Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2023 Jun 2;88(11):6827-6846.
doi: 10.1021/acs.joc.3c00126. Epub 2023 May 20.

Synthesis and Evaluation of Diguanosine Cap Analogs Modified at the C8-Position by Suzuki-Miyaura Cross-Coupling: Discovery of 7-Methylguanosine-Based Molecular Rotors

Blazej A Wojtczak  1 , Marcelina Bednarczyk  1   2 , Pawel J Sikorski  1 , Anna Wojtczak  2 , Piotr Surynt  1   2 , Joanna Kowalska  2 , Jacek Jemielity  1
Affiliations

Synthesis and Evaluation of Diguanosine Cap Analogs Modified at the C8-Position by Suzuki-Miyaura Cross-Coupling: Discovery of 7-Methylguanosine-Based Molecular Rotors

Blazej A Wojtczak et al. J Org Chem. .

Abstract

Chemical modifications of the mRNA cap structure can enhance the stability, translational properties, and half-life of mRNAs, thereby altering the therapeutic properties of synthetic mRNA. However, cap structure modification is challenging because of the instability of the 5'-5'-triphosphate bridge and N7-methylguanosine. The Suzuki-Miyaura cross-coupling reaction between boronic acid and halogen compound is a mild, convenient, and potentially applicable approach for modifying biomolecules. Herein, we describe two methods to synthesize C8-modified cap structures using the Suzuki-Miyaura cross-coupling reaction. Both methods employed phosphorimidazolide chemistry to form the 5',5'-triphosphate bridge. However, in the first method, the introduction of the modification via the Suzuki-Miyaura cross-coupling reaction at the C8 position occurs postsynthetically, at the dinucleotide level, whereas in the second method, the modification was introduced at the level of the nucleoside 5'-monophosphate, and later, the triphosphate bridge was formed. Both methods were successfully applied to incorporate six different groups (methyl, cyclopropyl, phenyl, 4-dimethylaminophenyl, 4-cyanophenyl, and 1-pyrene) into either the m7G or G moieties of the cap structure. Aromatic substituents at the C8-position of guanosine form a push-pull system that exhibits environment-sensitive fluorescence. We demonstrated that this phenomenon can be harnessed to study the interaction with cap-binding proteins, e.g., eIF4E, DcpS, Nudt16, and snurportin.

PubMed Disclaimer

Conflict of interest statement

The authors declare no competing financial interest.

Figures

Scheme 1
Scheme 1. Synthesis and Postsynthetic Cap Modification
(A) Synthesis of m7GpppG8Br and m2′-O,7GpppG8Br. Reagents and conditions: (i) ZnCl2, DMF. (B) Postsynthetic cap modification by Suzuki–Miyaura cross-coupling reaction. Reagents and conditions: (i) Pd(OAc)2/TPPTS complex, NaHCO3 buffer (100 mM, pH 8.5), R8-B(OH)2, 80–90 °C, 15 min.
Scheme 2
Scheme 2. Synthesis of 8-Substituted Monophosphates and 8-Substituted Cap Analogs
(A) Synthesis of 8-substituted monophosphates. Reagents and conditions: (i) Pd(OAc)2/TPPTS complex, Cs2CO3, R-B(OH)2, 90–95 °C, 15–60 min; (ii) MeI, DMSO/DMF (1:1). (B) Synthesis of 8-substituted cap analogs via phosphorimidazolide intermediates. Reagents and conditions: (i) ZnCl2, DMF, 24 h.
Figure 1
Figure 1
Absorption (solid line) and emission (dotted line) spectra of (A) 14b–d and (B) 15b–d in various solvents. The concentrations of all samples were 5 μM.
Figure 2
Figure 2
Absorption spectra of monophosphates (14b–d) and N7-monophosphates (15b–d) measured in phosphate buffer at pH 7 and 6, respectively. The concentrations of all samples were 5 μM.
Figure 3
Figure 3
(A) Comparison of fluorescence intensity of C8-modified cap analogs depending on the site of modification. (B) Normalized absorption (blue) and emission (green) spectra of C8-modified cap analogs. The concentrations of all samples were 5 μM.
Figure 4
Figure 4
Screening of C8-modified cap analogs with eIF4E protein.
Figure 5
Figure 5
Translation efficiency of mRNAs carrying various C8-modified cap analogs at the 5′ end, evaluated as total protein expression (cumulative luminescence) in HeLa cells.
Figure 6
Figure 6
Screening of C8-modified cap analogs with DcpS.
Figure 7
Figure 7
(A) Titration of 0.1 μM snurportin with compound 8 or unmodified TMGpppG. (B) Emission spectra of 1 μM compound 8 during titration with snurportin (from 0 to 2 μM). (C) Influence of snurportin concentration on the fluorescence intensity of compound 8.
See this image and copyright information in PMC

References

    1. Sonenberg N.; Rupprecht K. M.; Hecht S. M.; Shatkin A. J. Eukaryotic mRNA cap binding protein: purification by affinity chromatography on sepharose-coupled m7GDP. Proc. Natl. Acad. Sci. U. S. A. 1979, 76, 4345–4349. 10.1073/pnas.76.9.4345. - DOI - PMC - PubMed
    2. Mitchell S. F.; Walker S. E.; Algire M. A.; Park E. H.; Hinnebusch A. G.; Lorsch J. R. The 5′-7-methylguanosine cap on eukaryotic mRNAs serves both to stimulate canonical translation initiation and to block an alternative pathway. Mol. Cell 2010, 39, 950–962. 10.1016/j.molcel.2010.08.021. - DOI - PMC - PubMed
    1. Ramanathan A.; Robb G. B.; Chan S. H. mRNA capping: biological functions and applications. Nucleic Acids Res. 2016, 44, 7511–7526. 10.1093/nar/gkw551. - DOI - PMC - PubMed
    1. Miyagi Y.; Sugiyama A.; Asai A.; Okazaki T.; Kuchino Y.; Kerr S. J. Elevated levels of eukaryotic translation initiation factor eIF-4E, mRNA in a broad-spectrum of transformed-cell lines. Cancer Lett. 1995, 91, 247–252. 10.1016/0304-3835(95)03737-h. - DOI - PubMed
    2. De Benedetti A.; Harris A. L. eIF4E expression in tumors: its possible role in progression of malignancies. Int. J. Biochem. Cell Biol. 1999, 31, 59–72. 10.1016/s1357-2725(98)00132-0. - DOI - PubMed
    1. Singh J.; Salcius M.; Liu S. W.; Staker B. L.; Mishra R.; Thurmond J.; Michaud G.; Mattoon D. R.; Printen J.; Christensen J.; Bjornsson J. M.; Pollok B. A.; Kiledjian M.; Stewart L.; Jarecki J.; Gurney M. E. DcpS as a therapeutic target for spinal muscular atrophy. ACS Chem. Biol. 2008, 3, 711–722. 10.1021/cb800120t. - DOI - PMC - PubMed
    1. Ziemniak M.; Strenkowska M.; Kowalska J.; Jemielity J. Potential therapeutic applications of RNA cap analogs. Future Med. Chem. 2013, 5, 1141–1172. 10.4155/fmc.13.96. - DOI - PubMed

Publication types

MeSH terms