Highly regioselective methylation of inosine nucleotide: an efficient synthesis of 7-methylinosine nucleotide

Article abstract of DOI:10.1080/15257770.2020.1738457

A facile, straightforward, reliable, and an efficient chemical synthesis of inosine nucleotides such as 7-methylinosine 5′-O-monophosphate, 7-methylinosine 5′-O-diphosphate, and 7-methylinosine 5′-O-triphosphate, starting from the corresponding inosine nucleotide is delineated. The present methylation reaction of inosine nucleotide utilizes dimethyl sulfate as a methylating agent and water as a solvent at room temperature. It is noteworthy that the present methylation reaction proceeds smoothly under aqueous conditions that is highly regioselective to afford exclusive 7-methylinosine nucleotide in good yields with high purity (>99.5%).

Full text of DOI:10.1080/15257770.2020.1738457

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Highly regioselective methylation of inosine
nucleotide: an efficient synthesis of 7-
methylinosine nucleotide
Annamalai Senthilvelan, Muthian Shanmugasundaram & Anilkumar R. Kore
To cite this article: Annamalai Senthilvelan, Muthian Shanmugasundaram & Anilkumar
R. Kore (2020): Highly regioselective methylation of inosine nucleotide: an efficient
synthesis of 7-methylinosine nucleotide, Nucleosides, Nucleotides & Nucleic Acids, DOI:
10.1080/15257770.2020.1738457
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
https://doi.org/10.1080/15257770.2020.1738457
Highly regioselective methylation of inosine nucleotide:
an efficient synthesis of 7-methylinosine nucleotide
Annamalai Senthilvelan, Muthian Shanmugasundaram, and Anilkumar R. Kore
Life Sciences Solutions Group, Thermo Fisher Scientific, Austin, Texas, USA
ABSTRACT
ARTICLE HISTORY
Received 8 November 2019
Accepted 2 March 2020
A facile, straightforward, reliable, and an efficient chemical
synthesis of inosine nucleotides such as 7-methylinosine 50-O-
monophosphate, 7-methylinosine 50-O-diphosphate, and
7-methylinosine 50-O-triphosphate, starting from the corre-
sponding inosine nucleotide is delineated. The present methy-
lation reaction of inosine nucleotide utilizes dimethyl sulfate
as a methylating agent and water as a solvent at room
temperature. It is noteworthy that the present methylation
reaction proceeds smoothly under aqueous conditions that is
highly regioselective to afford exclusive 7-methylinosine
nucleotide in good yields with high purity (>99.5%).
KEYWORDS
Inosine; 7-methylinosine;
nucleotide; methylation;
regioselective
1. Introduction
The 50-end of cellular and eukaryotic viral RNAs that are synthesized by
various RNA polymerase contain distinct cap structure m7G[50]ppp[50]N,
where N is any nucleotide (N ¼ G, A, C or U).[1] The cap structure con-
taining 7-methylguanosine residue displays a wide spectrum of molecular
biology applications such as intracellular transport, splicing, translation,
and protection from degradation.[2–4] Notably, the cap structure is being
specifically recognized by eukaryotic initiation factor 4E (eIF4E) during the
initiation of translation,[5] vaccinia virus methyltransferase VP39,[6] and the
CBC80/20 nuclear cap-binding complex.[7] The presence of 7-methylguano-
sine moiety in the cap structure plays an important role for the recognition
as it forms Watson-Crick-like hydrogen bonds with the enzymes.[5] The
exploration of 7-methylguanosine nucleotide and cap analog as a potential
translation inhibitor is a powerful target for anti-cancer directed ther-
apy.[8–11] The elevated eIF4E levels in tumor cells is suppressed by the abil-
ity of these analogs to bind with eIF4E and acts as an anti-cancer agent. In
addition to the synthesis of capped mRNA containing 7-methylguanosine,
the synthesis of mRNA containing 50-terminal 7-methylinosine-capped was
achieved by the initiation of reovirus transcription using inosine 50-
CONTACT Anilkumar R. Kore
anil.kore@thermofisher.com
Life Sciences Solutions Group, Thermo Fisher
Scientific, 2130 Woodward Street, Austin, TX 78744-1832, USA.
ß 2020 Taylor & Francis Group, LLC

2
A. SENTHILVELAN ET AL.
triphosphate.[12] While the use of 7-methylguanosine capped mRNA has
been well documented in the literature for several molecular biology appli-
cations,[1,8] the utilization of 7-methylinosine capped mRNA is
under explored.
The use of methyl iodide has been the most commonly used methylating
agent for the synthesis of 7-methylguanosine nucleotide, starting from the
corresponding guanosine nucleotide.[3,13–15] However, the reaction requires
longer time to complete and often results in poor yields due to the poor
solubility of guanosine nucleotide in the presence of organic solvent.
Another method involves the use of methyl methanesulfonate as a methyl-
ating agent under aqueous conditions for the methylation of guanosine
moiety but a wide variety of generality has not been achieved using this
reagent.[16] We have developed an efficient synthesis of various substituted
7-methylguanosine nucleotides in good yields involving the use of dimethyl
sulfate as a methylating agent under aqueous conditions.[17–23] It has been
reported in the literature that 7-methylinosine was achieved by the reaction
of inosine using methyl iodide as a methylating agent.[24] While the synthe-
sis of 7-methylinosine and several types of substituted 7-methylguanosine
nucleotide have been reported,[17–24] to the best of our knowledge, no syn-
thesis of 7-methylinosine nucleotide has been reported in the literature.
Given the presence of delocalized positive charge to the nucleobase for
both 7-methylgunaosine and 7-methylinosine, we envisaged that the prop-
erties of 7-methylinosine nucleotide will be similar to the properties of 7-
methylguanosine nucleotide and these analogs will be used in the area of
mRNA cap analogs and cancer immunotherapy. Therefore, the develop-
ment of new chemical method for the synthesis of 7-methylinosine nucleo-
tide is demanded in order to explore the potential applications of these
analogs. Our continuous interest in the area of nucleic acid chemis-
try,[18–23,25–28] prompted us to develop a reliable chemical method for the
synthesis of inosine nucleotides. Herein, we report the first example of an
efficient chemical synthesis of 7-methylinosine nucleotides such as 7-meth-
ylinosine 50-O-monophosphate (m7IMP), 7-methylinosine 50-O-diphosphate
(m7IDP), and 7-methylinosine 50-O-triphosphate (m7ITP) in good yields
with high purity (>99.5%).
2. Results and discussion
The reaction pathway leading to the formation of 7-methylinosine 50-O-
monophosphate (3) is depicted in Scheme 1. The required starting material
(i.e.,) inosine 50-O-monophosphate (2) was obtained by the monophosphor-
ylation of inosine (1) using POCl3 as a phosphorylating agent and trimethyl
phosphate as a solvent.[29] The methylation reaction of 2 with dimethyl

NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
3
Scheme 1. Synthesis of 7-methylinosine 50-O-monophosphate (3).
sulfate as a methylating agent using water as a solvent at pH 4.0 afforded
the corresponding 7-methylinosine 50-O-monophosphate (3) in 80% yield.
It is noteworthy that the methylation reaction is highly regioselective. No
other regioisomers was formed as evidenced by HPLC of the crude reaction
1
mixture. The regiochemistry of 3 was confirmed H NMR based on the
1
disappearance of H-8 proton in 3. The H NMR spectrum of 2 showed a
characteristic singlet peak for H-8 proton at d 8.10,[29] whereas, in the case
of 3, no characteristic peak was seen for the H-8 proton probably due to
the presence of delocalized positive charge to the nucleobase. This observa-
tion is in agreement with our previously reported 7-methylguanosine ana-
1
logs in which there was no characteristic peak for H-8 proton in the H
NMR spectrum.[18–23] The regiochemistry of 3 was further corroborated
based on 2D-NOESY spectrum. The purity of final isolated product 3 was
1
checked by HPLC (Figure 1). The structure of 3 was confirmed by H and
13C NMR and mass data.
In a similar manner, the synthetic strategy for the formation of 7-methyl-
inosine 50-O-diphosphate (6) is shown in Scheme 2. The activation of
inosine monophosphate 2 with imidazole in the presence of triphenylphos-
phine and 2,20-dipyridyl disulfide, followed by the phosphorylation of
resulting imidazolide 4 with (Bu3NH)3PO4 in the presence of zinc chloride
as a catalyst afforded the corresponding inosine-50-diphosphate (5) in 75%
yield.[29] Next, treatment of inosine diphosphate 5 with dimethyl sulfate
under optimized aqueous conditions afforded the highly regioselective 7-
methylinosine 50-O-diphosphate (6) in 75% yield.
In addition to the synthesis of 7-methylinosine monophosphate 3 and
diphosphate 6, the present reaction strategy was successfully extended to the
synthesis of 7-methylinosine 50-O-triphosphate (8) as depicted in Scheme 3.
The inosine triphosphate 7 was achieved using “one pot, three-step” Ludwig
synthetic strategy.[29] The monophosphorylation reaction of inosine (1) with
phosphorus oxychloride using trimethyl phosphate as a solvent, followed by
the triphosphorylation reaction with tributylammonium pyrophosphate using

4
A. SENTHILVELAN ET AL.
Figure 1. HPLC spectrum of 7-methylinosine 50-O-monophosphate (3).
Scheme 2. Synthesis of 7-methylinosine 50-O-diphosphate (6).
acetonitrile as a solvent and tributylamine as a base afforded inosine-50-tri-
phosphate (7) in 62% yield. The methylation reaction of 7 with dimethyl sul-
fate under optimized aqueous conditions furnished the highly regioselective
7-methylinosine 50-O-triphosphate (8) in 74% yield.
There are several interesting features that merit comments for the pre-
sent methylation reaction. First, in all cases, the methylation reaction is
high regioselective affording the corresponding single regioisomer, 7-meth-
ylinosine nucleotide in good yields. The other possible regioisomer, 1-
methylinosine nucleotide was not formed under our optimized reaction
conditions. It is to be noted that the synthesis of 1-methylinosine was
achieved by the methylation reaction of inosine using trimethyloxosulfo-
nium hydroxide as a methylating agent.[30] Second, the present methylation
reaction proceeds smoothly under aqueous conditions. This is in striking
contrast with the literature method that utilizes organic solvent for the
methylation of purine nucleotides.[13–15] Finally, the purification procedure
for the synthesis of inosine nucleotide is simple, straightforward, and fur-
nishes a final pure product with extremely high purity (>99.5%) as evi-
denced by HPLC. It is to be noted that the successful molecular biology
applications depend on the high purity of nucleotide.

NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
5
Scheme 3. Synthesis of 7-methylinosine 50-O-triphosphate (8).
In conclusion, we have developed an efficient and reliable chemical
method for the synthesis of 7-methylinosine nucleotides such as 7-methyli-
nosine 50-O-monophosphate, 7-methylinosine 50-O-diphosphate, and 7-
methylinosine 50-O-triphosphate in good yields. It is noteworthy that the
present developed methylation reaction is highly regioselective and the final
product is isolated with extremely high purity (>99.5%) as evidenced by
HPLC. Further work is in progress to study the potential biological applica-
tions of these analogs and will be reported in due course.
2.1. Experimental
2.1.1. General
All of the commercial reagents and solvents are used as such without fur-
ther purification. Inosine was purchased from Sigma-Aldrich. Compounds
such as inosine-50-monophosphate (2), inosine-50-diphosphate (5), and
inosine-50-triphosphate (7) were prepared according to a reported
method.[29] 1H NMR spectra were recorded in D2O on a Bruker 400 MHz
and 31P NMR were recorded on a Bruker 162 MHz. Chemical shifts are
reported in ppm, and signals are described as s (singlet), d (doublet), t
(triplet), q (quartet), and m (multiplet). ESI mass was recorded on an
Applied Biosystems/Sciex MDX API 150 model. HPLC was run on a
Thermo Scientific DIONEX Ultimate 3000 using a Hypersil SAX column.
€
FPLC (fast protein liquid chromatography) was performed with an AKTA
purifier (GE Healthcare) using a DEAE Sepharose column.
2.2. Synthesis of 7-methylinosine 50-O-monophosphate (3)
To a stirred solution of inosine-50-monophosphate (2) (1.0 g, 2.70 mmol) in
20.0 mL of water, acetic acid was added slowly to adjust the pH of the solu-
tion to 4.0. To this mixture, dimethyl sulfate (3.8 mL) was added drop wise

6
A. SENTHILVELAN ET AL.
over a period of 30min. and the reaction mixture was allowed to stir at rt
for 4 h. As the methylation proceeds, the pH drops down to around 2.0 and
the pH was readjusted back to 4.0 using 1M NaOH solution. Then, the reac-
tion mixture was extracted with ethyl acetate (2ꢀ 25 mL) to remove
unreacted excess dimethyl sulfate. The collected aqueous solution was
adjusted to pH 6.5 and loaded on a DEAE Sepharose column. The desired
product was eluted using a linear gradient of 0-1M TEAB and the fractions
containing the product were pooled, evaporated, and co-evaporated with
water (3ꢀ 100 mL). The TEA salt of 7-methylinosine 50-O-monophosphate
thus obtained was subjected to ion-exchange with sodium perchlorate (5.0 g)
in acetone (100.0 mL) for two times to afford the sodium salt of 7-methyli-
nosine 50-O-monophosphate 3 as a fine white powder (Yield 0.83 g, 80%).
1H NMR (D2O, 400 MHz) d 8.36 (s, 1H, H-2), 6.26 (d, J ¼ 3.2 Hz, 1H, H-10),
4.70 (m, 1H), 4.41 (m, 2H), 4.23 (m, 1H), 4.20 (s, 3H, CH3), 4.10 (m, 1H);
31P NMR (D2O, 162 MHz) d 0.38 (s, 1P); MS (m/z): 362 [M-H]-.
2.3. Synthesis of 7-methylinosine 50-O-diphosphate (6)
To a stirred solution of inosine-50-diphosphate (5) (1.0 g, 2.12 mmol) in
20.0 mL of water, acetic acid was added to adjust the pH of the solution to
4.0. To this mixture, dimethyl sulfate (3.0 mL) was added drop wise over a
period of 30 min. and the reaction mixture was allowed to stir at rt for 5 h.
As the methylation proceeds, the pH drops down to around 2.0 and the
pH was readjusted back to 4.0 using 1M NaOH solution. After completion
of the reaction, it was extracted with ethyl acetate (2 ꢀ 25 mL) to remove
unreacted excess dimethyl sulfate. The collected aqueous solution was
adjusted to pH 6.5 and loaded on a DEAE Sepharose column. The desired
product was eluted using a linear gradient of 0-1M TEAB and the fractions
containing the product were pooled, evaporated and co-evaporated with
water (3 ꢀ 100 mL). The TEA salt of 7-methylinosine 50-O-diphosphate
thus obtained was subjected to ion-exchange with sodium perchlorate
(5.0 g) in acetone (100.0 mL) for two times to afford the sodium salt of 7-
methylinosine 50-O-diphosphate 6 as a fine white powder (Yield 0.77 g,
1
75%). H NMR (D2O, 400 MHz) d 8.28 (s, 1H, H-2), 6.19 (d, J ¼ 3.2 Hz,
1H, H-10), 4.63 (m, 1H), 4.34 (m, 2H), 4.24 (m, 1H), 4.13 (s, 3H, CH3),
4.07 (m, 1H); 31P NMR (D2O, 162 MHz) d ꢁ9.58 (d, J ¼ 21.0 Hz, 1P),
ꢁ11.03 (d, J ¼ 21.0 Hz, 1P); MS (m/z): 442 [M-H]-.
2.4. Synthesis of 7-methylinosine 50-O-triphosphate (8)
To a stirred solution of inosine-50-triphosphate (7) (1.0 g, 1.74 mmol) in
20.0 mL of water, acetic acid was added to adjust the pH of the solution to

NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
7
4.0. To this mixture, dimethyl sulfate (2.5 mL) was added drop wise over a
period of 30 min. and the reaction mixture was allowed to stir at rt for 5 h. As
the methylation proceeds, the pH drops down to around 2.0 and the pH was
readjusted back to 4.0 using 1 M NaOH solution. After completion of the
reaction, the mixture was extracted with ethyl acetate (2 ꢀ 25 mL) to remove
unreacted excess dimethyl sulfate. The collected aqueous solution was
adjusted to pH 6.5 and loaded on a DEAE Sepharose column. The desired
product was eluted using a linear gradient of 0-1M TEAB and the fractions
containing the product were pooled, evaporated and co-evaporated with
water (3 ꢀ 100 mL). The TEA salt of 7-methylinosine 50-O-triphosphate thus
obtained was subjected to ion-exchange with sodium perchlorate (5.0 g) in
acetone (100.0 mL) for two times to afford the sodium salt of 7-methylinosine
1
50-O-triphosphate 8 as a fine white powder (Yield 0.76 g, 74%). H NMR
(D2O, 400 MHz) d 8.29 (s, 1H, H-2), 6.17 (d, J ¼ 3.2 Hz, 1H, H-10), 4.62 (m,
1H), 4.38 (m, 2H), 4.27 (m, 1H), 4.15 (s, 3H, CH3), 4.11 (m, 1H); 31P NMR
(D2O, 162 MHz) d ꢁ8.78 (d, J ¼ 17.9 Hz, 1P), ꢁ11.15 (d, J ¼ 19.6 Hz, 1P),
ꢁ22.16 (t, J ¼ 19.6 Hz, 1P); MS (m/z): 522 [M-H]-.
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