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Link to original content: http://pubmed.ncbi.nlm.nih.gov/25150148
Synthesis, properties, and biological activity of boranophosphate analogs of the mRNA cap: versatile tools for manipulation of therapeutically relevant cap-dependent processes - PubMed Skip to main page content
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. 2014;42(16):10245-64.
doi: 10.1093/nar/gku757. Epub 2014 Aug 22.

Synthesis, properties, and biological activity of boranophosphate analogs of the mRNA cap: versatile tools for manipulation of therapeutically relevant cap-dependent processes

Joanna Kowalska  1 , Anna Wypijewska del Nogal  1 , Zbigniew M Darzynkiewicz  1 , Janina Buck  2 , Corina Nicola  2 , Andreas N Kuhn  3 , Maciej Lukaszewicz  1 , Joanna Zuberek  1 , Malwina Strenkowska  1 , Marcin Ziemniak  1 , Maciej Maciejczyk  4 , Elzbieta Bojarska  5 , Robert E Rhoads  6 , Edward Darzynkiewicz  7 , Ugur Sahin  3 , Jacek Jemielity  8
Affiliations

Synthesis, properties, and biological activity of boranophosphate analogs of the mRNA cap: versatile tools for manipulation of therapeutically relevant cap-dependent processes

Joanna Kowalska et al. Nucleic Acids Res. 2014.

Abstract

Modified mRNA cap analogs aid in the study of mRNA-related processes and may enable creation of novel therapeutic interventions. We report the synthesis and properties of 11 dinucleotide cap analogs bearing a single boranophosphate modification at either the α-, β- or γ-position of the 5',5'-triphosphate chain. The compounds can potentially serve either as inhibitors of translation in cancer cells or reagents for increasing expression of therapeutic proteins in vivo from exogenous mRNAs. The BH3-analogs were tested as substrates and binding partners for two major cytoplasmic cap-binding proteins, DcpS, a decapping pyrophosphatase, and eIF4E, a translation initiation factor. The susceptibility to DcpS was different between BH3-analogs and the corresponding analogs containing S instead of BH3 (S-analogs). Depending on its placement, the boranophosphate group weakened the interaction with DcpS but stabilized the interaction with eIF4E. The first of the properties makes the BH3-analogs more stable and the second, more potent as inhibitors of protein biosynthesis. Protein expression in dendritic cells was 2.2- and 1.7-fold higher for mRNAs capped with m2 (7,2'-O)GppBH3pG D1 and m2 (7,2'-O)GppBH3pG D2, respectively, than for in vitro transcribed mRNA capped with m2 (7,3'-O)GpppG. Higher expression of cancer antigens would make mRNAs containing m2 (7,2'-O)GppBH3pG D1 and m2 (7,2'-O)GppBH3pG D2 favorable for anticancer immunization.

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Figures

Figure 1.
Figure 1.
(A) Structure of the 5′-end of eukaryotic mRNA showing the cap and first three template nucleotide residues. (B) Structure of previously synthesized phosphorothioate cap analogs (S-analogs) that have favorable biological properties: m7GpSppG, a potent translational inhibitor, and m27,2′-OGppSpG, a reagent for enhancing the biological stability and translation efficiency of capped mRNAs.
Figure 2.
Figure 2.
Structural comparison of phosphorothioate and boranophosphate moieties. (A) Electronic structures; (B) stereochemical structures. Both O to BH3 and O to S substitutions may result in P-diastereoisomerism. It should be noted, however, that the same spatial arrangement of substituents around stereogenic phosphorus center for phosphorothioate and boranophosphate groups produces different absolute configurations (SP and RP) because of the different priority of BH3 and S substituents according to Cahn–Ingold–Prelog priority rules. (C) A representative RP HPLC chromatogram of a mixture of two diastereomeric BH3-analogs (m7GppBH3pG D1 and D2; 2a and 2b). D1 denotes the isomer eluting faster from a reversed-phase (RP) HPLC column.
Figure 3.
Figure 3.
Synthesis of cap BH3-analogs 1 and 5 modified at the α-position of the triphosphate bridge. Abbreviations—BSA: N,O-bis(trimethylilyl)acetamide; ACN: acetonitrile.
Figure 4.
Figure 4.
Synthetic routes for cap BH3-analog modified at the γ-position of the triphosphate bridge (3). (A) Attempted synthesis by coupling of 7-methylguanosine 5′-boranophosphate (10) and GDP imidazolide derivative. (B) A successful approach employing 7-methylguanosine 5′-(1-boranodiphosphate) (13) as the key intermediate.
Figure 5.
Figure 5.
Synthesis of cap BH3-analogs 2 (A), 4 (B) and 6 (C) modified at the β-position of the triphosphate bridge.
Figure 6.
Figure 6.
Synthesis of guanosine and adenosine α-boranotriphosphates and diadenosine 1,3-diboranotriphosphate by means of phosphorimidazolide chemistry.
Figure 7.
Figure 7.
Representative HPLC profiles from the DcpS-susceptibility assay at lower enzyme concentration (Assay I). BH3-analogs were incubated at 40 μM with 100-nM human or C. elegans DcpS and aliquots taken at different time points were analyzed by RP HPLC at 260 nm as described in the Materials and Methods section. The analogs that were hydrolyzed in less than 10% within 2 h were assumed to be resistant to DcpS (see Supplementary Table S2). The black arrow in each panel indicates the retention time at which the reaction substrate is eluted. The initial degradation products of β-modified analogs are m7GMP and guanosine β-boranodiphosphate (GDPβBH3); however, the latter is chemically labile and rapidly hydrolyses to GMP during high-temperature deactivation of the enzyme (data not shown).
Figure 8.
Figure 8.
The influence of different mRNA cap analogs on luciferase expression in human immature dendritic cells (hiDCs). After electroporation of respective 5′-capped mRNAs into hiDCs, luciferase activity was measured after 2, 4, 8, 24, 48 and 72 h (each experiment was performed in duplicate). The corresponding averaged bioluminescence signals are depicted as a function of time. The data are shown as mean ± SD.
Figure 9.
Figure 9.
Effects of non-bridging phosphate chain modifications on the susceptibility of cap dinucleotides to cleavage by human DcpS: comparison between BH3-analogs (A) and S-analogs (B). The schematic map of plausible protein–ligand interactions is based on crystallographic structure of a catalytically inactivated hDcpS mutant (His 277→Asp) in complex with m7GpppG (PDB entry 1ST0 (17)). The main difference between the O to BH3 and O to S substitutions is that the β-BH3 substitutions produce DcpS-resistant analogs, whereas corresponding β-S-analogs are good substrates for DcpS. A possible explanation is that the β-boranophosphate moiety, due to insufficient ability of the BH3 substituent to form hydrogen bonds, cannot be sufficiently stabilized as a leaving group by the basic amino acid residues in DcpS's cap-binding pocket. Some ribose and nucleobase interactions have been omitted for clarity.
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