Chemical Strategies for Controlling Sulfation in Biomacromolecules

Abstract

Sulfation is a fundamental post-translational modification that imparts negative charge and structural complexity to biomolecules, thereby regulating molecular recognition, signaling, and homeostasis across all domains of life. Yet, the ability to interrogate the biological functions of sulfation has long been hindered by the difficulty of constructing molecules with defined sulfation patterns. This Account summarizes our efforts to develop chemical strategies that enable precise control over sulfation in glycans and proteins. We describe an organobase-promoted sulfur(VI) fluoride exchange (SuFEx) chemistry that allows early-stage, chemoselective O-sulfation across a broad substrate scope, providing a general solution to sulfate installation in complex settings. Building on this foundation, we introduce an iterative “clickable disaccharide” platform for the programmable assembly of sequence-defined heparan sulfate glycomimetics, enabling systematic dissection of sulfation-dependent glycan–protein interactions. Extending these concepts to the protein realm, we developed a fluorosulfate tyrosine strategy that installs latent sulfates into peptides and proteins, which can be unmasked under physiological conditions or light control via hydroxamic acid-mediated Lossen rearrangement, offering spatiotemporal control of sulfation in living systems. Collectively, these approaches delineate a unified chemical framework for constructing and manipulating sulfated biomacromolecules with molecular precision, opening new opportunities to elucidate and engineer the biological roles of sulfation.

Graphical Abstract

1. INTRODUCTION

Sulfation is a fundamental post-translational modification that introduces structural and functional diversity into biomolecules through the covalent addition of sulfate groups. Sulfation profoundly alters the biophysical properties of biomolecules such as proteins, glycans, lipids, and small molecules in critical molecular characteristics including charge distribution, hydrogen bonding potential, and molecular recognition surfaces, thereby influencing the way biomolecules fold, interact, and localize. As a result, sulfation plays a vital role in the regulation of cellular functions. Sulfation occurs across virtually all domains of life, reflecting its deep evolutionary conservation and functional necessity. While often overshadowed by other negatively charged modifications such as phosphorylation, sulfation is also modulated by unique molecular mechanisms that act with precision to orchestrate context-dependent biological outcomes (Fig. 1).

Figure 1.

Sulfation in nature and its biological significance. a) Sulfotransferases in the Golgi apparatus transfer sulfate from the universal sulfate donor PAPS to a variety of biologically important molecules, including carbohydrates, lipids, and proteins; representative sulfated small molecules and a sulfotyrosine-containing protein (HCII) are shown. b) Sulfated molecules at the cell surface and in the extracellular space participate in key biological processes, such as cell signaling, viral infection, and immune responses. Created in BioRender. Niu, J. (2025) https://BioRender.com/i56ub84. c) Sulfation has also been reported to occur histone H3, and is implicated in transcriptional regulation.

The specificity of sulfation is mediated by an array of sulfotransferase enzymes, which catalyze the transfer of sulfate groups from the universal donor 3’-phosphoadenosine-5’-phosphosulfate (PAPS) to a diverse set of substrates in the Golgi apparatus, providing sulfated glycosaminoglycans (GAGs), steroids, liposacchatides, and proteins (Fig. 1a). These enzymes exhibit both tissue-specific expression and substrate selectivity, enabling fine-tuned regulation of sulfation of biomolecules. For example, GAGs regulate cell adhesion, migration, morphogen gradient, and intracellular signaling through sulfation-dependent interactions with growth factors and chemokines (Fig. 1b).^1–5 In host-pathogen interactions, sulfated GAGs and glycoproteins often serve as high-affinity binding partners for viral receptors, facilitating viral entry and tropism. Through these diverse contexts, sulfation acts as a dynamic and spatially resolved determinant of biological signaling. Due to their important roles in cell physiology, dysregulation of sulfation directly contributes to disease. In cancer, aberrant sulfation of proteoglycans has been shown to reshape the extracellular matrix, alter cell signaling, and promote metastatic behavior.^6–9 In neurodegenerative disorders such as Alzheimer’s and Parkinson’s disease, deviations in sulfation patterns of proteins and GAGs within the neural extracellular matrix impairs synaptic integrity, protein clearance, or neuroimmune regulation.^10–13 Tyrosine sulfation in peptides and proteins, meanwhile, mediates ligand-receptor affinities in immune recognition and platelet activation, with implications on processes ranging from leukocyte recruitment to blood coagulation.^14–16 In 2023, it was found that sulfation also occurred on histone H3 proteins, regulating proper protein complexing and gene transcription (Fig. 1c).¹⁷ Importantly, sulfation defects often manifest as both contributors to disease development and as biomarkers of diseased state, suggesting that controlling sulfation in biomolecules may offer utilities in both mechanistic biology and clinical applications.

Recent advances in proteomics, structural biology, and chemical probes have enabled increasingly detailed interrogation of sulfation-dependent processes. With the development of methods for synthesizing biomolecules with precise sulfation patterns at the level of individual residues and saccharide units, it is now possible to correlate the function of specific sulfation patterns in biomolecules with defined biological phenotypes.¹⁸ As a result, not only the rules that govern sulfation site specificity and turnover, but also the higher-order organization of sulfated domains in complex biomolecular assemblies and aggregates can be studied. New insights have made it increasingly clear that sulfation is not a static structural feature; rather in many contexts it is dynamically regulated. Therefore, new tools that allow tight spatial and temporal control of sulfation, particularly in tissues where rapid or localized signaling are urgently needed. Such new tools will provide a deeper understanding of sulfation, including its enzymatic logic, regulatory inputs, and pathophysiological consequences, essential for decoding its role in human health and for unlocking therapeutic strategies that restore or mimic its function in disease settings.

In this Account, we will provide an overview of our work in the context of the recent advances in the chemistry and chemical biology strategies for reproducing and engineering biomolecules with defined sulfation patterns. Rather than attempting a comprehensive review, we will focus on our perspectives in (1) synthetic analogs of GAGs with diverse defined sulfation patterns and (2) tools that enable spatiotemporal control over peptide and protein sulfation. Readers are directed to recent excellent reviews for other important aspects of sulfation control in biomolecules.^19–24

2. CONTROLLING SULFATION IN GLYCOSAMINOGLYCANS

2.1. Origins of sulfation patterns of GAGs

Naturally occurring polysulfated GAG glycans are synthesized in the Golgi apparatus through a non-template-driven process, resulting in extensive structural and sulfation heterogeneity.^{25, 26} Prominent examples include heparan sulfate (HS), heparin (HP), chondroitin sulfate (CS), dermatan sulfate (DS), and keratan sulfate (KS) (Fig. 2a–d). These sulfated glycoconjugates play critical roles in diverse physiological and pathological processes, such as cell–cell communication, regulation of cell fate, viral attachment and entry, and modulation of immune responses, including inflammation.²⁷ While sulfation of glycans is predominantly associated with the Golgi apparatus, emerging evidence suggests that sulfated glycans may also exert significant functions within the cytoplasm, influencing various intracellular processes.²⁸ Given the myriad critical functions and structural complexity sulfated GAGs, systematic evaluation of the structure-activity relationship of GAGs is of key significance. However, the high degree of complexity and heterogeneity of the naturally occurring GAGs make them too convoluted for detailed structure-function studies, and the synthesis of GAG molecules or their mimetics with well-defined skeletons and sulfation sites is desirable. The complexity and heterogeneity of GAGs arise from two main sources: the polysaccharide backbone and its sulfation patterns.^{29, 30} Unlike nucleic acids and proteins, construction of the GAG backbone and sulfation patterns is not template-directed. Instead, chains are assembled in the Golgi apparatus by sets of processing enzymes whose activities are governed by their intrinsic specificities and kinetics, as well as the local cellular environment.³¹ In the same compartment, sulfation is introduced by sulfotransferases in a similarly non-templated manner. Both features generate highly heterogeneous GAG populations and make rigorous structure–activity relationship (SAR) analysis challenging. Meanwhile, sulfate modifications can be removed by extracellular sulfatases (SULF1/2) for selective removal of 6-O-sulfate and lysosomal sulfatases for sequential desulfation during turnover, enabling continuous remodeling (Fig. 2e).^32–34 It is noteworthy that cell-based glycan arrays have also been developed to probe heparan sulfate (HS) structure–function relationships in the cellular context, using genetic perturbations in cell lines and providing a platform that presents diverse GAG structures.^35–38 For recent reviews, please see the references.^{39, 40} Here, we will focus on the chemical and chemoenzymatic methods.

Figure 2.

Sulfated GAG glycans in human and their repeating unit. a–c) Representative repeating disaccharide units of the principal sulfated GAG families found in humans: a) heparan sulfate/heparin (HS/HP), b) chondroitin sulfate/dermatan sulfate (CS/DS), and c) keratan sulfate (KS). d) General modes of sulfated GAG presentation. e) Schematic biosynthetic pathway highlighting linker assembly, chain elongation/epimerization, and stepwise sulfation to generate structurally diverse sulfated GAG domains. Created in BioRender. Niu, J. (2025) https://BioRender.com/v6q8pnt.

2.2. Chemical Synthesis

2.2.1. Synthesis of sulfated disaccharides and short oligosaccharides

Challenges in GAG synthesis largely stem from the installation of defined glycosidic linkages and the preparation of oligosaccharides with precise backbone sequences and sulfation patterns. For example, incorporation of L-iduronic acid (IdoA) residues often requires multistep, carefully orchestrated synthetic routes.^41–43 A second major hurdle is stereocontrol in glycosidic bond formation. Formation of 1,2-cis linkages from glucosamine donors is particularly demanding because neighboring-group participation cannot be exploited to direct stereoselectivity.^{44, 45} In addition, the development of efficient, regio- and chemoselective sulfation methods remains crucial for accessing well-defined GAG structures.^46–48 To date, chemical synthesis of sulfated glycosaminoglycan (GAG) oligosaccharides can be divided into convergent and iterative synthetic approaches (Fig. 3).⁴⁹ Within these paradigms, improved donors/activators, protecting-group strategies, and sulfate-installation protocols now enable routine preparation of sequence-defined sulfated glycans..^{19, 50–52} A prominent example is fondaparinux, a synthetic pentasaccharide approved by the FDA in 2001 and one of the most synthesized sulfated oligosaccharides (Fig. 4).^{53, 54} In a notable work in 2019, Zhao and coworkers reported a one-pot glycosylation method for quick access to fondaparinux (Arixtra) in a preactivated and highly stereoselective manner.⁵⁵ They first accessed three glucosamine building blocks (2–1, 2–2, and 2–3) using classical thioglycoside donors, followed by their assembly into higher-order structures, thereby significantly simplifying the overall preparation (Fig. 4, Route A). While in another notable work in 2021, the Hotha group reported the stepwise synthesis of the target, preparing five strategically designed monosaccharide building blocks(2–4 to 2–8) from common precursors, which were then sequentially coupled under silver-assisted gold-catalyzed conditions (Fig. 4, Route B).⁵⁶

Figure 3.

Strategies to access complex saccharides. a) Pre-built blocks (e.g., di-/trisaccharides) are united in fewer glycosylation events to reach the target length. b) Monosaccharide acceptors are iteratively glycosylated with activated donors (LG = leaving group) to extend the chain one residue at a time in the chemical synthesis of the sulfated glycans.

Figure 4.

Synthetic strategies to access Fondaparinux via Route A: convergent synthesis or Route B: iterative synthesis.

The synthesis of GAG glycan libraries with diverse and defined sulfation patterns is a significant goal toward the understanding of their structure-function relationship. In a landmark report in 2012, Huang et al. disclosed the synthesis of 48 HS-based disaccharides with various sulfation patterns (Fig. 5a).⁵⁷ By employing a divergent strategy from two orthogonally protected disaccharide precursors, the authors synthesized all 48 possible heparan sulfate disaccharides through skillful control of protecting group transformations. This library was used to investigate glycan–protein interactions with fibroblast growth factor-1 (FGF-1),^{58, 59} revealing specific sugar–protein binding motifs. Progressing from disaccharide to tetrasaccharide building blocks and utilizing a divergent modification strategy from orthogonally protected intermediates, Huang and Hsieh-Wilson groups independently created a complex library of 64 HS oligosaccharides, exhibiting distinct sulfation patterns at N-, 2-O-, and 6-O-positions (Fig. 5b).^{60, 61} Their streamlined approaches leveraged a fluorinated tag at the chain end for rapid purification of intermediates,⁶² accelerating the synthesis process and enabling high product purity. These works revealed the critical sequence-specific features of sulfated glycans for binding and molecular recognition towards FGFs and chemokines.⁶³ In another notable strategy, the Gardiner group reported an efficient two-step iterative assembly procedure for the preparation of a heparin dodecasaccharide by repeatedly linking pre-made tetrasaccharide units as building blocks. This method leveraged the incorporation of a latent aldehyde tag at the chain end as a generic group for labelling/conjugation (Fig. 5c).⁶⁴ Using this strategy, in 2015 the Gardiner group reported the iterative synthesis of heparin oligosaccharides ranging from 16 to 40 monomeric units in length.⁶⁵

Figure 5.

Different strategies to assemble sulfated saccharide libraries. a) Divergent synthesis of 48 heparan sulfate disaccharides. b) Divergent modification of 64 orthogonally protected tetrasaccharides. c) Tetrasaccharide-based approach for assembling heparin oligosaccharides with different lengths and patterns. NEt₃: triethylamine; Py: pyridine.

Despite these advances, the synthesis of longer oligosaccharides remains a considerable challenge, as biologically active sulfated GAGs glycans typically require domains of pentasaccharide or larger. Traditionally, sulfated GAGs have been prepared via late-stage sulfation (Fig. 5 and Fig. 6a). After introduction of orthogonal protecting groups and assembly of the glycosidic backbone, the desired hydroxyl or amino groups are selectively deprotected and then sulfated, typically using sulfur trioxide–amine complexes at the final stage of the synthesis. However, these late-stage sulfation approaches frequently suffer from harsh reaction conditions, the need for excessive amount of sulfation reagents (≥ 5 equivalents per sulfonation site), difficulty in purification, and the requirement of repetitive treatment if multiple sulfates are installed.^{66, 67} An alternative “early-stage sulfation” strategy involves the pre-installation of the sulfate groups onto monosaccharide building blocks before backbone glycosylation. While this approach simplifies downstream processing, the negatively charged sulfate group is generally incompatible with the glycosylation chemistries and protecting groups for sulfate are required.^68–74 However, early-stage sulfation approaches often suffer from low efficiency in installation, incompatibility with other protecting groups, and significantly reduced reactivity of the modified glycosyl donors/acceptors.^68–74 To tackle these challenges, our group developed an early-stage sulfation strategy based on sulfur(VI) fluoride exchange reaction (SuFEx),^75–77 in which hydroxyl groups at specific positions of a glycosyl donor/acceptor were converted into glycosyl aryl sulfate diesters via the SuFEx reaction.⁷⁸ The aryl protecting group was deprotected after the backbone assembly to generate O-sulfates in the final step (Fig. 6).

Figure 6.

Approaches for O-sulfation. a) Existing strategies. b) A general SuFEx approach for the early-stage O-sulfation in carbohydrates was developed by our group. Reproduced with permission from ref 78. Copyright 2020 John Wiley and Sons.

Using trimethylsilyl-protected galactose as a model, we identified (1,8-diazabicyclo(5.4.0)undec-7-ene (DBU) or triazabicyclodecene (TBD) as efficient organobase catalysts for SuFEx coupling with electronically diverse aryl sulfonyl fluorides, providing the corresponding aryl sulfate diesters in high yield. We found that the electron-deficient fluorosulfates are more susceptible to nucleophilic attack and therefore more reactive, giving substantially higher yields than electron-rich fluorosulfates. Furthermore, hexamethyldisilazane (HMDS) streamlines a one-pot silylation: a SuFEx sequence that converts free alcohols directly into aryl sulfate diesters within 2–3 hours and scales to gram quantities. This protocol generalizes across multiple substrate classes. In carbohydrates, we routinely install one or two sulfate units at defined positions on mono- and disaccharides and then carry those masked sulfates through assembly. Beyond sugars, steroids and amino-acid derivatives such as tyrosine tolerate the sequence, allowing late-stage installation of sulfate handles on peptide or steroidal frameworks without extensive route redesign. We propose the introduction of the sulfate diester onto sugar is not through a previously proposed concerted mechanism but through a six-membered ring system catalyzed by TBD, which was supported by another report using Barton’s base for SuFEx reaction (Fig. 7).⁷⁹

Figure 7.

Proposed mechanism of O-Sulfation of carbohydrates via SuFEx.

The aryl sulfate diester protecting group displays notable compatibility with acidic, basic, oxidative, and reductive conditions encountered in complex molecule synthesis. For example, tetrabutylammonium fluoride removes silyl groups on sugars within minutes without perturbing the sulfate diester—an advantage over classical 2,2,2-trichloroethyl sulfate protections.^{80, 81} Crucial for carbohydrate assembly, common glycosylation modalities (bromide, fluoride, phosphate, thioglycoside, and sulfoxide donors) proceed in the presence of aryl sulfate diester modification, enabling efficient preparation of mono- and disulfated disaccharides. Thioglycoside donors can be converted into sulfoxides to provide a robust donor that supported high-yield couplings.

Both hydrolytic and hydrogenolytic conditions selectively remove the aryl group to reveal the sulfate: base-promoted hydrolysis benefits from electron-withdrawing aryl substituents, or mild hydrogenolysis that proceeds efficiently across substitution patterns. For hydrogenolysis, experimental evidence suggests a mechanism involving an oxidative insertion of palladium followed by reductive elimination to result in the sulfate group, thereby revising the previous premise that catalytic hydrogenolysis converted the aryl ring to base-labile cyclohexyl group (Fig. 8).⁸² The hydrogenolysis procedure is highly scalable, and milligram- to gram-scale quantities of site-defined sulfated molecules could be readily generated from a simple reaction workup. From a practical bench perspective, keeping the sulfate masked improves not only chemoselectivity but also day-to-day workflow: intermediates behave like neutral organic compounds and can be readily purified by flash column chromatography. The strongly anionic functionality appears only when needed for the final structure or for biological evaluation.

Figure 8.

Potential pathways for the deprotection of aryl glycosyl sulfate diester via hydrogenolysis. a) Previously proposed mechanism. b) Our evidence-supported mechanism. Reproduced with permission from ref 78. Copyright 2020 John Wiley and Sons.

This SuFEx-based strategt provides a practical, chemoselective, and scalable approach to sulfated biomolecules. Conceptually, treating the aryl sulfate diester as a charge-masked “linchpin” lets us decouple molecular construction from the burdens of handling highly anionic intermediates. We anticipate that this strategy will accelerate systematic structure–activity studies of sulfation patterns in glycobiology and chemical biology, enable preparation of reference standards and probes, and open late-stage editing opportunities on complex scaffolds where sulfate installation is best reserved for the finish line.

2.2.2. Iterative Synthesis

The rapid evolution of iterative glycan synthesis with control over the regio- and stereoselectivity in glycosidic bond formation and the ability to customize monosaccharide sequence has enabled automation in glycan synthesis (Fig. 9).^{83, 84} The iterative synthesis of oligosaccharides involves a systematic sequence of organic reactions, where substrate molecules progressively elongate with each cycle, while the functional groups driving the growth are regenerated to enable continuous iteration.⁸⁵ Historically, the synthesis of oligosaccharides containing defined sulfation patterns required labor-intensive operations, involving assembly of the backbone via glycosylation, and site-specific deprotection followed by sulfation. The glycosylation step represents one of the most significant challenges in the overall synthetic pathway. Recent advancements, however, have introduced innovative strategies to significantly improve the efficiency of the glycosylation reactions to connect monosaccharide units through non-native linkages.^86–88

Figure 9.

Solution-phase routes to the sulfated pentasaccharide Fondaparinux: from one-pot synthesis to automated assembly. a) Chemical structure of the anticoagulant pentasaccharide Fondaparinux. b) One-pot solution-phase synthesis of Fondaparinux, providing 38.5 mg of product in 27% overall yield. c) Automated glycan assembly of the protected Fondaparinux pentasaccharide, affording 1.06 g of target compound in 62% overall yield. Created in BioRender. Niu, J. (2025) https://BioRender.com/9z0fxxa.

2.2.3. Automated glycan assembly (AGA)

The introduction of automated synthesis platforms offers the potential to dramatically improve the efficiency, scalability, and precision of oligosaccharide production. The automated glycan assembly (AGA) technology, as a next-generation platform, is promising in simplifying the synthesis of sulfated glycans (Fig. 9).⁸⁹ For example, adapting peptide synthesizer system to carbohydrate assembly via repetitive glycosylation and deprotection cycles, Seeberger et al. achieved rapid and efficient synthesis of complex oligosaccharides.^{90, 91} This approach has already enabled the construction of several complex and lengthy polysaccharides⁹², assembling the oligosaccharide backbone on solid-phase resin, followed by cleavage from beads and sulfation and global deprotection in solution.⁹³

Notably, in 2013, the Seeberger group reported the automated synthesis of a chondroitin sulfate hexasaccharide using a photocleavable linker, achieving sequential addition of glycosyl phosphates and automated sulfation with overall yields of 13% and 8% over 16 steps.⁹⁴ This work marked a significant step forward in integrating sulfation into the automated assembly process. Subsequently, the same group reported the synthesis of DS and KS oligosaccharides using similar approaches with distinct building blocks.⁸⁹ In 2024, the Seeberger group also reported an automated glycan assembly to produce fucoidan oligosaccharides, reaching lengths of up to 20-mers, with diverse branching patterns, and up to 11 sulfate esters.⁹⁵ The ease of purification and its readily programmable characteristics provides several advantages over the solution-phase assembly. For example, with a photolabile 2-nitrobenzyl type linker and judiciously chosen protecting groups on the modules, linear glycan equipped with various sulfation patterns was quickly made by AGA with differentiated procedure applied (Fig. 10).⁹⁶ Additionally, the terminal amine tail on the reducing end is particularly suitable for biological interaction studies like microarrays. One noteworthy example among these synthesized glycans is the KS tetrasaccharide III, which exhibited specific binding with adeno-associated virus AAVrh10, representing a potential glycan acceptor.

Figure 10.

On-resin synthesis of KS oligosaccharides. a) Bioactive KS oligosaccharide as target. b) Photolabile linker on beads and modular building block for AGA. c) AGA synthesis. Reaction conditions: (a) Acidic wash and glycosylation 1; (b) acidic wash and glycosylation 2; (c) Fmoc deprotection; (d) capping; (e) Nap deprotection; (f) Lev deprotection; (g) sulfation; (h) UV cleavage (305 nm); (i) NaOMe, MeOH, 40°C; and (j) Pd/C, H2, MeOH/triple-distilled water/AcOH (15:15:1).

Compared to the solid-phase strategy, solution-phase AGA strategies offer unique advantage of high efficiency and the ability to produce long polysaccharides. In 2022, the Ye group reported a remarkable 1080-mer polyarabinosides using an automated solution-phase glycan synthesizer, which exceedingly surpassed the artificial synthesis of nucleic acids and protein.^97–99 Based on their preactivation one-pot multicomponent and continuous multiplicative synthesis strategy, gram-scale (1.06 g) synthesis of a protected fondaparinux pentasaccharide in 62% isolated yield was achieved, although the final sulfation step was not included.

2.2.4. Iterative Synthesis of Sulfated Glycomimetics

Despite significant advancements in the synthesis of sulfated saccharides via both solution-phase and solid-phase strategies,¹⁰⁰ the synthesis of well-defined sulfated oligosaccharides continues to present considerable challenges. Glycosylation reactions required for the assembly of GAG backbone often yield suboptimal results, with low yields and incomplete stereocontrol necessitating continued efforts in reaction optimization. Drastically lower reactivity is often observed in the glycosylation reaction involving sulfated glycosyl donor or acceptors, due to the disarming effect of the sulfate modification. Furthermore, approaches effective for shorter oligosaccharides often require substantial adaptation for the successful construction of longer glycan chains.^101–103 In addition, with existing synthetic approaches typically geared toward specific sulfated glycan structures, it is often challenging to apply them toward the construction of libraries consisting of a large number of sulfated oligosaccharides. To address these limitations, glycomimetic oligomers and polymers have emerged as a promising solution (Fig. 11).^{104, 105} While promising, most early designs attach sulfated saccharides as side chains on synthetic polymers, or execute sulfation after polysaccharides assembled, providing limited control over sulfation sequence and spatial arrangement. To further improve the homogeneity, linking chemistries of higher efficiency compared to glycosylation, such as using the copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) (“click chemistry”) or amide bond formation, are used to assemble glycomimetic oligomers and polymers stepwise with defined sulfation patterns (Fig. 11c). In these approaches, sulfated saccharides are connected in a native-like linear “head-to-tail” fashion with a non-native linker. Such glycomimetic systems circumvent the need for repetitive glycosylation reactions, empowering researchers to rapidly access a large number of complex sulfation patterns.^106–108

Figure 11.

Representative strategies for synthesizing sulfated glycopolymer mimetics. a) Post-polymerization sulfation of pre-formed polymer backbones bearing reactive handles, without control over sulfation pattern. b) Polymerization to form polysaccharides with diverse linkages followed by sulfation. c) Iterative synthesis of sugar motifs to form polysaccharides with unnatural linkers.

In 2023, our group developed an iterative assembly platform that enables the synthesis of sequence-defined HS glycomimetics with precise sulfation control (Fig. 12).¹⁰⁹ Our approach uses five modular “clickable” disaccharide building blocks that faithfully mimic the iduronic acid–glucosamine repeat unit of native HS, with programmable sulfation at N-, 2-O-, and 6-O-positions. Each disaccharide carries orthogonal azide and triisopropylsilyl-protected alkyne termini, enabling successive elongation via CuAAC under mild conditions that preserve sulfate integrity. This design also allows incorporation of non-natural triazole linkers, expanding structural diversity beyond the native glycosidic framework.

Figure 12.

Sequence-defined HS-mimetics via iterative assembly of variably sulfated clickable disaccharides. a) Chemical structures of natural HS and the HS glycomimetics. b) The iterative assembly of the clickable disaccharides via CuAAC click chemistry. Reproduced with permission from ref 109. Copyright 2023 Royal Society of Chemistry.

Starting from Cbz-protected alkyne initiators, we established efficient routes to a panel of variably sulfated disaccharides (Fig. 13a), ensuring compatibility with iterative coupling–deprotection cycles. Using these modules, we constructed a library of 16 HS-mimetic oligomers, including disaccharides, tetrasaccharides and octasaccharides, with defined sequences and sulfation patterns (Fig. 13b). Uniquely, these synthetic oligomers can be fully sequenced by negative electron transfer dissociation (NETD) tandem mass spectrometry, which preserves sulfate groups and pinpoints their positions at the residue level—capabilities rarely accessible in HS glycomimetic analysis (Fig. 13c).

Figure 13.

Assembly and MS/MS sequencing of the HS-mimetic oligomers. a) Structures of variably sulfated clickable disaccharides. b) Sequences of the HS-mimetic oligomer library. A native HS hexasaccharide is used as a control in the SPR competitive binding assay. c) NETD MS/MS sequencing of tetrasaccharide H allowed for confident assignment of the sulfation pattern. Reproduced with permission from ref 109. Copyright 2023 Royal Society of Chemistry.

The biological relevance of these constructs was established through glycan microarray and surface plasmon resonance (SPR) binding assays with fibroblast growth factor 2 (FGF2), a prototypical HS-binding protein (Fig. 14). The results revealed a clear correlation between sulfation pattern and binding affinity: oligomers bearing more sulfation density and specific sulfation patterns exhibited the strongest binding, in some cases matching the potency of a native HS fragment.

Figure 14.

Characterization of the interactions between the HS-mimetic oligomers and FGF2. a) Workflow of the glycan microarray experiment and its binding profile of the HS-mimetic oligomer library microarray at 100 μM and 300 μM (n = 4). The concentration of FGF2 was 5 μg/mL. P: Buffer only. b) Workflow of the SPR experiment and the FGF2 binding of the HS-mimetic oligomers and native HS hexasaccharide in competition with the immobilized heparin in the SPR experiment. The results are fitted to the competitive inhibition model. Reproduced with permission from ref 109. Copyright 2023 Royal Society of Chemistry.

Molecular dynamics (MD) simulations provided complementary structural insights, indicating that the triazole spacer increases local flexibility and stabilizes conformations favorable for FGF2 engagement. Simulations further confirmed that high-affinity mimetics and native HS share similar sulfation-dependent binding modes, with adjacent N- and 2-O-sulfates serving as dominant interaction sites. These findings illuminated how these synthetic constructs closely recapitulate native HS recognition while offering greater structural versatility.

In summary, this iterative “clickable disaccharide” strategy delivers a scalable and programmable route to HS glycomimetics, enabling systematic interrogation of sulfation-dependent structure–function relationships and opening access to chemical space beyond what is found in natural glycosaminoglycans for both fundamental studies and translational applications.

2.3. Chemoenzymatic Synthesis

Compared to the chemical synthesis strategies, chemoenzymatic synthesis is a promising approach to sulfated glycans, achieving high efficiency and high specificity and requiring no protecting groups.^{21, 110, 111} Chemoenzymatic approaches have been developed to generate GAG backbones and sulfation patterns in vitro. These strategies exploit glycosyltransferases and sulfotransferases to introduce sulfate groups in a protecting-group-free and highly regioselective manner, making them well suited for the synthesis of oligosaccharides with defined backbone sequences and sulfation motifs. By reconstituting and engineering components of the natural biosynthetic pathway, sulfation can be installed in a programmed order: typically starting with N-sulfation and 2-O-sulfation and followed by 6-O-sulfation and 3-O-sulfation (Fig. 15a).^{112, 113} Liu and his collaborators paved the way to efficient construction of heparin pentasaccharide with ultralow molecular weight (ULMW) with minimum artificial protecting group manipulation (Fig. 15b).¹¹⁴ Two heparin ULMW heparin constructs 1 (MW = 1778.5) and 2 (MW = 1816.5) with discrete sulfation patterns were prepared in 10- and 12-steps with 45% and 37% overall yields, respectively. In comparison, chemical synthesis of fondaparinux, a pentasaccharide HS structure, required approximately 50 steps with an overall yield of ~0.1%.¹¹⁵ The molecules prepared by the chemoenzymatic approach demonstrated excellent anticoagulant activity in the in vitro experiment as its parent structure porcine and bovine heparin.¹¹⁶ Furthermore, in a rabbit model, these molecules exhibited pharmacokinetic properties similar to fondaparinux. Liu’s group also applied the chemoenzymatic strategy for the synthesis of chondroitin sulfate E (CS-E). They constructed a CS-E 19-mer, which was shown to protect the lung tissue against systemic inflammation by targeting extracellular histone.¹¹⁷ In 2024, the Sheng group developed a system combining engineered E. coli strains and protein-engineered N-sulfotransferases (NST) to overcome limitations in microbial heparin synthesis with a 2.6-fold increase in catalytic activity and 11.3-fold enhanced stability compared to wild-type enzymes.¹¹⁸ The Huang group applied a solid-phase-supported chemoenzymatic strategy to efficiently synthesize chondroitin sulfate proteoglycan (CSPG) glycopeptides.¹¹⁹ By immobilizing peptides on Sepharose and sequentially applying glycosyltransferases and sulfotransferases, they directly construct native tetrasaccharide linkages and extended CS chains without relying on glycosylated amino acid precursors. The method used a mild-cleavable squarate linker and reduced synthetic complexity, highlighting the utility of chemoenzymatic approaches for accessing well-defined, functionally relevant glycopeptides. Although current chemoenzymatic methods typically produce tens of milligrams of the target compounds, this rapidly evolving approach has great potential for scalable and broad substrate production.

Figure 15.

Chemoenzymatic synthesis of O-sulfated glycans. a) Sequential installation of O-sulfate groups on a defined heparan sulfate backbone using recombinant sulfotransferases. b) Chemoenzymatic synthesis of ULMW heparin.

3. CONTROLLING SULFATION IN PEPTIDES AND PROTEINS

Sulfation of the phenolic side chain of tyrosine residues to form sulfotyrosine (sTyr) is a widespread post-translational modification of extracellular and integral membrane proteins, influencing the activities of these proteins in cellular adhesion, blood clotting, inflammatory responses, and pathogen infection (Fig. 16).^{120, 121} However, purification of individual sulfoforms from natively heterogeneous sulfoproteome is impractical, given the high diversity and heterogeneity of the sulfoproteins and the large number of different sulfoforms of each sulfoprotein. To elucidate the influence of sulfation at various sites, homogeneously sulfated proteins (or peptides) were prepared through either chemical synthesis or genetic code expansion techniques.

Figure 16.

Schematic representation of protein sulfation processes in endothelial cells and their biological roles. Within endothelial cells, proteins synthesized in the endoplasmic reticulum are modified by tyrosyl protein sulfotransferase (TPST) in the trans-Golgi network, utilizing ATP to add sulfate groups. Sulfated proteins are directed toward secretory pathways, membrane incorporation, or intracellular signaling. These sulfated proteins are critically involved in virus infection, inflammation, and signal transduction pathways.

3.1. Chemical Synthesis

Chemical synthesis is a straightforward pathway for the investigation of the structure–activity relationship of sulfopeptides and sulfoproteins both in vitro and in vivo.¹²² For example, through solid-phase peptide synthesis (SPPS), the Payne group developed a strategy that involves two chemically synthesized sulfopeptide fragments, one of which contains a C-terminal thioester and the other one contains an N-terminal cysteine residue. The two fragments then underwent transthioesterification and S to N acyl shift to form native amide bond. Using this approach, they obtained larger sialoproteins-serial hirudin P6 variants. After testing folding and thrombin inhibition, they found that tyrosine sulfation, along with O-glycosylation, is important for thrombin inhibitory activity. Besides salivary sulfated peptides and sulfoproteins for blood clotting, the sulfation modifications are also found in the chemokine receptors such as CXCR4, glyco-CCR5 and glycol-CCR7.¹²³ The Payne group’s work demonstrated that chemokine dimerization can be manipulated by an allosteric mechanism through CXCL12 binding to the sTyr21 motif in CXCR4.^{124, 125}

Traditionally, Fmoc-Tyr(SO₃^-)-OH was directly coupled into the growing peptide chain in SPPS. However, the presence of the negative charge often impedes further amino acid coupling and compromises resin swelling. Furthermore, phenolic sulfate ester is labile to acidic conditions that are inevitable in either Boc-SPPS or Fmoc-SPPS chemistries, causing unspecific removal of the sulfate group from the product.^126–129

To overcome these challenges, suitably protected sulfotyrosine has grown to be the preferred method for sulfopeptide synthesis.¹³⁰ Ali and Taylor (2009) introduced trichloroethyl (TCE) and dichlorovinyl (DCV) protecting groups to stabilize sulfate esters during solid-phase peptide synthesis,¹³¹ allowing efficient sulfotyrosine incorporation. Liu et al. (2014) employed orthogonally protected tyrosine derivatives with groups such as o-nitrobenzyl, allyl, and tert-butyldimethylsilyl, enabling site-specific deprotection and selective sulfation for synthesizing diverse sulfopeptides, including CCR5 sulfopeptide libraries.¹³² Simpson and Widlanski (2006) contributed a comprehensive approach using neopentyl and isobutyl protecting groups, which offer stability under a wide range of conditions and enable precise control of sulfate ester deprotection.⁶⁸ The Payne group reported a divergent synthesis of eight distinct forms of N-terminal C-C chemokine receptor type 5 (CCR5, 2–22), each bearing discrete sulfation at Tyr10, Tyr14, and Tyr15 with three orthogonally protected Tyr building blocks on a single resin-bound intermediate (Fig. 17).¹³² With three preinstalled o-nitrobenzyl (o-NB), Allyl (All), and tert-butyl-dimethylsilyl (TBS) protecting groups of the phenolic alcohol of tyrosine that could be differentially removed, differential installation of TCE-protected sulfates at various sites was accomplished.¹³³ This synthetic approach allowed the authors to access a series of variably sulfated sulfopeptides. The importance of the sulfation in the obtained sulfopeptides was demonstrated in an enzyme-linked immunosorbent assay (ELISA)-based competitive binding assay between sulfated CCR5_2–22 and HIV-1 envelope glycoprotein gp120.¹⁶ Among all the sulfation sites, sY at position 14 were found critical for the HIV viral entry into cells. These methods collectively address critical synthesis challenges, allowing for the efficient, stable, and site-selective incorporation of sulfotyrosine into peptides for biological studies.

Figure 17.

Site-selective sulfation of a synthetic CCR5 library.

In 2015, Sharpless, Kelly, and coworker developed a fluorosulfated tyrosine precursor strategy, where the fluorosulfate group is integrated into peptides via SPPS and subsequently converted to sulfotyrosine using ethylene glycol.¹³⁴ In their work, the FsY residue could then be converted to sulfotyrosine (sY) via ethylene glycolysis. However, the ethylene glycolysis conditions are incompatible with living systems. In 2023, our group disclosed a novel method that enabled the conversion of the FsY residue in sulfopeptides and sulfoproteins into sY by hydroxamic acid (HA) reagents under physiologically relevant conditions (Fig. 18).¹³⁵ Using FsY-containing hexapeptide 1 [DADE(FsY)L-NH₂] as the model test substrate, the strong nucleophilicity of HA makes it prone to react with the fluorosulfate group on FsY, forming adduct 12. Real-time LC-MS monitoring revealed the isocyanate adduct with hydroxamic acid (13), providing evidence that a Lossen-like rearrangement is responsible for generating the desired sulfate. Furthermore, isotope labeling with H₂¹⁸O combined with LC-MS analysis showed no incorporation of ¹⁸O into the sulfate product, confirming that the reaction proceeds exclusively through the Lossen rearrangement pathway. Interestingly, nature employs a similar strategy: glucosinolates undergo a Lossen-like rearrangement to release thiocyanate, highlighting the biological relevance of our system.

Figure 18.

Lossen rearrangement for efficient conversion of FsY to sY. Reproduced with permission from ref 135. Copyright 2023 American Chemical Society.

We demonstrated that the FsY to sY conversion could be successfully carried out in the tsetse thrombin inhibitor (TTI) peptides and their derivatives TTI01, TTI02(FsY), TTI03(FsY), and TTI04(FsY) using HA reagent N-hydroxynicotinamide (3-PHA) under physiologically relevant conditions (Fig. 19a). Moreover, using the 2-nitrobenzyl-caged HA reagent N-(2-nitrophenoxy)nicotinamide (3-CPHA), FsY decaging could be controlled by light (370 nm) irradiation, which suggested the potential of spatiotemporal control of sulfation patterns in sulfopeptides (Fig. 19b). Then, we conducted the α-thrombin activity assay with Chromozym TH (Tosyl-Gly-Pro-Arg-4-nitranilide acetate), a chromophoric substrate, for the measurement of thrombin activity to determine the inhibitory effects of FsY (latent) and sY (active)-bearing TTI peptides. Our results indicated TTI peptides bearing sY possess superior inhibition activity than the unmodified or FsY-bearing peptides (Fig. 19b), which showcases that sulfation plays an important role in thrombin inhibition activity.

Figure 19.

FsY as a latent sulfate in sulfopeptides. a) Schematic illustration of blood coagulation by tsetse thrombin inhibitor (TTI) secreted by tsetse fly. b) Chemical synthesis of TTI variants containing FsY via SPPS.¹³⁵ c) and d) Light-mediated decaging of FsY-bearing TTI peptide T9–12(FsY) regulates its sulfation-dependent thrombin inhibitory activity.¹³⁵

We found the HA-mediated decaging conditions were compatible with live Staphylococcus aureus cells, as a FsY-bearing peptide that were grafted onto the cell surface could be decaged to sY residues (Fig. 20). After decaging reaction, this peptide was cleaved and the cleaved fragment was analyzed by LC-MS, confirming complete conversion of FsY to sY. Importantly, neither the sortase-mediated ligation nor the decaging step caused significant cytotoxicity, with S. aureus cells maintaining over 80% survival even at millimolar reagent concentrations. Together, these results establish a cytocompatible strategy for controlled decaging of FsY groups in peptides on live cell surface under physiologically relevant conditions.

Figure 20.

Cell surface decaging of FsY. a) Sortase A-mediated ligation of peptide 22 onto the S. aureus cell surface and its decaging followed by the TEV protease cleavage. b) Percent of S. aureus cell survived after sortase A-mediated ligation of 22 (Step I) and after fluorosulfate decaging by 3-PHA (Step II) compared to the cells treated with PBS. The average data of two trials were plotted. c) LC-MS analysis of samples after the TEV cleavage identified the decaged peptide (24, bottom) compared to the cleaved peptide before decaging (23, top). Reproduced with permission from ref 135. Copyright 2023 American Chemical Society.

3.2. Genetic Code Expansion

Chemical synthesis is generally limited to polypeptides or small proteins and cannot be applied to most full-length proteins.¹²⁶ Other methods, such as in vitro enzymatic sulfation by sulfotransferases, remain challenging to introduce sulfate groups site-specifically.^{136, 137} The ability to site-specifically incorporate sY into proteins using genetic code expansion (GCE) technology can overcome these challenges (Fig. 21a). In 2006, the Schultz group used an orthogonal aminoacyltRNA synthetase, evolved from Methanococcus jannaschii tyrosyl-tRNA synthetase (MjTyrRS), specifically paired with an engineered nonsense suppressor from Methanococcus jannaschii (${\mathrm{M}\mathrm{j}\mathrm{t}\mathrm{R}\mathrm{N}\mathrm{A}}_{\text{CUA}}^{\text{Tyr}}$), for site-specific incorporation of sY into hirudin proteins via TAG (amber) codon suppression in E. coli (Fig. 21a).¹³⁸ In 2020, Chatterjee et al. and W. Niu et al. each independently evolved a tyrosyl-tRNA synthetase (EcTyrRS) mutant, which enabled the incorporation of sY into mammalian proteins.^{139, 140} These tools were applied to investigate the role of sulfation in the activation of chemokine receptor CXCR4 and GAG-dependent thrombin inhibition by the two groups, respectively. Despite these advances, the poor cell permeability of sY remains a challenge for its incorporation into intracellularly expressed proteins via GCE. In 2022, the Han group reported the creation of autonomous prokaryotic and eukaryotic cells with the ability to biosynthesize sY and incorporate it into proteins via genetic code expansion.¹⁴¹ They chose both madanin-1 and chimadanin identified in the salivary gland of Haemaphysalis longicornis and verified that the function of sulfation was critical for both proteins. Taken together, the development of these GCE strategies allowed for the site-specific incorporation of sY for studying sulfation patterns in sulfoproteins.

Figure 21.

a) Genetically encoded sY and FsY and their corresponding synthetase/tRNA systems. b) Fluorosulfate decaging in FsY-bearing protein sfGFP-151-FsY and its corresponding high-resolution mass spectrometry.

Recent works highlight the regulatory roles of sulfation in determining protein-protein and protein-glycan interactions. Precise regulation of when and where sulfation occurs enables key knowledge into how cells dynamically modulate signaling pathways, immune responses, and developmental processes. FsY in proteins, as the latent sulfate, could be activated by HA reagents and enable the spatial or temporal release under physiologically relevant conditions (Fig. 21b).¹³⁵ The incorporation of FsY as a noncanonical amino acid into proteins through a bioorthogonal synthetase/tRNA pair was first reported by Wang et al.¹⁴² Following this seminal work, we mutated Methanosarcina mazei pyrrolysyl-tRNA synthetase (MmPylRS) at A302I/N346T/Y348I/C384L/W417K to form FSYRS, which specifically recognize pyrrolysyl-tRNA and charge FsY on the tRNA partner. Using this FSYRS/tRNA pair, superfolder green fluorescent protein(sfGFP) with TAG codon at position 3, 151 or 200 is as reporter to allow the FsY incorporated at the sites respectively. Upon treatment with HA reagents, FsY was efficiently converted to sY on physiologically relevant conditions (37 °C, pH 7.4), while the protein’s tertiary structure and fluorescence were retained. To identify the minimal mass difference between FsY and sY (2 Da), corresponding to the pre- and post-decaging states, we confirmed the sfGFP-151-sY generation by high-resolution mass spectrometry. LC-MS/MS, which is a powerful method for locating protein modification site, also proved the successful release of sulfate from FsY at corresponding position. From these characterization results, we demonstrated the excellent biocompatibility of our reagents and efficient FsY decaging in proteins, suggesting that these reagents could be used in experiments involving live systems.

4. CONCLUSION & FUTURE DIRECTIONS

Sulfation is a versatile regulatory post-translational modification with a variety of functional roles in signaling and immunity. Targeted control over when, where, and how sulfation occurs is crucial to unravel its functional roles and to enable new avenues in therapeutics. Toward this goal, we introduced a modular and efficient method for O-sulfation based on sulfur(VI) fluoride exchange chemistry, enabling the scalable synthesis of diverse sulfate esters across a broad range of substrates. This foundational work laid the groundwork for more complex, programmable approaches, including the iterative assembly of sequence-defined glycomimetics that mimic natural sulfation patterns. These synthetic glycan analogs replicated key biological functions and allow systematic exploration of sulfation-dependent interactions. To achieve spatiotemporal control of sulfation in proteins and peptides, the fluorosulfate group demonstrated unique reactivities that allow them to act as latent sulfate precursors that could be selectively activated by hydroxamic acid reagents under physiological conditions. Light-triggered decaging of the hydroxamic acid reagent highlighted the potential to dynamic modulate sulfation in living systems. Collectively, these approaches mark a shift toward precise control of sulfation in biomolecules, offering powerful tools to investigate the biological roles of sulfation and to engineer functional molecules for research and therapeutic applications.

Recent progress has significantly expanded the ability to construct sulfated biomolecules and regulate their sulfation patterns with precision. However, precisely controlling the position and pattern of sulfation on biomolecules continues to pose a formidable challenge.^{135, 143} Regioselective sulfation requires fine-tuned protection and deprotection strategies, which can be laborious and with limited scalability. Moreover, current chemical methods often struggle to reproduce the complexity and diversity found in natural sulfation patterns. Addressing these obstacles demands innovative synthetic methodologies that combine exquisite chemical reactivity with operational simplicity, such as catalyst-controlled regioselective sulfation.

Furthermore, the integration of high-throughput synthetic platforms with machine learning and computational modeling is on track to revolutionize the production, characterization, and functional studies of sulfated biomolecules. Automated synthesis accelerates the assembly of complex sulfation patterns, enabling rapid generation of diverse structures. Advances in chemoenzymatic synthesis and click chemistry hold promise for efficient, scalable, and combinatorial synthesis of sulfated biomolecules and their analogs. Meanwhile, computation-guide design of sulfation motifs and data-driven understanding and prediction can reveal unprecedented insights into how sulfation patterns regulate biological interactions in signaling and immunity. Together, these technologies expand the functional landscape and enable therapeutic potentials, such as immune modulation, viral entry inhibition, or regulation of growth factor signaling in cancer.