RETRACTED ARTICLE: Convenient synthesis of pyrimidine 2′-deoxyribonucleoside monophosphates with important epigenetic marks at the 5-position

Article abstract of DOI:10.1039/d0ob00884b

Methyl groups of thymine and 5-methylcytosine (5mC) bases in DNA undergo endogenous oxidation damage. Additionally, 5mC residues can be enzymatically deaminated or oxidized through either genetic alterations or the newly identified epigenetic reprogramming pathway. Several methods have been developed to measure the formation of modified DNA nucleobases including 32P-postlabeling. However, the postlabeling method is often limited by the absence of authentic chemical standards. The synthesis of monophosphate standards of nucleotide oxidation products is complicated by the presence of additional functional groups on the modified bases that require complex protection and deprotection strategies. Due to the emerging interest in the pyrimidine oxidation products, the corresponding protected 3′-phosphoramidites needed for solid-phase oligonucleotide synthesis have been reported, and several are commercially available. We report here an efficient synthesis of 3′-monophosphates from 3′-phosphoramidites and the subsequent enzymatic conversion of 3′-monophosphates to the corresponding 5′-monophosphates using commercially available enzymes. This journal is

Full text of DOI:10.1039/d0ob00884b

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Convenient synthesis of pyrimidine
2’-deoxyribonucleoside monophosphates with
important epigenetic marks at the 5-position†
Cite this: DOI: 10.1039/d0ob00884b
b,c
Song Zheng,a Ai Tran,a Alyson M. Curry,b Dawanna S. Whiteb and Yana Cen
*
Methyl groups of thymine and 5-methylcytosine (5mC) bases in DNA undergo endogenous oxidation
damage. Additionally, 5mC residues can be enzymatically deaminated or oxidized through either genetic
alterations or the newly identified epigenetic reprogramming pathway. Several methods have been devel-
oped to measure the formation of modified DNA nucleobases including 32P-postlabeling. However, the
postlabeling method is often limited by the absence of authentic chemical standards. The synthesis of
monophosphate standards of nucleotide oxidation products is complicated by the presence of additional
functional groups on the modified bases that require complex protection and deprotection strategies.
Due to the emerging interest in the pyrimidine oxidation products, the corresponding protected 3’-phos-
phoramidites needed for solid-phase oligonucleotide synthesis have been reported, and several are com-
mercially available. We report here an efficient synthesis of 3’-monophosphates from 3’-phosphoramidites
and the subsequent enzymatic conversion of 3’-monophosphates to the corresponding 5’-monophos-
phates using commercially available enzymes.
Received 28th April 2020,
Accepted 23rd June 2020
DOI: 10.1039/d0ob00884b
rsc.li/obc
(Scheme 1).8,9 In addition to the active demethylation pathway,
an alternative proposal for restoration of cytosine (C) involves
Introduction
The importance of DNA oxidation and hydrolysis in the devel- the direct C–C bond cleavage at the 5-position to remove the
opment of human diseases is increasingly recognized.1–3 substitution.15 The potential coexistence of the enzymatic 5mC
Recently a biochemical pathway has been identified in which modification pathway and established endogenous DNA damage
5-methylcytosine (5mC), an important component of the epige- and repair pathways creates significant analytical challenges.
netic landscape in higher organisms, can be enzymatically oxi-
Multiple methods have been developed to quantify the
dized to the corresponding 5-hydroxymethyl (5hmC), formyl abovementioned epigenetic modifications including chromato-
(5foC) and carboxyl (5caC) analogs, mediated by ten eleven graphic separation by HPLC, GC and TLC and identification
translocation (TET) enzymes (Scheme 1).4–9 This epigenetic and quantification by mass spectrometry, UV detection or 32P-
reprogramming pathway is essential to normal development labeling. The 32P-labeling approach has proven to be very sen-
and differentiation, yet, defective in most human cancer sitive for measuring a multitude of DNA damage products;16–19
cells.4–11 The spontaneous hydrolysis of 5mC to T at methylated however, this approach is often limited by the absence of auth-
CpG islands has long been considered the mechanism under- entic standards. While MS methods have emerged as extremely
lying the single most frequent type of single-base mutation powerful and sensitive approaches to the measurement and
found in human cancer cells.12–14 This pathway might also unequivocal identification of DNA damage products, 32P-label-
include enzymatic deamination to the corresponding uracil ing strategies provide an approach for following sequential
analogs followed by glycosylase removal and DNA repair conversions of specific nucleotides through multiple chemical
or enzymatic pathways in complicated matrices including
intact cells and cell extracts and at specific sequence positions
aDepartment of Pharmaceutical Sciences, Albany College of Pharmacy and Health
Sciences, Colchester, VT 05446, USA
bDepartment of Medicinal Chemistry, Virginia Commonwealth University, Richmond,
VA 23219, USA. E-mail: ceny2@vcu.edu; Tel: +1 804-828-7405
cInstitute for Structural Biology, Drug Discovery and Development, Virginia
Commonwealth University, Richmond, VA 23219, USA
within the oligonucleotides.
In the case of the modified pyrimidines examined here,
chemical synthesis of the 2′-deoxynucleosides can be challen-
ging, and conversion to the corresponding monophosphates is
complicated by the additional functional groups present on
the modified bases that must be selectively protected prior to
chemical phosphorylation.20–23 Due to the growing interest in
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
d0ob00884b
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Scheme 1 Endogenous chemical or enzymatic conversions among pyrimidine analogs. 5-Methylcytosine (5mC), which is formed in DNA during
epigenetic programming, can be oxidized to several 5-substituted cytosine derivatives. These cytosine derivatives can also undergo deamination to
form uracil derivatives. The enzyme-mediated oxidation and deamination of 5mC is part of an epigenetic reprogramming pathway in mammalian
cells. 5mC, 5-methylcytosine; 5hmC, 5-hydroxymethylcytosine; 5foC, 5-formylcytosine; 5caC, 5-carboxylcytosine; 5hmU, 5-hydroxymethyluracil; 5foU,
5-formyluracil; 5-caU, 5-carboxyluracil.
Table 1 Characterization of 3’-dNMPs
Elemental composition
Exact mass, m/z
[M − H]−
Measured mass, m/z
[M − H]−
λmax
(nm)
Extinction coefficient
Retention timea
(min)
3′-dNMPs
[M − H]−
(103 M−1 cm−1
)
3′-cadUMP
3′-cadCMP
3′-dCMP
C10H12N2O10P−
C10H13N3O9P−
C9H13N3O7P−
C10H15N3O8P−
C9H12N2O8P−
C10H14N2O9P−
C10H15N3O7P−
C10H12N2O9P−
C10H14N2O8P−
C10H13N3O8P−
351.0230
350.0389
306.0491
336.0597
307.0331
337.0437
320.0648
335.0280
321.0488
334.0440
351.0236
350.0390
306.0490
336.0586
307.0345
337.0444
320.0643
335.0289
321.0493
334.0424
271
280
269
274
261
263
277
275
267
283
12.3 0.5
10.6 0.5
9.7 0.7
12.5 0.7
13.7 0.8
11.9 0.5
10.2 0.3
17.2 0.3
10.3 0.6
14.1 0.3
3.25
3.52
5.78
6.22
6.71
3′-hmdCMP
3′-dUMP
3′-hmdUMP
3′-mdCMP
3′-fodUMP
3′-dTMP
6.97
10.17
10.88
11.47
13.27
3′-fodCMP
a The retention times were obtained on an Aquasil C18 column using an ammonium acetate (20 mM, pH 5.5) – methanol solvent system.
the modified pyrimidines, several groups have reported the presence of a weakly acidic azole catalyst and the subsequent
synthesis of phosphoramidite derivatives needed for the solid- trapping of the resulting tetrazolyl phosphoramidite inter-
phase assembly of oligonucleotides containing these modifi- mediate with different nucleophiles set the stage for a straight-
cations (Table 1), and some of these modified phosphorami- forward synthesis of phosphite triester. Indeed, when mixed
dites are now commercially available. The investment made in with 1H-tetrazole in CH3CN, the diisopropylamino group of
developing methods for the protection and deprotection of the phosphoramidite was readily protonated and rapidly displaced
3′-phosphoramidites could be leveraged to provide a con- by the hydroxyl group of 2-cyanoethanol despite the reduced
venient source for both the corresponding 3′- and 5′-mono- nucleophilicity due to the presence of the electron-withdraw-
phosphates. Here we report a novel and versatile synthesis of ing cyano group. This reaction proceeds extremely fast: com-
3′-dNMPs using nucleoside phosphoramidites as building plete consumption of the phosphoramidite starting material
blocks. With the availability of fully characterized dNMPs, not was observed after 15 to 30 min at room temperature (moni-
only the composition of synthetic oligonucleotides can be tored by TLC). Ease of purification is another important
determined, but also the 32P-labeling strategy can be applied feature of this coupling reaction: after regular aqueous
to this group of biologically important pyrimidine analogs.
workup, the organic solvent was removed under reduced
pressure to afford the desired phosphite triester as a white to
pale-yellow foam. These crude products were subjected to the
next step without further purification.
Oxidation and detritylation. In modern solid-phase oligo-
nucleotide synthesis, oxidation of phosphite triester is usually
Results and discussion
Synthesis of 3′-dNMPs
Chemical coupling of phosphoramidite with 2-cyanoetha- accomplished by aqueous iodine in combination with pyri-
nol. The facile activation of phosphoramidite monomer in the dine. However, for solution-phase reactions, separation of
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detritylation was carried out in 80% acetic acid for about
30 min, again at room temperature. Removal of the solvent
provided deoxynucleoside-3′-phosphate triester with a free 5′-
hydroxyl group. The decent solubility of these compounds in
organic solvent allows them to be chromatographically puri-
fied on silica gel using CH2Cl2 and methanol.
The formation of deoxynucleoside-3′-phosphate triester
with a free 5′-hydroxyl group was further confirmed by NMR
analysis. 1H NMR showed complete disappearance of signals
corresponding to trityl group protons, and 31P signals shifted
from around 148 ppm (doublet) in phosphoramidites to
−3.5 ppm (singlet) in deoxynucleoside-3′-phosphate triesters.
Deprotection strategies. Removal of cyanoethyl groups from
phosphate and removal of protecting group from bases can be
achieved in concentrated aqueous NH4OH at elevated tempera-
ture (60 °C). The solvent was then evaporated under reduced
pressure, and the crude product was ready for purification.
However, for several phosphoramidites, the presence of special
protecting groups or sensitive functionality requires alternative
Fig. 1 Conversion of a phosphoramidite to a 3’-dNMP. The phosphora-
midite is coupled with 2-cyanoethanol in the presence of tetrazole in
CH3CN to generate the phosphite triester A, which is then oxidized with
H2O2 and acetic acid to create the phosphate triester B. Alkaline de-
protection of B leads to the formation of the corresponding 3’-dNMP.
byproduct from phosphate triester could be problematic. We or additional deprotection strategies. For 5-carboxy-dC- and
thus seek a “traceless” yet mild oxidizing agent. H2O2 in dU-CE phosphoramidites, the carboxylate exists as either an
CH3CN has been used to oxidize thymidine phosphoramidites ethyl or a methyl ester. Deprotections were carried out in 0.4
to the corresponding phosphoramidates in quantitative yields. M methanolic NaOH (methanol/water = 4/1) for 17 h at room
This method was optimized for our 3′-dNMPs synthesis. temperature before being neutralized with acetic acid.
Deoxynucleoside-3′-phosphite triester was treated with 3% Commercially available 5-formyl-dC-CE phosphoramidite con-
H2O2 in CH3CN (v/v = 1/5) for 15 min at room temperature tains acetylated diol instead of a formyl group. NH4OH treat-
before the solvent was evaporated in vacuo, which led to the ment removes the acetyl group to generate 1,2-diol, which
formation of 3′-phosphate triester without further purification. requires further oxidative cleavage by NaIO4 to unmask the
The oxidation step allowed good functional group tolerance formyl functionality. This has been performed with 50 mM
(Table 1) with full conversion within minutes. The subsequent (final concentration) NaIO4 solution at 4 °C for 30 min. The
Fig. 2 Special deprotection strategies. (A) Commercially available 5-formyl-dC-CE phosphoramidite contains acetylated diol instead of a formyl
group. NH4OH treatment removes the acetyl group to generate 1,2-diol, which requires further oxidative cleavage by NaIO4 to unmask the formyl
functionality. (B) The 5-formyl-dU-CE phosphoramidite synthesized in our lab bears unprotected formyl group, which was compatible with concen-
trated NH4OH for 4 h at room temperature.
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reaction was then quenched with the addition of excess ethyl-
The aforementioned synthetic protocols of 3′-dNMPs have
ene glycol. The 5-formyl-dU-CE phosphoramidite synthesized several beneficial features. First of all, phosphoramidites are
in our lab bears unprotected formyl group, which was compati- either commercially available or easily accessible, which
ble with concentrated NH4OH for 4 h at room temperature.
enables a straightforward construction of 3′-dNMPs. Secondly,
Purification. Purification for most of the 3′-dNMPs was quite already-existing protecting groups in phosphoramidites
straightforward. The crude product was dissolved in water, prevent undesired side reactions. Reactions were easily moni-
washed with ethyl acetate to remove the side product from de- tored by TLC, and the 3′-dNMPs were obtained in 70–90%
protection. The aqueous layer was then concentrated to yields with minimal purification (most synthesis only requires
provide the desired 3′-dNMP. For 3′-cadCMP, 3′-cadUMP and one chromatographic isolation). The method is, therefore, suit-
3′-fodCMP, HPLC purification was performed to separate able for large scale synthesis for a broad class of compounds.
3′-monophosphate from the inorganic salt generated during Corresponding MS (Table 1), UV (Table 1), HPLC (Table 1,
deprotection step (see “Materials and methods” section for Fig. 3 and S1†) and NMR (ESI†) data are provided for each of
HPLC conditions).
the ten 3′-monophosphates prepared here.
Fig. 3 HPLC separation of the 3’-dNMPs and 5’-dNMPs. The HPLC chromatograms of (A) 3’-dNMPs and (B) 5’-dNMPs were obtained on an Aquasil
C18 column using ammonium acetate (20 mM, pH 5.5) – methanol solvent system. The above chromatograms were obtained with UV absorbance
at 275 nm.
Fig. 4 Enzymatic conversion of a 3’-dNMP to a 5’-dNMP: a chemically synthesized 3’-dNMP is initially converted to the 3’,5’-bisphosphate with T4
kinase. Following HPLC purification, the dephosphorylation of the bisphosphate by Nuclease P1 results in the formation of 5’-dNMP. Substitution
with 32P-enriched ATP in the T4 kinase step can be used to generate the corresponding labeled 5’-dNMP.
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Table 2 Characterization of 5’-dNMPs
Enzymatic conversion of 3′-dNMP to 5′-dNMP
The easy access to the 3′-dNMPs made the facile synthesis of
5′-dNMPs possible. This enzymatic conversion was inspired by
phosphoglycerate mutase, a glycolytic enzyme.24 It catalyzes
the conversion of 3-phosphoglycerate to 2-phosphoglycerate
through a 2,3-bisphosphoglycerate intermediate.24,25 In our
two-enzyme coupled synthesis, the 3′-dNMP was first incu-
bated with ATP and a T4 kinase lacking the 3′-phosphatase
activity to generate the corresponding 3′,5′-bisphosphate
(Fig. 4).26,27 Subsequently, the 3′,5′-bisphosphate was depho-
sphorylated by nuclease P1 to afford the 5′-dNMP.28 Both T4
kinase and nuclease P1 demonstrated excellent functional
group tolerance as 5′-dNMPs carrying various substituents
were generated in decent to good yields by this robust
Yielda
(%)
Retention
5′-dNMPs
timeb (min)
5′-cadUMP
5′-cadCMP
5′-dCMP
80.2
79.2
53.2
85.7
54.0
81.5
56.2
83.3
59.3
31.5
3.91
4.33
5.61
6.58
6.97
8.34
9.22
10.94
11.01
12.24
5′-hmdCMP
5′-dUMP
5′-hmdUMP
5′-mdCMP
5′-dTMP
5′-fodUMP
5′-fodCMP
a Yield for two steps starting from the 3′-dNMP. b The retention times
were obtained on an Aquasil C18 column using an ammonium acetate
(20 mM, pH 5.5) – methanol solvent system.
Fig. 5 Confirmation of 5hmU in an oligonucleotide sequence. (A) Sequence of the single-stranded oligonucleotide. HPLC chromatograms of the
dNMPs resulting from the digestion of 12mer with (B) snake venom phosphodiesterase I, or (C) bovine spleen phosphodiesterase II.
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sequence (Table 2). Similar to the 3′-dNMPs, the 5′-dNMPs can nuclease that can remove 3′-dNMPs sequentially from the 5′-
be readily resolved by HPLC (Fig. 3).
terminus.31 HPLC analysis suggested the formation of
3′-hmdUMP along with other nucleoside monophosphates
(Fig. 5C).
Determination of synthetic oligonucleotide composition using
dNMP standards
Probing conversions of nucleobase using dNMP standards
The presence of modified pyrimidines in synthetic oligonu-
cleotides can be confirmed using either the 3′- or 5′-dNMP The availability of the dNMP standards should facilitate experi-
standards. A 12mer oligonucleotide containing an internal ments probing both chemical and enzymatic conversions of
5hmU residue was generated by standard solid-phase DNA syn- target residues in oligonucleotides. We demonstrate this by
thesis (Fig. 5A). This oligonucleotide was incubated with snake examining the chemical reactivity of 5hmC in a duplex oligo-
venom phosphodiesterase I, an exonuclease capable of succes- nucleotide. Bisulfite sequencing has become extremely power-
sively hydrolyzing 5′-dNMPs from the 3′-terminus of a DNA ful in distinguishing cytosine from 5mC residues and thus
sequence.29,30 The digested sample was then injected onto tracking methylation patterns in genomic DNA.32–34 However,
HPLC for further analysis. Comparison with the 5′-dNMP stan- 5mC and 5hmC cannot be distinguished by this method.35 It
dards indicated that in addition to G, T, C and A, 5hmU was has been reported that 5hmC can be oxidized to 5foC by
also present in the sequence (Fig. 5B). The composition of the KRuO4.36–38 Subsequently, bisulfite treatment can convert 5foC
oligonucleotide can also be determined by treating the same to uracil (U).39 This two-step sequence could serve as an
12mer with bovine spleen phosphodiesterase II, an exo- alternative approach in detecting 5hmC level (Fig. 6A). In order
Fig. 6 Conversion of 5hmC in an oligonucleotide sequence. (A) Chemical modifications of the 5hmC-containing oligonucleotide. (B) Sequence of
the duplex. HPLC chromatograms of the 5’-dNMPs resulting from the digestion of (C) duplex, (D) duplex treated with KRuO4, and (E) duplex treated
with KRuO4 and bisulfite. The signals were detected by a radio-detector.
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to evaluate this conversion sequence directly, a 12mer oligo- dine, diisopropylethylamine, 2-cyanoethanol, 2-cyanoethyl
nucleotide containing a 5hmC residue at the 5′-position was N,N,N′,N′-tetraisopropylphosphordiamidite, and carbon mon-
32P-labeled, annealed to the unlabeled 24mer complementary oxide were purchased from Sigma-Aldrich (St Louis, MO).
strand and then ligated to an unlabeled 12mer oligonucleotide Commercially available phosphoramidites and sublimed 1H-
to form a duplex (Fig. 6B). A fraction of the duplex was tetrazole in anhydrous acetonitrile were purchased from Glen
digested with DNase I and snake venom phosphodiesterase I. Research (Sterling, VA). Thin layer chromatography (TLC) was
The resulting 5′-dNMPs were then resolved by HPLC with an performed on precoated silica gel 60 F254, 5 cm × 20 cm,
in-line radio-detector. The 32P label was found exclusively in 250 μM thick plates purchased from EMD (Gibbstown, NJ).
the 5′-hmdCMP (Fig. 6C). A second fraction of the duplex was Snake venom phosphodiesterase I and bovine spleen phospho-
treated with KRuO4 and digested as described above. The for- diesterase II were purchased from Worthington Biochemical.
mation of 5foC was readily detected (Fig. 6D). The complete T4 polynucleotide kinase and T4 DNA ligase were purchased
conversion of 5hmC to U was observed when the duplex was from New England Biolabs. Nuclease P1 was purchased from
incubated with KRuO4 followed by bisulfite treatment and US Biologicals.
enzymatic digestion. In this case, the radioactivity was
1H, 13C, and 31P NMR spectra were obtained using a Bruker
detected in 5′-dUMP exclusively (Fig. 6E). While analytical Ultrashield 300 MHz spectrometer (Manning Park, MA). HPLC
methods have been used to confirm the conversion of 5hmC to analysis was performed with a ThermoFinnigan (Waltham,
U,36 ours is the first study to directly follow the transform- MA) Surveyor HPLC system with a photodiode array and a
ations in on oligonucleotide with 32P-labeling. The selective single quad mass spectrometer detector. For the analysis of
32P-labeling of a target base, followed by enzymatic digestion 32P-labeled monophosphates, a similar HPLC system was inter-
and HPLC analysis, provides a method to monitor either enzy- faced with a Berthold Technologies FlowStar LB513 radioac-
matic or chemical conversions, even with crude cellular tivity detector (Wildbad, Germany). Oligonucleotides were syn-
extracts.
thesized with a Millipore Expedite nucleic acids synthesis
system (Billerica, MA). High-resolution mass spectra were
obtained with either
a Waters Micromass Q-tof Ultima
(Milford, MA) or a Thermo Scientific Q-Exactive Hybrid
Quadrupole Orbitrap (Waltham, MA).
Conclusions
Phosphoramidites prepared for the synthesis of oligonucleo-
tides containing biologically important modified bases can be
conveniently converted to the corresponding 3′-dNMPs.
5′-dNMPs are then obtained through enzyme-mediated phos-
General procedures for deoxynucleoside-3′-monophosphate
synthesis
Coupling. To a solution of phosphoramidite in anhydrous
phorylation and dephosphorylation. Authentic 3′- and CH3CN were added 0.45 M tetrazole in CH3CN (1.5 equiv.) and
5′-dNMP standards, such as those described here, are essential 2-cyanoethanol (1.5 equiv.). The reaction was stirred at room
for the validation of highly sensitive analytical methods using temperature and monitored by TLC. Upon completion (usually
32P-labeling. We have demonstrated this approach by following 15 to 30 min), the reaction mixture was diluted with ethyl
the conversion of a selectively-labeled 5hmC residue in an acetate with 1% TEA. The organic phase was washed with 5%
oligonucleotide to 5foC, and then on to U, using Rh oxidation NaHCO3, water, brine and dried over anhydrous Na2SO4. The
and bisulfite conversion methods currently employed to solvent was removed under reduced pressure to afford phos-
sequence 5mC and 5hmC residues in DNA. This approach can phite triester A (Fig. 1).
be extended to study future base modification strategies
Oxidation and detritylation. Phosphite triester A was dis-
needed to follow additional base conversions in DNA, includ- solved in a mixture of CH3CN and 3% H2O2 (v/v = 5/1), stirred
ing those involved with epigenetic reprogramming.
at room temperature for 15 min before the solvent was evapor-
ated under reduced pressure. The residue was redissolved in
80% acetic acid, kept at room temperature for another 30 min
before the solvent was evaporated. The crude product was puri-
fied by column chromatography to afford phosphate triester B.
Deprotection. Phosphate triester B was treated with conc.
Materials and methods
Materials
Reagent grade solvents including dichloromethane, methanol, NH4OH at 60 °C overnight. The solvent was evaporated under
and ethyl acetate were purchased from Fisher Scientific reduced pressure. The residue was redissolved in water, and
(Pittsburgh, PA) and used without further purification. extracted with ethyl acetate 3 times. The aqueous layer was
Anhydrous grade solvents including tetrahydrofuran, pyridine, concentrated in vacuo to afford the desired deoxynucleoside-3′-
trimethylamine, and acetonitrile were purchased from Sigma- monophosphate.
Aldrich (St Louis, MO) and used as received unless otherwise
noted. Di-tert-butylsilyl bis(trifluoromethanesulfonate), di-
Special deprotection strategies
5-Carboxyl-2′-deoxycytidine (5cadC) and 5-carboxyl-2′-deox-
methylformamide, 5-iodo-2′-deoxyuridine, 4,4′-dimethoxytrityl
chloride, tris(dibenzylideneacetone)dipalladium(0), triphenyl- yuridine (5cadU). For 5cadC or 5cadU, intermediate B was
phosphine, toluene, imidazole, tributyltin hydride, HF-pyri- treated with 0.4 M of NaOH in methanol/water (v/v = 4/1) at
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room temperature for 17 h before it was neutralized with system (Thermo Finnigan, Waltham, MA) with a flow of 1 mL
acetic acid. The solvent was then evaporated under reduced min−1 over an Aquasil C18 column (Thermo Scientific,
pressure. The residue was redissolved in water and extracted Waltham, MA). Eluting buffers were buffer A (20 mM NH4OAc,
with ethyl acetate 3 times. The aqueous layer was concentrated pH 5.5) and buffer B (methanol). The gradient was 0 →
in vacuo to afford 3′-cadCMP or 3′-cadUMP.
10 min, 100% → 90% buffer A, 0 → 10% buffer B; 10 →
5-Formyl-2′-deoxycytidine (5fodC). After NH4OH de- 15 min, 90% → 80% buffer A, 10% → 20% buffer B; 15 →
protection, the 1,2-diol requires further oxidation with NaIO4 21 min, 80% → 52% buffer A, 20 → 48% buffer B; 21 →
to unveil the aldehyde functional group (Fig. 2A). It was dis- 22 min, 52% → 40% buffer A, 48 → 60% buffer B; 22 →
solved in cold distilled water (4 °C) and treated with an equal 24 min, 40% buffer A, 60% buffer B; 24 → 25 min, 40% →
volume of cold (4 °C) 100 mM of NaIO4 solution for 30 min at 100% buffer A, 60% → 0% buffer B; 25 → 35 min, 100% buffer
4 °C before it was quenched with 8 equiv. of ethylene glycol A and 0% buffer B. The elution was scanned from 210 nm to
with respect to NaIO4.
600 nm (Surveyor PDA Detector, Thermo Finnigan, Waltham,
5-Formyl-2′-deoxyuridine (5fodU). The NH4OH deprotection MA). The chromatographic eluent was directly injected into
was carried out at room temperature for 4 h before the solvent the ion source with a 1/5 splitting. Ions were scanned by use of
was evaporated under reduced pressure (Fig. 2B). This mild de- a negative polarity mode over a full-scan range of m/z
protection allows the convenient isolation of sensitive 100–500.40 The separation of the 3′-dNMPs and 5′-dNMPs is
3′-fodUMP.
shown in Fig. 3, and the retention times are given in Tables 1
and 2. The nucleotides were detected with a diode array detec-
tor that provided the UV spectrum of each analyte.
Enzymatic conversion of a 3′-dNMP to the corresponding
5′-dNMP
Determination of extinction coefficient
Each 3′-dNMP was enzymatically converted to the corres-
ponding 5′-dNMP using a series of two enzymatic reactions. In
the first step, the 3′-dNMP was converted to the 3′,5′-bispho-
sphate. In this reaction, 400 nmol of the 3′-dNMP was com-
bined with 20 μL 1 M Tris-HCl, pH 7.0, 20 μL 0.1 M MgCl2,
20 μL 50 mM DTT, 40 μL 20 mM ATP, and 20 μL T4 kinase
(10 U μL−1) lacking 3′-phosphatase activity in a total volume of
200 μL as described previously.26,27 The reaction mixture was
incubated at 37 °C, and progress was followed by HPLC ana-
lysis of a 5 μL aliquot of the reaction. In some cases, the
3′-dNMP co-eluted with ATP. However, the ATP can be reduced
by incubation with 1 μL of 1 U μL−1 apyrase for 10 min prior to
HPLC analysis.
The 3′,5′-bisphosphate generated in the above reaction was
purified by HPLC as described below and converted to the
5′-dNMP with nuclease P1 as described previously.28 HPLC
fractions containing the bisphosphate were combined and
dried. The bisphosphate was incubated in water (50 μL), buffer
(25 μL 0.25 M NaOAc, pH 5.0, 15 μL 2 mM ZnCl2) and nuclease
P1 (10 μL 5 μg μL−1) at 37 °C. The reaction was monitored by
HPLC, and the final product 5′-dNMP isolated by HPLC and
characterized by UV and mass spectrometry as described
below.
Determination of 3′-dNMP concentration by 1H NMR. In a
typical measurement, 3′-dNMP was dissolved in 700 μL of
99.96% D2O. To this solution was added 1 or 2 μL of 99.9%
DMSO as an internal standard. The sample was then trans-
ferred to a Wilmad 528 NMR tube capped with a rubber
septum and sealed with parafilm. Proton NMR spectra were
recorded on a Bruker Ultrashield 300 MHz spectrometer using
a published protocol with modifications. In brief, spectra were
acquired with 64 scans, 90° pulse, 35 s relaxation delay, SW =
6000 Hz. Integration was carried out using Bruker XWin NMR
software. For 3′-dNMPs, base proton(s) and H1′ signals were
used for quantitative comparison; thus, several measurements
contribute to the average. The DMSO methyl integral was set
to be 1.000, the averaged relative intensity of 3′-dNMP peaks
gave the concentration of 3′-dNMP.
Determination of extinction coefficient. UV absorbance was
recorded on a Cary 100 Bio UV/Vis spectrophotometer blanked
against 100 mM phosphate buffer (pH 7). NMR samples were
diluted with 100 mM phosphate buffer (pH 7) so that Amax
readings were between 0.5 and 1.0 to ensure high sensitivity.
UV measurements were performed on the same day as NMR
measurements to minimize the possibility of evaporation or
sample degradation. The extinction coefficient was calculated
using eqn (1):
High-resolution mass spectrometry (HRMS) analysis. The 3′-
dNMPs were characterized by high-resolution mass spec-
trometry on a Thermo Scientific Q-Exactive Hybrid Quadrupole
Orbitrap mass spectrometer operating in the negative ion
mode. The 3′-dNMPs were dissolved in acetonitrile/water (v/v =
Amax ꢀ D
ε ¼
ð1Þ
C ꢀ l
1/1) to a final concentration of 20 μM and directly infused into where ε is the extinction coefficient at optimal wavelength,
the ESI source at a rate of 5 μL min−1. The source temperature Amax is the absorbance at optimal wavelength, D is the dilution
was at 320 °C and electrospray high voltage was −3.5 kV. In factor, C is the concentration of 3′-dNMP, and l is the path
each case, the measured mass was within 5 ppm of the calcu- length of the sample, which is 1 cm.
lated (theoretical) mass. The results are given in Table 1.
Oligonucleotide synthesis and purification. Oligonucleotides
HPLC analysis and purification. The 3′-dNMPs, 5′-dNMPs were prepared by solid-phase synthesis method as described
and 3′,5′-bisphosphates were analyzed by LC-ESI-MS on previously.41–43 Following synthesis and deprotection, they
Surveyor MSQ and were chromatographed by Surveyor LC were purified by HPLC using Hamilton PRP-1 column and a
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gradient of 0 to 25% CH3CN in 10 mM of potassium phos-
phate buffer (pH 6.8). Oligonucleotides were then detritylated
with 80% aqueous acetic acid at room temperature for
30 min. Following detritylation, they were purified by
HPLC using a Vydac C18 column and a gradient of 0 to 70%
CH3CN in water. Oligonucleotide purity was examined by
MALDI-TOF.44
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