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

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.

Scheme 1. Synthesis and Postsynthetic Cap Modification

(A) Synthesis of m⁷GpppG^8Br and m^2′-O,7GpppG^8Br. 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. 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

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

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

(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

Screening of C8-modified cap analogs with eIF4E protein.

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

Screening of C8-modified cap analogs with DcpS.

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.