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Analogs of the mRNA cap are widely employed to study processes involved in mRNA metabolism as well as being useful in biotechnology and medicinal applications. Here we describe synthesis of six dinucleotide cap analogs bearing a single phosphorothioate modification at either the α, β, or γ position of the 5′,5′-triphosphate chain. Three of them were also modified with methyl groups at the 2′-O position of 7-methylguanosine to produce anti-reverse cap analogs (ARCAs). Due to the presence of stereogenic P centers in the phosphorothioate moieties, each analog was obtained as a mixture of two diastereomers, D1 and D2. The mixtures were resolved by RP HPLC, providing 12 different compounds. Fluorescence quenching experiments were employed to determine the association constant (KAS) for complexes of the new analogs with eIF4E. We found that phosphorothioate modifications generally stabilized the complex between eIF4E and the cap analog. The most strongly bound phosphorothioate analog (the D1 isomer of the β-substituted analog m7GppSpG) was characterized by a KAS that was more than fourfold higher than that of its unmodified counterpart (m7GpppG). All analogs modified in the γ position were resistant to hydrolysis by the scavenger decapping pyrophosphatase DcpS from both human and Caenorhabditis elegans sources. The absolute configurations of the diastereomers D1 and D2 of analogs modified at the α position (i.e., m7GpppSG and m27,2′-OGpppSG) were established as SP and RP, respectively, using enzymatic digestion and correlation with the SP and RP diastereomers of guanosine 5′-O-(1-thiodiphosphate) (GDPαS). The analogs resistant to DcpS act as potent inhibitors of in vitro protein synthesis in rabbit reticulocyte lysates.

Enzymatic cleavage of the P-chiral diastereoisomers of the 5' mRNA cap analogue bearing phosphorothioate moiety in alfa position of 5',5'-triphosphate bridge (m(7)Gppp(S)G D1 and D2) was performed by human Decapping Scavenger (DcpS) enzyme. Analysis of the degradation products allowed to estimate the absolute configuration at the asymmetric phosphorus atoms in examined compounds via correlation with the R(P) and S(P) diastereoisomers of guanosine 5'-O-(1-thiodiphosphate) (GDPalphaS).

The unique structure of 5' mRNA cap from Trypanosomatids is the most modified cap found in nature. Here we present the synthesis of cap-4 (m(7)Gpppm(3)(6,6,2')Apm(2')Apm(2')Cpm(2)(3,2')Up) on a disulfide-tethered solid support. This approach allows obtaining cap-4 more efficiently then previously described. Moreover such modified resin could be a useful tool for affinity purification of Leishmania proteins interacting with cap-4. For the final step of synthesis, namely coupling of phosphorylated tetranucleotide with activated 7-methylguanosine 5'-diphosphate two systems were compared. Surprisingly, the coupling in water with Mn(2+) as a catalyst, gave better results than usually more effective coupling in DMF with ZnCl(2).

A straightforward method for the synthesis of nucleotide analogues bearing a phosphorothioate moiety at the terminal position of the polyphosphate chain is described. Several nucleoside 5′-(2-thiodiphosphates) and 5′-(3-thiotriphosphates) were synthesized by treatment of the appropriate nucleotide imidazolide derivative with a ca. 4-fold excess of thiophosphate triethylammonium salt in DMF in the presence of zinc chloride. The HPLC reaction yields varied from 80% to 100%, in the majority of cases exceeding 90%. Separation was accomplished by Sephadex ion-exchange chromatography or reverse-phase HPLC with preparative yields of about 70%.

Synthesis of Leishmania mRNA 5'-cap analogs, m(7)Gpppm(2)(6)AmpAm (cap-2), and m(7)Gpppm(2)(6)AmpAmpCm (cap-3) is reported. Binding affinities of those cap analogs for LeishIF4E proteins were determined using fluorescence spectroscopy. Cap-3 showed similar affinity to LeishIF4Es compared to the mature trypanosomatids cap structure (cap-4).

The 5′ cap structure of trypanosomatid mRNAs, denoted cap 4, is a complex structure that contains unusual modifications on the first four nucleotides. We examined the four eukaryotic initiation factor 4E (eIF4E) homologues found in the Leishmania genome database. These proteins, denoted LeishIF4E-1 to LeishIF4E-4, are located in the cytoplasm. They show only a limited degree of sequence homology with known eIF4E isoforms and among themselves. However, computerized structure prediction suggests that the cap-binding pocket is conserved in each of the homologues, as confirmed by binding assays to m 7 GTP, cap 4, and its intermediates. LeishIF4E-1 and LeishIF4E-4 each bind m 7 GTP and cap 4 comparably well, and only these two proteins could interact with the mammalian eIF4E binding protein 4EBP1, though with different efficiencies. 4EBP1 is a translation repressor that competes with eIF4G for the same residues on eIF4E; thus, LeishIF4E-1 and LeishIF4E-4 are reasonable candidates for serving as translation factors. LeishIF4E-1 is more abundant in amastigotes and also contains a typical 3′ untranslated region element that is found in amastigote-specific genes. LeishIF4E-2 bound mainly to cap 4 and comigrated with polysomal fractions on sucrose gradients. Since the consensus eIF4E is usually found in 48S complexes, LeishIF4E-2 could possibly be associated with the stabilization of trypanosomatid polysomes. LeishIF4E-3 bound mainly m 7 GTP, excluding its involvement in the translation of cap 4-protected mRNAs. It comigrates with 80S complexes which are resistant to micrococcal nuclease, but its function is yet unknown. None of the isoforms can functionally complement the Saccharomyces cerevisiae eIF4E, indicating that despite their structural conservation, they are considerably diverged.

Nucleosides and oligonucleotides with an oxygen replaced by sulfur atom are an interesting class of compounds because of their improved stability toward enzymatic cleavage by nucleases. We have synthesized several dinucleotide mRNA cap analogs containing a phosphorothioate moiety in the alpha, beta, or gamma position of 5',5'-triphosphate chain [m7Gp(s)ppG, m7Gpp(s)pG, and m7Gppp(s)G]. These are the first examples of the biologically important 5'mRNA cap analogs containing a phosphorothioate moiety, and these compounds may be useful in a variety of biochemical and biotechnological applications. Incorporation of a sulfur atom in the alpha or gamma position within the dinucleotide cap analog was achieved using PSCl3 in a nucleoside phosphorylation reaction followed by coupling the phosphorothioate of nucleoside with a second nucleotide. Synthesis of cap analogs with the phosphorothioate moiety in beta position was performed using an organic phosphorothioate salt in a coupling reaction with an activated nucleotide. The structures of newly synthesized compounds was confirmed using MS and 1H and 31P NMR spectroscopy. We present here the results of preliminary studies on their interaction with translation initiation factor eIF4E and enzymatic hydrolysis with human and nematode DcpS scavengers.

Leishmania and other trypanosomatids are early eukaryotes that possess unusual molecular features, including polycistronic transcription and trans-splicing of pre-mRNAs. The spliced leader RNA (SL RNA) is joined to the 5′ end of all mRNAs, thus donating a 5′ cap that is characterized by complex modifications. In addition to the conserved m7GTP, linked via a 5′-5′-triphosphate bound to the first nucleoside of the mRNA, the trypanosomatid 5′ cap includes 2′-O methylations on the first four ribose moieties and unique base methylations on the first adenine and the fourth uracil, resulting in the cap-4 structure, m7 Gpppm36,6,2′Apm2′Apm2′ Cpm23,2′U, as reported elsewhere previously. A library of analogs that mimic the cap structure to different degrees has been synthesized. Their differential affinities to the cap binding proteins make them attractive compounds for studying the molecular basis of cap recognition, and in turn, they may have potential therapeutic significance. The interactions between cap analogs and eIF4E, a cap-binding protein that plays a key role in initiation of translation, can be monitored by measuring intrinsic fluorescence quenching of the tryptophan residues. In the present communication we describe the multistep synthesis of the trypanosomatid cap-4 structure. The 5′ terminal mRNA tetranucleotide fragment (pm36,6,2′Apm2′Apm2′ Cpm23,2′U) was synthesized by the phosphoramidite solid phase method. After deprotection and purification, the 5′-phosphorylated tetranucleotide was chemically coupled with m7GDP to yield the cap-4 structure. Biological activity of this newly synthesized compound was confirmed in binding studies with eIF4E from Leishmania that we recently cloned (LeishIF4E-1), using the fluorescence time-synchronized titration method.

Studies on the interaction of the murine translation initiation factor 4E with two new-synthesized cap-analogues, modified at C2′ of 7-methylguanosine, have been performed by means of the fluorescence titration method. No difference in the binding affinity for eIF4E was observed compared with the “anti reversed” cap analogues, possessing the analogous modifications at C3′. Potential significance of the novel caps as research tools for examination of the nuclear cap binding complex CBC80/20 has been discussed.

Synthetic analogs of the 5′-terminal caps of eukaryotic mRNAs and snRNAs are used in elucidating such physiological processes as mRNA translation, pre-mRNA splicing, intracellular transport of mRNA and snRNAs, and mRNA turnover. Particularly useful are RNAs capped with synthetic analogs, which are produced by in vitro transcription of a DNA template using a bacteriophage RNA polymerase in the presence of ribonucleoside triphosphates and a cap dinucleotide such as m7Gp3G. Unfortunately, because of the presence of a 3′-OH on both the m7Guo and Guo moieties, up to half of the mRNAs contain caps incorporated in the reverse orientation. Previously we designed and synthesized two “anti-reverse” cap analogs (ARCAs), m73′dGp3G and m27,3′-OGp3G, that cannot be incorporated in the reverse orientation because of modifications at the C3′ position of m7Guo. In the present study, we have synthesized seven new cap analogs modified in the C2′ and C3′ positions of m7Guo and in the number of phosphate residues, m27,2′-OGp3G, m72′dGp3G, m72′dGp4G, m27,2′-OGp4G, m27,3′-OGp4G, m7Gp5G, and m27,3′-OGp5G. These were analyzed for conformation in solution, binding affinity to eIF4E, inhibition of in vitro translation, degree of reverse capping during in vitro transcription, capping efficiency, and the ability to stimulate cap-dependent translation in vitro when incorporated into mRNA. The results indicate that modifications at C2′, like those at C3′, prevent reverse incorporation, that tetra- and pentaphosphate cap analogs bind eIF4E and inhibit translation more strongly than their triphosphate counterparts, and that tetraphosphate ARCAs promote cap-dependent translation more effectively than previous cap analogs.

The cap structure is a specific feature of the 5 � end of mRNA which plays an important role in the post-transcriptional control in gene expression. A major step of gene regulation occurs at the level of mRNA turnover. Degradation of most eukaryotic mRNAs entails the removal of the cap structure in the various pathways. A human scavenger decapping enzyme (hDcpS) catalyses the cleavage of the residual cap structure m 7 GpppN and/or short oligonucleotides after the 3 � → 5 � exosom mediated digestion. In this paper we report a fluorescence study of association process of hDcpS with m 7 GMP, m 7 GDP and selected dinucleotide cap analogues resistant to enzymatic hydrolysis. The calculated values of association constants (Kas) and corresponding Gibbs free energies (� G ◦ ) depend on the type of substituents and their positions in the cap molecule, indicating which structural modifications are crucial for the catalysis.