Synthesis of 13C- and 14C-labeled dinucleotide mRNA cap analogues for structural and biochemical studies

Article abstract of DOI:10.1016/j.bmcl.2012.04.120

Herein we describe the first simple and short method for specific labeling of mono- and trimethylated dinucleotide mRNA cap analogues with ¹³C and ¹⁴C isotopes. The labels were introduced within the cap structures either at the N7 for monomethylguanosine cap or N7 and N2 position for trimethylguanosine cap. The compounds designed for structural and biochemical studies will be useful tools for better understanding the role of the mRNA cap structures in pre-mRNA splicing, nucleocytoplasmic transport, translation initiation and mRNA degradation.

Full text of DOI:10.1016/j.bmcl.2012.04.120

Bioorganic & Medicinal Chemistry Letters 22 (2012) 4391–4395
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of 13C- and 14C-labeled dinucleotide mRNA cap analogues
for structural and biochemical studies
Karolina Piecyk a, Richard E. Davis b, Marzena Jankowska-Anyszka a,
⇑
a Faculty of Chemistry, University of Warsaw, Pasteura 1, 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
Article history:
Herein we describe the first simple and short method for specific labeling of mono- and trimethylated
dinucleotide mRNA cap analogues with 13C and 14C isotopes. The labels were introduced within the
cap structures either at the N7 for monomethylguanosine cap or N7 and N2 position for trimethylguano-
sine cap. The compounds designed for structural and biochemical studies will be useful tools for better
understanding the role of the mRNA cap structures in pre-mRNA splicing, nucleocytoplasmic transport,
translation initiation and mRNA degradation.
Received 4 April 2012
Revised 26 April 2012
Accepted 29 April 2012
Available online 4 May 2012
Keywords:
Cap analogue
TMG cap
13C label
14C label
mRNA
Ó 2012 Elsevier Ltd. All rights reserved.
Gene expression in eukaryotes is a complex process that in-
volves several steps and requires numerous protein factors. One
common RNA element involved in mRNA splicing, export, transla-
tion, and stability is the unusual structure at the 50 end of mRNA
known as a ‘cap’.1 This unique structure consist of 7-methylguano-
sine linked via a 50,50-triphosphate bridge to the first transcribed
nucleotide (MMG cap). For many years, numerous studies have
been conducted to describe the molecular mechanism of the inter-
action of the cap with eukaryotic initiation factor 4E (eIF4E) that is
a key step in translation initiation. Structural requirements for the
cap–eIF4E interaction were elucidated by biophysical and bio-
chemical methods involving several chemically prepared cap ana-
logues and subsequently confirmed and further defined by
multidimensional nuclear magnetic resonance and X-ray crystal-
lography.2 In some cases, for example in nematodes such as Caeno-
rhabditis elegans and Ascaris suum, in addition to regular MMG cap,
an atypical hypermethylated form of the cap, with two additional
methyl groups at the N2 position of 7-methylguanosine, is present
(TMG cap).3 This cap is added to the 50 end of a large percentage of
mRNAs by RNA trans-splicing of a short 22-nt spliced leader se-
quence (SL).4 In general, eIF4E proteins from higher eukaryotes
are unable to effectively recognize and bind the TMG cap.2d,5 How-
ever, nematodes efficiently translate TMG-capped mRNAs.3b,6
Identification and exploration of five eIF4E isoforms in C. elegans
provided evidence that the 4E isoforms vary in their ability to dis-
tinguish between MMG and TMG caps suggesting that eIF4E of
these organisms may differ from that of other species.7 Apart from
three C. elegans isoforms (IFE-1, -2, -5) which are able to bind MMG
and TMG caps, other eIF4E orthologs recognizing the two mRNA
caps have been described, for example Ascaris suum eIF4E-36a
and the sole Schistosoma mansoni eIF4E isoform.8 Biochemical, bio-
physical, theoretical, and structural data are available for different
4E proteins (C. elegans,9 A. suum,6a,10 S. mansoni,8 murine11). Based
on these data, models for binding the two caps have been pro-
posed.6a,8–11 Recently the first crystal structure of Ascaris eIF4E-3
in complex with MMG and TMG cap has been published.10 While
the data indicate how Ascaris eIFE-3 interacts with the MMG and
TMG cap, the exact coherent molecular mechanism describing
the interaction of other isoforms binding either cap is not com-
pletely understood.
Herein we describe the synthesis of 13C and 14C labeled mono-
and trimethylated cap analogues as tools that will facilitate analy-
sis of the specificity of nematode eIF4E isoforms for the MMG and
TMG caps and help to examine proteins interactions with the two
caps in a more detailed way.
A well-defined synthetic pathway to prepare dinucleotide cap
analogues involves obtaining imidazolide derivative of one nucleo-
tide in a reaction with 2,20-ditiopyridine and triphenylphosphine
prior to coupling with a second nucleotide in the presence of an ex-
cess of double-charged ions (such as Zn2+ or Mn2+) in anhydrous or
aqueous condition.12 Nevertheless, for the synthesis of 13C/14
C
MMG and TMG caps, the procedure needed to be modified to min-
imize the number of steps with the radioactive materials and due
⇑
Corresponding author. Tel.: +48 22 822 0211; fax: +48 22 822 5996.
E-mail address: marzena@chem.uw.edu.pl (M. Jankowska-Anyszka).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.04.120

4392
K. Piecyk et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4391–4395
to the cost of isotopically labeled substrates (13C or 14C methyl io-
dide). Our method to synthesize the MMG dinucleotide cap ana-
logues labeled with either 13C or 14C was accomplished
(Scheme 1) by direct methylation of the dinucleotide GpppG that
was prepared by coupling of guanosine 50-diphosphate (GDP) with
an imidazolide derivative of guanosine 50-monophosphate
(imGMP).13 As GpppG is symmetric and can be methylated on both
guanine residues, the procedure was optimized and evaluated
using unlabeled methyl iodide and numerous reaction conditions
(variation of reaction time, excess of methyl iodide, temperature)
and HPLC (data not shown) to define conditions that prevent over
methylation. The best results were obtained with twofold molar
excess of 13CH3I (Sigma–Aldrich) or 14CH3I (ViTrax) at room tem-
perature.14 The synthesis of 13C labeled MMG cap has been de-
scribed previously15 but it was achieved by a longer two-step
procedure.
added to the reaction mixture to remove the NPE group followed
by the addition of a mixture of 0.5 M NaOH in THF/MeOH/NaOHaq
in order to complete acetyl deprotection.16 The nucleoside (4) was
purified on silica gel and then phosphorylated using the Yoshikawa
method with phosphorus oxide trichloride in trimethyl phosphate
(5) and then methylated with fivefold excess of 13CH3I in anhy-
13
drous DMSO (6). The C trimethylated guanosine 50- monophos-
phate was further coupled with the imidazolide derivative of
GDP (7) under anhydrous conditions (DMF) in the presence of zinc
ions.15,17 This route was chosen as it is routinely used by us for the
preparation of unlabeled TMG cap analogues.
We chose to introduce the carbon-14 labeled methyl group into
the N7 position of guanine as this reaction is relatively simple and
with only one labeled methyl group in the cap moiety, leads to a
relatively high specific activity of the cap. 14C TMG cap was ob-
tained as described for the preparation of 13C labeled TMG cap ex-
cept that the substitution step leading to N2,N2-dimethylguanosine
was carried out with unlabeled dimethylamine. All four cap ana-
logues were purified by ion-exchange chromatography on DEAE-
Sephadex A-25 (HCOꢀ3 form), the relevant fractions collected,
pooled, and evaporated repeatedly with cold ethanol. The fractions
with both high UV absorbance and high specific activity were com-
bined and the final specific activity of 14C labeled cap analogues
was determined after lyopilization: 0.82 and 1.44 GBq/mmol for
MMG and TMG cap, respectively. MMG and TMG cap analogues la-
beled with 14C and 13C isotopes were converted into sodium salts
using Dowex 50 WX8 ion-exchange resin. The structure and homo-
geneity of final products were confirmed by HPLC, mass spectrom-
etry, 1H NMR, 31P NMR and 13C NMR. 1H NMR spectra of 13C-
labeled MMG and TMG caps revealed no signals corresponding to
unlabeled methyl group (Fig. 1). All methyl groups in the spectra
were 13CH3 observed as a doublet with the expected large coupling
constant. 13C labeling of 1 and 8 was also confirmed by a single-fre-
quency proton decoupled 13C NMR spectra. Extremely strong sig-
nals from one (1) or three (8) methyl carbons with appropriate
coupling pattern were also observed.
In contrast to MMG cap, a TMG cap labeled with 13C and 14C iso-
topes required a multistep procedure (Scheme 2). To our knowl-
edge this is the first report of such a synthesis. The first step
required transformation of guanosine to the corresponding
20,30,50-O-acetylguanosine using acetic anhydride (Ac2O) in the
presence of triethylamine (TEA) and N,N-(dimethylamino) pyridine
(DMAP). The obtained derivative was further protected at the O6
position of guanosine using the Mitsunobu reaction with p-
nitrophenylethanol (NPE) under anhydrous conditions. The prod-
uct 3 was then converted to its 2-fluoro derivative 3 by performing
non-aqueous diazotization and fluoridation at low temperature
with t-butyl nitrite as the diazotizing agent and HF in pyridine as
the fluoride source. The purified 2-fluoro nucleoside was then trea-
ted with a fourfold molar excess of (13CH3)2NH. As the dimethyl-
amine labeled with 13C carbon can only be obtained as
a
hydrochloride, a nucleophilic substitution was performed in aque-
ous solution of DMSO/AcCN/H2O/Et3N in a temperature-controlled
oil bath at 60 °C until the fluoronucleoside had completely disap-
peared, as evaluated by TLC analysis. After completion of the sub-
stitution reaction, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) was
Scheme 1. Synthesis of 13C and 14C MMG cap analogues. Reagents: (i) 13CH3I or 14CH3I, DMSO.

K. Piecyk et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4391–4395
4393
Scheme 2. Synthesis of 13C and 14C TMG cap analogues. Reagents and conditions: (i) (a) acetic anhydride, DMAP, Et3N, AcCN, 4 °C to rt, (b) NPE, PPh3, DIAD, toluene, rt, (c) HF/
pyridine, tBuONO, pyridine, ꢀ40 °C; (ii) (a) (13CH3)2NH, DMSO/AcCN/H2O/Et3N, 60 °C or (CH3)2NH, DMSO, 60 °C, (b) THF/MeOH/NaOHaq; (iii) POCl3, trimethylphosphate, 4 °C;
(iv) 13CH3I or 14CH3I, DMSO, rt (v) ZnCl2, DMF.
Figure 1. Portions of the 1H NMR spectra of the synthesized 13C-labeled MMG (A) and TMG (B) caps illustrating the characteristic 13CH3 signals. (A) N7 13C methyl group of
the MMG cap, J1H–13C = 145 Hz (B) N7 13C methyl group, J1H–13C = 145 Hz and two N2 13C methyl groups of the TMG cap, J1H–13C = 140 Hz.

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K. Piecyk et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4391–4395
McFarland, C.; Stepinski, J.; Jankowska-Anyszka, M.; Darzynkiewicz, E.; Davis,
R. E. Mol. Cell. Biol. 2010, 30, 1958.
6. (a) Lall, S.; Friedman, C. C.; Jankowska-Anyszka, M.; Stepinski, J.;
Darzynkiewicz, E.; Davis, R. E. J. Biol. Chem. 2004, 279, 45573; (b) Cheng, G.;
Cohen, L.; Mikhli, C.; Jankowska-Anyszka, M.; Stepinski, J.; Darzynkiewicz, E.;
Davis, R. E. Mol. Biochem. Parasitol. 2007, 153, 95.
7. (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.
8. 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.
9. Miyoshi, H.; Dwyer, D. S.; Keiper, B. D.; Jankowska-Anyszka, M.; Darzynkiewicz,
E.; Rhoads, R. E. EMBO J. 2002, 21, 4680.
10. 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, 39, 8820.
11. Rutkowska-Wlodarczyk, I.; Stepinski, J.; Dadlez, M.; Darzynkiewicz, E.;
Stolarski, R.; Niedzwiecka, A. Biochemistry 2008, 47, 2710.
12. Burgess, K.; Cook, D. Chem. Rev. 2000, 100, 2047.
Figure 2. DcpS hydrolysis of labeled cap analogues. DcpS reactions were carried out
as previously described and the labeled substrates and products were separated by
PEI-cellulose thin layer chromatography (TLC), plates developed in 0.45 M ammo-
nium sulfate, and labeled substrate and products detected by autoradiography.18
The plates correspond to (A) 14C-labeled MMG cap, (B) 14C-labeled TMG cap; lane
13. (a) Fukuoka, K.; Suda, F.; Suzuki, R.; Takaku, H.; Ishikawa, M.; Hata, T.
Nucleosides Nucleotides 1994, 13, 1557; (b) Stepinski, J.; Bretner, M.; Jankowska,
M.; Felczak, K.; Stolarski, R.; Wieczorek, Z.; Cai, A. L.; Rhoads, R. E.; Temeriusz,
A.; Haber, D.; Darzynkiewicz, E. Nucleosides Nucleotides 1995, 14, 717.
14. General procedure for a synthesis of m7⁄GpppG cap analogues labeled with 13C or
14C. GpppG was prepared by coupling of imidazolide derivative of guanosine
50-monophosphate (GMP) (21 mg, 0.05 mmol) with guanosine 50-diphosphate
(27 mg, 0.05 mmol) in anhydrous conditions in the presence of ZnCl2 (50 mg,
0.37 mmol) as previously described.13 GpppG (20 mg, 0.02 mmol) was further
methylated with appropriate methyl iodide, labeled with 13C or 14C
(0.04 mmol) in anhydrous DMSO at room temperature for 2 h. The reaction
mixture was poured into water and extracted three times with diethyl ether.
Aqueous phase was purified on DEAE–Sephadex using a 0–0.8 M gradient of
TEAB. Final products were obtained as colorless crystals ([13C] m7⁄GpppG, (1)
8.4 mg, 0.01 mmol, 52%, [14C] m7⁄GpppG, (2) 7.8 mg, 0.0096 mmol, 48%); [13C]
m7⁄GpppG MS-ESI: m/z calcd. 804.1024, Found: 804.1183; 1H NMR (D2O,
500 MHz) d 8.03 (s, 1H; H-8, G), 5.91 (d,1H; H-10, m7G), 5.82 (d, 1H; H-10, G),
4.67 (t, 1H, H-20, G), 4.51–4.48 (m, 2H; H-20, m7G; H-30, G), 4.43–4.35 (m, 4H;
H-30, m7G, H-40, G, H-40, m7G, H-50, G), 4.32–4.26 (m, 3H; H-50, H-500, m7G; H-
500, G), 4.06 (d, 3H; 7-CH3, J1H,13C = 145 Hz); 13C NMR (D2O, 125 MHz, H1
1—14
C C cap analogue treated with C. elegans DcpS
cap analogue, lane 2—14
(expressed and purified as described18), lane 3—14C cap analogue treated with A.
suum DcpS (GenBank Accession number (ADB92583), expressed and purified as
described18). ⁄indicates the position of the label.
To evaluate the 14C cap analogues in a functional assay, we car-
ried out decapping experiments (Fig. 2) using A. suum and C. ele-
gans DcpS, the scavenger decapping enzymes. DcpS cleaves
mono- as well as trimethylated cap regioselectively between b
and
c
phosphates of the 50,50-triphosphate bridge to release
m7GMP or m32,2,7GMP and a downstream oligonucleotide. This
simple experiment indicated that incubation of nematode DcpS
with the labeled m7⁄GpppG or m32,2,7⁄GpppG led to labeled
m7⁄Gp and m32,2,7⁄Gp cap-derived products, respectively, as illus-
trated by TLC and autoradiography (Fig. 2).
decouple 500 MHz) d 36.24; 31P NMR (D2O, 202 MHz) d ꢀ10.31 (1P,
a), ꢀ10.40
(1P
c), ꢀ21.91 (1P, b).
15. Jemielity, J.; Stolarski, R.; Darzynkiewicz, E. Nucloesides, Nucleotides, Nucleic
Acids 2007, 26, 1315.
16. N2-fluoro-20,30,50-O-triacetyl-O6-[2-(4-nitrophenyl)ethyl]inosine
(3)
The
In conclusion we have developed a simple and short method for
20,30,50-tri-O-acetylguanosine was prepared from guanosine using acetic
anhydride in the presence of triethylamine and N,N-(dimethylamino)
pyridine using a procedure modified from Nair et al. (J. Am. Chem. Soc., 1987,
109, 7223). In the next step 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-
nitrophenyl)ethanol (1.45 g, 8.7 mmol) in anhydrous toluene was stirred for
30 min and diisopropylazodicarboxylate (1.4 mL) was added dropwise over a
period of 45 min. The reaction mixture was kept for 12 h at rt. The solvent was
evaporated and the residual oil was purified by column chromatography on
silica gel with chloroform to produce a pure product, 20,30,50-tri-O-acetyl-O6-
[2-(4-nitrophenyl)ethyl]guanosine, of yellowish crystals, 2.26 g (70%).1H NMR
(200 MHz, CDCl3) d 8.17 (d, 2H), 7.72 (s, 1H), 7.49 (d, 2H), 6.05–5.91 (m, 2H),
5.87–5.75 (m, 1H), 4.73 (t, J = 6.7 Hz, 2H), 4.50–4.36 (m, 3H), 3.28 (t, 6.7 2H),
2.14 (s, 3H), 2.09 (s, 3H), 2.08 (s, 3H); m/z: calcd for C24H26N6O10(M+H)+:
559.1783, found: 559.1784.
specific labeling of mono- and trimethylated caps with 13C and 14
C
isotopes. The compounds will be extremely useful as tools for NMR
studies (13C), for monitoring chemical and enzymatic reactions
(
14C), or to synthesize RNA containing the 14C labeled cap struc-
tures by in vitro transcription.
Acknowledgment
This work was partially supported by a Grant N N301 096339
from the Ministry of Science and Higher Education, Poland and
Grants R0149558 and AI080805 from National Institutes of Health,
USA. (to R.E.D.). We thank Dr Weizhi Liu for providing DcpS en-
zymes and members of the Davis lab for their help with the DcpS
assays. We are also grateful to Prof. Darzynkiewicz for all kind of
support.
In the next step dry 20,30,50-tri-O-acetyl-O6-[2-(4-nitrophenyl)ethyl]guanosine
(1 g, 1.79 mmol) in polypropylene tube under nitrogen was dissolved 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 1 h, the reaction was quenched at 0 °C by slowly pouring 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 chromatography using as eluate 60:1 CH2Cl2:MeOH
References and notes
1. (a) Furuichi, Y.; Shatkin, A. J. Adv. Virus Res. 2000, 55, 135; (b) Topisirovic, I.;
Svitkin, Y. V.; Sonenberg, N.; Shatkin, A. J. WIREs RNA 2011, 2, 277.
gave 0.85 g (85%) of product (3); TLC silica gel, CH2Cl2/MeOH, 60:1 RF = 0.4; 1
H
2. (a) Matsuo, H.; Li, H.; McGuire, A. M.; Fletcher, C. M.; Gingras, A.-C.; Sonenberg,
N.; Wagner, G. Nat. Struct. Mol. Biol. 1997, 4, 717; (b) Marcotrigiano, J.; Gingras,
A.-C.; Sonenberg, N.; Burley, S. K. Cell 1997, 89, 951; (c) Cai, A.; Jankowska-
Anyszka, M.; Centers, A.; Chlebicka, L.; Stepinski, J.; Stolarski, R.;
Darzynkiewicz, E.; Rhoads, R. E. Biochemistry 1999, 38, 8538; (d)
Niedzwiecka, A.; Marcotrigiano, J.; Stepinski, J.; Jankowska-Anyszka, M.;
Wyslouch-Cieszynska, A.; Dadlez, M.; Gingras, A.-C.; Mak, P.; Darzynkiewicz,
E.; Sonenberg, N.; Burley, S.; Stolarski, R. J. Mol. Biol. 2002, 319, 615.
3. (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.
4. (a) Van Doren, K.; Hirsh, D. Mol. Cell. Biol. 1990, 10, 1769; (b) Maroney, P. A.;
Hannon, G. J.; Nilsen, T. W. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 709.
NMR (700 MHz, CDCl3) d 8.21–8.15 (m, 2H), 8.08 (s, 1H), 7.50 (d, 2H), 6.13 (d,
J = 5.6 Hz, 1H), 5.82 (t, J = 4.9 Hz, 1H), 5.60–5.55 (m, 1H), 4.84 (t, J = 6.7 Hz, 2H),
4.46–4.42 (m, 2H), 4.38–4.36 (m, 1H), 3.32 (t, J = 6.7 Hz, 2H), 2.15 (s, 6H), 2.08
(s, 3H).m/z: calcd for C24H24FN5O10 (M+H)+: 562.1579, found: 562.1581.
Synthesis of 13C labeled m32,2,7⁄GpppG (8) N2-fluoro-20,30,50-O-triacetyl-O6-[2-(4-
nitrophenyl)ethyl]inosine (3, 250 mg, 0.45 mmol) was dissolved in 2 mL of
aqueous solution of DMSO/AcCN/H2O/Et3N and
(
13CH3)2NH (1.8 mmol)
was added. The reaction mixture was stirred at 60 °C for 4 h until the
fluoronucleoside completely disappeared. After completion of the substitution
reaction, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) was added to the reaction
mixture followed by the addition of 2.75 mL a mixture of 0.5 M NaOH in
THF/MeOH/NaOHaq (5:4:2). The solvent was evaporated and the residual oil
was purified by column chromatography on silica gel with CH2Cl2/MeOH (6:1)
5. (a) Darzynkiewicz, E.; Stepinski, J.; Ekiel, I.; Haber, D.; Sijuwade, T.; Tahara, S.
M. Nucleic Acids Res. 1988, 16, 8953; (b) Wallace, A.; Filbin, M. E.; Veo, B.;

K. Piecyk et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4391–4395
4395
to produce
(75 mg, 0.24 mmol) was in the next step phosphorylated (Tetrahedron Lett.
1967, 8, 5065) with phosphorus trichloride oxide (100 L) in trimethyl
a
pure product (4) (100 mg, 56%). N2⁄,N2⁄-dimethylguanosine
chromatography on DEAE–Sephadex. Pure product (8) was obtained as plain
17
crystals (29 mg, 0.035 mmol, 70%)
MS-ESI: m/z: calcd 834.1337, found
l
834.1749; 1H NMR (D2O, 500 MHz) d 7.97 (s, 1H; H-8, G), 5.95 (d,1H; H-10, m7G),
5.76 (d, 1H; H-10, G), 4.55–4.52 (m, 2H, H-20, G, m7G), 4.44–4.25 (m, 8H; H-20, H-
30, H-40, H-50, H-500, G, m7G), 4.07 (d, 3H; 7-CH3, J1H,13C = 145 Hz), 3.10 (dd, 6H,
N2-CH3, J1H,13C = 140 Hz); 13C NMR (D2O, 125 MHz, H1 decouple 500 MHz) d
phosphate (2.35 mL) at 4 °C and methylated with fivefold excess of 13CH3I
(0.075 mL, 1.2 mmol) in anhydrous DMSO (0.5 mL). The reaction mixture
was poured into water (5 mL) and extracted three times with diethyl ether.
Aqueous phase was concentrated and applied on DEAE–Sephadex. The product
was eluted using a 0–0.8 M gradient of TEAB. m32,2,7⁄GMP was obtained as a
colorless crystals (6) (44 mg, 0.09 mmol, 60%). The 13C trimethylated guanosine
50-monophosphate (6) (0.05 mmol, TEA salt) was further coupled with the
imidazolide derivative of GDP (27 mg, 0.05 mmol) (7) in anhydrous DMF
(0.5 mL) in the presence of zinc ions (50 mg, 0.37 mmol). The reaction mixture
was poured into a solution of EDTA (110 mg, 0.46 mmol) in water (1.5 mL),
neutralized to pH 7 by addition of 1 M TEAB.and separated by ion-exchange
37.46, 36.14; 31P NMR (D2O, 202 MHz) d ꢀ10.31 (1P,
a), ꢀ10.41 (1P c), ꢀ21.93
(1P, b).
17. (a) Kadokura, M.; Wada, T.; Urashima, C.; Sekine, M. Tetrahedron Lett. 1997, 38,
8359; (b) Jankowska-Anyszka, M.; Piecyk, K.; Samonina-Kosicka, J. Org. Biomol.
Chem. 2011, 9, 5564.
18. Cohen, L. S.; Mikhli, C.; Friedman, C.; Jankowska-Anyszka, M.; Stepinski, J.;
Darzynkiewicz, E.; Davis, R. E. RNA 2004, 10, 1609.