Post-translational modifications (PTMs) of proteins, such as methylation, phosphorylation, and glycosylation, have a profound impact on proteins’ properties and functions, including protein–protein interactions, cellular targeting, stability, and signaling [1]. Among these PTMs, glycosylation is one of the most abundant and complicated modifications in all eukaryotic cells [2], playing a significant role in a wide range of biological events [3], for example, cell recognition and adhesion, signaling, immune evasion, and tumor metastasis. Naturally, protein glycosylation occurs in two main forms, glycosylation of Ser/Thr (O-glycan) [4] and glycosylation of Asn (N-glycan) [5]. In the case of O-glycosylation, glycans are typically attached to Ser/Thr residues [6] and occasionally to Tyr residues [7] and hydroxylysine residues [8], influencing proteins both intracellularly and extracellularly and contributing to the regulation of protein function and stability [9].

The diversity of O-glycans derives from their processing in the endoplasmic reticulum (ER) and Golgi apparatus [2]. The most common O-glycosylation pathway is GalNAc-O-glycosylation, which occurs in the Golgi apparatus. In mammalian systems, O-glycans are classified into four major O-GalNAc cores (cores 1–4) and four rare cores (cores 5–8) (Figure 1a) [10]. These core structures serve as foundational building blocks that undergo further modifications and elongations, leading to the diverse array of O-glycan structures observed in biological systems. As a result, naturally occurring glycoproteins can exist in various forms, sharing the same peptide backbone but exhibiting differentiation in the type and complexity of their glycosylation patterns [11].

Due to the complexity of glycosylation and its diverse roles in biological processes, it is challenging to identify the essential glycan involved in specific biological events. Therefore, it is strongly required for the preparation of glycoproteins with homogenous glycan structures. To achieve this goal, chemical synthesis and semi-synthesis have emerged as fundamental approaches to generate homogeneous glycoproteins [12, 13, 14, 15, 16, 17]. By employing these strategies, researchers have made significant progress in the synthesis of O-linked glycopeptides and glycoproteins, enabling detailed investigations into the structure–activity relationships (SARs) and functional implications of specific glycan structures.

The key challenges in synthesizing O-linked glycopeptides and glycoproteins involve achieving precise glycan attachment, selective glycosylation, and maintaining structural complexity while controlling stereochemistry and regiochemistry. Herein, we present a concise summary of recently developed strategies for the synthesis of homogeneous O-linked glycopeptides and glycoproteins, along with several illustrative examples. These strategies have paved the way for significant advancements in the field of glycoprotein research and offer valuable insights into the functional implications of specific glycan structures in various biological processes. The preparation of N-linked glycoproteins is not within the scope of this article, and we suggest recent reviews by experts in this field [18, 19, 20∗, 21∗].