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. Author manuscript; available in PMC: 2013 Jul 1.
Published in final edited form as: Bioorg Med Chem Lett. 2012 May 4;22(13):4391–4395. doi: 10.1016/j.bmcl.2012.04.120

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,*
PMCID: PMC3652009  NIHMSID: NIHMS382201  PMID: 22658555

Abstract

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.

Keywords: Cap analogue, TMG cap, 13C label, 14C label, mRNA

Gene expression in eukaryotes is a complex process that involves several steps and requires numerous protein factors. One common RNA element involved in mRNA splicing, export, translation, and stability is the unusual structure at the 5′ end of mRNA known as a ‘cap’.1 This unique structure consist of 7-methylguanosine linked via a 5′,5′-triphosphate bridge to the first transcribed nucleotide (MMG cap). For many years, numerous studies have been conducted to describe the molecular mechanism of the interaction 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 biochemical methods involving several chemically prepared cap analogues 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 Caenorhabditis 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 5′ end of a large percentage of mRNAs by RNA trans-splicing of a short 22-nt spliced leader sequence (SL).4 In general, eIF4E proteins from higher eukaryotes are unable to effectively recognize and bind the TMG cap.2d,5 However, 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 distinguish 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, biophysical, 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 proposed.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 completely understood.

Herein we describe the synthesis of 13C and 14C labeled mono- and trimethylated cap analogues as tools that will facilitate analysis 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 nucleotide in a reaction with 2,2′-ditiopyridine and triphenylphosphine prior to coupling with a second nucleotide in the presence of an excess of double-charged ions (such as Zn2+ or Mn2+) in anhydrous or aqueous condition.12 Nevertheless, for the synthesis of 13C/14C MMG and TMG caps, the procedure needed to be modified to minimize the number of steps with the radioactive materials and due to the cost of isotopically labeled substrates (13C or 14C methyl iodide). Our method to synthesize the MMG dinucleotide cap analogues labeled with either 13C or 14C was accomplished (Scheme 1) by direct methylation of the dinucleotide GpppG that was prepared by coupling of guanosine 5′-diphosphate (GDP) with an imidazolide derivative of guanosine 5′-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 temperature.14 The synthesis of 13C labeled MMG cap has been described previously15 but it was achieved by a longer two-step procedure.

Scheme 1.

Scheme 1

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Synthesis of 13C and 14C MMG cap analogues. Reagents: (i) 13CH3I or 14CH3I, DMSO.

In contrast to MMG cap, a TMG cap labeled with 13C and 14C isotopes required a multistep procedure (Scheme 2). To our knowledge this is the first report of such a synthesis. The first step required transformation of guanosine to the corresponding 2′,3′,5′-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 product 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 treated with a fourfold molar excess of (13CH3)2NH. As the dimethylamine labeled with 13C carbon can only be obtained as a hydrochloride, a nucleophilic substitution was performed in aqueous solution of DMSO/AcCN/H2O/Et3N in a temperature-controlled oil bath at 60 °C until the fluoronucleoside had completely disappeared, as evaluated by TLC analysis. 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 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 anhydrous DMSO (6). The 13C trimethylated guanosine 5′- monophosphate 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.

Scheme 2.

Scheme 2

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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.

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 obtained as described for the preparation of 13C labeled TMG cap except that the substitution step leading to N2,N2-dimethylguanosine was carried out with unlabeled dimethylamine. All four cap analogues were purified by ion-exchange chromatography on DEAE-Sephadex A-25 (HCO3− form), the relevant fractions collected, pooled, and evaporated repeatedly with cold ethanol. The fractions with both high UV absorbance and high specific activity were combined 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 labeled with 14C and 13C isotopes were converted into sodium salts using Dowex 50 WX8 ion-exchange resin. The structure and homogeneity of final products were confirmed by HPLC, mass spectrometry, 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-frequency proton decoupled 13C NMR spectra. Extremely strong signals from one (1) or three (8) methyl carbons with appropriate coupling pattern were also observed.

Figure 1.

Figure 1

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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.

To evaluate the 14C cap analogues in a functional assay, we carried out decapping experiments (Fig. 2) using A. suum and C. elegans DcpS, the scavenger decapping enzymes. DcpS cleaves mono- as well as trimethylated cap regioselectively between β and γ phosphates of the 5′,5′-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 illustrated by TLC and autoradiography (Fig. 2).

Figure 2.

Figure 2

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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 ammonium 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 1—14C cap analogue, lane 2—14C cap analogue treated with C. elegans DcpS (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.

In conclusion we have developed a simple and short method for specific labeling of mono- and trimethylated caps with 13C and 14C 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 structures 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 enzymes 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.

References and notes

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