Synthesis of Glycosylated 3-(3-Amino-3-carboxypropyl)uridine: A Minimum Unit of GlycoRNA

Abstract

N-Glycosylated RNA (glycoRNA) has been identified on the cell surface, and 3-(3-amino-3-carboxypropyl)uridine has been reported as a conjugation site of N-glycans on RNA. Although the association of glycoRNAs with various diseases has been reported, their biosynthetic mechanisms and biological roles remain unexplored. Here, we report the preparation of two species of N-glycan-conjugated 3-(3-amino-3-carboxypropyl)uridine as the minimal units of glycoRNA. Our synthesized glycoRNA unit would contribute to future biochemical research on glycoRNAs.

Introduction

Glycosylation plays a pivotal role in several biological processes.¹⁾ Traditionally, proteins and lipids are recognized as substrates for glycosylation, giving rise to glycoproteins and glycolipids, respectively. However, in 2021, it was reported that RNA can also serve as a substrate for glycosylation.²⁾ The biosynthesis, metabolism, and biological significance of N-glycosylated RNAs (glycoRNAs) remain largely unexplored, although recent studies have highlighted their association with various diseases. GlycoRNAs are predominantly located on the cell surface and interact with immune cell receptors, particularly members of the sialic acid-binding immunoglobulin-like lectin (Siglec) receptor family. Neutrophil surface glycoRNAs are critical for regulating adhesion to endothelial cells, facilitating transendothelial migration, and controlling neutrophil recruitment.³⁾ Furthermore, surface glycoRNA expression inversely correlates with tumor malignancy and metastasis in breast cancer cell lines.⁴⁾ Specific glycosylated microRNAs have also been implicated as regulators of pancreatic cancer cell growth and proliferation.⁵⁾ Similar to neuronal glycoproteins, glycoRNAs may serve as ligands for microglial Siglec-11 receptors, polarizing them toward an anti-inflammatory phenotype by reducing cytokines such as interleukin-1β (IL-1β) and nitric oxide-producing nitric oxide synthase 2 (NOS2).^2,6)

In the respiratory system, glycoRNA has emerged as a novel component of the alveolar epithelial glycocalyx, regulating epithelial barrier function and potentially influencing influenza A virus infection.⁷⁾ Additionally, glycoRNAs on alveolar epithelial cell surfaces have been identified as potential ligands for immune cell receptors, such as Siglec-11 and Siglec-14, and lung collections like surfactant proteins A and D, highlighting their potential role in modulating immune responses in inflammatory lung diseases. GlycoRNA is classified into glycoRNA-L, glycoRNA-M, and glycoRNA-S based on their size. GlycoRNA-L is predominantly expressed in most tissues and cell lines, whereas glycoRNA-S is expressed only in certain tissues and cell lines, including adipose tissue and human monocytes. Siglec-5 preferentially binds to glycoRNA-L over glycoRNA-S.⁸⁾

Recently, 3-(3-amino-3-carboxypropyl)uridine (acp³U) was identified as a site for N-glycan attachment in glycoRNAs.⁹⁾ Advanced detection systems for glycoRNA^4,10) have revealed that while sialic acid-containing glycans were initially considered the primary glycan structures, subsequent studies have unveiled substantial glycan diversity.¹¹⁾ To elucidate the biological functions of glycoRNA and develop glycoRNA-based therapeutics, a scalable synthesis of glycoRNA is imperative. In this study, we present the first successful synthesis of glycosylated acp³U 1 and 2 (Fig. 1), representing the minimal functional unit of glycoRNA.

Fig. 1. Structure of Synthesized GlycoRNAs

Results and Discussion

The coupling reaction between galactose-terminated G2 glycan-NH₂ 3¹²⁾ and acp³U 5⁹⁾ was conducted using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (WSCDI·HCl) and 1-hydroxybenzotriazole (HOBt) in 56% yield to give compound 6 (Chart 1). The stereochemistry at the anomeric position of the major product was determined to be β, as evidenced by the ¹H chemical shift (4.92 ppm) and coupling constant (9.7 Hz) at the anomeric position of a reducing terminal GlcNAc residue. Then, the N-tert-butoxycarbony (N-Boc) group was removed under acidic conditions to give glycoRNA without N-acetylneuraminic acid 1 in 89% yield. In the synthesis of glycoRNA containing N-acetylneuraminic acid, a concern arises that N-acetylneuraminic acid is sensitive to acidic conditions. However, protecting the carboxyl group of sialic acid with a benzyl group enhances the stability of sialic acid linkages under acidic conditions.¹³⁾ After coupling the benzyl-protected N-acetylneuraminic acid carrying A2 glycan-NH₂ 4¹²⁾ and 5 in 53% yield, the Boc group of compound 7 was removed under 95% aqueous trifluoroacetic acid. After hydrolysis of the benzyl ester, glycoRNA with N-acetylneuraminic acid 2 was prepared.

Chart 1. Synthetic Route of GlycoRNAs 1 and 2

(i) WSCDI·HCl, HOBt, DMSO, 56% for 6, 53% for 7; (ii) 95% TFA aq., 89% for 1, 87% for 8; (iv) 0.1 M aq. NaOH, 88%. DMSO, dimethyl sulfoxide; TFA, trifluoroacetic acid.

Conclusion

Now, glycoRNA is attracting attention for its potential as a novel diagnostic marker and therapeutic target. The development of detection methods has expanded our understanding of the structural and functional roles of glycoRNAs. Given that a library of N-glycans is commercially available, our method enables the synthesis of glycoRNAs with diverse structures. Preparation of glycoRNAs is expected to contribute to the future medical applications of glycoRNA.

Acknowledgments

We thank Dr. Mayumi Ikegami for her technical assistance in NMR support. This work was partially supported by JSPS KAKENHI (Grant Number: 24K02157).

Conflict of Interest

K.I. is an employee of Glytech. H. Y. and S. M. declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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