Synthesis of N2-modified 7-methylguanosine 5′- monophosphates as nematode translation inhibitors

Article abstract of DOI:10.1016/j.bmc.2012.05.078

Preparative scale synthesis of 14 new N²-modified mononucleotide 5′ mRNA cap analogues was achieved. The key step involved use of an S_NAr reaction with protected 2-fluoro inosine and various primary and secondary amines. The derivatives were tested in a parasitic nematode, Ascaris suum, cell-free system as translation inhibitors. The most effective compound with IC₅₀ ～0.9 μM was a N²-p-metoxybenzyl-7- methylguanosine-5′-monophosphate 35.

Full text of DOI:10.1016/j.bmc.2012.05.078

Bioorganic & Medicinal Chemistry 20 (2012) 4781–4789
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of N2-modified 7-methylguanosine 50-monophosphates
as nematode translation inhibitors
Karolina Piecyk a, Richard E. Davis b, Marzena Jankowska-Anyszka a,
⇑
a Faculty of Chemistry, University of Warsaw, 02-093 Warsaw, Poland
b Department of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, Aurora, CO 80045, USA
a r t i c l e i n f o
a b s t r a c t
Preparative scale synthesis of 14 new N2-modified mononucleotide 50 mRNA cap analogues was achieved.
The key step involved use of an SNAr reaction with protected 2-fluoro inosine and various primary and
secondary amines. The derivatives were tested in a parasitic nematode, Ascaris suum, cell-free system
Article history:
Received 4 April 2012
Revised 25 May 2012
Accepted 30 May 2012
Available online 10 June 2012
as translation inhibitors. The most effective compound with IC50 ꢀ0.9
l
M was a N2-p-metoxybenzyl-7-
methylguanosine-50-monophosphate 35.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Cap analogue
mRNA
Translation inhibitor
Nematode
Ascaris suum
1. Introduction
is associated with a variety of human cancers.8 Consequently, cap
analogs as specific inhibitors to counteract elevated eIF4E level in
tumor cells have been explored.9
Methylation of the exocyclic amino group of guanosine is an
important biological modification of RNAs. Modified nucleosides
occur frequently in RNAs including N2-methyl-(m2G) and N2,N2-
dimethylguanosine (m22,2G) within ribosomal (rRNA)1 and transfer
(tRNA)2 RNAs, respectively. N2,N2-dimethylG as a part of the RNA
cap structure is also found at the 50 end of small nuclear RNAs (U
snRNA) and some messenger RNAs (mRNA). Eukaryotic mRNAs
possess a 50 terminal cap structure consisting of a 7-methylguano-
sine linked via a 50,50-triphosphate bridge to the first transcribed
nucleotide (m7GpppN, where N=G, A, C or U; MMG cap).3 In nem-
atodes, however, two different mRNAs co-exist in cells. In addition
to mRNAs with the common MMG cap, about 70% of the mRNAs
possess an atypical, hypermethylated cap. This cap (TMG-cap,
m32,2,7GpppG) has the N7-methyl as well as N2,N2-dimethyl in its
structure and is added along with a 22 nt spliced leader sequence
during trans-splicing.4
The cap plays an essential role in several processes during gene
expression5 by interacting with proteins that specifically recognize
its structure.6 The best-characterized cap-binding protein is the
translation initiation factor eIF4E. Recognition of the mRNA cap
by eIF4E is the critical, rate limiting step for efficient translation
initiation, and it is a major target for translational control.7 It has
been shown that eIF4E is a potent oncogene and its overexpression
Many nematodes, including Ascaris suum, are parasitic, infect
over 2 billion people, and remain a significant health problem.
The role of trans-splicing as a mechanism of nematode gene
expression and the effect of the TMG cap and a spliced leader addi-
tion on mRNA metabolism have been intensively investigated.10
Ascaris cap-binding proteins must deal with two distinct popula-
tions of mRNA suggesting these proteins are unique and may be
targets for the discovery of novel cap analogs that can specifically
block parasite gene expression. We recently prepared and analyzed
a guanosine derivative with a benzyl at N2 position (bn2m7GMP,
32) to examine the intermolecular interaction of MMG/TMG caps
with Ascaris eIF4E-3.11 The addition of an N2-benzyl substituent
on a monophosphate led to significant translation inhibition in
an Ascaris suum embryo cell-free system. Therefore, we chose to
extend these analyses to several other N2 modified derivatives.
In the present study we designed and synthesized a series of N2-
substituted 7-methylguanosine 50-monophosphates. This type of
cap analog has not been widely studied due to difficulties with
the development of a good method to efficiently introduce substit-
uents at the N2 position of guanosine. To achieve our goal we used
a six step strategy starting from guanosine using an SNAr reaction
with fully protected 2-fluoro inosine and various primary and
secondary amines. The synthesized mononucleotide TMG cap
analogues were tested in a parasitic nematode, Ascaris suum, cell-
free system as translation inhibitors.
⇑
Corresponding author.
E-mail address: marzena@chem.uw.edu.pl (M. Jankowska-Anyszka).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.05.078

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K. Piecyk et al. / Bioorg. Med. Chem. 20 (2012) 4781–4789
guanosine (2) was transformed into N2-fluoro-20,30,50-O-triacetyl-
2. Results and discussion
2.1. Chemistry
O6-[2-(4-nitrophenyl)ethyl]inosine (3). We tested three methods
for diazotiation and fluorination of nucleosides. The first was to
prepare the 2-fluoro derivative using aqueous diazotization of gua-
nosine in the presence of fluoroboric acid.17 These conditions, how-
ever, were too harsh and led to depurination. A second approach
was the introduction of the fluorine atom under anhydrous condi-
tions with t-butyl nitrite as the diazotizing agent and HF in pyri-
dine as the fluoride source.18 The reaction proceeded smoothly in
very good yield (85%), and the main product remained fully pro-
tected. However, without careful monitoring, this reaction led to
depurination or even failure to obtain product unless very high
quality HF/pyridine reagent was used and a low temperature was
maintained during the course of the reaction. Consequently, we
also explored milder fluorination conditions using polyvinylpyrid-
inium polyhydrogenfluoride (PVPHF) reagent developed by Olah
and Li.19 The advantages of this method were easy handling and
an extremely convenient work-up, however, yields were limiting.
Consequently, the most efficient way to prepare N2-fluoro-
20,30,50-O-triacetyl-O6-[2-(4-nitrophenyl)ethyl]inosine (3) is the
procedure that involves using HF/pyridine for the substitution
step.
In order to synthesize N2-substituted mononucleotide cap ana-
logues, we adopted and modified a method for the preparation of
the N2,N2-dimethylguanosine by Eritja and co-workers.12 The syn-
thesis was carried out in six steps using inexpensive, commercially
available guanosine as the starting material (Scheme 1). The syn-
thesis began with the triacetylation of guanosine using acetic
anhydride (Ac2O) in the presence of triethylamine (TEA) and N,N-
(dimethylamino)pyridine (DMAP) using a procedure modified
from Nair et al.13 By carrying out the addition of the Ac2O at 0 °C,
N-acetylation was suppressed and 20,30,50-tri-O-acetylguanosine
(1) was obtained in a near quantitative yield (98%). In the next step,
protection of the O6 group of guanosine was carried out with a p-
nitrophenylethyl (NPE) group as it is believed to be stable under
mild acid and base hydrolyzes, and it can be easily cleaved by
DBU or DBN in a b-elimination reaction. The reaction was carried
out via the Mitsunobu reaction with p-nitrophenylethanol, diiso-
propyl azodicarboxylate (DIAD), and triphenylphosphine (TPP) in
anhydrous toluene at room temperature.14 A long standing prob-
lem with the Mitsunobu reaction is the purification of the desired
product from the reaction mixture. We have tried to replace
triphenylphosphine and diisopropyl azodicarboxylate (which are
converted during the reaction into triphenylphosphine oxide and
related dialkylhydrazinodicarboxylate, respectively) by other re-
agents to facilitate the isolation and purification processes.15 Our
observations showed that obtaining pure product was possible
using standard reagents but the purification required a very long
chromatography process. In order to shorten the isolation of the
20,30,50-tri-O-acetyl-O6-[2-(4-nitrophenyl)ethyl]guanosine (2), we
partially purified the final product and then used 2 with some
impurities (mainly triphenylphosphine oxide) in the next step.
By-products remaining after the Mitsunobu reaction were easily
removed during the purification of 3. This faster procedure allowed
us to obtain a pure fluorinated derivative with good yield (85%).
The main route to N2-substituted guanosine analogues is via
nucleophilic displacement of a halogen at the 2 position of inosine.
Since fluoro derivatives are more reactive toward nucleophilic
displacement in a SNAr reaction than other halogen derivatives,16
the fully protected 20,30,50-tri-O-acetyl-O6-[2-(4-nitrophenyl)ethyl]
The 2-fluoro intermediate was used directly in a SNAr reaction
by treating it with two-fold excess of primary or secondary, ali-
phatic or aromatic amines varying in size and steric branching in
anhydrous DMSO (Scheme 2). Incorporated amines were chosen
to produce several N2 substituents (aliphatic, cyclic, or aromatic)
in the cap to vary the size and steric branching of aliphatic N2 sub-
stituents (compounds 11–17) and to influence the electron density
of the aromatic ring through the introduction of various substitu-
ents, such as metoxy (7), chloro (8), nitro (9), or phenyl (10), into
the benzene ring. In addition to the mono substituted analogues,
we also used some secondary amines and synthesized compounds
bearing additional methyl (5, 11, 13, 16), ethyl (15) or benzyl (6)
groups at the N2 position to check the effectiveness of these sub-
stituents on the cap as translation inhibitors. The nature of the
nucleophile is the most critical parameter in the SNAr reaction.
Therefore, for the simple primary amines, the substitution reaction
occurred rapidly and the fluorine derivative was used up in less
than 30 min at temperature not higher than 50 °C. The reaction
with sterically hindered and secondary amines such as dibenzyl-
amine required longer time (in some cases even 2 days) and a
Scheme 1. Preparation of N2-fluoro-20,30,50-O-triacetyl-O6-[2-(4-nitrophenyl)ethyl]inosine; (a) acetic anhydride, DMAP, Et3N, AcCN, 4 °C to rt; (b) NPE, PPh3, DIAD, toluene, rt;
(c) HF/pyridine, tBuONO, pyridine, ꢁ40 °C.

K. Piecyk et al. / Bioorg. Med. Chem. 20 (2012) 4781–4789
4783
Scheme 2. Preparation of N2-modified guanosine derivatives via a SNAr reaction; (a) amine, DMSO, 50 °C to 65 °C. aNumbers in brackets correspond to compounds on
Scheme 3.
higher temperature (65 °C). After completion of the substitution
reaction 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) was added to
the reaction mixture to remove the NPE group followed by the
addition of a mixture of THF/MeOH/NaOHaq in order to complete
acetyl deprotection. Chromatographic purification on silica gel
yielded pure nucleoside derivatives 4–17 in good yield. It is worth
mentioning, that in course of the reaction progress, we observed
that all protecting groups (acetyl and NPE) were slowly lost if the
substitution reaction was carried out under prolonged heating in
the presence of excess amine. This observation led us to a simpler
purification procedure in which full deprotection took place with-
out any DBU and final N2-substituted guanosine derivatives were
easily crystallized from the reaction mixture by the addition of
CH2Cl2.
RT21 (Scheme 3). The reaction progress of the last step was
monitored by HPLC to avoid over methylation. Purification of the
analogues was done using ion-exchange chromatography on
DEAE–Sephadex A-25 (HCOꢁ3 form) and the derivatives were sub-
sequently converted into the sodium salt using Dowex 50WX8.
The structure and homogeneity of final products were confirmed
by HPLC, mass spectrometry, 1H NMR and 31P NMR.
2.2. Biology
One of the synthesized compounds, bz2m7GMP (32), has been
recently used in Ascaris suum eIF4E-3/m7G- and m32,2,7G-cap bind-
ing studies.11 It was shown that bz2m7GMP binds to the protein
with a 10-fold higher affinity compared with m7GMP and is a
strong inhibitor of either a m7G- or m32,2,7G-capped mRNA transla-
tion in a cell-free Ascaris suum translation system. To evaluate the
ability of newly synthesized N2-modified 7-methylguanosine 50-
monophosphates to inhibit cap-dependent translation in a cell-free
All the N2-modified guanosine analogues were further 50 phos-
phorylated using the Yoshikawa method20 with phosphorus oxide
trichloride in trimethyl phosphate at 4 °C and subsequently meth-
ylated at the N7 position of the guanine ring with CH3I in DMSO at

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K. Piecyk et al. / Bioorg. Med. Chem. 20 (2012) 4781–4789
shown, the inhibitory potency of cap analogues generally increases
with the length of polyphosphate bridge.9a Therefore, in order to
determine the strength of our new mononucleotide cap analogues
we also compared them to m7GMP which is known as a weak
translation inhibitor. The results (Fig. 1, Table 1) indicated that
any mono substitution at the N2 position of guanine (alkyl–ali-
phatic, cyclic, or aryl) produced inhibitory compounds with IC50
similar to m7GTP. All alkyl substituents led to similar levels of inhi-
bition regardless of their size and steric branching. Aryl substituted
compounds were more effective as translation inhibitors. The
strongest inhibitor, about 7 times stronger than m7GTP, was deriv-
ative 35 with an IC50 of 0.9
ties of para substituents (NO2 > Cl > Phe > OCH3) were roughly
correlated with the IC50 of the compounds (12.6 M, 3.8 M,
4.3 M, 0.9 M, respectively). The enhanced electron density of
lM. The electron withdrawing proper-
l
l
l
l
p
the benzene ring increases the effectiveness of cap analogues as
translational inhibitors, but other factors such as the size of substi-
tuent may also play an important role. Recent studies on the co-
crystal structure of m32,2,7GTP with Ascaris eIF4E-311 indicated that
the two methyl groups at the N2 position of the base are solvent ex-
posed and do not interact directly with the protein. The N2 substit-
uents may form additional interactions with eIF4E influencing
conformational changes known to form the binding pocket24 or
may change the thermodynamic aspects of the protein binding to
the 50 cap.
Our results also showed that the addition of a second substitu-
ent at the N2 position of guanosine decreased the level of inhibition
significantly (e.g. compounds 32 versus 33 or 39 versus 40). These
data are consistent with a comparison of the crystal structures of
Ascaris eIF4E-3 with m7GTP or m32,2,7GTP.11 These studies
Scheme 3. Preparation of N2-modified mononucleotide cap analogues (a) POCl3,
trimethyl phosphate, 4 °C; (b) methyl iodide, DMSO, rt. aR corresponds to various
substituents at the N2 position of guanosine (see Scheme 2, numbers in brackets).
translation system where naturally occurring mono- and trimethy-
lated mRNAs are translated, we tested 14 cap analogues (32–45) in
the same manner as previously reported.10c,11 Two standard cap
analogues were used as internal controls in the experiments:
m7GTP as a positive control as it is an effective translation inhibitor
in various cell-free systems (rabbit reticulocyte,9a wheat germ,23
Ascaris suum10c) and ApppG as a negative control that have very lit-
tle or no inhibitory efficacy due to lack of the positive charge on
guanine that is generated by the N7 methyl group. As previously
Figure 1. Translation inhibition assay in A. suum extract. The synthesized cap analogues were assayed for their ability to inhibit cap-dependent translation of an m7GpppG-
capped Renilla luciferase mRNA in an Ascaris suum embryo cell-free translation system. 22The measurements were carried out as previously described and the% translation
activity plotted against the inhibitor concentration.10c All measurements were made in triplicate in several preparations of extracts. Data presented are representative
experiments.

K. Piecyk et al. / Bioorg. Med. Chem. 20 (2012) 4781–4789
4785
Table 1
Inhibition of translation in A. suum extract by N2-modified cap analogues
R =
IC50
2.9
(l
M)
R =
IC50
>50
(lM)
32
33
39
40
>50
>50
3.9
34
41
>50
35
36
37
38
0.9
42
43
44
45
4.5
3.8
>50
13.9
5.7
12.6
4.3
m7GTP
m7GTP
6.1
>50
m7GMP
IC50 values were extracted directly from the plotted of % translation activity values against compound concentration. IC50 values >50 lM—no measurable effect.
demonstrated that the addition of the second substituent at the N2
position of guanosine leads to the loss of one hydrogen bond
between the amine group of guanine and the carboxyl group of
Glu116. This loss of a hydrogen bond likely influences the effec-
tiveness of analogues as translation inhibitors.
subsequently converted into the sodium salt using Dowex
50WX8 (Na+ form). Homogeneity of the final analogues was
checked by reversed-phase analytical HPLC. HPLC was performed
using a Supelcosil LC-18-T RP column (4.6 ꢂ 250 mm, flow rate
1.0 mL/min) with: Method A — a linear gradient of methanol from
0% to 50% (v/v) in 0.05 M ammonium acetate (pH 5.9) in 20 min, an
isocratic elution of 50% methanol (v/v) in 0.05 M ammonium ace-
tate (pH 5.9) till 30 min, Method B — a linear gradient of methanol
from 0% to 50% (v/v) in 0.05 M ammonium acetate (pH 5.9) in
10 min and then an isocratic elution of 50% methanol (v/v) in
0.05 M ammonium acetate (pH 5.9) till 30 min, Method C — an iso-
cratic elution of 50% methanol (v/v) in 0.05 M ammonium acetate
(pH 5.9), on a Knauer instrument, with UV detection at 254 nm. MS
spectra were acquired using Waters Micromass Q-TOF Premier
spectrometer with positive electrospray ionization source. 1H and
13C NMR spectra of intermediate derivatives were obtained with
a Varian UnityPlus 200 MHz spectrometer. 1H and 31P NMR spectra
of the final compounds were recorded on a Varian INOVA 700 MHz
spectrometer.
3. Conclusions
We have prepared 14 novel N2 modified trimethylguanosine
cap analogues via a six-step synthesis from guanosine. This general
strategy provides a new approach to scale-up the synthesis of a
large number of modified cap analogues that should be useful in
studying eIF4E function and cap-dependent translation. In addition
the idea of exploring cap analogues that possess only one phos-
phate that typically have little inhibitory activity offers an oppor-
tunity to explore compounds that are not highly charged and can
be good candidates for new drug development.
4. Materials and methods
4.1. Experimental
All reagents were the highest available purity and purchased
from Sigma–Aldrich Chemical Co. Triethylammonium bicarbonate
(TEAB) buffer was prepared by bubbling CO2 through an ice-cold
aqueous solution of redistilled triethylamine. Intermediate nucleo-
tides were separated by ion-exchange chromatography on a DEAE-
Sephadex A-25 (HCOꢁ3 form) using a linear gradient of TEAB buffer,
pH 7.6. Fractions containing products were combined and evapo-
rated under reduced pressure with several additions of ethanol
and isolated as triethylammonium salts (TEA salts) and
4.1.1. 20,30,50-Tri-O-acetylguanosine (1)
2.2 mL of acetic anhydride (22 mmol) was added dropwise to a
suspension of guanosine (2 g, 7.0 mmol) (dried for 2 days over
P4O10 in high vacuum), triethylamine (7.7 mL, 55.2 mmol) and
N,N-(dimethylamino)pyridine (92 mg, 0.75 mmol) in 27 mL of ace-
tonitrile at 0 °C. The mixture was stirred until it became homoge-
neous and kept an additional 3 h at room temperature. The

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K. Piecyk et al. / Bioorg. Med. Chem. 20 (2012) 4781–4789
reaction was quenched with methanol (2.3 mL). The volume was
reduced to 1/3 using a rotary evaporator and diethyl ether was
added dropwise to induce precipitation of a fine white powder.
The product was collected by filtration, washed with diethyl ether,
and then stirred for 2 hr with acetone (30 mL) at 50 °C. The filtrate
produced 2.8 g (98%) of a fine white powder. 1H NMR (200 MHz,
DMSO) d 10.54 (s, 1H, NH) 7.93 (s, 1H, H-8), 6.34 (br, 2H, NH2),
5.98 (d, J = 6.1 Hz, 1H, H-10), 5.79 (t, J = 5.8 Hz, 1H, H-20), 5.51–
5.48 (m, 1H, H-30), 4.40–4.37 (m, 1H, H-40), 4.33–4.31 (m, 1H, H-
50), 4.23–4.28 (m, 1H, H-500), 2.11 (s, 3H, CH3, acetyl), 2.05 (s, 3H,
CH3, acetyl), 2.04 (s, 3H, CH3, acetyl); 13C NMR (200 MHz, CDCl3)
d 172.1 (C@O), 171.0 (C@O), 169.9 (C@O), 156.3 (C-6), 152.8 (C-
2), 151.2 (C-4), 137.6 (C-8), 119.6 (C-5), 86.1 (C-10), 82.3 (C-40),
73.6 (C-20), 72.3 (C-30), 61.8 (C-50), 21.1 (C, acetyl), 20.6 (C, acetyl),
20.2 (C, acetyl); m/z: calcd for C18H21N5O5 (M+H)+: 410.1306,
found: 410.1305.
4.1.4. General procedure for the synthesis of N2-substitutiuted
derivatives 4-17
N2-fluoro-20,30,50-O-triacetyl-O6-[2-(4-nitrophenyl)ethyl]ino-
sine (250 mg, 0.45 mmol) was dissolved in 2 mL of anhydrous
dimethylsulfoxide (DMSO) and then an amine (0.9 mmol) was
added. The reaction mixture was stirred at 60 °C from 30 min to
2 days until the fluoronucleoside completely disappeared (based
on TLC) followed by addition of 0.5 M NaOH in THF/MeOH/H2O
(5/4/2). The solvent was removed under high vacuum and the
resulting oily residue was treated with CH2Cl2 to induce precipita-
tion of a fine white powder. The product was collected by filtration
and washed several times with methanol and dried over P4O10 to
yield:
4.1.4.1. N2-Benzylguanosine (4). 134 mg, 66%; 1H NMR (200 MHz,
CDCl3) d 7.69 (s, 1H, H-8), 7.38–7.19 (m, 5H, Ph), 5.66 (d, J = 5.6 Hz,
1H, H-10), 4.64–4.62 (m, 1H, H-20), 4.55–4.47 (m, 2H, CH2Ph), 4.12–
4.10 (m, 1H, H-30), 3.89–3.84 (m, 1H, H-40), 3.69–3.57 (m, 2H, H-50);
13C NMR (50 MHz, CDCl3) d 157.4 (C-6), 153.8 (C-2), 150.6 (C-4),
140.4 (Ph) 134.6 (C-8), 128.0 (Ph), 127.2 (Ph), 126.2 (Ph), 117.1
(C-5), 87.5 (C-10), 85.1 (C-40), 73.0 (C-20), 70.6 (C-30), 61.7 (C-50),
44.2 (N-CH2Ph); m/z: calcd for C17H19N5O5 (M+H)+: 374.1458,
found: 374.1453.
4.1.2. 20,30,50-Tri-O-acetyl-O6-[2-(4-nitrophenyl)ethyl]guanosine
(2)
A
suspension of 20,30,50-tri-O-acetylguanosine (2.37 g,
5.8 mmol), triphenylphosphine (2.28 g, 8.7 mmol) and 2-(4-nitro-
phenyl)ethanol (1.45 g, 8.7 mmol) in anhydrous toluene was stir-
red for 30 min and diisopropyl azodicarboxylate (1.4 mL) was
added dropwise over a period of 45 min. The reaction mixture
was kept for 12 h at rt. Then the solvent was evaporated and the
residual oil was purified by column chromatography on silica gel
with chloroform to produce a pure product of yellowish crystals,
2.26 g (70%). 1H NMR (200 MHz, CDCl3) d 8.17 (d, 2H, Ph), 7.72
(s, 1H, H-8), 7.49 (d, 2H, Ph), 6.05–5.91 (m, 2H, H-10, H-20), 5.87–
5.75 (m, 1H, H-30), 4.73 (t, J = 6.7 Hz, 2H, O–CH2, NPE), 4.50–4.36
(m, 3H, H-40, H-50, H-500), 3.28 (t, 6.7 2H, CH2Ph, NPE), 2.14 (s, 3H,
CH3, acetyl), 2.09 (s, 3H, CH3, acetyl), 2.08 (s, 3H, CH3, acetyl); 13C
NMR (200 MHz, CDCl3) d 171.7 (C@O), 171.0 (C@O), 169.7 (C@O),
161.9, 155.5 (C-6), 154.5 (C-2), 148.8, 148.6, 138.9 (C-8), 129.3
(C, Ph), 124.8 (C, Ph), 113.6 (C-5), 86.8 (C-10), 82.3 (C-40), 73.4 (C-
20), 70.6 (C-30), 67.1 (OCH2, NPE), 62.5 (C-50), 35.5 (CH2Ph), 21.2
(C, acetyl), 20.6 (C, acetyl), 20.2 (C, acetyl); m/z: calcd for
4.1.4.2. N2-Benzyl-N2-methylguanosine (5). 109 mg, 52%; 1H
NMR (200 MHz, CDCl3) d 7.66 (s, 1H, H-8), 7.26–7.14 (m, 5H, Ph),
5.64 (d, J = 5.5 Hz, 1H, H-10), 4.62–4.60 (m, 1H, H-20), 4.50–4.42
(m, 2H, CH2Ph), 4.13–4.11 (m, 1H, H-30), 3.81–3.74 (m, 1H, H-40),
3.62–3.51 (m, 2H, H-50), 2.88 (s, 3H, N7-CH3); 13C NMR (50 MHz,
CDCl3) d 157.6 (C-6), 151.1 (C-2), 149.4 (C-4), 140.7, 137.2, 128.6
(Ph), 128.5 (Ph), 127.2 (Ph), 119.3 (C-5), 87.9 (C-10), 84.6 (C-40),
73.2 (C-20), 71.9 (C-30), 62.3 (C-50), 44.8 (N-CH2Ph), 37.0 (N-CH3);
m/z: calcd for C18H21N5O5 (M+H)+: 388.1615, found: 388.1613.
4.1.4.3. N2,N2-Dibenzylguanosine (6). 103 mg, 41%,1H NMR
(200 MHz, CDCl3) d 7.59 (s, 1H, H-8), 7.36–7.29 (m, 10H, Ph),
5.67 (d, J = 5.6 Hz, 1H, H-10), 4.68 (s, 4H, CH2Ph), 4.61 (t,
J = 5.0 Hz, 1H, H-20), 4.16–4.13 (m, 1H, H-30), 3.87–3.83 (m, 1H,
H-40), 3.60–3.43 (m, 2H, H-50); 13C NMR (50 MHz, CDCl3) d 155.8
(C-6), 152.2 (C-2), 150.3 (C-4), 139.8, 137.3, 128.5 (Ph), 128.2
(Ph), 127.1 (Ph), 119.6 (C-5), 88.9 (C-10), 85.8 (C-40), 74.4 (C-20),
72.0 (C-30), 62.3 (C-50), 54.3 (N-CH2Ph). m/z: calcd for C24H25N5O5
(M+H)+: 464.1928, found: 464.1918.
C
24H26N6O10 (M+H)+: 559.1783, found: 559.1784.
4.1.3. N2-Fluoro-20,30,50-O-triacetyl-O6-[2-(4-nitrophenyl)ethyl]
inosine (3)
Dry 20,30,50-tri-O-acetyl-O6-[2-(4-nitrophenyl)ethyl]guanosine
(1 g, 1.79 mmol) in polypropylene tube under nitrogen was dis-
solved in anhydrous pyridine (6.75 mL, 0.082 mol). The tube was
placed in a dry ice/acetonitrile cooling bath (ꢁ35 to ꢁ45 °C) and
70% HF/pyridine solution (12 mL, 0.42 mol) was added dropwise
over a period of 5 min to 45% final HF. The reaction mixture was
stirred for 15 min and t-butyl nitrite (0.54 mL, 4.5 mmol) was
added. After 1hr, the reaction was quenched at 0 °C by slowly pour-
ing the reaction mixture into an aqueous K2CO3 solution (28.5 g in
25 mL of water) and then extracted three times with ethyl acetate.
The organic layers were collected, dried over anhydrous Na2SO4
and evaporated to dryness. Purification by column chromatogra-
phy using as eluate 60:1 CH2Cl2: MeOH gave 0.85 g (85%) of prod-
uct; TLC silica gel, CH2Cl2: MeOH, 60:1 RF = 0.4; 1H NMR (700 MHz,
CDCl3) d 8.21–8.15 (m, 2H, Ph), 8.08 (s, 1H, H-8), 7.50 (d, 2H, Ph),
6.13 (d, J = 5.6 Hz, 1H, H-10), 5.82 (t, J = 4.9 Hz, 1H, H-20), 5.60–
5.55 (m, 1H, H-30), 4.84 (t, J = 6.7 Hz, 2H, O–CH2, NPE), 4.46–4.42
(m, 2H, H-40, H-50), 4.38–4.36 (m, 1H, H-500), 3.32 (t, J = 6.7 Hz,
2H, CH2Ph, NPE), 2.15 (s, 6H, CH3, acetyl), 2.08 (s, 3H, CH3, acetyl).
13C NMR (200 MHz, CDCl3) d 170.2 (C@O), 169.5 (C@O), 169.3
(C@O), 160.3, 159.3 (C-6), 154.2 (C-2), 147.1, 145.1, 140.8, 129.9
(Ph), 123.8 (Ph), 86.4 (C-10), 80.5 (C-40), 73.0 (C-20), 72.4 (C-30),
67.7 (OCH2, NPE), 62.8 (C-50), 34.9 (CH2Ph), 20.7 (C, acetyl), 20.5
(C, acetyl), 20.3 (C, acetyl); m/z: calcd for C24H24FN5O10 (M+H)+:
562.1579, found: 562.1581.
4.1.4.4. N2-p-Metoxybenzylguanosine (7). 140 mg, 64%,1H NMR
(200 MHz, CDCl3) d 8.02 (s, 1H, H-8), 7.27 (d, 2H, Ph), 6,85 (d, 2H,
Ph), 5.92 (d, J = 5.4 Hz, 1H, H-10), 4.73 (t, J = 4.9 Hz, 1H, H-20), 4.48
(s, 2H, CH2Ph) 4.21–4.18 (m, 1H, H-30), 3.93–3.90 (m, 1H, H-40),
3.72–3.68 (m, 2H, H-50), 3,59 (s, 3H, O-CH3); 13C NMR (50 MHz,
CDCl3) d 158.9 (C-6), 155.9, 154.1, 150.2, 137.8, 132.8, 127.9,
118.9, 113.9, 88.9 (C-10), 86.1 (C-40), 74.3 (C-20), 72.0 (C-30), 62.7
(C-50), 56.0 (OCH3), 44.6 (N-CH2Ph). m/z: calcd for C18H21N5O6
(M+H)+: 404.1564, found: 404.1560.
4.1.4.5. N2-p-Chlorobenzylguanosine (8). 153 mg, 69%,1H NMR
(200 MHz, CDCl3) d 8.04 (s, 1H, H-8), 7.36–7.30 (m, 4H, Ph), 5.90
(d, J = 5.3 Hz, 1H, H-10), 4.65 (t, J = 4.8 Hz, 1H, H-20), 4.35 (s, 2H,
CH2Ph), 4.18–4.15 (m, 1H, H-30), 3.91–3.88 (m, 1H, H-40), 3.70–
3.67 (m, 2H, H-50); 13C NMR (50 MHz, CDCl3) d 157.2 (C-6), 154.8
(C-2), 150.4 (C-4), 137.9, 136.7, 131.3, 129.2 (Ph), 128.3 (Ph),
119.8 (C-5), 88.6 (C-10), 85.5 (C-40), 74.3 (C-20), 71.7 (C-30), 62.4
(C-50), 44.8 (N-CH2Ph). m/z: calcd for C17H18ClN5O5 (M+H)+:
408.1069, found: 408.1073.
4.1.4.6. N2-p-Nitrobenzylguanosine (9). 134 mg, 59%,1H NMR
(200 MHz, CDCl3) d 8.04 (s, 1H, H-8), 8.22 (d, 2H, Ph), 7.63 (d, 2H,

K. Piecyk et al. / Bioorg. Med. Chem. 20 (2012) 4781–4789
4787
Ph), 5.84 (d, J = 5.2 Hz, 1H, H-10), 4.63 (s, 2H, CH2Ph) 4.66–4.64 (m,
1H, H-20), 4.18–4.15 (m, 1H, H-30), 3.98–3.95 (m, 1H, H-40), 3.75–
3.71 (m, 2H, H-50); 13C NMR (50 MHz, CDCl3) d 156.6 (C-6), 153.7
(C-2), 150.9, 147.8, 146.4, 136.9 (C-8), 128.2 (Ph), 123.9 (Ph),
118.8 (C-5), 88.4 (C-10), 86.8 (C-40), 72.7 (C-20), 71.2 (C-30), 62.2
(C-50), 43.8 (N-CH2Ph). m/z: calcd for C17H18N6O7 (M+H)+: 419.
1309, found: 419.1312.
4.63 (br, 1H, H-20), 4.15 (br, 1H, H-30), 3.83–3.78 (m,,3H, H-40, H-
50), 3.69–3.57 (m, 4H, CH2 ethyl), 1.06 (t, 6H, CH3 ethyl); 13C
NMR (50 MHz, CDCl3) d 156.8 (C-6), 151.8 (C-2), 141.6 (C-4),
136.9 (C-8), 117.3 (C-5), 87.5 (C-10), 86.2 (C-40), 74.2 (C-20), 71.9
(C-30), 61.8 (C-50), 42.6 (N-CH2 ethyl), 13.1 (CH3 ethyl). m/z: calcd
for C14H21N5O5 (M+H)+: 340.1615, found: 340.1610.
4.1.4.13. N2-Ethyl-N2-methylguanosine (16). 100 mg, 56%,1H
NMR (200 MHz, CDCl3) 7.79 (s, 1H, H-8), 5.67 (d, J = 5.1 Hz, 1H, H-
10), 4.72–4.69 (t, J = 5.2, 1H, H-20), 4.16 (br, 1H, H-30), 3.83–3.78 (m,
1H, H-40), 3.69–3.57 (m, 2H, H-50), 3.62 (q, J = 6.8 Hz, 2H, CH2 ethyl),
3.12 (s, 3H, N-CH3), 1.22 (t, J = 7.1 Hz, 3H, CH3 ethyl); d 13C NMR
(50 MHz, CDCl3) d 157.7 (C-6), 152.4 (C-2), 140.2 (C-4), 137.5 (C-8),
119.2 (C-5), 88.8 (C-10), 85.9 (C-40), 75.0 (C-20), 71.3 (C-30), 63.4 (C-
50), 44.4 (N-CH2 ethyl), 37.5 (N-CH3), 11.9 (CH3 ethyl). m/z: calcd
for C13H19N5O5 (M+H)+: 326.1458, found: 326.1463.
4.1.4.7. N2-p-Phenylbenzylguanosine (10). 134 mg, 55%,1H NMR
(200 MHz, CDCl3) d 7.98 (s, 1H, H-8), 7.75–7.71 (m, 9H, Ph), 5.89
(d, J = 5.3 Hz, 1H, H-10), 4.69–4.65 (m, 2H, CH2Ph), 4.61 (t,
J = 4.9 Hz, 1H, H-20), 4.16–4.14 (m, 1H, H-30), 3.89–3.81 (m, 1H, H-
40), 3.64–3.41 (m, 2H, H-50); 13C NMR (50 MHz, CDCl3), d 158.7 (C-
6), 153.2 (C-2), 151.8 (C-4), 140.9, 140.8, 138.1, 137.5, 129.1 (Ph),
128.5 (Ph), 127.7 (Ph), 127.6 (Ph), 127.3 (Ph), 117.4 (C-5), 88.3 (C-
10), 87.5 (C-40), 75.6 (C-20), 72.8 (C-30), 63.7 (C-50), 43.7 (N-CH2Ph);
m/z: calcd for C23H23N5O5 (M+H)+: 450.1771, found: 450.1773.
4.1.4.14. N2-Cyclohexylmethylguanosine (17). 144 mg, 70%,1H
NMR (200 MHz, CDCl3) d 7.82 (s, 1H, H-8),5.68 (d, J = 3.8 Hz, 1H,
H-10), 4.72 (t, J = 4.9 Hz, 1H, H-20), 4.15 (t, J = 5.7 Hz, 1H, H-30),
3.85–3.80 (m, 1H, H-40), 3.61–3.52 (m, 2H, H-50), 3.32–3.28 (m,
2H, N-CH2), 1.82–0.98 (m, 11H, cyclohexyl); 13C NMR (50 MHz,
CDCl3) d 156.1 (C-6), 153.2 (C-2), 150.4 (C-4), 137.9 (C-8), 120.1
(C-5), 87.9 (C-10), 86.6 (C-40), 73.9 (C-20), 71.9 (C-30), 62.3 (C-50),
44.9 (N-CH2), 35.7 (cyclohexyl), 29.9 (cyclohexyl), 25.8 (cyclo-
hexyl), 25.1 (cyclohexyl). m/z: calcd for C17H25N5O5 (M+H)+:
380.1928, found: 380.1925;
4.1.4.8. N2-Butyl-N2-methylguanosine (11). 92 mg, 48%,1H NMR
(200 MHz, CDCl3) d 8.11 (s, 1H, H-8), 5.72 (d, J = 4.9 Hz, 1H, H-10),
4.69 (t, J = 5.3 Hz, 1H, H-20), 4.13 (t, J = 3.9 Hz, 1H, H-30), 3.76–
3.73 (m, 1H, H-40), 3.63–3.61 (m, 2H, N-CH2), 3.58–3.46 (m, 2H,
H-50), 3.09 (s, 3H, N-CH3), 1.60–1.52 (m, 2H, CH2 butyl), 1.38–
1.31 (m, 2H, CH2CH3 butyl), 0.90 (t, J = 7.3 Hz, 3H, CH3 butyl); 13C
NMR (50 MHz, CDCl3), d 158.2 (C-6), 152.3 (C-2), 140.3 (C-4),
136.9 (C-8), 118.5 (C-5), 87.9 (C-10), 85.6 (C-40), 74.3 (C-20), 71.4
(C-30), 62.4 (C-50), 51.2 (N-CH2 butyl), 38.5 (N-CH3), 27.8 (CH2 bu-
tyl), 20.3 (CH2 butyl), 13.7 (CH3 butyl). m/z: calcd for C15H23N5O5
(M+H)+: 354. 1771, found: 354.1768.
4.1.5. General procedure for the synthesis of N2-substitutiuted
50-monophosphates 32–45
4.1.4.9. N2-Butylguanosine (12). 144 mg, 78%, 1H NMR (200 MHz,
CDCl3) d 8.09 (s, 1H, H-8), 5.68 (d, J = 4.8 Hz, 1H, H-10), 4.64–4.59
(m, 1H, H-20), 4.11–4.08 (m, 1H, H-30), 3.76–3.73 (m, 1H, H-40),
3.63 (t, J = 6.2, 2H, N-CH2), 3.58–3.46 (m, 2H, H-50), 1.63–1.54 (m,
2H, CH2 butyl), 1.28–1.25 (m, 2H, CH2CH3 butyl), 0.93 (t,
J = 7.3 Hz, 3H, CH3 butyl); 13C NMR (50 MHz, CDCl3) d 157.6 (C-
6), 154.4 (C-2), 150.3 (C-4), 137.8 (C-8), 117.8 (C-5), 86.8 (C-10),
84.9 (C-40), 74.0 (C-20), 72.3 (C-30), 62.1 (C-50), 42.3 (N-CH2 butyl),
30.7 (CH2 butyl), 20.4 (CH2 butyl), 14.3 (CH3 butyl). m/z: calcd for
Phosphorus oxide trichloride (POCl3) (3.5 equiv) in trimethyl
phosphate (0.05 equiv) was cooled to 0 °C and added to the dried
over P4O10 compound 4–17 (1 equiv). The reaction mixture was
stirred at 0 °C. After 2 h, 1 M aqueous TEAB was added to neutralize
the pH of the reaction mixture. The product was purified by ion-ex-
change chromatography on a DEAE-Sephadex A-25 column using a
linear 0–1 M TEAB gradient to produce products 18-31 as TEA salts.
Methyl iodide (7 equiv) was added to a suspension of mononucle-
otide derivative (18–31) in 1.5 mL anhydrous dimethylsulfoxide
(DMSO) and stirred at room temperature for 2 h. The mixture
was poured into water and extracted three times with diethyl
ether. The aqueous phase was purified on DEAE–Sephadex using
a linear 0–0.8 M gradient of TEAB. Final products were converted
into sodium salts using DOWEX 50WX8.
C
14H21N5O5 (M+H)+: 340. 1615, found: 340.1619.
4.1.4.10. N2-isoButyl-N2-methylguanosine (13). 100 mg, 52%, 1H
NMR (200 MHz, CDCl3) d 7.55 (s, 1H, H-8), 5.64 (d, J = 5.2 Hz, 1H,
H-10), 4.64 (t, J = 5.0 Hz, 1H, H-20), 4.16 (t, J = 3.8 Hz, 1H, H-30),
3.86–3.81 (m, 1H, H-40), 3.74–3.71 (m, 2H, N-CH2), 3.60–3.45 (m,
2H, H-50), 2.97 (s, 3H, N-CH3), 1.89–1.85 (m, 1H, CH isobutyl),
0.94 (d, J = 6.9, 6H, CH3 isobutyl); 13C NMR (50 MHz, CDCl3) d
156.8 (C-6), 152.4 (C-2), 141.9 (C-4), 136.7 (C-8), 119.8 (C-5),
89.3 (C-10), 85.1 (C-40), 74.0 (C-20), 72.3 (C-30), 62.8 (C-50), 52.5
(N-CH2 isobutyl), 38.6 (N-CH3), 25.2 (CH isobutyl), 20.4 (CH3 isobu-
4.1.5.1.
N2-Benzyl-7-methylguanosine-50-monophosphate
(32). 129 mg, 65%, sodium salt; 1H NMR (700 MHz, D2O) d 7.46–
7.32 (m, 5H, Ph), 6.07 (d, J = 3.4 Hz, 1H, H-10), 4.61 (s, 2H, CH2Ph),
4.58 (t, J = 4.7 Hz, 1H, H-20), 4.43 (t, J = 5.7 Hz, 1H, H-30), 4.35–
4.33 (m, 1H, H-40), 4.15–4.11 (m, 1H, H-50), 4.09 (s, 3H, N7-CH3),
4.00–3.97 (m, 1H, H-500); 31P NMR (283 MHz, D2O) 3.234; m/z:
calcd for C18H23N5O8P: 468.1127, found: 468.1131; HPLC (Method
B) tR 9 min.
tyl). m/z: calcd for
C
15H23N5O5 (M+H)+: 354. 1771, found:
354.1777.
4.1.4.11. N2-isoButylguanosine (14). 147 mg, 80%,1H NMR
(200 MHz, CDCl3) d 7.64 (s, 1H, h-8), 5.61 (d, J = 5.5 Hz, 1H, H-10),
4.64–4.61 (m, 1H, H-20), 4.15 (t, J = 3.9 Hz, 1H, H-30), 3.87–3.83
(m, 1H, H-40), 3.61–3.58 (m, 2H, N-CH2), 3.58–3.47 (m, 2H, H-50),
1.90–1.86 (m, 1H, CH isobutyl), 0.92 (d, J = 6.5, 6H, CH3 isobutyl);
13C NMR (50 MHz, CDCl3) d 155.9 (C-6), 151.9 (C-2), 149.2 (C-4),
137.5 (C-8), 119.8 (C-5), 87.7 (C-10), 85.6 (C-40), 74.6 (C-20), 72.8
(C-30), 62.5 (C-50), 50.3 (N-CH2 isobutyl), 28.7 (CH isobutyl), 19.2
(CH3 isobutyl). m/z: calcd for C14H21N5O5 (M+H)+: 340.1615, found:
340.1615.
4.1.5.2. N2-Benzyl-N2,7-dimethylguanosine-50-monophosphate
(33). 110 mg, 68%, sodium salt; 1H NMR (700 MHz, D2O) d 7.45–
7.31 (m, 5H, Ph), 6.11 (d, J = 3.5 Hz, 1H, H-10), 4.68 (s, 2H,CH2Ph),
4.62 (t, J = 4.7 Hz, 1H, H-20), 4.44 (t, J = 5.4 Hz, 1H, H-30), 4.35–
4.32 (m, 1H, H-40), 4.15–4.11 (m, 1H, H-50), 4.10 (s, 3H, N7-CH3),
4.01–3.97 (m, 1H, H-500), 3.17 (s, 3H, N2-CH3); 31P NMR
(283 MHz, D2O) 3.139; m/z: calcd for C19H25N5O8P: 482.1284,
found: 482.1287; HPLC (Method B) tR 13 min.
4.1.5.3.
N2,N2-Dibenzyl-7-dimethylguanosine-50-monophos-
4.1.4.12. N2,N2-Diethylguanosine (15). 101 mg, 55%,1H NMR
(200 MHz, CDCl3) d 7.58 (s, 1H, H-8), 5.65 (d, J = 5.0 Hz, 1H, H-10),
phate (34). 84 mg, 58%, sodium salt; 1H NMR (700 MHz, D2O) d
7.42–7.28 (m, 10H, 2 ꢂ Ph), 6.07 (d, J = 3.6 Hz, 1H, H-10), 4.82 (s,

4788
K. Piecyk et al. / Bioorg. Med. Chem. 20 (2012) 4781–4789
4H, CH2Ph), 4.56 (t, J = 4.9 Hz, 1H, H-20), 4.42 (t, J = 5.6 Hz, 1H, H-
30), 4.31–4.28 (m, 1H, H-40), 4.09 (s, 3H, N7-CH3), 4.08–4.06 (m,
1H, H-50), 3.97–3.93 (m, 1H, H-500); 31P NMR (283 MHz, D2O)
3.085; m/z: calcd for C25H29N5O8P: 558.1597, found: 558.1592;
HPLC (Method B) tR 20.5 min.
H-50), 4.09 (s, 3H, N7-CH3), 4.07–4.03 (m, 1H, H-500), 3.45–3.40
(m, 2H, N-CH2), 3.17 (s, 3H, N2-CH3), 2.15–2.05 (m, 1H, CH isobu-
tyl), 0.92 (d, J = 6.5 Hz, 6H, CH3 isobutyl); 31P NMR (283 MHz,
D2O) 1.646; m/z: calcd for C16H27N5O8P: 448.1440 found:
448.1442; HPLC (Method A) tR 21.5 min.
4.1.5.4. N2-p-Metoxybenzyl-7-methylguanosine-50-monophos-
phate (35). 134 mg, 66%, sodium salt; 1H NMR (700 MHz, D2O) d
7.39 (d, 2H, Ph), 6.95 (d, 2H, Ph), 6.08 (d, J = 3.4 Hz, 1H, H-10),
4.60 (t, J = 4.7 Hz, 1H, H-20), 4.51 (s, 2H, CH2Ph), 4.44 (t, J = 5.7 Hz,
1H, H-30), 4.36–4.34 (m, 1H, H-40), 4.17–4.13 (m, 1H, H-50), 4.07
(s, 3H, N7-CH3), 4.02–3.98 (m, 1H, H-500), 3.79 (s, 3H, OCH3); 31P
NMR (283 MHz, D2O) 3.011; m/z: calcd for C19H25N5O9P:
498.1233, found: 498.1237; HPLC (Method B) tR 12.25 min.
4.1.5.11.
N2-isoButyl-7-methylguanosine-50-monophosphate
(42). 130 mg, 57%, sodium salt; 1H NMR (700 MHz, D2O) d 6.12
(d, J = 3.3 Hz, 1H, H-10), 4.67 (t, J = 4.9 Hz, 1H, H-20), 4.47 (t,
J = 5.6 Hz, 1H, H-30), 4.38–4.36 (m, 1H, H-40), 4.20–4.16 (m, 1H,
H-50), 4.09 (s, 3H, N7-CH3), 4.06–4.02 (m, 1H, H-500), 3.32–3.20
(m, 2H, N-CH2), 1.98–1.89 (m, 1H, CH isobutyl), 0.93 (d,
J = 6.7 Hz, 6H, CH3 isobutyl); 31P NMR (283 MHz, D2O) 2.061; m/
z: calcd for C15H25N5O8P: 434.1284, found: 434.1287; HPLC (Meth-
od A) tR 16 min.
4.1.5.5. N2-p-Chlorobenzyl-7-methylguanosine-50-monophos-
phate (36). 119 mg, 53%, sodium salt; 1H NMR (700 MHz, D2O) d
7.40 (s, 4H, Ph), 5.90 (d, J = 3.6, 1H, H-10), 4.66–4.62 (m, 1H, H-
20), 4.50 (s, 2H, CH2Ph), 4.35–4.32 (m, 1H, H-30), 4.29–4.26 (m,
1H, H-40), 4.09 (s, 3H, N7-CH3), 4.07–3.97 (m, 2H, H-50, H-500); 31P
NMR (283 MHz, D2O) 2.590; m/z: calcd for C18H22N5O8PCl:
502.0894, found: 502.0897; HPLC (Method B) tR 15 min.
4.1.5.12. N2,N2-Diethyl-7-methylguanosine-50-monophosphate
(43). 83 mg, 52%, sodium salt; 1H NMR (700 MHz, D2O) d 6.12 (d,
J = 3.1 Hz, 1H, H-10), 4.70 (t, J = 4.9 Hz, 1H, H-20), 4.46 (t,
J = 5.5 Hz, 1H, H-30), 4.37–4.35 (m, 1H, H-40), 4.22–4.18 (m, 1H,
H-50), 4.08 (s, 3H, N7-CH3), 4.07–4.04 (m, 1H, H-500), 3.60 (q,
J = 6.1 Hz, 4H, CH2 ethyl), 1.22 (t, J = 7.1 Hz, 6H, CH3 ethyl); 31P
NMR (283 MHz, D2O) 1.299; m/z: calcd for C15H25N5O8P:
434.1284, found: 434.1291; HPLC (Method A) tR 14.2 min.
4.1.5.6.
N2-p-Nitrobenzyl-7-methylguanosine-50-monophos-
phate (37). 96 mg, 49%, sodium salt; 1H NMR (700 MHz, D2O) d
8.25 (d, 2H, Ph), 7.62 (d, 2H, Ph), 6.01 (d, J = 3.5 Hz, 1H, H-10),
4.68 (s, 2H, CH2Ph), 4.52–4.50 (m, 1H, H-20), 4.41–4.39 (m, 1H,
H-30), 4.32–4.29 (m, 1H, H-40), 4.10–4.09 (m, 1H, H-50), 4.07 (s,
3H, N7-CH3), 3.98–3.94 (m, 1H, H-50000); 31P NMR (283 MHz, D2O)
3.339; m/z: calcd for C18H22N6O10P: 513.0978, found: 513.0976;
HPLC (Method C) tR 4.5 min.
4.1.5.13. N2-Ethyl-N2,7-dimethylguanosine-50-monophosphate
(44). 109 mg, 70%, sodium salt; 1H NMR (700 MHz, D2O) d 6.12
(d, J = 3.4 Hz, 1H, H-10), 4.70 (t, J = 4.8 Hz, 1H, H-20), 4.46 (t,
J = 5.8 Hz, 1H, H-30), 4.38–4.35 (m, 1H, H-40), 4.23–4.19 (m, 1H,
H-50), 4.08 (s, 3H, N7-CH3), 4.07–4.05 (m, 1H, H-500), 3.64 (dd,
J = 14.3, 7.1 Hz, 2H), 3.15 (s, 3H), 1.20 (t, J = 7.1 Hz, 3H); 31P NMR
(283 MHz, D2O) 0.866; m/z: calcd for C14H23N5O8P: 420.1127,
found: 420.1128; HPLC (Method A) tR 13.9 min.
4.1.5.7. N2-p-Phenylbenzyl-7-methylguanosine-50-monophos-
phate (38). 114 mg, 60%, sodium salt; 1H NMR (700 MHz, D2O) d
7.84–7.41 (m, 9H, Ph–Ph), 6.09 (d, J = 3.5 Hz, 1H, H-10), 4.69 (s,
2H, CH2Ph), 4.64 (t, J = 4.7 Hz, 1H, H-20), 4.46–4.44 (m, 1H, H-30),
4.39–4.32 (m, 1H, H-40), 4.19–4.16 (m, 1H, H-50), 4.08 (s, 3H, N7-
CH3), 4.05–4.01 (m, 1H, H-500); 31P NMR (283 MHz, D2O) 3.121;
m/z: calcd for C24H25N5O8P: 542.1440, found: 542.1443; HPLC
(Method C) tR 15.4 min.
4.1.5.14.
N2-Cyclohexylmethyl-7-methylguanosine-50-mono-
phosphate (45). 122 mg, 56%, sodium salt; 1H NMR (700 MHz,
D2O) d 6.12 (d, J = 3.6 Hz, 1H, H-10), 4.66 (t, J = 4.8 Hz, 1H, H-20),
4.47 (t, J = 5.7 Hz, 1H, H-30), 4.38–4.35 (m, 1H, H-40), 4.16–4.12
(m, 1H, H-50), 4.10 (s, 3H, N7-CH3), 4.02–3.98 (m, 1H, H-500), 3.30–
3.27 (m, 2H, N-CH2), 1.79–0.95 (m, 11H, cyclohexyl); 31P NMR
(283 MHz, D2O) 3.218; m/z: calcd for C18H29N5O8P: 474.1597,
found: 474.1592; HPLC (Method A) tR 15.6 min.
4.1.5.8.
N2-Butyl-N2,7-dimethylguanosine-50-monophosphate
(39). 78 mg, 55%, sodium salt; 1H NMR (700 MHz, D2O) d 6.11
(d, J = 3.2 Hz, 1H, H-10), 4.69 (t, J = 4.7 Hz, 1H, H-20), 4.46 (t,
J = 5.9 Hz, 1H, H-30), 4.38–4.35 (m, 1H, H-40), 4.22–4.18 (m, 1H,
H-50), 4.09 (s, 3H, N7-CH3), 4.06–4.03 (m, 1H, H-500), 3.68–3.60
(m, 2H, CH2 butyl), 3.15 (s, 3H, N2-CH3), 1.66–1.60 (m, 2H, CH2
butyl), 1.37–1.28 (m, 2H, CH2CH3 butyl), 0.92 (t, J = 7.4 Hz, 3H,
CH3 butyl). 31P NMR (283 MHz, D2O)1.548; m/z: calcd for
C16H27N5O8P: 448.1440, found: 448.1445; HPLC (Method A) tR
18.7 min.
Acknowledgments
This work was partially supported by Grants N N301 096339
from the Ministry of Science and Higher Education, 02/EuroNano-
Med/2011 from the National Centre for Research and Develop-
ment, Poland and National Institutes of Health Grants R0149558
and AI080805 (to R.E.D.). We gratefully acknowledge Professor
Edward Darzynkiewicz from the Department of Biophysics, Insti-
tute of Experimental Physics, Warsaw University for all kind of
support. We also thank members of the Davis lab for their help
with biological experiments.
4.1.5.9.
N2-Butyl-7-methylguanosine-50-monophosphate
(40). 134 mg, 60%, sodium salt; 1H NMR (700 MHz, D2O) d 6.11
(d, J = 3.0 Hz, 1H, 1H, H-10), 4.69 (t, J = 4.8 Hz, 1H, H-20), 4.46 (t,
J = 5.8 Hz, 1H, 1H, H-30), 4.40–4.36 (m, 1H, H-40), 4.24–4.20 (m,
1H, H-50), 4.08 (s, 3H, N7-CH3), 4.07–4.05 (m, 1H, H-500), 3.45 –
3.40 (m, 2H, CH2 butyl), 1.64–1.55 (m, 2H, CH2 butyl), 1.42–1.32
(m, 2H, CH2CH3 butyl), 0.91 (t, J = 7.4 Hz, 3H, CH3 butyl); 31P
NMR (283 MHz, D2O) 0.770; m/z: calcd for C15H25N5O8P:
434.1284, found: 434.1285; HPLC (Method A) tR 13.2 min.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.05.078.
References and notes
N2-isoButyl-N2,7-dimethylguanosine-50-monophos-
1. Andersen, N. M.; Douthwaite, S. J. Mol. Biol. 2006, 359, 777.
2. (a) Edqvist, J.; Straby, K. B.; Grosjean, H. Biochimie 1995, 77, 54; (b)Modification
and editing of RNA; Grosjean, H., Benne, R., Eds.; ASM Press: Washington, DC,
1998.
4.1.5.10.
phate (41). 78 mg, 51%, sodium salt; 1H NMR (700 MHz, D2O) d
6.11 (d, J = 3.2 Hz, 1H, H-10), 4.67 (t, J = 4.8 Hz, 1H, H-20), 4.46 (t,
J = 5.9 Hz, 1H, H-30), 4.37–4.35 (m, 1H, H-40), 4.22–4.18 (m, 1H,
3. Furuichi, Y.; Shatkin, A. J. Adv. Virus Res. 2000, 55, 135.

K. Piecyk et al. / Bioorg. Med. Chem. 20 (2012) 4781–4789
4789
4. (a) Thomas, J. D.; Conrad, R. C.; Blumenthal, T. Cell 1988, 54, 533; (b) Liou, R. F.;
Blumenthal, T. Mol. Cell. Biol. 1990, 10, 1764.
5. (a) Sharp, P. A. Cell 1994, 77, 805; (b) Lewis, J. D.; Izaurralde, E. Eur. J. Biochem.
1997, 247, 461; (c) Rhoads, R. E. Prog. Mol. Subcell. Biol. 1985, 9, 104; (d) Gingras,
A. C.; Raught, B.; Sonenberg, N. Annu. Rev. Biochem. 1999, 68, 913.
6. (a) Sonenberg, N.; Hinnebusch, A. Cell 2009, 136, 731; (b) Von Der Haar, T.;
Gross, J.; Wagner, G.; McCarthy, J. Nat. Struct. Mol. Biol. 2004, 11, 503; (c)
Izaurralde, E.; Lewis, J.; McGuigan, C.; Jankowska, M.; Darzynkiewicz, E.;
Majtaj, I. W. Cell 1994, 78, 657; (d) Calero, G.; Wilson, K. F.; Ly, T.; Rios-Steiner,
J. L.; Clardy, J. C.; Cenione, R. A. Nat. Struct. Biol. 2002, 9, 912; (e) Huber, J.;
Cronshagen, U.; Kadokura, M.; Marshallsay, C.; Wada, T.; Sekine, M.; Luhrmann,
R. EMBO J. 1998, 17, 4114; (f) Strasser, A.; Dickmanns, A.; Luhrmann, R.; Ficner,
R. EMBO J. 2005, 24, 2235; (g) Liu, H.; Rodgers, N. D.; Jiao, X.; Kiledjian, M. EMBO
J. 2002, 21, 4699; (h) van Dijk, E.; Cougot, N.; Meyer, S.; Babajko, S.; Wahle, E.;
Seraphin, B. EMBO J. 2002, 21, 6915; (i) Wang, Z.; Jiao, X.; Carr-Schmid, A.;
Kiledjian, M. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 12663.
Biochem. Parasitol. 2007, 153, 95; (e) Liu, W.; Zhao, R.; McFarland, C.; Kieft, J.;
Niedzwiecka, A.; Jankowska-Anyszka, M.; Stepinski, J.; Darzynkiewicz, E.;
Jones, D. N. M.; Davis, R. E. J. Biol. Chem. 2009, 284, 31333; (f) Wallace, A.; Filbin,
M.; Veo, B.; McFarland, C.; Jankowska-Anyszka, M.; Stepinski, J.;
Darzynkiewicz, E.; Davis, R. E. Mol. Cell. Biol. 2010, 30, 1958; (g)
Ruszczynska-Bartnik, K.; Maciejczyk, M.; Stolarski, R. J. Mol. Model. 2011, 17,
727; (h) Lasda, E. L.; Blumenthal, T. Wiley Interdiscip. Rev. RNA 2011, 2, 417; (i)
Allen, M. A.; Hillier, L. W.; Waterston, R. H.; Blumenthal, T. Genome Res. 2011,
21, 255; (j) Zaslaver, A.; Baugh, L. R.; Sternberg, P. W. Cell 2011, 145, 981.
11. Liu, W.; Jankowska-Anyszka, M.; Piecyk, K.; Dickson, L.; Wallace, A.;
Niedzwiecka, A.; Stepinski, J.; Stolarski, R.; Darzynkiewicz, E.; Kieft, J.; Zhao,
R.; Jones, D. N. M.; Davis, R. E. Nucleic Acids Res. 2011, 20, 8820.
12. Aviñó, A.; Mayordomo, A.; Espuny, R.; Bach, M.; Eritja, R. Nucleosides
Nucleotides 1995, 7, 1613.
13. Nair, V.; Turner, G. A.; Chamberlain, S. D. J. Am. Chem. Soc. 1987, 109, 7223.
14. Himmelsbach, F.; Schultz, S.; Trichtinger, T.; Charubala, R.; Pfleiderer, W.
Tetrahedron 1984, 40, 59.
15. (a) Harned, A. M.; SongHe, H.; Toy, P. H.; Flynn, D. L.; Hanson, P. R. J. Am. Chem.
Soc. 2005, 127, 52; (b) Lan, P.; Porco, J. A.; South, M. S.; Parlow, J. J. J. Comb.
Chem. 2003, 5, 660; (c) Fleckenstein, C. A.; Plenio, H. Adv. Synth. Catal. 2006,
348, 1058; (d) Dandapani, S.; Curran, D. P. Tetrahedron 2002, 58, 3855.
16. Liu, J.; Robins, M. J. J. Am. Chem. Soc. 2007, 129, 5962.
17. Acedo, M.; Fabrega, C.; Aviñó, A.; Fagan, P.; Wammer, D.; Eritja, R. Nucleic Acids
Res. 1994, 22, 2982.
18. (a) Woo, J.; Sigurdsson, T.; Hopkins, P. B. J. Am. Chem. Soc. 1993, 115, 3407; (b)
Allerson, C. R.; Chen, S. L.; Verdine, G. L. J. Am. Chem. Soc. 1997, 119, 7423.
19. Olah, A. G.; Li, X. Synlett 1990, 267.
20. Yoshikawa, M.; Kato, T.; Takenishi, T. Tetrahedron Lett. 1967, 50, 5065.
21. (a) Adams, B. L.; Morgan, M.; Shatkin, A. J. J. Biol. Chem. 1978, 253, 2589; (b)
Darzynkiewicz, E.; Dekiel, I.; Lassota, P.; Tahara, S. M. Biochemistry 1987, 26.
4372.
22. Cohen, L. S.; Mikhli, C.; Friedman, C.; Jankowska-Anyszka, M.; Stepinski, J.;
Darzynkiewicz, E.; Davis, R. E. RNA 2004, 10, 1609.
7. (a) McKendrick, L.; Pain, V. M.; Morley, S. J.; Int, J. Biochem. Cell Biol. 1999, 31,
31; (b) Sonenberg, N.; Gingras, A. C. Curr. Opin. Cell Biol. 1998, 10, 268; (c) von
der Haar, T.; Gross, J. D.; Wagner, G.; McCarthy, J. E. G. Nat. Struct. Mol. Biol.
2004, 11, 503.
8. (a) Clemens, M. J.; Bommer, U. A. Int. J. Biochem. Cell Biol. 1999, 31, 1; (b) De
Benedetti, A.; Graff, J. R. Oncogene 2004, 23, 3189; (c) Mamane, Y.; Petroulakis,
E.; Rong, L.; Yoshida, K.; Ler, L. W.; Sonenberg, N. Oncogene 2004, 23, 3172.
9. (a) Cai, A.; Jankowska-Anyszka, M.; Centers, A.; Chlebicka, L.; Stepinski, J.;
Stolarski, R.; Darzynkiewicz, E.; Rhoads, R. E. Biochemistry 1999, 38, 8538; (b)
Ghosh, P.; Park, C.; Peterson, M. S.; Bitterman, P. B.; Polunovsky, V. A.; Wagner,
C. R. Bioorg. Med. Chem. Lett. 2005, 15, 2177; (c) Jia, Y.; Chiu, T.-L.; Amin, E. A.;
Polunovsky, V.; Bitterman, P. B.; Wagner, C. R. Eur. J. Med. Chem. 2010, 45, 1304;
(d) Jemielity, J.; Kowalska, J.; Rydzik, A. M.; Darzynkiewicz, E. New J. Chem.
2010, 34, 829.
10. (a) Jankowska-Anyszka, M.; Lamphear, B. J.; Aamondt, E. J.; Harrington, T.;
Darzynkiewicz, E.; Stolarski, R.; Rhoads, R. E. J. Biol. Chem. 1998, 273, 10538; (b)
Keiper, B. D.; Lamphear, B. J.; Deshpande, A. M.; Jankowska-Anyszka, M.;
Aamodt, E. J.; Blumenthal, T.; Rhoads, R. E. J. Biol. Chem. 2000, 275, 10590; (c)
Lall, S.; Friedman, C. C.; Jankowska-Anyszka, M.; Stepinski, J.; Darzynkiewicz,
E.; Davis, R. E. J. Biol. Chem. 2004, 279, 45573; (d) Cheng, G.; Cohen, L.; Mikhli,
C.; Jankowska-Anyszka, M.; Stepinski, J.; Darzynkiewicz, E.; Davis, R. E. Mol.
23. Lax, S.; Fritz, W.; Browning, K.; Ravel, J. Proc. Natl. Acad. Sci. U.S.A. 1985, 82, 330.
24. Volpon, L.; Osborne, M. J.; Topisirovic, I.; Siddiqui, N.; Borden, K. L. EMBO J.
2006, 25, 5138.