Photoinduced site-specific chemical glycosylation of proteins

Abstract

A non-enzymatic method of synthesizing a glycosylated protein is described. The method involves a glycosylation reaction, where a thioglycosyl donor / glycosylating agent is reacted with a protein substrate in the presence of a catalyst / activator / promoter and a photoillumination source. The thioglycosyl donor / glycosylating agent converts the protein substrate to synthesize the glycosylated protein. In particular, the thioglycosyl donor / glycosylating agent is a 2, 3,5,6- tetrafluoropyridine-4-thiogylcoside derivative of a native sugar. The method can be employed to synthesize C- glycosylproteins or S-glycosylproteins from native sugars or derivatives thereof.

Description

PHOTOINDUCED SITE-SPECIFIC CHEMICAL GLYCOSYLATION OF PROTEINSTECHNICAL FIELD

[0001] The present invention relates to chemical glycosylation of proteins. More specifically, the invention describes a photoinduced site-specific chemical glycosylation to synthesize C-glycoproteins and S-glycoproteins.BACKGROUND OF THE INVENTION

[0002] Post-translational glycosylation of proteins is a ubiquitous biological phenomenon occurring in various living systems. Glycoproteins resulting from this modification mediate a wide array of biological processes, including cell adhesion, signal transduction, host-pathogen interactions, and immune response. However, dissecting the precise biological roles of these glycoproteins remains a great challenge due to the dearth of methods for generating homogenous glycoproteins. In natural biosynthetic pathways, glycoproteins are assembled in multiple steps involving complex biological machinery regulated by dozens of glycotransferases and glycosidases. Unfortunately, biosynthesis cannot realize the stepwise enzymatic glycosylation in a controlled manner, which ultimately delivers a mixture of inseparable glycoforms due to the inherent promiscuity, thereby hampering further biological studies.

[0003] Early-stage disclosures typically involve the incorporation of noncanonical amino acids into the expressed protein using genetically encoded technologies, providing anchoring points for site-specific chemical ligation with sugar-derived reagents to generate neoglycoproteins. This method requires sophisticated biotechnological manipulation, and extending this strategy to another protein often necessitates re-optimization. Additionally, the unnatural glycosidic linkages introduced by these methods deviate from their native counterparts in chemical structure, size and stereochemistry, potentially leading to immunogenicity and false positive phenomena.

[0004] In nature, glycoproteins are typically formed by linking sugar units to O- or N-containing side chains of amino acid residues serine, threonine or asparagine using glycosyltransferases, such as the attachment of O-linked-p-D-N-acetylglucosamine (O-GIcNAc) to serine / threonine residues by O-GIcNAc transferase. However, this glycosylation can be reversed by intracellular glycosidases, and such a “write and erase” dynamic process makes it challenging to probe the biological functions of glycoproteins. In this context, chemical approaches to generate hydrolytically resistant glycosidic linkage (such as C / S- glycosylproteins) offer alternative and promising strategies for systematically investigating glycoprotein functions.SUMMARY OF THE INVENTION

[0005] This summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This summary is not intended to identify key aspects or essential aspects of the claimed subject matter.

[0006] A non-enzymatic method of synthesizing a glycosylated protein or protein glycosylation is described. The method involves reacting a thioglycosyl donor / glycosylating agent with a protein substrate in the presence of a catalyst / activator / promoter and a photoillumination source, wherein the thioglycosyl donor / glycosylating agent converts the protein substrate to synthesize or produce the glycosylated protein.

[0007] Furthermore, a method of protein glycosylation according to Scheme 1 is provided.

[0008] Furthermore, a method of synthesizing C-glycoproteins / C-glycosylproteins according to Scheme 2 is provided.

[0009] Furthermore, a method of synthesizing C-glycoproteins / C-glycosylproteins according to Scheme 3 is provided.

[0010] Furthermore, a method of synthesizing C-glycoproteins / C-glycosylproteins according to Scheme 4 is provided.

[0011] Furthermore, a method of synthesizing S-glycoproteins / S-glycosylproteins according to Scheme5 is provided.

[0012] All features of embodiments which are described in this disclosure and are not mutually exclusive can be combined with one another. Elements of one embodiment can be utilized in the otherembodiments without further mention. Other aspects, features, advantages, and benefits of the pharmaceutical compositions, formulations, and methods of the present disclosure will become apparent to those ordinarily skilled in the art upon review of the following description of specific embodiments in conjunction with the accompanying Figures.BRIEF DESCRIPTION OF FIGURES

[0013] For a better understanding of the various embodiments described herein, and to show more clearly how these various embodiments may be carried into effect, reference will be made, by way of example, to the accompanying drawings which show at least one example embodiment, and which are now described. The drawings are not intended to limit the scope of the teachings described herein.

[0014] The above and other aspects, features and advantages of the invention will become more readily apparent from the following description, reference being made to the accompanying drawings in which:

[0015] In the drawings, exemplary embodiments are illustrated by way of example. It is to be expressly understood that the description and drawings are only for the purpose of illustrating certain embodiments and are an aid for understanding. They are not intended to be a definition of the limits of the invention.

[0016] Figure 1 provides a depiction of Scheme 1 .

[0017] Figure 2 provides a depiction of Scheme 2.

[0018] Figure 3 shows an exemplary post-translational chemical glycosylation of C-Glycoproteins using native sugars via 2,3,5,6-tetrafluoro-4-pyridiyl glycosyl sulfides.

[0019] Figure 4 to Figure 9 provides LC-MS data and yields of various glycoprotein conjugates.

[0020] Figure 15 - Figure 32 provides conversion rates of glycoproteins with mass spectrometry analysis.

[0021] Figure 33 to Figure 39 provides fragmentation spectra of various glycoprotein conjugates.

[0022] Figure 40 provides a proposed mechanism for photoinduced C-glycosylation per Scheme 4.

[0023] Figure 41 provides a depiction of prior art and proposed S-glycosylation techniques.

[0024] Figure 42 provides a depiction of Scheme 5.

[0025] Figure 43 shows optimization of glycosylation conditions.

[0026] Figure 44 provides time-course studies of the proposed S-glycosylation reaction.

[0027] Figure 45 provides mechanistic studies of S-glycoprotein synthesis.

[0028] Figure 46 to Figure 58 provides LC-MS analysis and results of various proteins.

[0029] Figure 59 to Figure 92 provides thioglycosyl donor / sugar donor scope anaylsis, including various examples of glycoprotein synthesis using different sugar sources and ESI-MS spectrum data of the resulting glycoproteins.

[0030] Figure 93 provides the scope of various thioglycosyl donors / sugars for glycosylation.

[0031] Figure 94 to Figure 1 19 provides protein substrate scope anaylsis, including various examples of glycoprotein synthesis from different protein substrates and and ESI-MS spectrum data of the resulting glycoproteins.

[0032] Figure 120 provides the scope of various protein substrates for glycosylation.DETAILED DESCRIPTION

[0033] The present technology is explained in greater detail below. This description is not intended to be a detailed catalog of all the different ways in which the technology may be implemented, or all the features that may be added to the instant technology. For example, features illustrated with respect to one embodiment may be incorporated into other embodiments, and features illustrated with respect to a particular embodiment may be deleted from that embodiment. In addition, numerous variations and additions to the various embodiments suggested herein will be apparent to those skilled in the art considering the instant disclosure which variations and additions do not depart from the present technology. Hence, the following description is intended to illustrate some embodiments of the technology, and not to exhaustively specify all permutations, combinations, and variations thereof. The following description is of preferred embodiments by way of example only and without limitation to the combination of features necessary for carrying the invention into effect.

[0034] All terms are intended to be understood as they would be understood by a person skilled in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0035] Although various features of the present disclosure can be described in the context of a single embodiment, the features can also be provided separately or in any suitable combination. Conversely,although the present disclosure can be described herein in the context of separate embodiments for clarity, the present disclosure can also be implemented in a single embodiment.

[0036] The following definitions supplement those in the art and are directed to the current application. Accordingly, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0037] In this application, the use of the singular includes the plural unless specifically stated otherwise. It must be noted that, as used in the specification, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise.

[0038] In this application, the use of "or" means "and / or" unless stated otherwise. The terms "and / or" and "any combination thereof and their grammatical equivalents as used herein, can be used interchangeably. These terms can convey that any and all combinations are specifically contemplated. The term "or" can be used conjunctively or disjunctively, unless the context specifically refers to a disjunctive use.

[0039] Furthermore, use of the term "including" as well as other forms, such as "include", "includes," and "included," is not limiting.

[0040] Reference in the specification to "some embodiments," "an embodiment," "one embodiment" “alternate embodiment”, or "other embodiments" means that a particular feature, structure, or characteristic described in connection with the embodiments is included in at least some embodiments, but not necessarily all embodiments, of the present disclosures.

[0041] As used in this specification and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the present disclosure, and vice versa. Furthermore, compositions of the present disclosure can be used to achieve methods of the present disclosure.

[0042] The term "about" in relation to a reference numerical value and its grammatical equivalents as used herein can include the numerical value itself and a range of values plus or minus 10% from that numerical value. The term "about" or "approximately" means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about"can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, "about" can mean a range of up to 20%, up to 10%, up to 5%, or up to 1 % of a given value. In another example, the amount "about 10" includes 10 and any amounts from 9 to 1 1 .

[0043] Glycosylation refers to the reaction in which a carbohydrate, i.e. a glycosyl donor, is attached to a hydroxyl or other functional group of another molecule in order to form a glycoconjugate.

[0044] Glycoproteins are proteins which contain oligosaccharide chains covalently attached to amino acid side chains. The carbohydrate is attached to the protein in a cotranslational or posttranslational modification such as by glycosylation.

[0045] Glycoproteins are abundant biomolecules that mediate numerous biological processes in living organisms which are known to mediate numerous essential biological processes. However, these biologically important glycoproteins are often produced as heterogenous mixtures through natural biological machinery, which hampers further studies of glycosylation-dependent biological activities. Therefore, developing chemical methods for glycosylating canonical amino acids with high fidelity to native glycosidic linkages holds great promises for probing the biological roles of glycoproteins.

[0046] In nature, glycoproteins are typically formed by linking sugar units to O- or N-containing side chains of amino acid residues serine, threonine or asparagine using glycosyltransferases, such as the attachment of O-linked-p-d-N-acetylglucosamine (O-GIcNAc) to serine or threonine residues by O- GIcNAc transferase. However, this glycosylation can be reversed by intracellular glycosidases, and such a write-and-erase dynamic process makes it challenging to probe the biological functions of glycoproteins. In this context, chemical approaches to generate non-cleavable glycoproteins (such as C-glycosylproteins or S-glycosylproteins) offer alternative and promising strategies for systematically investigating glycoprotein functions.

[0047] Chemical glycosylation of canonical amino acids offers an invaluable alternative to access homogenous glycoproteins with precise site-selectivity and high fidelity. Post-translational chemical glycosylation of proteins, particularly the attachment of sugar units to proteins via direct anomeric functionalization, is largely unexplored in synthetic carbohydrate and protein chemistry. This may be ascribed to the lack of suitable unprotected glycosyl precursors that are stable yet sufficiently reactive, as well as the stringent requirements for biocompatible conditions including water compatibility which quenches heterolytic chemical glycosyl donors ability to remain non-destructive to biological substrates and low reactivity towards the biogenic functional groups that are present in most biological environments.

[0048] Embodiments

[0049] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the invention shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

[0050] In an embodiment of the invention, a non-enzymatic method of synthesizing a glycosylated protein or protein glycosylation is described. The method involves reacting a thioglycosyl donor / glycosylating agent with a protein substrate in the presence of a catalyst / activator / promoter and a photoillumination source, wherein the thioglycosyl donor / glycosylating agent converts the protein substrate to synthesize or produce the glycosylated protein.

[0051] In a further embodiment of the invention, the method involves a thioglycosyl donor / glycosylating agent such as a native sugar or a derivative thereof.

[0052] In a further embodiment, method of claim 2, the method involves a thioglycosyl donor / glycosylating agent such as 2,3,5,6-tetrafluoropyridine-4-thiogylcoside derivative of the native sugar.

[0053] In a further embodiment of the invention, the method of claim 3, the method involves obtaining a 2,3,5,6-tetrafluoropyridine-4-thiogylcoside derivative of the native sugar by cross-coupling the native sugar.

[0054] In a further embodiment, the method involves native sugars such as D-galactose, D-mannose, D-GIcNAc, D-GalNAc, D-ManNAc, D-xylose, D-glucose, L-rhamnose, L-glucose, D-allose, D-lyxose, cellobiose, lactose, maltose, melibiose, maltotriose, and oligosaccharide acarbose.

[0055] In a further embodiment, the method involves protein substrates such as histone, pre-SUMO1 , PstS, NpP, SspG, cabVCAM, AcrA and GFP.

[0056] In a further embodiment, the method involves a protein substrate that is tagged with an amino acid.

[0057] In a further embodiment, the method involves a protein substrate tagged with an aliphatic amino acid to synthesize a C-glycoprotein.

[0058] In a further embodiment, the method involves a protein substrate tagged with alanine or dehydro alanine (DHA) to synthesize a C-glycoprotein.

[0059] In a further embodiment, the method involves a catalyst / activator / promoter which is a reductant.

[0060] In a further embodiment, the method involves a catalyst / activator / promoter such as 4- methylbenzethiol or bis(catecholato)diboron (BgCatg).

[0061] In a further embodiment, the method or the glycosylation reaction is carried out at a temperature ranging from about 2°C to about 6°C.

[0062] In a further embodiment, the method or the glycosylation is carried out at a pH ranging from about 6 to about 10.

[0063] In a further embodiment, the method or the glycosylation reaction is carried out in the presence of a buffer such as TBS buffer, PBS buffer, and Bis-Tris buffer.

[0064] In a further embodiment, the method is carried out under a photoillumination source such as UV light, blue LED, or a light source with a wavelength ranging from 250nm to 500nm.

[0065] In a further embodiment, the method is carried out under a photoillumination source with a wavelength of 500 nm or lower.

[0066] In an alternate embodiment, the method involves a protein substrate tagged with a nucleophilic amino acid to synthesize an S-glycoprotein.

[0067] In the alternate embodiment, the method involves a protein substrate tagged with cysteine, threonine, serine, tyrosine, glutamic acid, aspartic acid, lysine, arginine, histidine or methionine to synthesize an S-glycoprotein.

[0068] In the alternate embodiment, the method involves a catalyst / activator / promoter which is a reductant.

[0069] In the alternate embodiment, the method involves a catalyst / activator / promoter such as 4- methylbenzethiol or Lithium Iodide.

[0070] In the alternate embodiment, the method or the glycosylation reaction is carried out at a temperature ranging from about 2°C to about 6°C.

[0071] In the alternate embodiment, the method or the glycosylation reaction is carried out at a pH ranging from about 6 to about 10.

[0072] In the alternate embodiment, the method or the glycosylation reaction is carried out in the presence of a buffer such as TBS buffer, PBS buffer, and Bis-Tris buffer.

[0073] In the alternate embodiment, the method or the glycosylation reaction is carried out with a photoillumination source such as UV light, blue LED, or a light source with a wavelength ranging from 250nm to 500nm.

[0074] In the alternate embodiment, the method or the glycosylation reaction is carried out with a photoillumination source at a wavelength of 365 nm or lower.

[0075] In any of the above embodiments, the method involves using the thioglycosyl donor / glycosylating agent and the catalyst / activator / promoter in equal proportions.

[0076] In any of the above embodiments, the method involves using the thioglycosyl donor / glycosylating agent and the catalyst / activator / promoter in unequal proportions.

[0077] In an embodiment of the invention, a method of protein glycosylation according to Scheme 1 is provided.

[0078] In an embodiment of the invention, a method of synthesizing C-glycoproteins according to Scheme 2 is provided.blue LED C-giyeosyiprote in

[0079] In an embodiment of the invention, a method of synthesizing C-glycosylprotein according to Scheme 3 is provided.

[0080] In an embodiment of the invention, a method of synthesizing C-glycosylprotein according to Scheme 4 is provided.

[0081] In an embodiment or the invention, a method of synthesizing S-glycosylproteins according toScheme 5 is provided.

[0082] Technical Discussion

[0083] To address these challenges, post-translational chemical glycosylation of expressed proteins would offer a more streamlined and concise protocol to produce glycoproteins for biofunction-oriented studies.

[0084] The following examples describe some modes of synthesizing glycosyl proteins that are described herein. These examples are for illustrative purposes only and are not meant to limit the scope of the reactants, techniques, operational conditions or methods described herein.

[0085] SYNTHESIS OF C-GLYCOPROTEINS

[0086] Inventors hypothesized that a protecting-group-free approach can be adopted for synthesis of diverse classes of glycoproteins (as shown in Scheme 1 ). Scheme 1 provides a non-enzymatic chemical synthesis of unprotected glycoproteins using native sugars. Figure 1 provides a depiction of Scheme 1 .

[0087] Previously, Hantzsch ester was used in protecting-group-free small-molecule glycosylation. Due to the insolubility of Hantzsch ester in the necessary aqueous medium, the inventors sought to develop photoinduced cross-coupling conditions based on the formation of charge-transfer complexes between a thioglycosyl donor or a glycosylating agent such as 2,3,5,6-tetrafluoropyridine-4-thioglycoside and a reductant such as bis(catecholato)diboron (BsCats).

[0088] A photoinduced, site-specific glycosylation of alanine or dehydroalanine-containing proteins (protein substrate) to afford diversified C-glycoproteins was tested by the inventors, specifically the inventors attempted the ‘cap and glycosylate’ protocol to synthesize glycoproteins as shown in scheme 2. Figure 2 provides a depiction of Scheme 2. An exemplary post-translational chemical glycosylation of C-Glycoproteins using native sugars via 2,3,5,6-tetrafluoro-4-pyridiyl glycosyl sulfides is shown in Figure 3.

[0089] Glycosylation of proteins by cross-coupling of representative native sugars (via capping as thioglycosyl donors) afforded unprotected C-alkyl glycoproteins as shown in Scheme 2. Yields were determined by LC-MS analysis based on conversion of the protein substrate. The yields in parentheses as shown in Scheme 2 denotes reactions with in situ-generated and unpurified thioglycosyl donors. The reaction mixture included Tris buffer, 2-amino-2-(hydroxylmethyl)-propane-1 ,3-diol, reductant B2Cat2, bis(catecholato)diboron with various native sugars such as Mannose, D-mannosyl, Galactose, D- galactosyl, and GIcNAc, N-acetyl-D-glucosaminyl.

[0090] Notably, histone H3-GlcNAc-Ala10 generated a non-cleavable mimetic of the reported epigenetic mark GlcNAc-Ser10. Similar efficiencies were also observed in the reactions of different thioglycosyl donors with each given protein (~85% conversion for eH3-Dha9, ~80% conversion for H3- Dha10, -55% conversion for TEV H3-Dha2, ~80% conversion for PanC-Dha44 and ~70% conversion for PstS-Dha57). About 5% of a minor product featuring two units of GIcNAc addition was detected for PstS-GlcNAc-Ala57, which the inventors ascribed to non-specific glycosylation of lysine residues. Traceless activation via in situ formation of the S-glycosyl intermediates could be implemented without compromising protein glycosylation efficiency, thereby exemplifying the power of protecting-group-free “cap and glycosylate” approach allowing native sugars to be directly utilized for post-translational glycosylation of proteins.

[0091] Methods: Sample proteolysis: For in-solution proteolytic digestion, samples were buffer- exchanged into 100 mM ammonium bicarbonate (urea was added to Ss£G samples at 6M finalconcentration), reduced with 10 mM tris(2-carboxyethyl)phosphine (Thermo Fisher) for 30 min at room temperature and alkylated with 30 mM 2-chloroacetamide (Sigma Aldrich) at room temperature for 30 min in the dark. LysC (Fujifilm Wako) was added to histone sample solutions and trypsin (Pierce) was added to the solutions of other proteins for a 6-hour incubation at 37°C with 1 :25 protease:protein ratio (w / w). SspG sample was diluted to 1.3M urea prior to adding trypsin. The samples were desalted by Oasis HLB cartridges (Waters) and reconstituted in water containing 5% formic acid, 5% DMSO right before the LC-MS analysis.

[0092] LCMS: Samples were subjected to LC-MS / MS using an UltiMate 3000 nanoUHPLC system (Thermo Fisher Scientific) coupled to an Orbitrap QExactive (Thermo Fisher Scientific). The peptides were trapped on a C18 PepMapl OO pre-column (300 pm i.d. x 5 mm, 100 A, Thermo Fisher Scientific) using solvent A (0.1 % formic acid in water), then separated on an in-house packed analytical column (50 pm i.d. x 50 cm in-house packed with ReproSil Gold 120 C18, 1.9 pm, Dr. Maisch GmbH) and utilized a 15 minute gradient (12% to 40%B where B is 0.1% formic acid in acetonitrile) at a flow rate of 100 nL / min. Full scan MS spectra were acquired in the Orbitrap (scan range 350-1400 m / z, resolution 70000, AGO target 3e6). Five most abundant peptides were selected each round for stepped HOD fragmentation using 20, 30, and 40% normalized collision energy, and fragmentation products were mass-analyzed in the Orbitrap (scan range 200-2000 m / z, resolution 17500, AGO target 5e4, maximum injection time 128 ms).

[0093] Data analysis: Spectra were searched using FragPipe (v20.0) MSFragger 3.813 with standard ‘open’ search settings against corresponding bespoke. FASTA files containing proteins of interest and potential contaminants. Data was filtered using the inbuilt tools within FragPipe to an FDR of below 1%. Modified peptides were discerned by filtering the resulting dataset using the expected changes in mass caused by each modification.

[0094] LC-MS data and yields of various glycoprotein conjugates are provided below from Figure 4 - Figure 9. Conversion rates of glycoproteins with mass spectrometry analysis are provided from Figure 15 - Figure 32. Fragmentation spectra of various glycoprotein conjugates are provided from Figure 33 to Figure 39.

[0095] Histone eH3-Dha9 [Figure 4]ARTKQTARDhaSTGGKAPRKQLATKAARKSAPATGGVKKPHRYRPGTVALREIRRYQKSTELLIRKLP FQRLVREIAQDFKTDLRFQSSAVMALQEAAEAYLVGLFEDTNLAAIHAKRVTIMPKDIQLARRIRGERA GGDYKDDDDKSAAGGYPYDVPDYACalculated mass = 17686Observed mass = 17686

[0096] Histone H3-Dha10 [Figure 5]ARTKQTARKDhaTGGKAPRKQLATKAARKSAPATGGVKKPHRYRPGTVALREIRRYQKSTELLIRKLP FQRLVREIAQDFKTDLRFQSSAVMALQEASEAYLVALFEDTNLAAIHAKRVTIM PKDIQLARRIRGERACalculated mass = 15221Observed mass = 15221

[0097] Histone H3-TEV-Dha2 [Figure 6]ADhaENLYFQGTKQTARKSTGGKAPRKQLATKAARKSAPATGGVKKPHRYRPGTVALREIRRYQKST ELLIRKLPFQRLVREIAQDFKTDLRFQSSAVMALQEASEAYLVALFEDTNLAAIHAKRVTIMPKDIQLAR RIRGERACalculated mass = 16004Observed mass = 16004

[0098] PanC-Dha44 [Figure 7]TIPAFHPGELNVYSAPGDVADVSRALRLTGRRVMLVPTMGALDhaEGHLALVRAAKRVPGSVVVVSIFVNPMQFGAGEDLDAYPRTPDDDLAQLRAEGVEIAFTPTTAAMYPDGLRTTVQPGPLAAELEGGPRPT HFAGVLTVVLKLLQIVRPDRVFFGEKDYQQLVLIRQLVADFNLDVAVVGVPTVREADGLAMSSRNRYL DPAQRAAAVALSAALTAAAHAATAGAQAALDAARAVLDAAPGVAVDYLELRDIGLGPMPLNGSGRLLVAARLGTTRLLDNIAIEIGTFAGTDRPDGYRA ILESHWRNKLAAALEHHHHHHCalculated mass = 33998Observed mass = 33998

[0099] PstS-Dha57 [Figure 8]MEASLTGAGATFPAPVYAKWADTYQKETGNKVNYQGIGSSGGVKQIIANTVDFGASDhaAPLSDEKLAQEGLFQFPTVIGGVVLAVNIPGLKSGELVLDGKTLGDIYLGKIKKWDDEAIAKLNPGLKLPSQNIAVVR RADGSGTSFVFTSYLAKVNEEWKNNVGTGSTVKWPIGLGGKGNDGIAAFVQRLPGAIGYVEYAYAKQ NNLAYTKLISADGKPVSPTEENFANAAKGADWSKTFAQDLTNQKGEDAWPITSTTFILIHKDQKKPEQGTEVLKFFDWAYKTGAKQANDLDYASLPDSVVEQVRAAWKTNIKDSSGKPLYCalculated mass = 34507Observed mass = 34507

[0100] SspG-Dha7:C344S:E387C [Figure 9]MYSFPNDhaFRFGWSQAGFQSEMGTPGSEDPNTDWYKWVHDPENMAAGLVSGDLPENGPGYWG NYKTFHDNAQKMGLKIARLNVEWSRIFPNPLPRPQNFDESKQDVTEVEINENELKRLDEYANKDALNH YREIFKDLKSRGLYFILNMYHWPLPLWLHDPIRVRRGDFTGPSGWLSTRTVYEFARFSAYIAWKFDDL VDEYSTMNEPNVVGGLGYVGVKSGFPPGYLSFELSRRAMYNIIQAHARAYDGIKSVSKKPVGIIYANS SFQPLTDKDMEAVEMAENDNRWWFFDAIIRGEITRGNEKIVRDDLKGRLDWIGVNYYTRTVVKRTEK GYVSLGGYGHGSERNSVSLAGLPTSDFGWEFFPEGLYDVLTKYWNRYHLYMYVTCNGIADDADYQR PYYLVSHVYQVHRAINSGADVRGYLHWSLADNYEWASGFSMRFGLLKVDYNTKRLYWRPSALVYRE IATNGAITDEIEHLNSVPPVKPLRHHHHHHHCalculated mass = 57454Observed mass = 57454

[0101] The inventors tested various reaction conditions for optimization. For instance, the reaction was carried out in the absence and presence of photoillumination at 365 nm, 385 nm, 405 nm, 420 nm, and 440 nm. Various catalysts (also referred as activators, promoters or reductants), e.g. bis(catecholato)diboron (BgCatg), Ru(bpy3)Cl2 / FeSO4*7H2O, 4-Methylbenzethiol, and 4- (Dimethylamino)benzenethiol; and buffers such as Tris buffer (TBS), PBS buffer, and Bis-Tris buffer, were also tested. Figure 10 provides screening results of various activators. As can be seen in the Figure, optimum results were observed with Activators A5 [4-(Dimethylamino)benzenethiol] and A6 [BzCats].

[0102] Figure 1 1 provides the screening results of various illumination wavelengths. Figure 12 provides a further screening of illumination wavelengths. As shown in the figures, with 200 equiv. of both the native sugar and the activator, optimum results were observed at an illumination wavelength of 365nm - 385nm. However, with 500 equiv. of both the native sugar and the activator, optimum results were observed at an illumination wavelength of 405nm - 445nm.

[0103] It was observed that B2Cat2 in the presence of photoillumination at 500nm or lower provided optimal results. It was further observed that replacing B2Cat2 with other reductants commonly employed in bioconjugation reactions, such as Ru(bpy3)CI2 / FeSO4*7H2O, were detrimental to the reaction. However, 4-methylbenzethiol proved to be a competent alternative reductant for facilitating glycosylation. The reaction efficiency remained consistent across various media and pH ranges. However, eliminating photoillumination or switching to wavelengths above 500 nm led to unproductive results. Wavelength of 500 nm or lower offered optimal results. A pH range of 6.3 to 7.4 was evaluatedand the reaction efficiency remained consistent throughout that range. After judicious evaluation of a series of these reaction parameters, inventors identified optimal glycosylation conditions which included equal proportions of the thioglycosyl donor (glycosylating reagent) with the catalyst (reductant) used in a media and photo illuminated at approximately 500 nm or lower for approximately an hour.

[0104] Scheme 3 provides a general procedure for photoinduced glycosylation of proteins as shown in Figure 13. As a general procedure, to a solution of Dha-tagged Protein (50 pM, 30 pL, 1 .00 equiv.) in TBS buffer (Tris 20 mM, NaCI 150 mM, pH = 8.0) was added to a solution of sugar donor (500 equiv., 0.2 M in H2O) and B2Cat2 (500 equiv., 1 M in DMSO) in glovebox. The mixture was shaken for 60 min at 4oC under 420 nm illumination. After completed, the reaction mixture was subjected to LC-MS for analysis. (*Note: 0.5 M in DMSO was used for GIcNAc donor). Conversions were calculated based on total ion count of thresholder signal peaks indicated in line with prior calibrations. Perturbed and unperturbed impurities are noted.

[0105] Conversion rates of glycoproteins were revealed with mass spectrometry analysis, results of which are provided below.

[0106] Histone eH3-Man-Ala9 [Figure 14]: 83% conversion; Calculated mass = 17850; Observed mass = 17850. In-situ glycosylation (without isolating sugar donor): 82% conversion; Observed mass = 17850 (shown in Figure 15).

[0107] Histone eH3-Gal-Ala9 [Figure 16]: 88% conversion; Calculated mass = 17850; Observed mass = 17850. In-situ glycosylation (without isolating sugar donor): 90% conversion; Observed mass = 17850 (shown in Figure 17).

[0108] Histone eH3-GlcNAc-Ala9 [Figure 18]: 70% conversion; Calculated mass = 17891 ; Observed mass = 17891 .

[0109] Histone H3-Man-Ala10 [Figure 19]: 80% conversion; Calculated mass = 15385; Observed mass = 15385.

[0110] Histone H3-Gal-Ala10 [Figure 20]: 83% conversion; Calculated mass = 15385; Observed mass = 15385.

[0111] Histone H3-GlcNAc-Ala10 [Figure 21 ]: 77% conversion; Calculated mass = 15426; Observed mass = 15426.

[0112] Histone H3-TEV-Man-Ala2 [Figure 22]: 52% conversion; Calculated mass = 16167; Observed mass = 16167.

[0113] Histone H3-TEV-Gal-Ala2 [Figure 23]: 56% conversion; Calculated mass = 16167; Observed mass = 16167.

[0114] Histone H3-TEV-GlcNAc-Ala2 [Figure 24]: 64% conversion; Calculated mass = 16208; Observed mass = 16208.

[0115] PanC-Man-Ala44 [Figure 25]: 81% conversion; Calculated mass = 34162; Observed mass = 34162; (Peak mass 34064 and 34242 are unperturbed impurities from PanC-Dha44).

[0116] PanC-GlcNAc-Ala44 [Figure 26] 78% conversion; Calculated mass = 34203; Observed mass = 34203; (Peak mass 34063 and 34242 are unperturbed impurities from PanC-Dha44).

[0117] PstS-Man-Ala57 [Figure 27]: 66% conversion; Calculated mass = 34671 ; Observed mass = 34672; (Peak mass 34811 ~ 34648 + 164, perturbed impurity from PstS-Dha57). Figure 28 shows in- situ glycosylation (without isolating sugar donor): 77% conversion; Observed mass = 34671. (Peak mass 34812 = 34648 + 164, perturbed impurity from PstS-Dha57).

[0118] PstS-Gal-Ala57 [Figure 29]: 80% conversion; Calculated mass = 34671 ; Observed mass = 34671 ; (Peak mass 34811 = 34648 + 164, perturbed impurity from PstS-Dha57). Figure 30 shows in- situ glycosylation (without isolating sugar donor): 75% conversion; Observed mass = 34671. (Peak mass 34812 = 34648 + 164, perturbed impurity from PstS-Dha57).

[0119] PstS-GlcNAc-Ala57 [Figure 31]: 78% conversion; Calculated mass = 34712; Observed mass = 34712; (Peak mass 34853 = 34648 + 205, perturbed impurity from PstS-Dha57); (Peak mass 34914 ~ 34507 + 205 + 203; ~ 5% two sugar unit addition glycoprotein was detected, which the inventors ascribe to non-specifical glycosylation of lysine residues; see Fragmentation spectrum of [QIIANTVDFGASA(HexNAc)APLSDEK]2+ originating from PstS-GlcNAc-Ala57, and Table 1 below for details).

[0120] SspG-GlcNAc-Ala7 [Figure 32]: 72% conversion; Calculated mass = 57659; Observed mass = 57659; (Peak mass 57681 « 57475 + 205, perturbed impurity from SspG-Dha7).

[0121] Fragmentation spectrum of [Acetyl-AA(Hex)ENLYFQGTK]2+originating from TEV-Histone-H3- Man-Ala2 is provided in Figure 33.

[0122] Fragmentation spectrum of [Acetyl-AA(Hex)ENLYFQGTK]2+originating from TEV-Histone-H3- Gal-Ala2 is provided in Figure 34.

[0123] Fragmentation spectrum of [Acetyl-AA(HexNAc)ENLYFQGTK]2+originating from TEV-Histone- H3-GlcNAc-Ala2 is provided in Figure 35.

[0124] Fragmentation spectrum of [QIIANTVDFGASA(Hex)APLSDEK]2+originating from PstS-Man- Ala57 is provided in Figure 36.

[0125] Fragmentation spectrum of [QIIANTVDFGASA(Hex)APLSDEK]z+originating from PstS-Gal- Ala57 is shown in Figure 37.

[0126] Fragmentation spectrum of [QIIANTVDFGASA(HexNAc)APLSDEK]2+originating from PstS- GlcNAc-Ala57 in shown in Figure 38.

[0127] Fragmentation spectrum of [MYSFPNA(HexNAc)FR]2+originating from Ss[3G-GlcNAc-Ala7 is provided in Figure 39.

[0128] A list of peptides with the mass shift corresponding (within the mass accuracy of 0.005 Da) to HexNAc was identified in the tryptic digest of PstS-GlcNAc-Ala57. Possible additional non-specific modification sites were labelled with red colour. The modification of the lysine residues in these peptides led to missed cleavages by trypsin at these sites. See Table 1 below.DQKKPEQGTEVLKDSSGKPLYETGNKVNYQGIGSSGGVKFFDWAYKTGAKGADWSKTFAQDLTNQKKPEQGTEVLKLISADGKPVSPTEENFANAAKLISADGKPVSPTEENFANAAKGADWSKLNPGLKLPSQNIAVVRMEASLTGAGATFPAPVYAKNNVGTGSTVKWPIGLGGKQIIANTVDFGASAAPLSDEKQNNLAYTKLISADGKPVSPTEENFANAAKSGELVLDGKTLGDIYLGKTLGDIYLGKIKTNIKDSSGKPLY

[0129] Coverage of the amino-acid sequence of PstS-GlcNAc-Ala57 by peptides listed in the Table below. Modification sites are highlighted below.

[0130] Proposed mechanism for photoinduced C-glycosylation is shown in Scheme 4 as shown in Figure 40. A thioglycosyl donor / glycosylating agent in the presence of a reductant (such as B2Cat2) forms a charge-transfer complex. The charge transfer complex undergoes homolysis in the presence of photo illumination (photoinduced electron transfer) and desulfurative fragmentation to form a glycosyl radical. The glycosyl radical reacts with the DHA-tagged protein / protein substrate to produce C- glycoprotein.

[0131] SYNTHESIS OF S-GLYCOPROTEINS

[0132] A photoinduced, site-specific anomeric glycosylation of cysteine-containing proteins to afford diversified S-glycoproteins was tested by the inventors. This protocol employs unprotected tetrafluoropyridyl glycosyl sulfides as the glycosylating reagents, activated via homolytic processes by iodide anions under photo illumination. Unlike prior reports that require chemical manipulation of proteins to introduce tailed tags for subsequent glycosylation, the proposed strategy streamlines the assembly of glycoproteins with broader application scope. Mechanistic studies indicate the thiol group on proteins is prone to oxidative dimerization, potentially mediated by glycosyl radical oxidation, forming the disulfide-linked dimerized protein. The labile disulfide bond then serves as the acceptor of an SH2- type glycosyl radical attack, ultimately affording a-S-glycoprotein. The configuration of a representative S-glycoprotein (H3-Glc-Cys10) was unambiguously confirmed by anomeric C1-13C labelled NMR studies.

[0133] Among 20 canonical amino acids, cysteine has garnered significant attention due to its strong nucleophilicity and low abundance, making it an ideal target for site-selective bioconjugation. Beyond general activity-based bioconjugation, cysteine-derived S-glycoproteins are often used as the mimetics of native serine-based O-glycoproteins in interrogating their biological functions. This is attributed to sulfur and oxygen atoms are both chalcogen elements, which endows them structural and electronic similarities. Moreover, S-glycosylation is also a kind of rare endogenous post-translational modification, this can arise when glycotransferases, such as O-GIcNAc transferase mistakenly target cysteine insteadof serine, due to their comparable size and binding affinity. This observation demonstrates the biochemical relevance of S-glycosylation and supports the feasibility of treating S-glycoproteins as reasonable surrogates of O-glycoproteins in biological studies.

[0134] Nevertheless, to realize the targeted S-glycosylation is untrivial. Despite the prosperity of smallmolecule glycosylation chemistry, successful extension of these glycosylating patterns to biomolecules remains scarce, owing to the difficulty of identifying competent glycosylating reagents with balanced stability and reactivity in aqueous solutions, as well as the potential interference from the other nucleophilic residues on proteins.

[0135] The depiction below shows the pre-functionalization approach and the “tag and modify” approach (see Figure 41 (a)) to access glycoproteins which have been used to overcome the previously discussed limitations. Previous approaches to access S-glycoproteins have capitalized on a two-step synthetic strategy (as shown in Figure 41 (b) below). In these methods, the cysteine thiol first undergoes nucleophilic attack of a thiolating electrophile forming a disulfide bridged protein. Depending on the ligated reagents, the embedded disulfide linkage can then be manipulated in two ways: (1 ) phosphinemediated intramolecular desulfurization to afford a-carbon stereocenter erased S-glycoprotein, or (2) homolytic fragment by intercepting an external glycosyl radical to deliver S-glycoprotein under photooxidation conditions. However, this pre-functionalization strategy has only been demonstrated with a limited number of protein examples and generally yields moderate conversion rates. The current proposed technique is depicted in Figure 41 (c).

[0136] The more general chemical post-translational mutagenesis method has been developed based on a ‘tag-and-modify’ strategy. In this protocol, a cysteine residue is first converted into dehydroalanine (Dha), a versatile acceptor for subsequent thiol-ene addition of glycosyl thiols to deliver S-glycoproteins. This protocol allows the modification of a wide range of proteins, and the resulting S-glycoproteins have been employed as surrogates of native O-glycoproteins in studying the biological roles of O- GIcNAcylation.

[0137] Despite the promising potential, the unselective thiol-ene addition produces a pair of epimeric mixtures in this vein, which has a high likelihood of ambiguity in followed analysis of biological tests. Taking these constraints into considerations, identifying a general strategy for glycosylating cys-proteins in a stereodefined manner, devoid of pre-functionalization of proteins, would offer an advanced toolkit for interrogating glycosylation-dependent biological activities. Through rigorous experimentation, the inventors found that a photoinduced approach can enable site-specific glycosylation of cysteine residues on proteins with defined stereochemistry. This report streamlines the synthesis of S- glycoproteins featuring a broad scope of saccharides and proteins.

[0138] Reaction in general is shown in Scheme 5 (Figure 42)Scheme 5

[0139] Photoinduced synthesis of S-glycoprotein: The protein glycosylation technology to cysteine- tagged proteins (protein substrate) containing nucleophilic thiol groups. For this endeavor, the inventors chose to make use of charge-transfer complexes between 2,3,5,6-tetrafluoropyridine-4-thioglycoside and iodide as the reductant. After identifying the optimal reaction conditions (200-500 equiv. of thioglycosyl donor, 200-1000 equiv. of Lil, 4 °C, 1 h, pH 8.0 in Tris buffer, 365 nm) as shown in Scheme 5, the inventors examined the glycosylation of histone H3 with a wide array of 2,3,5,6-tetrafluoropyridine- 4-thioglycosides derived from various mono- and oligosaccharides, including D-glucose, D-galactose, D-mannose, D-GIcNAc, D-GalNAc, D-ManNAc, D-xylose, L-rhamnose, L-glucose, D-allose, D-lyxose, cellobiose, lactose, maltose, melibiose, maltotriose, and oligosaccharide acarbose. The desired S- glycosylproteins were obtained as a anomers in good to excellent yields across the board (84%-98%). Preliminary mechanistic evidence also suggested that a disulf ide-linked protein is formed through the oxidative dimerization of the on-protein thiol, which serves as the intermediate for accepting a glycosyl radical in S-glycosylation.

[0140] In glovebox, stock solutions of sugar donor (200 - 500 equiv., 0.1 M in TBS buffer or 0.3 M in DMSO) and the reductant / catalyst Lil (200 -1000 equiv., 0.5 M in TBS) were prepared and added to a solution of protein (1 .00 equiv., 50 pM, 30 pL) in TBS buffer (Tris 20 mM, NaCI 150 mM, pH = 8.0). The mixture was shaken for 60 min at 4°C under 365 nm illumination. After completed, the reaction mixture was subjected to LC-MS for analysis. (*Note: 0.3 M in DMSO was used for GIcNAc and GalNAc donor).

[0141] Reaction optimization: The inventors initiated this study by selecting a thioglycosyl donor or a glycosylating reagent, such as a native sugar or a derivative thereof. Tetrafluoropyridyl thioglycoside (Glc-SpyF) was tested as the glycosylating reagent due to its testified biocompatibility under photochemical conditions, and a mutant histone H3-S10C as the model protein. Optimization of glycosylation conditions is shown in Figure 43.

[0142] The inventors tested various reaction conditions prior for optimization. For instance, the reaction was carried out in the absence and presence of photoillumination at 365 nm, 385 nm, 405 nm,420 nm, and 440 nm. Various catalysts (also referred as activators, promoters or reductants), e.g. Lithium Iodide (Lil), bis(catecholato)diboron (B2Cat2), Ru(bpy3)Cl2 / FeS04’7H20-Methylbenzethiol, and 4-(Dimethylamino)benzenethiol; and buffers such as Tris buffer (TBS), PBS buffer, and Bis-Tris buffer, were also tested.

[0143] It was observed that Lithium Iodide in the presence of photoillumination provided optimal results. It was further observed that replacing Lil with other reductants commonly employed in bioconjugation reactions, such as B2Cat2, Ru(bpy3)Cl2 / FeS04*7H2O, were detrimental to the reaction. However, 4-methylbenzethiol proved to be a competent alternative reductant for facilitating glycosylation. The reaction efficiency remained consistent across various media and pH ranges. However, eliminating photoillumination or switching to wavelengths above 365 nm led to unproductive results. Wavelength of 365 nm or lower offered optimal results. Strikingly, the absence of Lil also offered H3-Glc-Cys10 in moderate conversion, likely due to the inherent photosensitivity of GlcSPyF under 365 nm illumination. A pH range of 6.3 to 7.4 was evaluated and the reaction efficiency remained consistent throughout that range. After judicious evaluation of a series of these reaction parameters, inventors identified optimal glycosylation conditions which included equal proportions of the thioglycosyl donor (glycosylating reagent) with the catalyst (reductant) used in a media and photo illuminated at approximately 365 nm or lower for approximately an hour. For instance, when mutant histone H3-S10C was reacted with 200 equivalents of Glc-SPyF and 200 equivalents of Lithium Iodide (Lil) used in a given media (e.g. TBS buffer (Tris 20 mM, NaCI 150 mM, pH = 8) or PBS buffer) at 4°C under 365 nm illumination for approximately 1 hour, provided glycoprotein H3-Glc-Cys10 in full conversion.

[0144] Mechanistic studies: The ambiguous stereochemistry of the glycosidic linkage within H3-Glc- Cys10 prompted the inventors to perform systematic mechanistic studies. As shown in Figure 44, timecourse studies of the reaction reveal that the formation of dimerized H3-Cys10 serves as an intermediate for subsequent glycosylation. Dimerized H3-Cys10 was observed in the early-stage of the reaction, which continues to accumulate and peak at 83% conversion in 20 minutes. In contrast, transformative conversion to H3-Glc-Cys10 remains sluggish during this period. After the induction phase, the production of H3-Glc-Cys10 was accelerated, reflected by the rapid consumption of H3-Cys10 dimer between 20 to 40 minutes. This observation may arise from a relatively high concentration of Lil, which facilitates the reduction of GlcSPyF, generating glucosyl radical for subsequent glycosylation processes. The beneficial effects of Lil were testified by boosted reaction kinetics and conversion rate, compared to that of the reaction eliminates. The reaction continues to progress till complete conversion of the dimer at 1 h, generating H3-Glc-Cys10 in nearly full conversion with high purity.

[0145] To probe the involvement of glycosyl radical species in the reaction, a radical-clock study was conducted. Subjecting C2-OAIIyl tethered GalSPyF to co-solvents of MeCN and D2O with Lil as thepromoter under photoillumination afforded radical cyclized glycosyl sulfide in 34% yield. This product likely forms via 5-exo-trig radical cyclization between the a-configured galactosyl radical and C2-OAIIyl motif, delivering a ring-cyclized methyl radical. This radical intermediate reassociates with the homolytically dissociated tetrafluoropyridyl thiol radical, yielding the galactosyl derived sulfide. In the absence of Lil, the reaction was significantly suppressed, and only 5% of product was observed. Based on the time course studies and radical-clock experiments, the inventors hypothesized the reaction mechanism as follows: 1 ) glycosylating reagents are reduced by Lil under photoillumination, generating glycosyl radicals. These radicals undergo single-electron oxidation of cysteine thiols to form an on- protein thiol radical. 2) The resulting radical is susceptible to oxidative dimerization, forming a disulfide- bridged intermediate. 3) The transformable disulfide linkage interacts with another molecule of glycosyl radical via a SH2 process, eventually producing a a-S-glycoprotein, as well as a molecule of an on- protein cysteine radical X amenable to further derivatization.

[0146] To validate the stereochemistry of glycosidic linkage deduced from mechanistic studies, the inventors prepared C113C-labelled GlcSPyF as the alternative glycosylating reagent. Corresponding13C isotope labelled H3-Glc-Cys10 was obtained in complete conversion and subjected to 2D NMR studies. From HSQC spectrum, the inventors were able to identify the anomeric proton with the chemical shift of 5.45 ppm (3J = 4.77 Hz), and this value was consistent with reported a-S-glycopeptides with slight differences in coupling consistent (A = -0.7 Hz). These results were further proved by HSQC diffusion studies, which confirmed that the observed signal was from an intact H3-Glc-Cys10 instead of small-molecule glucosyl residues. This observation aligned well with mechanistic studies and supported the involvement of a-configured glucosyl radical during the reaction process. Mechanistic studies of S- glycoprotein synthesis are shown in Figure 45.

[0147] All of these glycoproteins exhibited excellent site selectivity, as confirmed by LC-MS / MS studies. Furthermore, the stereochemistry of these newly formed glycosidic linkages was suggested to be consistent with that of H3-Glc-Cys10, as glycosyl radicals often adapt a-configured geometry during the reaction process.

[0148] The LC-MS results of various proteins are provided in Figure 46 - Figure 58.Histone H3-S10C [Figure 46]ARTKQTARKCTGGKAPRKQLATKAARKSAPATGGVKKPHRYRPGTVALREIRRYQKSTELLIRKLPFQ RLVREIAQDFKTDLRFQSSAVMALQEASEAYLVALFEDTNLAAIHAKRVTIM PKDIQLARRIRGERACalculated Mass = 15255Observed Mass = 15255Histone TEV-H3-R2C [Figure 47]ACENLYFQGTKQTARKSTGGKAPRKQLATKAARKSAPATGGVKKPHRYRPGTVALREIRRYQKSTELLIRKLPFQRLVREIAQDFKTDLRFQSSAVMALQEASEAYLVALFEDTNLAAIHAKRVTIMPKDIQLARRIRGERACalculated Mass = 16038Observed Mass = 16038Histone eH3-K4C [Figure 48]ARTCQTARKSTGGKAPRKQLATKAARKSAPATGGVKKPHRYRPGTVALREIRRYQKSTELLIRKLPFQRLVREIAQDFKTDLRFQSSAVMALQEAAEAYLVGLFEDTNLAAIHAKRVTIMPKDIQLARRIRGERAGGDYKDDDDKSAAGGYPYDVPDYACalculated Mass = 17720Observed Mass = 17720Histone H3-K9C [Figure 49]ARTKQTARCSTGGKAPRKQLATKAARKSAPATGGVKKPHRYRPGTVALREIRRYQKSTELLIRKLPFQRLVREIAQDFKTDLRFQSSAVMALQEASEAYLVALFEDTNLAAIHAKRVTIM PKDIQLARRIRGERACalculated Mass = 15214Observed Mass = 15214Histone H3-K18C [Figure 50]ARTKQTARKSTGGKAPRCQLATKAARKSAPATGGVKKPHRYRPGTVALREIRRYQKSTELLIRKLPFQRLVREIAQDFKTDLRFQSSAVMALQEASEAYLVALFEDTNLAAIHAKRVTIMPKDIQLARRIRGERACalculated Mass = 15214Observed Mass = 15213Histone H3-K27C [Figure 51]ARTKQTARKSTGGKAPRKQLATKAARCSAPATGGVKKPHRYRPGTVALREIRRYQKSTELLIRKLPFQ RLVREIAQDFKTDLRFQSSAVMALQEASEAYLVALFEDTNLAAIHAKRVTIM PKDIQLARRIRGERACalculated Mass = 15214Observed Mass = 15214 pre-SUMO1-wt (C51) [Figure 52]ADQEAKPSTEDLGDKKEGEYIKLKVIGQDSSEIHFKVKMTTHLKKLKESYCQRQGVPMNSLRFLFEGQ RIADNHTPKELGMEEEDVIEVYQEQTGGHSTVLEHHHHHHCalculated Mass = 12475Observed Mass = 12474PstS_D178C [Figure 53]MEASLTGAGATFPAPVYAKWADTYQKETGNKVNYQGIGSSGGVKQIIANTVDFGASDAPLSDEKLAQ EGLFQFPTVIGGVVLAVNIPGLKSGELVLDGKTLGDIYLGKIKKWDDEAIAKLNPGLKLPSQNIAWRRA DGSGTSFVFTSYLAKVNEEWKNNVGTGSTVKWPIGLGGKGNCGIAAFVQRLPGAIGYVEYAYAKQNNLAYTKLISADGKPVSPTEENFANAAKGADWSKTFAQDLTNQKGEDAWPITSTTFILIHKDQKKPEQGT EVLKFFDWAYKTGAKQANDLDYASLPDSVVEQVRAAWKTNIKDSSGKPLYCalculated Mass = 34541Observed Mass = 34540 cAbVCAM_Q1 N_Q118C [Figure 54]NVQLQESGGGSVQTGGSLRLSCAASGYTNSIMYMAWFRQAPGKKREGVAAIRFPDDSAYYAGSVK GRFTISHDNAKNTVYLQMNNLNPEDTAMYYCAARSSPYSFAWNDPSNYNYWGCGTQVTVSSHHHH HHCalculated Mass = 14619Observed Mass = 14619AcrA-Cys123 [Figure 55]SKEEAPKIQMPPQPVTTMSAKSEDLPLSFTYPAKLVSDYDVIIKPQVSGVIVNKLFKAGDKVKKGQTLFIIEQDKFKASVDSAYGQALMAKATFENASKDFCRSKALFSKSAISQKEYDSSLATFNNSKASLASARAQLANARIDLDHTEIKAPFDGTIGDALVNIGDYVSASTTELVRVTNLNPIYADFFISDTDKLNLVRNTQSGKWDLDSIHANLNLNGETVQGKLYFIDSVIDANSGTVKAKAVFDNNNSTLLPGAFATITSEGFIQKNGFKV PQIGVKQDQNDVYVLLVKNGKVEKSSVHISYQNNEYAIIDKGLQNGDKIILDNFKKIQVGSEVKEIGAQL EHHHHHHCalculated Mass = 38817Observed Mass = 38817Npp-Cys61 [Figure 56]MFSSHHHHHHSSGLVPRGSHIDVGKLRQLYAAGERDFSIVDLRGAVLENINLSGAILHGACLDEANLQ QANLSRADLSGATLNGADLRGANLSKADLSDAILDNAILEGAILDEAVLNQANLKAANLEQAILSHANIR EADLSEANLEAADLSGADLAIADLHQANLHQAALERANLTGANLEDANLEGT ILEGGNNNLATCalculated Mass = 21031Observed Mass = 21031SspG_S7C [Figure 57]MYSFPNCFRFGWSQAGFQSEMGTPGSEDPNTDWYKWVHDPENMAAGLVSGDLPENGPGYWGNYKTFHDNAQKMGLKIARLNVEWSRIFPNPLPRPQNFDESKQDVTEVEINENELKRLDEYANKDALNHYREIFKDLKSRGLYFILNMYHWPLPLWLHDPIRVRRGDFTGPSGWLSTRTVYEFARFSAYIAWKFDDLVDEYSTMNEPNVVGGLGYVGVKSGFPPGYLSFELSRRAMYNIIQAHARAYDGIKSVSKKPVGIIYANSSFQPLTDKDMEAVEMAENDNRWWFFDAIIRGEITRGNEKIVRDDLKGRLDWIGVNYYTRTVVKRTEKGYVSLGGYGHGSERNSVSLAGLPTSDFGWEFFPEGLYDVLTKYWNRYHLYMYVTCNGIADDADYQRPYYLVSHVYQVHRAINSGADVRGYLHWSLADNYEWASGFSMRFGLLKVDYNTKRLYWRPSALVYREIAT NGAITDEIEHLNSVPPVKPLRHHHHHHHCalculated Mass = 57488Observed Mass = 57489GFP_S175C [[Figure 58]MHHHHHHSSGVDLGTDNLYFQSMRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFKIRHNVEDGCVQLAD HYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEFVTAAG ITHGMDELYKCalculated Mass = 29361Observed Mass = 29341 , weight loss of 20 Da due to chromophore formation in GFP protein.

[0149] Reaction scope: With the optimal glycosylation conditions in hands, the inventors evaluated the generality of the reaction across various glycosylating reagents, including those derived monosaccharides-, disaccharides- and oligosaccharides. The proposed protocol well accommodated to all kinds of tested sugars and corresponding glycoproteins were obtained in excellent conversions. Sugar motifs commonly found in glycoproteins from mammalian cells, such as D-galactose, D-mannose, D-GIcNAc, D-GalNAc, D-ManNAc and D-xylose, were efficiently incorporated into the desired S- glycoproteins in full conversions. Notably, H3-GlcNAc-Cys10 was obtained as the a-epimeric mimetic of the endogenous epigenetic mark GlcNAc-Ser10. Additionally, several glycosylating reagents derived from unnatural and rare sugars, including L-rhamnose, L-glucose, D-allose and D-lyxose, were also compatible under the established glycosylation conditions, yielding S-glycoproteins with comparable efficiency. To further showcase the robustness of this method, the inventors extended this strategy to incorporate more complex disaccharides and oligosaccharides. Glycoproteins bearing cellobiose, lactose, maltose, melibiose, maltotriose and acarbose units, were successfully yielded without noticeable comprised productivity.

[0150] Sugar donor scope for S-glycoprotein synthesis:

[0151] Histone H3-Glc-C10: Glycoprotein H3-Glc-C10 was prepared according to the general procedure shown earlier with S1 (200 equiv.), Lil (200 equiv.) as shown in Figure 59.

[0152] After completed, the reaction mixture was subjected to LC-MS analysis for determining the conversion. ESI-MS spectrum for H3-Glc-C10: 98% conversion; Calculated Mass = 15417; Observed Mass = 15417 [Figure 60].

[0153] Histone H3-Gal-C10: Glycoprotein H3-Gal-C10 was prepared according to the General procedure shown earlier with S2 (200 equiv.), Lil (200 equiv.), as shown in Figure 61 . After completed, the reaction mixture was subjected to LC-MS analysis for determining the conversion. ESI-MS spectrum for H3-Gal-C10: 98% conversion; Calculated Mass = 15417; Observed Mass = 15416 [Figure 62],

[0154] Histone H3-Man-C10: Glycoprotein H3-Man-C10 was prepared according to the General procedure B with S3 (200 equiv.), Lil (200 equiv.), as shown in Figure 63. After completed, the reaction mixture was subjected to LC-MS analysis for determining the conversion. ESI-MS spectrum for H3-Man- C10: 98% conversion; Calculated Mass = 15417; Observed Mass = 15416 [Figure 64],

[0155] Histone H3-GlcNAc-C10: Glycoprotein H3-GlcNAc-C10 was prepared according to the General procedure B with S4 (200 equiv.), Lil (200 equiv.) as shown in Figure 65. After completed, the reaction mixture was subjected to LC-MS analysis for determining the conversion. ESI-MS spectrum for H3-GlcNAc-C10: 98% conversion; Calculated Mass = 15458; Observed Mass = 15458 [Figure 66].

[0156] Histone H3-GalNAc-C10: Glycoprotein H3-GalNAc-C10 was prepared according to the General procedure B with S5 (500 equiv.), Lil (1000 equiv.), as shown in Figure 67. After completed, the reaction mixture was subjected to LC-MS analysis for determining the conversion. ESI-MS spectrum for H3-GalNAc-C10: 98% conversion; Calculated Mass = 15458; Observed Mass = 15458 [Figure 68].

[0157] Histone H3-ManNAc-C10: Glycoprotein H3-ManNAc-C10 was prepared according to the General procedure B with S6 (500 equiv.), Lil (1000 equiv.), as shown in Figure 69. After completed, the reaction mixture was subjected to LC-MS analysis for determining the conversion. ESI-MS spectrum for H3-ManNAc-C10: 98% conversion; Calculated Mass = 15458; Observed Mass = 15458 [Figure 70],

[0158] Histone H3-Xyl-C10: Glycoprotein H3-Xyl-C10 was prepared according to the General procedure B with S7 (200 equiv.), Lil (200 equiv.), as shown in Figure 71 . After completed, the reaction mixture was subjected to LC-MS analysis for determining the conversion. ESI-MS spectrum for H3-Xyl- C10: 98% conversion; Calculated Mass = 15387; Observed Mass = 15386 [Figure 72].

[0159] Histone H3-LRha-C10: Glycoprotein H3-LRha-C10 was prepared according to the General procedure B with S8 (200 equiv.), Lil (200 equiv.), as shown in Figure 73. After completed, the reaction mixture was subjected to LC-MS analysis for determining the conversion. ESI-MS spectrum for H3- LRha-C10 is shown below: 98% conversion; Calculated Mass = 15401 ; Observed Mass = 15401 as shown in Figure 74.

[0160] Histone H3-LGIc-C10: Glycoprotein H3-LGIc-C10 was prepared according to the General procedure B with S9 (200 equiv.), Lil (200 equiv.), as shown in Figure 75. After completed, the reaction mixture was subjected to LC-MS analysis for determining the conversion. ESI-MS spectrum for H3- LGIc-C10: 98% conversion; Calculated Mass = 15417; Observed Mass = 15417, as shown in Figure 76.

[0161] Histone H3-AII-C10: Glycoprotein H3-AII-C10 was prepared according to the General procedure B with S10 (500 equiv.), Lil (1000 equiv.), as shown in Figure 77. After completed, the reaction mixture was subjected to LC-MS analysis for determining the conversion. ESI-MS spectrum for H3-AII-C10: 98% conversion; Calculated Mass = 15417; Observed Mass = 15417, as shown in Figure 78.

[0162] Histone H3-Lyx-C10: Glycoprotein H3-Lyx-C10 was prepared according to the General procedure B with S11 (200 equiv.), Lil (200 equiv.), as shown in Figure 79. After completed, the reactionmixture was subjected to LC-MS analysis for determining the conversion. ESI-MS spectrum for H3-Lyx- C10: 98% conversion; Calculated Mass = 15387; Observed Mass = 15386, as shown in Figure 80.

[0163] Histone H3-Cel-C10: Glycoprotein H3-Cel-C10 was prepared according to the General procedure B with S12 (200 equiv.), Lil (200 equiv.), as shown in Figure 81 . After completed, the reaction mixture was subjected to LC-MS analysis for determining the conversion. ESI-MS spectrum for H3-Cel- C10: 84% conversion; Calculated Mass = 15579; Observed Mass = 15579, as shown in Figure 82.

[0164] Histone H3-Lac-C10: Glycoprotein H3-Lac-C10 was prepared according to the General procedure B with S13 (200 equiv.), Lil (200 equiv.), as shown in Figure 83. After completed, the reaction mixture was subjected to LC-MS analysis for determining the conversion. ESI-MS spectrum for H3-Lac- C10: 91 % conversion; Calculated Mass = 15579; Observed Mass = 15579, as shown in Figure 84.

[0165] Histone H3-Mal-C10: Glycoprotein H3-Mal-C10 was prepared according to the General procedure B with S14 (200 equiv.), Lil (200 equiv.), as shown in Figure 85. After completed, the reaction mixture was subjected to LC-MS analysis for determining the conversion. ESI-MS spectrum for H3-Mal- C10: 85% conversion; Calculated Mass = 15579; Observed Mass = 15579, as shown in Figure 86.

[0166] Histone H3-Mel-C10: Glycoprotein H3-Mel-C10 was prepared according to the General procedure B with S15 (200 equiv.), Lil (200 equiv.), as shown in Figure 87. After completed, the reaction mixture was subjected to LC-MS analysis for determining the conversion. ESI-MS spectrum for H3-Mel- C10 is shown below: 90% conversion; Calculated Mass = 15579; Observed Mass = 15579, as shown in Figure 88.

[0167] Histone H3-Malt-C10: Glycoprotein H3-Malt-C10 was prepared according to the General procedure B with S16 (200 equiv.), Lil (200 equiv.), as shown in Figure 89. After completed, the reaction mixture was subjected to LC-MS analysis for determining the conversion. ESI-MS spectrum for H3-Malt- C10: 98% conversion; Calculated Mass = 15741 ; Observed Mass = 15741 , as shown in Figure 90.

[0168] Histone H3-Acar-C10: Glycoprotein H3-Acar-C10 was prepared according to the General procedure B with S17 (200 equiv.), Lil (200 equiv.), as shown in Figure 91 . After completed, the reaction mixture was subjected to LC-MS analysis for determining the conversion. ESI-MS spectrum for H3- Acar-C10 is shown below: 98% conversion; Calculated Mass = 15882; Observed Mass = 15882, as shown in Figure 92.

[0169] Scope of various sugars for glycosylation is shown in Figure 93.

[0170] Encouraged by these successful examples on sugar scope, the inventors further attempted to extend this protocol to other proteins to expand the repertoire of synthesized glycoproteins. Threerepresentative glycosylating reagents, GIcNAc-SPyF, Man-SPyF and Gal-SPyF, were selected to interact with different proteins. These sugar units were successfully incorporated into glycoproteins in high efficiency regardless of their size and folding. For instance, Histone homologs eH3 and H3 (small a-helical nuclear proteins) harbour a series of mutated cysteine residues at site 2, 4, 9, 18, 27, were glycosylated by GIcNAc-SPyF in full conversion. Similarly, glycosylation of pre-SUMO1 (SUMO, small ubiquitin like modifier), a small globular protein containing a-helices and p-sheets, also showed high efficiency. However, 24% of a two-unit GIcNAc addition product was detected from MS spectrum, likely due to competitive interference from nucleophilic lysine residues. S-GIcNAcylation of the other four more sizable proteins resulted in moderate to good conversions. These proteins included PstS (involved in bacteria phosphate transport), Npp (a p-helical pentapeptide repeat), SspG (an apsTIM barrel enzyme) and GFP (a green fluorescent protein). Additionally, cabVCAM (a cross-reactive nanobody again human and murine VCAM1) and AcrA (a membrane protein) were glycosylated by Man-SPyF and Gal-SPyF, yielding cabVCAM-Man-Cys1 18 and Acra-Gal-Cys123 with conversions of 90% and 86%, respectively. The stereochemistry of these on-protein sugars was presumed to be identical to that of glycosylated histones.

[0171] Protein scope for S-glycoprotein synthesis is provided below.

[0172] Histone TEV-H3-GlcNAc-C2: Glycoprotein TEV-H3-GlcNAc-C2 was prepared according to the General procedure B with S4 (200 equiv.), Lil (200 equiv.), as shown in Figure 94. After completed, the reaction mixture was subjected to LC-MS analysis for determining the conversion. ESI-MS spectrum for TEV-H3-GlcNAc-C2: 98% conversion; Calculated Mass = 17923; Observed Mass = 17923, as shown in Figure 95.

[0173] Histone eH3-GlcNAc-C4: Glycoprotein eH3-GlcNAc-C4 was prepared according to the General procedure B with S4 (500 equiv.), Lil (1000 equiv.), as shown in Figure 96. After completed, the reaction mixture was subjected to LC-MS analysis for determining the conversion. ESI-MS spectrum for eH3-GlcNAc-C4 is shown below: 98% conversion; Calculated Mass = 17923; Observed Mass = 17923, as shown Figure 97.

[0174] Histone H3-GlcNAc-C9: Glycoprotein H3-GlcNAc-C9 was prepared according to the General procedure B with S4 (500 equiv.), Lil (1000 equiv.), as shown in Figure 98. After completed, the reaction mixture was subjected to LC-MS analysis for determining the conversion. ESI-MS spectrum for H3- GlcNAc-C9 is shown below: 98% conversion; Calculated Mass = 15417; Observed Mass = 15416, Figure 99.

[0175] Histone H3-GlcNAc-C18: Glycoprotein H3-GlcNAc-C18 was prepared according to the General procedure B with S4 (200 equiv.), Lil (200 equiv.), as shown in Figure 100. After completed,the reaction mixture was subjected to LC-MS analysis for determining the conversion. ESI-MS spectrum for H3-GlcNAc-C18: 98% conversion; Calculated Mass = 15417; Observed Mass = 15416, as shown in Figure 101 .

[0176] Histone H3- GlcNAc-C27: Glycoprotein H3-GlcNAc-C27 was prepared according to the General procedure B with S4 (200 equiv.), Lil (200 equiv.), as shown in Figure 102. After completed, the reaction mixture was subjected to LC-MS analysis for determining the conversion. ESI-MS spectrum for H3-GlcNAc-C27: 98% conversion; Calculated Mass = 15417; Observed Mass = 15416, as shown in Figure 103.

[0177] Histone eH3- GlcNAc-C27: Glycoprotein eH3-GlcNAc-C27 was prepared according to the General procedure B with S4 (200 equiv.), Lil (500 equiv.), as shown in Figure 104. After completed, the reaction mixture was subjected to LC-MS analysis for determining the conversion. ESI-MS spectrum for eH3-GlcNAc-C27: 98% conversion; Calculated Mass = 1792; Observed Mass = 17923, as shown in Figure 105.

[0178] preSUMO1-GlcNAc-C51 : Glycoprotein pre-SUMO1 -GlcNAc-C51 was prepared according to the General procedure B with S4 (200 equiv.), Lil (200 equiv.), as shown in Figure 106. After completed, the reaction mixture was subjected to LC-MS analysis for determining the conversion. ESI-MS spectrum for H3-GlcNAc-C27: 98% conversion (24% two units of GIcNAc addition), Observed Mass = 12677; peak 12881 denotes two units of GIcNAc addition, as shown in Figure 107.

[0179] PstS-GlcNAc-C178: Glycoprotein PstS-GlcNAc-C178 was prepared according to the General procedure B with S4 (200 equiv.), Lil (200 equiv.), as shown in Figure 108. After completed, the reaction mixture was subjected to LC-MS analysis for determining the conversion. ESI-MS spectrum for PstS- GlcNAc-C178: 57% conversion; Calculated Mass = 34744; Observed Mass = 34743, as shown in Figure 109.

[0180] cabVCAM-Man-C118: Glycoprotein cabVCAM-Man-C1 18 was prepared according to the General procedure B with S3 (500 equiv.), Lil (1000 equiv.), as shown in Figure 1 10. After completed, the reaction mixture was subjected to LC-MS analysis for determining the conversion. ESI-MS spectrum for cabVCAM-Man-C1 18: 90% conversion; Calculated Mass = 14781 ; Observed Mass = 14781 , as shown in Figure 1 1 1.

[0181] Npp-GlcNAc-C61 : Glycoprotein Npp-GlcNAc-C61 was prepared according to the General procedure B with S4 (500 equiv.), Lil (1000 equiv.), as shown in Figure 1 12. After completed, the reaction mixture was subjected to LC-MS analysis for determining the conversion. ESI-MS spectrum forNpp-GlcNAc-C61 : 78% conversion; Calculated Mass = 21234; Observed Mass = 21233, as shown in Figure 1 13.

[0182] AcrA-Gal-C123: Glycoprotein AcrA-Gal-C123 was prepared according to the General procedure B with S2 (500 equiv.), Lil (1000 equiv.), as shown in Figure 1 14. After completed, the reaction mixture was subjected to LC-MS analysis for determining the conversion. ESI-MS spectrum for AcrA-Gal-C123: 86% conversion; Calculated Mass = 38979; Observed Mass = 38979, as shown in Figure 1 15.

[0183] Ss G-GlcNAc-C7: Glycoprotein SspG-GlcNAc-C7 was prepared according to the General procedure B with S4 (500 equiv.), Lil (1000 equiv.), as shown in Figure 1 16. After completed, the reaction mixture was subjected to LC-MS analysis for determining the conversion. ESI-MS spectrum for Ss[3G-GlcNAc-C7 : 71% conversion; Calculated Mass = 57691 ; Observed Mass = 57692, as shown in Figure 1 17.

[0184] GFP-GlcNAc-C175: Glycoprotein GFP-GlcNAc-C175 was prepared according to the General procedure B with S4 (500 equiv.), Lil (1000 equiv.), as shown in Figure 1 18. After completed, the reaction mixture was subjected to LC-MS analysis for determining the conversion. ESI-MS spectrum for GFP-GlcNAc-C175: 76% conversion; Calculated Mass = 29544; Observed Mass = 29542, as shown in Figure 1 19.

[0185] The microenvironment of protein can affect the pH and the nucleophilicity of lysine residues, and models such as AlphaFold can be used to make predictions. However, side reactions of lysine glycosylation cannot be fully avoided, as they are determined by the protein’s structure.

[0186] Scope of proteins for glycosylation is provided in Figure 120.

[0187] In summary, the inventors established a general platform for the synthesis of a wide range of S-glycoproteins. Mechanistic studies reveal that a disulfide-bridged dimerized protein serves as the critical intermediate in the radical S-glycosylation process, leading to the formation of a-configured S- glycoproteins. The stereochemistry of a representative S-glycoprotein, H3-Glc-Cys10, was further validated using NMR analysis. With its operational simplicity and precise site specificity, the inventors anticipate this method will have broad utility in developing glycosylated biotherapeutics and providing powerful tools to unravel glycosylation-dependent biological processes.References:1 . Floyd, N., Vijayakrishnan, B., Koeppe, J. R. & Davis, B. G. Thiyl glycosylation of olefinic proteins: S- linked glycoconjugate synthesis. Angew. Chem. Int. Ed. 48, 7798-7802 (2009).2. Mollner, T. A. et al. Post-translational insertion of boron in proteins to probe and modulate function. Nat. Chem. Biol. 17, 1245-1261 (2021 ).3. Kong, A. T., Leprevost, F.V., Avtonomov, D. M., Mellacheruvu, D. & Nesvizhskii, A. I. MS Fragger: ultrafast and comprehensive peptide identification in mass spectrometry-based proteomics. Nat. Methods 14, 513-520 (2017).4. Moremen, K. W.; Tiemeyer, M.; Nairn, A. V. Vertebrate protein glycosylation: diversity, synthesis and function. Nat. Rev. Mol. Cell Biol. 2012, 13, 448-462.5. Rich, J. R.; Withers, S. G. Emerging methods for the production of homogeneous human glycoproteins. Nat. Chem. Biol. 2009, 5, 206-215.6. Howard C. Hang, C. R. B. Chemoselective Approaches to Glycoprotein Assembly. Acc. Chem. Res. 2001 , 34, 727-736.7. Van Kasteren, S. I.; Kramer, H. B.; Gamblin, D. P.; Davis, B. G. Site-selective glycosylation of proteins: creating synthetic glycoproteins. Nat. Protoc. 2007, 2, 3185-3194.8. David P. Gamblin, E. M. S., and Benjamin G. Davis Glycoprotein Synthesis: An Update. Chem. Rev. 2009, 109, 131 -163.9. Schjoldager, K. T.; Narimatsu, Y.; Joshi, H. J.; Clausen, H. Global view of human protein glycosylation pathways and functions. Nat. Rev. Mol. Cell Biol. 2020, 21 , 729-749.10. Sinclair, A. M.; Elliott, S. Glycoengineering: the effect of glycosylation on the properties of therapeutic proteins. J Pharm Sci 2005, 94, 1626-1635.1 1 . Reily, C.; Stewart, T. J.; Renfrow, M. B.; Novak, J. Glycosylation in health and disease. Nat. Rev. Nephrol. 2019, 15, 346-366.12. Wang, L. X.; Lomino, J. V. Emerging technologies for making glycan-defined glycoproteins. ACS Chem. Biol. 2012, 7, 1 10-122.13. Haitian Liu, L. W., Ansgar Brock, Chi-Huey Wong, Peter G. Schultz A Method for the Generation of Glycoprotein Mimetics. J. Am. Chem. Soc. 2003, 125, 1702-1703.14. Hudak, J. E.; Yu, H. H.; Bertozzi, C. R. Protein glycoengineering enabled by the versatile synthesis of aminooxy glycans and the genetically encoded aldehyde tag. J. Am. Chem. Soc. 2011 , 133, 16127- 16135.15. Berti, F.; Adamo, R. Antimicrobial glycoconjugate vaccines: an overview of classic and modern approaches for protein modification. Chem. Soc. Rev. 2018, 47, 9015-9025.16. Anish, C.; Beurret, M.; Poolman, J. Combined effects of glycan chain length and linkage type on the immunogenicity of glycoconjugate vaccines. NPJ Vaccines 2021 , 6, 150.17. Li, C.; Wang, L. X. Chemoenzymatic Methods for the Synthesis of Glycoproteins. Chem. Rev. 2018, 1 18, 8359-8413.18. Groenevelt, J. M.; Corey, D. J.; Fehl, C. Chemical Synthesis and Biological Applications of O- GIcNAcylated Peptides and Proteins. Chembiochem 2021 , 22, 1854-1870.19. Bak, D. W.; Bechtel, T. J.; Falco, J. A.; Weerapana, E. Cysteine reactivity across the subcellular universe. Curr. Opin. Chem. Biol. 2019, 48, 96-105.20. Spears, R. J.; McMahon, C.; Chudasama, V. Cysteine protecting groups: applications in peptide and protein science. Chem. Soc. Rev. 2021 , 50, 11098-1 1155.21 . Chen, F. J.; Gao, J. Fast Cysteine Bioconjugation Chemistry. Chem. Eur. J. 2022, 28, e202201843.22. Lercher, L.; Raj, R.; Patel, N. A.; Price, J.; Mohammed, S.; Robinson, C. V.; Schofield, C. J.; Davis, B. G. Generation of a synthetic GIcNAcylated nucleosome reveals regulation of stability by H2A-Thr101 GIcNAcylation. Nat. Commun. 2015, 6, 7978.23. Raj, R.; Lercher, L.; Mohammed, S.; Davis, B. G. Synthetic Nucleosomes Reveal that GIcNAcylation Modulates Direct Interaction with the FACT Complex. Angew. Chem. Int. Ed. 2016, 55, 8918-8922.24. De Leon, C. A.; Levine, P. M.; Craven, T. W.; Pratt, M. R. The Sulfur-Linked Analogue of O-GIcNAc (S-GIcNAc) Is an Enzymatically Stable and Reasonable Structural Surrogate for O-GIcNAc at the Peptide and Protein Levels. Biochemistry 2017, 56, 3507-3517.25. Gorelik, A.; Bartual, S. G.; Borodkin, V. S.; Varghese, J.; Ferenbach, A. T.; van Aalten, D. M. F. Genetic recoding to dissect the roles of site-specific protein O-GIcNAcylation. Nat. Struct. Mol. Biol. 2019, 26, 1071 -1077.26. Maynard, J. C.; Burlingame, A. L.; Medzihradszky, K. F. Cysteine S-linked N-acetylglucosamine (S- GIcNAcylation), A New Post-translational Modification in Mammals. Mol. Cell. Proteom. 2016, 15, 3405- 341 1 .27. Xiao, H.; Wu, R. Global and Site-Specific Analysis Revealing Unexpected and Extensive Protein S- GIcNAcylation in Human Cells. Anal. Chem. 2017, 89, 3656-3663.28. Jiang, Y.; Zhang, Y.; Lee, B. C.; Koh, M. J. Diversification of Glycosyl Compounds via Glycosyl Radicals. Angew. Chem. Int. Ed. 2023, 62, e202305138.29. Shang, W.; Niu, D. Radical Pathway Glycosylation Empowered by Bench-Stable Glycosyl Donors. Acc. Chem. Res. 2023, 56, 2473-2488.30. Wadzinski, T. J.; Steinauer, A.; Hie, L.; Pelletier, G.; Schepartz, A.; Miller, S. J. Rapid phenolic O- glycosylation of small molecules and complex unprotected peptides in aqueous solvent. Nat. Chem. 2018, 10, 644-652.31 . Jiang, Y.; Wei, Y.; Zhou, Q. Y.; Sun, G. Q.; Fu, X. P.; Levin, N.; Zhang, Y.; Liu, W. Q.; Song, N.; Mohammed, S.; Davis, B. G.; Koh, M. J. Direct radical functionalization of native sugars. Nature 2024, 631 , 319-327.32. Bernardes, G. J.; Grayson, E. J.; Thompson, S.; Chalker, J. M.; Errey, J. C.; El Oualid, F.; Claridge, T. D.; Davis, B. G. From disulfide- to thioether-linked glycoproteins. Angew. Chem. Int. Ed. 2008, 47, 2244-2247.33. Wan, L. Q.; Zhang, X.; Zou, Y.; Shi, R.; Cao, J. G.; Xu, S. Y.; Deng, L. F.; Zhou, L.; Gong, Y.; Shu, X.; Lee, G. Y.; Ren, H.; Dai, L.; Qi, S.; Houk, K. N.; Niu, D. Nonenzymatic Stereoselective S- Glycosylation of Polypeptides and Proteins. J. Am. Chem. Soc. 2021 , 143, 1 1919-1 1926.34. Justin M. Chalker, G. J. L. B., Benjamin G. Davis A “Tag-and-Modify” Approach to Site-Selective Protein Modification. Acc. Chem. Res. 201 1 , 44, 730-741 .35. Dadova, J.; Galan, S. R.; Davis, B. G. Synthesis of modified proteins via functionalization of dehydroalanine. Curr. Opin. Chem. Biol. 2018, 46, 71 -81.36. Fu, X., et al. ACS Cent. Sci. 9, 405-416 (2023).37. Josephson, B. et al. 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Claims

1. CLAIMS1 . A non-enzymatic method of synthesizing a glycosylated protein / protein glycosylation comprising: reacting a thioglycosyl donor / glycosylating agent with a protein substrate in the presence of a catalyst I activator / promoter and a photoillumination source, wherein the thioglycosyl donor / glycosylating agent converts the protein substrate to produce the glycosylated protein.

2. The method of claim 1 , wherein the thioglycosyl donor / glycosylating agent is a native sugar or a derivative thereof.

3. The method of claim 2, wherein the thioglycosyl donor is 2,3,5,6-tetrafluoropyridine-4- thiogylcoside derivative of the native sugar.

4. The method of claim 3, wherein the 2,3,5,6-tetrafluoropyridine-4-thiogylcoside derivative of the native sugar is obtained by cross-coupling of the native sugar.

5. The method of claim 4, wherein the native sugar is D-galactose, D-mannose, D-GIcNAc, D- GalNAc, D-ManNAc, D-xylose, D-glucose, L-rhamnose, L-glucose, D-allose, D-lyxose, cellobiose, lactose, maltose, melibiose, maltotriose, and oligosaccharide acarbose.

6. The method of claim 1 , wherein the protein substrate is histone, pre-SUMO1 , PstS, Npp, SspG, cabVCAM, AcrA and GFP.

7. The method of any one of claims 1 -6, wherein the protein substrate is tagged with an amino acid.

8. The method of claim 7, wherein the protein substrate is tagged with an aliphatic amino acid to synthesize a C-glycoprotein.

9. The method of claim 8, wherein the protein substrate is tagged with alanine or dehydro alanine (DHA).

10. The method of claim 8 or 9, wherein the catalyst / activator / promoter is a reductant.1 1 . The method of claim 10, wherein the catalyst / activator / promoter is 4- (Dimethylamino)benzenethiol, 4-methylbenzethiol or bis(catecholato)diboron (E^Catg).

12. The method of any one of claims 8-1 1 , wherein the reaction is carried out at a temperature ranging from about 2°C to about 6°C.

13. The method of any one of claims 8-12, wherein the reaction is carried out at a pH ranging from about 6 to about 10.

14. The method of any one of claims 8-13, wherein the reaction is carried out in the presence of a buffer such as TBS buffer, PBS buffer, and Bis-Tris buffer.

15. The method of any one of claims 8-14, wherein the photoillumination source is UV light, blue LED, or a light source with a wavelength ranging from 250nm to 500nm.

16. The method of any one of claims 8-15, wherein the photoillumination source has a wavelength of 500 nm or lower.

17. The method of claim 7, wherein the protein substrate is tagged with a nucleophilic amino acid to synthesize an S-glycoprotein.

18. The method of claim 17, wherein the protein substrate is tagged with cysteine, threonine, serine, tyrosine, glutamic acid, aspartic acid, lysine, arginine, histidine or methionine.

19. The method of claim 17 or 18, wherein the catalyst / activator / promoter is a reductant.

20. The method of claim 19, wherein the catalyst / activator / promoter is 4-methylbenzethiol or Lithium Iodide.21 . The method of any one of claims 17-20, wherein the reaction is carried out at a temperature ranging from about 2°C to about 6°C.

22. The method of any one of claims 17-21 , wherein the reaction is carried out at a pH ranging from about 6 to about 10.

23. The method of any one of claims 17-22, wherein the reaction is carried out in the presence of a buffer such as TBS buffer, PBS buffer, and Bis-Tris buffer.

24. The method of any one of claims 17-23, wherein the photoillumination source is UV light, blue LED, or a light source with a wavelength ranging from 250nm to 500nm.

25. The method of any one of claims 17-24, wherein the photoillumination source has a wavelength of 365 nm or lower.

26. The method of any one of claims 1 -25, wherein the thioglycosyl donor / glycosylating agent and the catalyst / activator / promoter are in equal proportions.

27. The method of any one of claims 1 -25, wherein the thioglycosyl donor / glycosylating agent and the catalyst / activator / promoter are in unequal proportions.

28. A method of protein glycosylation according to Scheme 1 :

29. A method of synthesizing C-glycoproteins according to Scheme 2:blue LED C-giyeosyiprote in30. A method of synthesizing C-glycosylprotein according to Scheme 3:31 . A method of synthesizing C-glycosylprotein according to Scheme 4:

32. A method of synthesizing S-glycosylproteins according to Scheme 5:

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