutes and loaded on gel.
Acknowledgements
The authors thank Alberta Innovates Technology Futures (AITF)
iCORE chair funding, National Institute of NanoTechnology
Facilities and Valentyna Semenchenko for preparing blunt-end 750bp
DNA initiator and gel electrophoresis.
Notes and references
1
2
R. Dahm, Hum. Genet, 2008, 122, 565.
F.S. Collins, E.D. Green, A.E. Guttmacher M.S. Guyer,
Nature, 2003, 422, 835.
3
J. Guo, N. Xu, Z. Li, S. Zhang, J. Wu, D.H. Kim, M.S. Marma,
Q. Meng, H. Cao, X. Li, S. Shi, L. Yu, S. Kalachikov, J.J.
Russo, N.J. Torro, J. Ju, PNAS, 2008, 105, 9145.
G.T. Hermanson, Bioconjugate technologies- 3rd edition, 2013,
Chapter 23, 960, Elsevier.
M. L. Metzker, R. Raghavachari, S. Rchards, S. E. Jacutin, A.
Civitello, K. Burgess, R. A. Gibbs, Nucleic Acid Research,
1994, 22, 4259.
4
5
6
7
8
J. T. Sczepanski, G. F. Joyce, Chemistry and Biology, 2012,
19, 1324–1332.
A.P. Pelliccioli, J. Wirz, Photochem. Photobiol. Sci., 2002, 1,
441-458.
M. L. Metzker, R.Raghavachari, S. Richards, S. E.Jacutin, A.
Civitello, K. Burgess, R.A.Gibbs, Nucleic Acids Research,
1994, 22, (20) 4259-4267
9
W. Wu, B. P. Stupi, V. A. Litosh, D. Mansouri, D. Farley, S.
Morris, S. Metzker, M. L. Metzker, Nucleic Acids Research,
2007, 35, (19) 6339–6349
10 J. Wu, S. Zhang, Q. Meng, H. Cao, Z. Li, X. Li, S. Shi, D. H.
Kim, L, Bi, N. J. Turro, J. Ju, PNAS, 2007, 104, 16462
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