Chemical approaches to explore ubiquitin-like proteins

492 | RSC Chem. Biol., 2025, 6, 492–509 © 2025 The Author(s). Published by the Royal Society of Chemistry Cite this: RSC Chem. Biol., 2025, 6, 492 Chemical approaches to explore ubiquitin-like proteins Reem Mousa, Dana Shkolnik,†Yam Alalouf†and Ashraf Brik * Chemical protein synthesis has emerged as a powerful approach for producing ubiquitin (Ub) and ubiquitin-like modifiers (Ubls) in both their free and conjugated forms, particularly when recombinant or enzymatic strategies are challenging. By providing precise control over the assembly of Ub and Ubls, chemical synthesis enables the generation of complex constructs with site-specific modifications that facilitate detailed functional and structural studies. Ub and Ubls are central regulators of protein homeostasis, regulating a wide range of cellular processes such as cell cycle progression, transcription, DNA repair, and apoptosis. Ubls share an evolutionary link with Ub, resembling its structure and following a parallel conjugation pathway that results in a covalent isopeptide bond with their cellular substrates. Despite their structural similarities and sequence homology, Ub and Ubls exhibit distinct functional differences. Understanding Ubl biology is essential for unraveling how cells maintain their regulatory networks and how disruptions in these pathways contribute to various diseases. In this review, we highlight the chemical methodologies and strategies available for studying Ubls and advancing our comprehensive understanding of the Ubl system in health and disease. Introduction Chemical protein synthesis and semi-synthesis have emerged as revolutionary tools in protein research, enabling the precise preparation of proteins and their analogs with atomic-level control. 1–3 Solid-phase peptide synthesis (SPPS) facilitates the assembly of peptides with defined sequences and modifications (Fig. 1a). 4 When combined with chemoselective ligation methods such as native chemical ligation (NCL), it allows the generation of full-length proteins (Fig. 1b). 5,6 In NCL, a Cterminal thioester of unprotected peptide is joined with an Nterminal cysteine/selenocysteine 7 peptide under mild, aqueous conditions to form native amide bonds. NCL has been further broadened by introducing desulfurization 8,9 and deselenization 10–12 reactions, which expands its applicability to the preparation of proteins lacking native cysteine residues (Fig. 1c). Additionally, other ligation strategies including serine/threonine 13 and a-ketoacid-hydroxylamine (KAHA) 14 ligation have expanded the scope of reactions to access diverse complex proteins (Fig. 1b). Semi-synthesis further extends the capabilities of these methods by combining chemically synthesized fragments with recombinantly expressed protein domains. 15,16 This hybrid approach enables the preparation of large proteins with different modifications, including non-canonical amino acids, isotopic labels, and post-translational modifications (PTMs). Chemical synthesis of proteins has enabled researchers to investigate their biochemical, structural, and functional properties in ways that are challenging to achieve using traditional molecular biology and enzymatic approaches. It also allowed the incorporation of specific and unique modifications to facilitate various studies such as the generation of activitybased probes (ABPs) designed to unravel a protein’s interactome, expression level, and cellular localization. 1–3 Chemical and semi-synthetic methods have been extensively applied to study ubiquitination and deubiquitination, 17–19 key post-translational modifications that maintain protein homeostasis and regulate cellular processes. 20 Ubiquitination involves attaching ubiquitin (Ub), a small, conserved protein with a b-grasp fold and a flexible C-terminal diglycine motif to substrates 21 through an isopeptide bond with a lysine residue, facilitated by a cascade of E1, E2, and E3 enzymes. 22 This process is counter reacted by deubiquitinating enzymes (DUBs) that remove Ub or Ub chains, modulating cellular signaling. 23–25 Ubiquitin-like proteins (Ubls), which are structurally similar to Ub, can also be conjugated to protein substrates via a similar mechanism, affecting various cellular processes. 26 Humans have eighteen conjugatable Ubls, including five SUMO paralogs, NEDD8, UFM1, URM1, ISG15, ATG12, and FAT10, and seven ATG8 paralogs (Fig. 2). 27 Research on Ubls focuses on their conjugation mechanisms, substrate recognition, Schulich Faculty of Chemistry, Technion-Israel Institute of Technology, Haifa 3200008, Israel. E-mail: [email protected]c.il †These authors contributed equally. Received 15th September 2024, Accepted 21st January 2025 DOI: 10.1039/d4cb00220b rsc.li/rsc-chembio RSC Chemical Biology REVIEW Open Access Article. Published on 12 February 2025. Downloaded on 11/6/2025 8:09:08 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. View Article Online View Journal | View Issue © 2025 The Author(s). Published by the Royal Society of Chemistry RSC Chem. Biol., 2025, 6, 492–509 | 493 specificity, interactions with other PTMs, and their roles in health and disease. 28 Relying solely on enzymatic methods to prepare homogeneous Ubl-based conjugates presents challenges, such as limited availability of enzymatic machinery and difficulties in achieving site-specific modifications in sufficient quantities and homogeneity for biochemical and functional analyses. Chemical synthesis offers solutions to these challenges, enabling deeper exploration of Ubl biology and their involvement in various diseases. In this review, we focus exclusively on UBLs that have been studied using chemical or semi-synthetic methods, except for FAT10, which has not been synthetically prepared. We highlight how the different approaches have contributed to our understanding of the various biochemical, structural, and functional aspects of Ubls. By providing this review, we offer a valuable resource for researchers to encourage them to use these methods to explore Ubls biology and understand their role in health and disease, potentially leading to new therapeutic applications. A brief overview SUMO In humans, small ubiquitin-related modifiers (SUMOs) are a family of five small proteins ranging from 93–97 residues (Fig. 2a and b), 29–32 which covalently modify their substrates in a process called SUMOylation, which regulates diverse cellular processes such as transcriptional regulation, DNA repair, and apoptosis. 33 SUMO 1–3 are the most extensively studied paralogs and exhibit varying sequence homology, where SUMO-1 shares 18% homology with Ub and SUMO-2, and SUMO-3 shares 45% homology with SUMO-1. 29–32 While Ub and SUMO share comparable enzymatic cascades, SUMOylation employs only one E2 enzyme i.e. Ubc9, 34,35 while Ub has about 40 E2 enzymes (Table 1). 36 SUMO is conjugated to its substrates through an isopeptide bond between its C-terminal Gly and a substrate’s Lys residue, leading to a single SUMO or poly-SUMO chain that is internally linked via an isopeptide bond(s) (Table 1 and Fig. 3). 37 These Fig. 1 Schematic representation of (a) SPPS on a rink amide resin where activation, coupling, deprotection and cleavage steps are shown. (b) The different chemical ligation approaches, highlighting NCL. (c) The desulfurization reaction in the presence of the radical initiator 2,20-azobis[2-(2imidazolin-2-yl)propane] dihydrochloride (VA-044) and the reducing agent tris(2-carboxyethyl)phosphine (TCEP). Review RSC Chemical Biology Open Access Article. Published on 12 February 2025. Downloaded on 11/6/2025 8:09:08 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. View Article Online 494 | RSC Chem. Biol., 2025, 6, 492–509 © 2025 The Author(s). Published by the Royal Society of Chemistry modifications can alter protein stability, sub-cellular localization, and their intercoms. For instance, SUMOylation of RanGTPase-activating protein 1 (RanGAP1) targets it to the nuclear pore complex, 38 while SUMOylation of promyelocytic leukemia (PML) assists in the assembly and stabilization of PML nuclear bodies (NBs), involved in DNA damage repair and antiviral responses. 39 The Lys residue in substrates is typically located within a distinctive motif known as the SUMO consensus motif, featuring the sequence CKX(E/D), where Crepresents hydrophobic amino acid, K is the modified lysine, X is any amino acid and E/D represents a negatively charged amino acid (either glutamate or aspartate). 40 In addition to the covalent linkage, SUMO can interact with other proteins non-covalently through a SUMO-interacting motif (SIM) of a substrate. 41 SUMOylation is known to be a reversible process where SUMO is cleaved from its substrate by SUMO-specific proteases (SENPs) (Table 1 and Fig. 3). SENPs are a family of cysteine Fig. 2 Representative conjugatable Ubl members. (a) Sequences of Ub and Ubls highlighting the diglycine motif and their length. (b) The tertiary structures of Ub and Ubls are also presented, highlighting several crucial structural elements (hydrophobic patches, Gly–Gly motif.). The PDB code for: Ub-1UBQ, NEDD8-1NDD, SUMO-1-4WJQ, UFM1-5IA7, ISG15-1Z2M, ATG8-2KQ7, FAT10-6GF1, and URM1-2QJL. RSC Chemical Biology Review Open Access Article. Published on 12 February 2025. Downloaded on 11/6/2025 8:09:08 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. View Article Online © 2025 The Author(s). Published by the Royal Society of Chemistry RSC Chem. Biol., 2025, 6, 492–509 | 495 proteases, comprising of seven members: SENP1–3 and SENP5– 7. 42 They are primarily localized in the nucleus, with certain members present in sub-nuclear compartments like the nucleolus and PML-NBs. SENPs play an additional role in the maturation process of SUMO where they cleave its tail, an extension of amino acids at the C-terminus, thus producing the mature SUMO with its di-Gly motif exposed and ready to initiate the cycle (Fig. 3). 42 Since SUMO is involved in numerous cellular processes, aberration in SUMOylation can contribute to various diseases including cancer, neurological disorders, infections, diabetes, and others. 43 NEDD8 Within the family of Ubls, the 76 amino acid NEDD8 (neural precursor cell expressed, developmentally downregulated 8) has the highest sequence identity with Ub (B59%), yet it possesses its own unique set of enzymes that ensure distinct conjugation pathways (Fig. 2 and Table 1). 44 NEDDylation occurs via activation of NEDD8 by E1 (NAE1: APPBP1-Uba3 dimer), transfer to E2 (Ubc12), which is known to be highly specific and achieved by the presence of Ala72 in NEDD8 that allows the specific activation by the E1 and the consequent interaction of the N-terminus of E2 (Ubc12). The final step is a covalent conjugation to the substrate by an E3 ligase (RBX1, RBX2, and others). 45 Like the majority of Ubls, NEDD8 is synthesized with a Cterminal tail that is cleaved by specific C-terminal hydrolases known as NEDP1, DEN1, SENP8, and the Ub hydrolase UCH-L3, to expose the diglycine motif, through which NEDD8 is covalently linked to different substrates. 46,47 Like ubiquitination, NEDDylation is a reversible process where the proteases Table 1 Summary of enzymes involved in Ubls conjugation and deconjugation and the chemical synthesis approachesused for their preparation in the free form or as conjugates Family name Enzymatic machinery Proteases Chemical synthesis approaches and applicationsE1 E2 E3 SUMO family SUMO-1 SUMO-2 SUMO-3 SUMO-4 SUMO-5 SAE1 SAE2 UBC9 RanBP2 PIAS 1–4 ZNF451 Others SENP 1–3 SENP 5–7 DeSI-1,2 USPL1 NCL (SEA thioester): SUMO-1, SUMO-1-P53 peptide conjugate, SUMO-2, SUMO-3, SUMO-2 dimer, SUMO-3 dimer KAHA ligation: SUMO-2, SUMO-3 Direct SPPS (aggregation breaker): SUMO-2, SUMO-3 NCL (Phcam linker): di-Ub(K63)-Lys11-SUMO-2, di-Ub(K63)- Lys33-SUMO-2 and di-Ub(K63)-Lys42-SUMO-2) Click chemistry: SUMO-1-RanGAP-1, SUMO-1-Ubc9, SUMO-2 -PML peptide conjugates NEDD8 NAE1 UBC12 RBX1/2 DCN1 NEDP1 NCL ([Pd(allyl)Cl] 2 ): NEDD8-cullin peptide Others DEN1 KAHA ligation SENP8 Direct SPPS (backbone amide propargylation) UFM1 UBA5 UFC1 UFL1 UFSP1 KAHA ligation UFSP2 ISG15 UBE1L UBCH8 HERC5 USP18 NCL UBA7 HERC6 NCL (Acm-NMe 2 ): ISGylated-Ub EFP ATG8 family ATG7 ATG3 ATG5 ATG4 EPL: lipidated-LC3 LC3A ATG10 ATG12 LC3B ATG16 LC3B2 Complex GABARAP GABARAPL1 GABARAPL2 ATG12 FAT10 Uba6 USE1 Parkin Not reported Not reported URM1 MOCS ? ? Not reported NCL (Cys alkylation to mimic Gln (C-Gln)) Fig. 3 The reversible SUMOylation pathway, illustrating the covalent attachment and removal (deSUMOylation) of SUMO from its targets. This process comprises three key steps: E1 activation, E2 conjugation, and E3 ligation. The enzymes participating in each step are highlighted, along with the SUMO-specific proteases (SENP) responsible for deSUMOylation. Additionally, the distinct SUMO tails are depicted. Review RSC Chemical Biology Open Access Article. Published on 12 February 2025. Downloaded on 11/6/2025 8:09:08 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. View Article Online 496 | RSC Chem. Biol., 2025, 6, 492–509 © 2025 The Author(s). Published by the Royal Society of Chemistry NEDP1/DEN1/SENP8 promote deconjugation of NEDD8 from its target (Table 1). 46,47 The most well-characterized substrates for the NEDD8 are the cullin protein family, which serves as a scaffold for Ub ligase complexes and promotes ubiquitination and proteasomal degradation. The cullin family regulates proteins involved in the cell-cycle, transcription, signal transductions, regulation of O 2 , centrosomes, and cytoskeleton. 48–50 NEDDylation can also modulate P53’s stability by modifying its E3 Ub ligase Mdm2, leading to increased ubiquitination and degradation. The direct NEDDylation of P53 inhibits its transcriptional activity by prompting or inhibiting its ability to activate or repress target genes. 51 UFM1 The ubiquitin fold modifier 1 (UFM1) exhibits structural homology but lacks any obvious sequence identity with Ub. 52 Composed of 83 amino acids (Fig. 2a and b), UFM1 is synthesized as an inactive precursor which undergoes maturation by two specific proteases – UFSP1 and UFSP2, to expose its C-terminal Gly residue. 53 The covalent attachment of UFM1 to its substrates is termed UFMylation and occurs through an enzymatic cascade like Ub, activated by E1 (UBA5), transferred to an E2 (UFC1), and ligated to its substrate(s) by E3 (Ufl1) (Table 1). 53 UFM1 can form polymeric chains through any of its six Lys residues, yet poly-UFMylation is reported to proceed predominantly via Lys69. Recent evidence suggests that UFMylation is involved in endoplasmic reticulum (ER) phagy, with findings showing that UFMylation on the ER surface acts as a signal for this process. In ER-phagy damaged or excess portions of the ER are targeted for degradation, ensuring the organelle remains functional and free of accumulated damage. DDRGK1, an adapter protein of the UFMylation system, facilitates the recruitment of the UFMylation machinery to the ER surface for conjugation to various proteins embedded in the ER membrane such as RPN1, RPL26, and CYB5R3. 54,55 UFMylation of these substrates induces recruitment of the ATG8 family, initiating autophagy of the UFMylated ER. Downregulation of UFM1-mediated ER-phagy leads to ER stress and accumulation of misfolded proteins. 55 Like other Ubls, UFMylation is a reversible PTM, where UFSP2 is known to be involved in de-UFMylation. Loss of function of the UFM1 pathway is implicated in various diseases such as cancer, diabetes, schizophrenia, and ischemic heart disease. Additionally, it plays a crucial role in embryonic development and hematopoiesis due to its tight relationship with the ER stress response. 56 ISG15 Interferon-stimulated gene 15 (ISG15) is a small protein made of 157 amino acids that exists only in vertebrates, and is characterized by a unique structure wherein two Ub-like domains are linked together through a hinge region (Fig. 2a and b). 57,58 ISG15 is initially synthesized as an inactive precursor which is proposed to undergo maturation to expose the C terminal Gly residue by two specific proteases: USP18, also known as UBP43, and the Ubp1-related protein. 59 ISG15 is linked to its substrate through an ISGylation process that occurs via a known enzymatic cascade involving activation by E1 (UBA7/UBE1L), transfer to an E2 (UBCH8), and ligation to its substrate(s) by E3 (HERC5, HERC6, EFP). 60,61 The reversibility of ISGylation is achieved by USP18, which is the only protease known so far for deISGylation (Table 1). ISG15 serves a dual function: intracellularly as a protein modifier and extracellularly as a cytokine that is highly expressed and secreted upon IFN stimulation. 62 As a protein modifier, ISGylation modulates various biological processes and displays an intricate interplay with ubiquitination. While in some cases upregulation of ISGylation inhibits Ub-mediated proteasomal degradation due to competition over Ub binding domains, in other cases, ISGylation can lead to Ub-mediated proteasomal degradation. 63,64 Controversial results of the connection between ubiquitination and ISGylation suggest contextdependent outcomes. Moreover, while Ub-ISG15 hybrid chains are known to exist, their recognition by other proteins remains poorly understood (Table 1). Notably, the expression of ISG15’s E1, E2, and E3 is also highly induced by type 1 interferon (IFN), influenza B virus, lipopolysaccharide, and genotoxic stress. Aberration in ISGylation is associated with cancer, neurodegenerative diseases, and problems in response to pathogen infections, while normal ISGylation is crucial in embryonic development. 65 ATG8 and ATG12 Mammalians have six autophagy-related genes (ATG8 proteins) which are subdivided into the microtubule-associated protein light chain 3 – LC3 (LC3A, LC3B, LC3C/LC3B2) and GABARAP (GABARAP, GABARAPL1, GABARAPL2) members (Table 1). Each one of these proteins is composed of B120 amino acids (Fig. 2). 66 The ATG8 C-terminus tail is cleaved by ATG4 protease to expose the Gly residue, 67 and subsequently conjugated to its substrates through the cascade of E1-like ATG7, E2-like ATG3 and E3-like ATG12-ATG5:ATG16 complex (Table 1). 68 Formation of the E3-like complex itself requires another Ubl conjugation pathway, where the Ubl ATG12 conjugates to the Lys residue of ATG5 in sequential reactions catalyzed by the specific E1 and E2. This conjugate further reacts with ATG16 to form the E3like complex. 69 ATG8 plays a crucial role in autophagosomal membrane formation, where its C-terminus is covalently linked to the phospholipid, phosphatidylethanolamine (PE), through the enzymatic cascade described before, forming a lipidated ATG8–PE. 70 This lipidated form serves as a scaffold to recruit other autophagy-related proteins that are necessary for autophagosome formation. ATG8–PE also ensures the specificity and selectivity of proteins, organelles, and cellular components that are targeted for degradation. This interaction is generally mediated by LC3-interacting regions (LIRs), located in the unstructured region of the ATG8interacting proteins, and is composed of negatively charged amino acids followed by two hydrophobic amino acids spaced by two random residues. 70 The attachment of ATG8 proteins to RSC Chemical Biology Review Open Access Article. Published on 12 February 2025. Downloaded on 11/6/2025 8:09:08 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. View Article Online © 2025 The Author(s). Published by the Royal Society of Chemistry RSC Chem. Biol., 2025, 6, 492–509 | 497 PE is reversed by the ATG4 proteases, which regulate its turnover on the autophagosomal membrane and the autophagy dynamics. 67 Dysregulation of the ATG8–PE interaction is associated with various diseases such as cancer, infections, inflammation, and neurodegenerative disorders. 71 FAT10 Like ISG15, the human leukocyte antigen F-adjacent transcript 10 (FAT10) has a structure of two Ub-like motifs linked together through a hinge region 72 (Fig. 2a and b), therefore it is also named diubiquitin or Ub D. FAT10 consists of 165 amino acids and is expressed as a mature protein that contains a free di-Gly motif necessary for conjugation (Fig. 2). While proteolytic activation is not required for FAT10, specific proteases may play roles in its deconjugations, yet no specific proteases involved in these processes have been reported. The constitutive expression of FAT10 is restricted to immune system tissues, but its presence in other tissues can be induced by pro-inflammatory cytokines like interferon (IFN)-gand tumor necrosis factor (TNF). 73 Covalent attachment of FAT10 to substrates is termed FATylation and occurs through an enzymatic cascade like Ub, including activation by E1 (UBA6) transfer to an E2 (USE1) and ligation to its substrate(s) by E3 (suggested to be PARKIN) (Table 1). 74–76 In addition to its involvement in immune responses and inflammation regulation, FAT10 directly targets its substrates for degradation by the 26S proteasome, making it unique amongst the Ubls. 77 FAT10 is upregulated in various cancer types, such as gastrointestinal cancer, hepatocellular carcinoma (HCC), pancreatic ductal adenocarcinoma, and human glioma. 78 URM1 URM1 is among the least studied Ubls, also featuring the bgrasp fold and the C-terminal di-glycine motif (Fig. 2a and b). 79,80 URM1 was identified through BLAST analysis for prokaryotic sulfur carrier proteins, noting its high sequence similarity to the proteins ThiS and MoaD. 81 While URM1 shares similar structural features with Ub and other Ubls, it is expressed as a mature protein and is activated by an unusual mechanism leading to the formation of a unique C-terminus thiocarboxylate (–COSH). 82 The URMylation pathway starts with the adenylation of the carboxylate (–COOH) by ATP-dependent E1 (Uba4 in yeast and MOCS in humans) forming an acyl disulfide bond. URM1 is then attached to a Lys residue on the substrate forming an isopeptide bond yet without evidence for the presence of E2 and E3 enzymes (Table 1). 83 In addition to its role as a protein modifier, URM1 acts as a sulfur carrier essential for the 2-thiolation of wobble uridines (S2U34), a universal tRNA modification essential for coordinating translation and protein synthesis. 84 In yeast, peroxiredoxin Ahp1 is the most studied substrate for URM1, 85 suggesting its potential role in regulating cell redox status. This is further confirmed by the detection of 21 human proteins modified by URM1, under oxidative stress conditions. 86 Recently, it was discovered that URMylation promotes the stress-dependent phase separation of target proteins aiding in stress resilience and cell survival. 87 Since deUrmylases have not been identified yet, it remains unclear whether deURMylation occurs and if so, how this process might be reversed. Synthesis of Ubls and conjugates Since Ubls in their free forms are relatively small proteins composed of 70–150 amino acids, they are accessible through chemical protein synthesis employing either direct SPPS or ligation approaches. Furthermore, combining these approaches with the semisynthetic one could also allow for the preparation of their conjugates. Here we describe, briefly, these methods and their application for the synthesis of various Ubls in their native or modified forms and their conjugates. Readers are also encouraged to peruse other comprehensive reviews of these methodologies. 1,2,18,88,89 Native chemical ligation (NCL) Since the introduction of NCL by Kent and his coworker, this method has been widely used to prepare hundreds of native and/or modified proteins. In this approach, chemoselective ligation of two unprotected peptides, one bearing a Cterminal thioester functionality and the other an N-terminal Cys residue, are ligated to form a native amide bond. 5 Our group utilized NCL and desulfurization together with Pd chemistry to assemble a NEDDylated peptide derived from the cullin protein (Fig. 4). 90 In this study, NEDD8 was prepared in its conjugated form with the 26-mer derived from cullin1 (703–728), a known substrate for NEDDylation. NEDD8 was prepared from two segments employing one ligation step at position 57, where Ala was mutated to Cys. First, the C-terminus of NEDD8 was prepared using Fmoc-SPPS where it was directly attached to Lys720 in the cullin1’s C-terminus fragment on resin (Fig. 4a). This was achieved by using the alloc protecting group on Lys720, allowing for resin selective removal and peptide elongation to generate peptide 1(Fig. 4a), 91 which was ligated with peptide 2to form the full-length NEDD8. After assembly of NEDD8, the NEDDylated peptide was treated with [Pd(allyl)Cl] 2 , for thiazolidine (Thz) deprotection to form 3. This complex was demonstrated to be an excellent reagent for effective unmasking of Thz, enabling its removal within 15 min under NCL conditions. 90 This intermediate was either ligated with the N-terminal peptide of cullin 4, followed by desulfurization to give the NEDDylated cullin conjugate 5 (Fig. 4b, path A), or directly deprotected and desulfurized to yield NEDDylated cullin conjugate 6(Fig. 4b, path B). Conjugate 6exhibited the secondary structure known for NEDD8 and was cleaved by the known ubiquitin C-terminal hydrolase isozyme 3 (UCH-L3). 92 It should be noted that UCH-L3 is not the endogenous enzyme responsible for removing NEDD8 from cullins and was used solely as a model to provide evidence on the integrity of our synthetic conjugates. Review RSC Chemical Biology Open Access Article. Published on 12 February 2025. Downloaded on 11/6/2025 8:09:08 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. View Article Online 498 | RSC Chem. Biol., 2025, 6, 492–509 © 2025 The Author(s). Published by the Royal Society of Chemistry Melnyk’s group reported a synthetic approach for the preparation of SUMO-1, using the bis(2-sulfanylethyl) amido (SEA) thioester surrogate (Fig. 5a). 93 SUMO-1(2–50) 7and SUMO1(51–97) 8bearing a SEA off (the cyclic disulfide form) were used for the assembly of full-length SUMO-1 9(Fig. 5a). Nto Csequential ligation was initiated by thioesterification of the SUMO-1(2–50)-SEA off 7by mercaptopropionic acid at pH 4 in the presence of TCEP, followed by NCL with SUMO-1(51–97)- SEA off 8to give 9. The full-length SUMO-1-SEA off 9was further activated to SEA on allowing further attachment to a model peptide featuring the SUMO consensus motif CKX(E/D). The Lys residue in this peptide was modified with Cys to facilitate the SEA ligation with SUMO-1 (Fig. 5a). The folded domain of SUMO-1 in the synthetic conjugate 10 was confirmed by CD spectrum and the cleavage assay using Ulp1, a known Cys protease. Using this strategy, the group prepared a SUMO1-P53 protein–peptide conjugate employing a one-pot approach based on three segments (7,8, and P53 peptide). The SUMO-1-SEA off 9 thioester was assembled using a single NCL reaction and further conjugated to P53 peptide through d-mercapotlysine residue (Fig. 5b). 94 To study the effect of Cys52 in SUMO-1 properties, the wild-type and Cys52Ala SUMO-1 analogs were prepared by selective and non-selective desulfurization, controlled by the absence or presence of denaturants in the reaction. These synthetic conjugates helped elucidate the important role of Cys52 in maintaining SUMO-1’s structure, thermal stability, and functionality. SEA linker was also employed for the preparation of SUMO-2 and SUMO-3 employing SEA-mediated ligation. 95 The sequence homology and the inability to distinguish between SUMO-2 and -3 encouraged Melnyk and coworkers to investigate the role of the conserved Cys residue on SUMO-2 and SUMO-3 domain’s stability and properties. Cys to Ala mutation was achieved by radical desulfurization under denaturing conditions. Both the secondary structure and the thermal stability analyses together with the conjugation and deconjugation studies revealed that mutating the conserved Cys47 in SUMO-3 must be considered with caution as the fold of SUMO-3 is significantly affected. Notably, this mutation interrupted the cleavage rate of the SUMO-3 conjugate by SENP1 and SENP2. This study highlighted that SUMO-2 and -3 are distinct proteins and should not be considered identical. Following these studies, Ovaa and coworkers employed NCL to prepare another Ubl, ISG15. ISG15 was considered as a linear dimer of two Ub-like modules and therefore it was divided into two domains at the native Cys76 which were ligated to give the full-length ISG15. 96 Recently, our group was able to access URM1 for the first time via a single NCL. 97 Since URM1 lacks a Cys residue and its Ala residues are not suitably positioned for ligation and desulfurization, an alternative method was used. Glutamine at position 32 was substituted with Cys, which was then alkylated with bromoacetamide to produce pseudo-glutamine (C-Gln), a mimic of Gln with a single atom difference (Fig. 6a). Using this approach three tetramethylrhodamine (TAMRA)-labeled URM1 analogs (Fig. 15c) were prepared containing different C-terminus modifications, carboxylic acid (URM1-COOH), hydrazide moiety (URM1-CONHNH 2 ) and deleted glycine at position 101 (URM1-DG101-COOH). All URM1 analogs were Fig. 4 Schematic presentation for (a) the synthesis of NEDD8 (57–76) attached to cullin (714–728) through Lys720, where the alloc protecting group was incorporated during SPPS. Selective Alloc removal using the [Pd(allyl)Cl] 2 complex allowed resin chain elongation of NEDD8 (57–76) to yield conjugate 1. (b) The preparation of NEDDylated cullin conjugate through NCL between (1)and(2), followed by Thz opening using [Pd(allyl)Cl] 2 . Direct ligation with cullin (703–713)-MeNbz (4) and desulfurization gave conjugate 5. Fig. 5 Schematic illustration for (a) the synthetic scheme of SUMO-1 conjugates starting from functionalized SEA-trityl-OH resin to synthesize 7and 8which further undergo sequential NCL reactions to form 10, and (b) the formation of SUMO1-P53 peptide conjugate based on the previous strategy. RSC Chemical Biology Review Open Access Article. Published on 12 February 2025. Downloaded on 11/6/2025 8:09:08 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. View Article Online © 2025 The Author(s). Published by the Royal Society of Chemistry RSC Chem. Biol., 2025, 6, 492–509 | 499 delivered into cells using a newly developed method termed suspension bead loading (SBL), requiring only small quantities of protein compared to other delivery methods (Fig. 6b). As URM1 is a synthetically challenging protein, SBL provided an efficient, economical, minimally cytotoxic delivery platform. We found that URM1 localizes mainly in the nucleolus under normal conditions and diffuses out in response to oxidative stress. Additionally, we have demonstrated that regardless of URM1’s C-terminus, its localization and degree of conjugation are oxidative stress dependent. KAHA ligation KAHA ligation was developed by Bode and his coworkers to overcome the necessity of Cys (or thiol-modified amino acids) 8 and a complementary thioester peptide in NCL. 98 This ligation occurs between a peptide bearing a-ketoacids, and a peptide with N-terminal hydroxylamine, that undergoes chemoselective and reagent-less ligation. Substituted hydroxylamine, 5oxaproline, was developed later to allow for effective ligation in acidic aqueous conditions. After ligation, the pH is adjusted to basic conditions, to facilitate an Oto N-acyl shift, leading to a homoserine residue at the ligation site (Fig. 7). 14,99 KAHA ligation was used in the preparation of several medium-sized proteins, including Ubls. For example, UFM1 was the first Ubl to be synthesized using this ligation, where three analogs with different C-terminus modifications were prepared, carboxylic acid, amide, and thioester (Fig. 7a). 100 These analogs were prepared from three peptides in two ligation steps, where the ligation sites were selected to be phe29Thr30 and Ala60-Gln61. 5-oxaproline (Opr) was introduced for sequential KAHA ligation in peptides 11 and 13, where Fmoc protection was needed for the middle peptide 11 which was also equipped with a-ketoacids. The N-terminal peptide 12 was also synthesized with the a-ketoacid functionality. In addition, peptide 13 was functionalized differently at its C-terminus. Employing KAHA ligation between these fragments gave the desired protein 14 (Fig. 7a). CD analysis confirmed its secondary structure featuring multiple b-strands and a-helices. This synthetic method for UFM1, specifically the thioester analog, could potentially enable labeling with tags and site-specific conjugation to protein substrates. Notably, the KAHA ligation process at its initial development required a key step where cyanosulfurylide had to be oxidized by oxone to form a-ketoacid. However, this step was incompatible with residues such as Cys, Met, and Trp, leading to undesired oxidation. 101 Therefore, the group introduced a protected form of AA suitable for obtaining enantiopure peptide a-ketoacid 15 directly upon cleavage from resin (Fig. 7b). This method, which is compatible with all amino acids, was successfully applied to the preparation of SUMO-2/3 from three segments. 102 Biochemical studies were performed to verify the structure and function of SUMO-2/3 that have homoserine residues due to the ligation requirement. Using SENP2, as a SUMO protease, successful cleavage of SUMO-2’s tail to expose the di-Gly motif was observed. Additionally, the SUMOylation reaction on the substrate RanGAP1 demonstrated SUMO-2/3 activity. Both experiments illustrated that the homoserine residue does not affect the in vitro recognition and processing by the SUMOylation machinery. NEDD8 has also been accessed using KAHA ligation. Investigating the NEDDylation process and identifying new substrates has always been challenging due to difficulties in its expression. NEDD8 synthesis involved a newly developed photolabile protecting group incorporated in the a-ketoacid to facilitate one-pot multiple KAHA ligation (Fig. 8a). 103 The photo-protected a-ketoacid in the desired peptide was unmasked under mild conditions through irradiation at 365 nm. Three distinct strategies were applied to prepare NEDD8, with two involving a three-segment process. The first strategy proceeded from the N-to-C-direction (Fig. 8b) using the Fig. 6 Schematic representation of (a) the URM1 synthetic approach. (b) The SBl delivery method utilizes glass beads to create physical disruptions in cell membranes, allowing protein molecules to enter suspended cells with minimal stress. Fig. 7 General schematic presentation for KAHA ligation applied for both UFM1 and NEDD8 syntheses, wherein (a) UFM1 was synthesized from three segments and two NCL reactions using Fmoc-Opr 11. The ligation conditions are also presented. (b) UFM1 synthesized similarly however, aketoacid 15 was prepared on resin. Review RSC Chemical Biology Open Access Article. Published on 12 February 2025. Downloaded on 11/6/2025 8:09:08 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. View Article Online 500 | RSC Chem. Biol., 2025, 6, 492–509 © 2025 The Author(s). Published by the Royal Society of Chemistry photo-protected tyrosine a-ketoacid in peptide 16, which reacted with 17. The ligation product was directly irradiated to unmask the a-ketoacid. Peptide 18 was subsequently ligated with the C-terminal peptide 19, yielding NEDD8 (3–76) (20) (Fig. 8b). The second approach operated from the C-to-N-direction using photo-protected oxaproline. It is worth noting, that in both strategies the segments were involved in sequential onepot KAHA ligations including photocleavage of the appropriate protecting group under the ligation conditions. The third strategy is based on four segments applying three ligation steps and only one HPLC purification step. The synthetic NEDD8 was obtained in good purity and acceptable yield without intermediate handling or isolation steps. Expressed protein ligation (EPL) EPL combines synthetic peptides with a recombinant large polypeptide often bearing a thioester moiety for the assembly of full-length proteins. The thioester fragment is obtained using intein technology while the synthetic peptide is prepared chemically and may contain various chemical modifications. 104 Lipidated LC3 was prepared for the first time using a synthetic lipidated peptide and EPL to study its role in autophagosome formation. 105,106 The LC3 fragment (1–114) was fused to an intein domain and a maltose binding protein (MBP), as a solubility tag, at the N-terminus (Fig. 9). Under folding conditions, the semisynthetic LC3(1–114)-thioester was ligated to the lipidated peptide in the presence of 4-mercaptophenylacetic acid (MPAA) as a thiol additive (Fig. 9), followed by the removal of the MBP tag using TEV protease. In vitro activity of the semisynthetic LC3-PE was assessed using an ATG4 cleavage assay, demonstrating cleavage within 1 h. Furthermore, the function of LC3-PE in membrane tethering and fusion was also examined to underscore the significance of LC3 lipidation for membrane association and fusion promotion. Additionally, mutants at the C-terminus of LC3 assisted in understanding the structure–function relationship of the deconjugation specificities of ATG4 and RavZ proteases. 107 Direct SPPS Although Fmoc-based SPPS is often limited to medium-length peptides (30–50 residues), Ovaa and his coworkers have succeeded in performing a direct SPPS to synthesize SUMO-1/2 and 3, without employing ligation approaches. Aggregation breakers such as pseudoproline and dimethoxybenzyl (DMB) were incorporated at different positions to improve synthesis (Fig. 10). 108 All SUMO paralogs were obtained in very good purity, featuring the correct fold. NEDD8 was also synthesized without the need for ligation reaction and/or pseudoproline dipeptides. Instead, direct synthesis was employed (71AA), combining Fmoc-SPPS and backbone amide propargylation to act as a disrupting element, which is removed on demand using AuCl (Fig. 10). 109 Click chemistry Click chemistry between azides and alkynes using the copper(I)- catalyzed azide alkyne cycloaddition (CuAAC) reaction has been widely used for bioconjugation to prepare complex Fig. 8 General schematic presentation for (a) the preparation of the photo-protected a-ketoacid and (b) NEDD8 synthesis through Nto CNCL reaction, using three segments and two ligations where photoprotected a-ketoacid was introduced to peptide 16. Fig. 9 EPL strategy for the preparation of lipidated LC3, where intein based technology and Fmoc SPPS were used. CBD refers to the chitin binding domain and MBP refers to maltose-binding protein (cleaved by TEV). RSC Chemical Biology Review Open Access Article. Published on 12 February 2025. Downloaded on 11/6/2025 8:09:08 AM. 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