Controlled glycan metabolic engineering systems and applications thereof

Abstract

Compositions are provided according to aspects of the present disclosure which include: a controllably-releasable glycan precursor, the controllably-releasable glycan precursor having a photocage component or a chemocage component covalently bonded to a glycan precursor component. Methods of controlled metabolic engineering in a cell are provided which include introducing a controllably-releasable glycan precursor of the present disclosure into a cell; and exposing the controllably-releasable glycan precursor in the cell to a stimulus effective to release the glycan precursor from the photocage component or chemocage component, thereby providing the glycan precursor to the cell, and providing controlled metabolic engineering in the cell.

Description

CONTROLLED GLYCAN METABOLIC ENGINEERING SYSTEMS AND APPLICATIONS THEREOF REFERENCE TO RELATED APPLICATION

[0001] This application claims priority from U.S. Provisional Patent Application Serial No. 63 / 524,319, filed June 30, 2023, the entire content of which is incorporated herein by reference. GOVERNMENT FUNDING CLAUSE

[0002] This invention was made with government support under R35GM142637 awarded by the National Institute of General Medicine Sciences. The government has certain rights in the invention. FIELD OF THE INVENTION

[0003] According to general aspects, compositions and methods for controlled glycan metabolic engineering are provided by the present disclosure. According to specific aspects, compositions are provided which include: a controllably-releasable glycan precursor, the controllably-releasable glycan precursor having a photocage component or a chemocage component covalently bonded to a glycan precursor component, along with methods of their use. BACKGROUND OF THE INVENTION

[0004] Sugar additions to biomolecules, or glycans, are some of the most abundant biomolecule modifications in biology because they enable cells to adapt to changing nutrient and stress conditions. A key sugar-driven pathway in cells is the glycosylation of biomolecules. In mammalian cells, glycosylation of proteins makes up a major class of post- translational modifications (PTMs) via N- or O-atom linkages to proteins.9Glycolipids and glycoRNAs are also biomolecules modified by carbohydrates.10Glycan precursors involved in biomolecule glycosylation include a relatively small number of acetylated sugar monosaccharides, N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), and sialic acids derived from N-acetylmannosamine (ManNAc).9Each of these N-acetyl sugars isnot used for metabolism, but rather become activated as nucleotide sugar phosphates for biomolecule glycosylation via a diverse class of glycosyltransferase enzymes (Fig.1A).11

[0005] In cells, the concentration of nucleotide sugar donors like uridine diphosphate- GlcNAc (UDP-GlcNAc) is critical for regulating glycan formation.12A key example is the reversible attachment of O-linked GlcNAc (O-GlcNAc) to thousands of proteins in mammalian cells.13–14The concentration of UDP-GlcNAc directly regulates O-GlcNAc dynamics.12,15–18Additionally, both cellular UDP-GalNAc and sialic acid glycans correlate with hyperglycemic conditions.19–20Aberrant glycosylation patterns of proteins play critical roles in the progression of diseases such as diabetes21, cancer,22rheumatoid arthritis23, and neurodegenerative disorders12,24because sugar metabolism can be altered in these pathological cell states. However, there is limited knowledge regarding the spatiotemporal dynamics and regulatory mechanisms that are affected by these deviations from the normal glycosylation patterns in these diseases. Thus, an unmet challenge for the field of glycobiology is the study of glycan biosynthetic pathways with chemical control, especially in live cell settings.

[0006] There is a continuing need for compositions and methods relating to biocompatible glycan precursors with controlled release properties. SUMMARY OF THE INVENTION

[0007] Compositions are provided according to aspects of the present disclosure which include: a controllably-releasable glycan precursor, the controllably-releasable glycan precursor having a photocage component or a chemocage component covalently bonded to a glycan precursor component.

[0008] Compositions are provided according to aspects of the present disclosure which include: a controllably-releasable glycan precursor, the controllably-releasable glycan precursor having a photocage component or a chemocage component covalently bonded to a glycan precursor component, wherein the glycan precursor component is an N-acetyl sugar.

[0009] Compositions are provided according to aspects of the present disclosure which include: a controllably-releasable glycan precursor, the controllably-releasable glycan precursor having a photocage component or a chemocage component covalently bonded to a glycan precursor component, wherein the glycan precursor component is selected from the group consisting of: GlcNAc, GalNAc, ManNAc, and a derivative of any thereof.

[0010] Compositions are provided according to aspects of the present disclosure which include: a controllably-releasable glycan precursor, the controllably-releasable glycanprecursor having a photocage component or a chemocage component covalently bonded to a glycan precursor component, wherein the photocage component or the chemocage component includes: an o-nitrobenzyl, a p-hydroxyphenyl, a coumarinyl, a nitrodibenzofuranyl, a nitrophenylbenzofuran (NPDF), or a derivative of any thereof.

[0011] Compositions are provided according to aspects of the present disclosure which include: a controllably-releasable glycan precursor, the controllably-releasable glycan precursor having a photocage component or a chemocage component covalently bonded to a glycan precursor component, wherein the glycan precursor component is an N-acetyl sugar, and wherein the photocage component or the chemocage component includes: an o- nitrobenzyl, a p-hydroxyphenyl, a coumarinyl, a nitrodibenzofuranyl, a nitrophenylbenzofuran (NPDF), or a derivative of any thereof.

[0012] Compositions are provided according to aspects of the present disclosure which include: a controllably-releasable glycan precursor, the controllably-releasable glycan precursor having a photocage component or a chemocage component covalently bonded to a glycan precursor component, wherein the glycan precursor component is selected from the group consisting of: GlcNAc, GalNAc, ManNAc, and a derivative of any thereof, and wherein the photocage component or the chemocage component includes: an o-nitrobenzyl, a p-hydroxyphenyl, a coumarinyl, a nitrodibenzofuranyl, a nitrophenylbenzofuran (NPDF), or a derivative of any thereof.

[0013] Compositions are provided according to aspects of the present disclosure which include: a controllably-releasable glycan precursor, the controllably-releasable glycan precursor having a photocage component covalently bonded to a glycan precursor component, wherein the photocage component comprises a member of the group consisting of: nitrophenylethanol (NPE), diethylaminocoumarin (DEAC), 2-nitrobenzodioxyethanol (NBDE), and a derivative thereof.

[0014] Compositions are provided according to aspects of the present disclosure which include: a controllably-releasable glycan precursor, the controllably-releasable glycan precursor having a photocage component covalently bonded to a glycan precursor component, wherein the glycan precursor component is an N-acetyl sugar, and wherein the photocage component comprises a member of the group consisting of: nitrophenylethanol (NPE), diethylaminocoumarin (DEAC), 2-nitrobenzodioxyethanol (NBDE), and a derivative thereof.

[0015] Compositions are provided according to aspects of the present disclosure which include: a controllably-releasable glycan precursor, the controllably-releasable glycanprecursor having a photocage component covalently bonded to a glycan precursor component, wherein the glycan precursor component is selected from the group consisting of: GlcNAc, GalNAc, ManNAc, and a derivative of any thereof, and wherein the photocage component comprises a member of the group consisting of: nitrophenylethanol (NPE), diethylaminocoumarin (DEAC), 2-nitrobenzodioxyethanol (NBDE), and a derivative thereof.

[0016] Compositions are provided according to aspects of the present disclosure which include: a controllably-releasable glycan precursor, the controllably-releasable glycan precursor having a chemocage component covalently bonded to a glycan precursor component, wherein the chemocage component comprises a member of the group consisting of: an aryl tetrazine, a trans-cyclooctene, or a derivative of any thereof

[0017] Compositions are provided according to aspects of the present disclosure which include: a controllably-releasable glycan precursor, the controllably-releasable glycan precursor having a chemocage component covalently bonded to a glycan precursor component, wherein the glycan precursor component is an N-acetyl sugar, and wherein the chemocage component comprises a member of the group consisting of: an aryl tetrazine, a trans-cyclooctene, or a derivative of any thereof.

[0018] Compositions are provided according to aspects of the present disclosure which include: a controllably-releasable glycan precursor, the controllably-releasable glycan precursor having a chemocage component covalently bonded to a glycan precursor component, wherein the glycan precursor component is selected from the group consisting of: GlcNAc, GalNAc, ManNAc, and a derivative of any thereof, and wherein the chemocage component comprises a member of the group consisting of: an aryl tetrazine, a trans- cyclooctene, or a derivative of any thereof

[0019] Compositions are provided according to aspects of the present disclosure which include: a controllably-releasable glycan precursor, wherein the controllably-releasable glycan precursor is a photo-releasable glycan precursor compound is selected from the group consisting of: compound 1a, compound 2a, compound 3a, compound 1b, compound 2b, compound 3b, compound 4, compound 5, compound 6, compound 7, and compound 8, or a derivative of any thereof.

[0020] Compositions are provided according to aspects of the present disclosure which include: a controllably-releasable glycan precursor, wherein the controllably-releasable glycan precursor is a bioorthogonal chemistry-releasable glycan precursor compound selectedfrom the group consisting of: compound 28, compound 32, compound 35, compound 36, or a derivative of any thereof.

[0021] Compositions are provided according to aspects of the present disclosure which include: a controllably-releasable glycan precursor, wherein the controllably-releasable glycan precursor is 6-NBE-GlcNAc, or a derivative thereof.

[0022] Compositions are provided according to aspects of the present disclosure which include: a controllably-releasable glycan precursor, wherein the controllably-releasable glycan precursor includes:, where the glycan precursor component is selected from the group consisting of: GlcNAc, GalNAc, ManNAc, and a derivative of any thereof; and where R is aryl, alkyl, or H.

[0023] Compositions are provided according to aspects of the present disclosure which include: a controllably-releasable glycan precursor, wherein the controllably-releasable glycan precursor includes:where the glycan precursor component is selected from the group consisting of: GlcNAc, GalNAc, ManNAc, and a derivative of any thereof.

[0024] Compositions are provided according to aspects of the present disclosure which include: a controllably-releasable glycan precursor, the controllably-releasable glycanprecursor having a photocage component or a chemocage component covalently bonded to a glycan precursor component, wherein the controllably-releasable glycan precursor includes: ,, where PG is an ester-based protecting group selected from the group consisting of: an acetyl ester, an ethyl ester, a propyl ester, a butyl ester, a pentyl ester, and a hexyl ester; where R is selected from the group consisting of: an azide, C2-C8 linear or cyclic alkene, and C2-C8 terminal or cyclic alkyne; and where R’ is selected from the group consisting of: H, C1-C6 ester, and a sugar phosphate in alpha or beta configuration.

[0025] Compositions are provided according to aspects of the present disclosure which include: a controllably-releasable glycan precursor, the controllably-releasable glycan precursor having a photocage component or a chemocage component covalently bonded to a glycan precursor component, wherein the controllably-releasable glycan precursor includes:and ,, where PG is an ester-based protecting group selected from the group consisting of: an acetyl ester, an ethyl ester, a propyl ester, a butyl ester, a pentyl ester, and a hexyl ester; where R is selected from the group consisting of: an azide, C2-C8 linear or cyclic alkene, and C2-C8 terminal or cyclic alkyne; and where R’ is selected from the group consisting of: H, C1-C6 ester, and a sugar phosphate in alpha or beta configuration.

[0026] Compositions are provided according to aspects of the present disclosure which include: a controllably-releasable glycan precursor shown or described herein and further including one or more of: a carrier, a buffer, and a salt.

[0027] Compositions are provided according to aspects of the present disclosure which include: a controllably-releasable glycan precursor shown or described herein and further including a pharmaceutically acceptable carrier.

[0028] Methods of of controlled metabolic engineering in a cell are provided according to aspects of the present disclosure which include: providing a controllably-releasable glycan precursor shown or described herein; introducing the controllably-releasable glycan precursorinto a cell; and exposing the controllably-releasable glycan precursor in the cell to a stimulus effective to release the glycan precursor from the photocage component or chemocage component, thereby providing the glycan precursor to the cell, and providing controlled metabolic engineering in the cell. According to aspects of the present disclosure, the cell is in vitro, in vivo, or ex vivo. According to aspects of the present disclosure, the cell is a human cell.

[0029] Methods of of controlled metabolic engineering in a cell are provided according to aspects of the present disclosure which include: providing a controllably-releasable glycan precursor shown or described herein; introducing the controllably-releasable glycan precursor into a cell; and exposing the controllably-releasable glycan precursor in the cell to a stimulus effective to release the glycan, wherein the controllably-releasable glycan precursor comprises a photocage component covalently bonded to a glycan precursor component and wherein the stimulus is light having a wavelength is in the range of about 200 nm to about 750 nm, thereby releasing the precursor from the photocage component component, and thereby providing the glycan precursor to the cell, and providing controlled metabolic engineering in the cell. According to aspects of the present disclosure, the cell is in vitro, in vivo, or ex vivo. According to aspects of the present disclosure, the cell is a human cell.

[0030] Methods of of controlled metabolic engineering in a cell are provided according to aspects of the present disclosure which include: providing a controllably-releasable glycan precursor shown or described herein; introducing the controllably-releasable glycan precursor into a cell; and exposing the controllably-releasable glycan precursor in the cell to a stimulus effective to release the glycan, wherein the controllably-releasable glycan precursor comprises a photocage component covalently bonded to a glycan precursor component and wherein the stimulus is light having a wavelength is in the range of about 350 nm to about 365 nm, thereby releasing the precursor from the photocage component component, and thereby providing the glycan precursor to the cell, and providing controlled metabolic engineering in the cell. According to aspects of the present disclosure, the cell is in vitro, in vivo, or ex vivo. According to aspects of the present disclosure, the cell is a human cell.

[0031] Methods of of controlled metabolic engineering in a cell are provided according to aspects of the present disclosure which include: providing a controllably-releasable glycan precursor shown or described herein; introducing the controllably-releasable glycan precursor into a cell; and exposing the controllably-releasable glycan precursor in the cell to a stimulus effective to release the glycan, wherein the controllably-releasable glycan precursor comprises a photocage component covalently bonded to a glycan precursor component andwherein the stimulus is light having a wavelength is in the range of about 350 nm to about 365 nm for NPE or NBDE, thereby releasing the precursor from the photocage component component, and thereby providing the glycan precursor to the cell, and providing controlled metabolic engineering in the cell. According to aspects of the present disclosure, the cell is in vitro, in vivo, or ex vivo. According to aspects of the present disclosure, the cell is a human cell.

[0032] Methods of of controlled metabolic engineering in a cell are provided according to aspects of the present disclosure which include: providing a controllably-releasable glycan precursor shown or described herein; introducing the controllably-releasable glycan precursor into a cell; and exposing the controllably-releasable glycan precursor in the cell to a stimulus effective to release the glycan, wherein the controllably-releasable glycan precursor comprises a photocage component covalently bonded to a glycan precursor component and wherein the stimulus is light having a wavelength is in the range of about 400 nm to about 420 nm for DEAC, thereby releasing the precursor from the photocage component component, and thereby providing the glycan precursor to the cell, and providing controlled metabolic engineering in the cell. According to aspects of the present disclosure, the cell is in vitro, in vivo, or ex vivo. According to aspects of the present disclosure, the cell is a human cell.

[0033] Methods of of controlled metabolic engineering in a cell are provided according to aspects of the present disclosure which include: providing a controllably-releasable glycan precursor shown or described herein; introducing the controllably-releasable glycan precursor into a cell; and exposing the controllably-releasable glycan precursor in the cell to a stimulus effective to release the glycan precursor from the chemocage component, wherein the stimulus is exposure to a bioorthogonal chemical reaction effective to release the glycan precursor from the chemocage component, thereby providing the glycan precursor to the cell, and providing controlled metabolic engineering in the cell. According to aspects of the present disclosure, the cell is in vitro, in vivo, or ex vivo. According to aspects of the present disclosure, the cell is a human cell. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figures 1A and 1B show hexosamine biosynthetic pathways leading to donor sugars for glycosylation. Figure 1A) Full biosynthetic pathways to each of the sugar nucleotides used in mammalian glycan synthesis. The boxes indicate where photoactivatablesugar analogs can enter these pathways in this report. Figure 1B) Examples of protein glycosylation motifs, O-linked GlcNAc (O-GlcNAc) or GalNAc-based O-glycosylation. Abbreviations: HK = hexokinase; GPI = glycose phosphate isomerase; GFAT = glutamine fructose-6-phosphate amidotransferase; GNPNAT = glycosamine 6-phosphate N- acetyltransferase; PGM3 = phosphoglucomutase 3; UAP / AGX1 = uridine diphosphate (UDP) N-acetylglucosamine pyrophosphorylase; GALE = UDP-galactose-4-epimerase. GK2 = glycerol kinase 2; MNK = ManNAc kinase; GNE = UDP-GlcNAc 2-epimerase; NANS = sialic acid synthase; NANP = N-acylneuraminate-9-phosphatase; CSS = cytidine 5′ - monophosphate-N-acetylneuraminic acid synthetase; OGT = O-GlcNAc transferase.

[0035] Figures 2A, 2B, and 2C are diagrams showing structures of photo-releasable glycan precursors according to aspects of the present disclosure.

[0036] Figures 3A, 3B, 3C, and 3D show characterization of decaging of glycan precursors from photo-releasable glycan precursors of the present disclosure by NMR and UV–vis. Figure 3A)31P NMR shifts of compound 4 with 350 nm light exposure at the indicated time points (solvent: 25% D2O in DMSO-d6). Fully caged compound 4 (right box), is mono-deprotected (middle box), and rapidly di-deprotected (left box) Figure 3B) UV–vis data showing release of sugar phosphate + free photocage (λmax = 325 nm) for compound 4 treatment with 350 nm light for the indicated time points (solvent: tissue culture-grade Dulbecco’s Modified Eagles Medium, DMEM). Figure 3C)31P NMR shifts of compound 7 with 350 nm light exposure at the indicated time points (solvent: 25% D2O in DMSO-d6). Fully caged compound 7 (right box), is mono-deprotected (middle box), and rapidly di- deprotected (left box) Figure 3D) UV–vis data showing release of sugar phosphate + free photocage for compound 7 treatment with 350 nm light for the indicated time points (solvent: DMEM). Small text on NMR indicates ppm shift values.

[0037] Figures 4A, 4B, 4C, 4D, 4E, and 4F show results of evaluation of photo- releasable glycan precursors and decaging conditions on cell viability in HeLa cells. Figure 4A) MTT viability assay of compounds 4, 6, 7, and 8 at concentrations of 12.5, 25, 50, 100, 200, and 400 μM for 24 h compared to the DMSO control. Figure 4B) MTT assay of 350 nm and 410 nm light compared to a non-light-exposed control. Figures 4C-4D) De-caging conditions study: compound 4, 6, 7, or 8 was incubated with cells for 16 h, the media was exchanged, and the cells were exposed to 350 nm light for 5 min. Cell viability was then determined after 1 h (Figure 4C) or 24 h (panel Figure 4D). Figures 4E-4F) Multiplexed cell viability (black) and toxicity (checkered) data for independent measures of cellular effects of compound caged 1-phospho-GlcNAc, 4 (Figure 4E) and caged 6-phospho-GlcNAc, 7 (Figure4F). All assays were performed in triplicate with a representative biological replicate shown of at least two biological replicates per assay. Error bars represent standard error of the mean. Full data for all compounds shown in Figs.6–9.

[0038] Figures 5A and 5C show results of HPLC measurements to estimate photo- decaging conversion of compound 4. Figure 5A is a series of traces showing absorbance chromatograms (200 nm) for samples at increasing UV exposure time points. Figure 5B is a diagram showing a scheme for the decaging reaction. Figure 5C is a graph showing the estimated conversion from intact photocaged GlcNAc-1-phosphate (compound 4, gray bars) to the decaged sugar, accompanied by formation of 2-nitrosoacetophenone byproduct (black bars). Data analyzed by integrating the 200 nm peaks normalized release points to 0 min (ca. 100% of compound 4) and 30 min (ca.100% of 2-nitrosoacetophenone).

[0039] Figures 6A and 6B: Multiplexed cell viability (black triangles) and toxicity (gray squares) for photo-release of 1P-diNPE-GlcNAc (compound 4) Figure 6A) 1 h after photo- release. Figure 6B) 24 h after photo-release.

[0040] Figure 7A and 7B: Multiplexed cell viability (black triangles) and toxicity (gray squares) for photo-release of 1P-diNPE-GalNAc (compound 6) Figure 7A) 1 h after photo- release. Figure 7B) 24 h after photo-release.

[0041] Figures 8A and 8B: Multiplexed cell viability (black triangles) and toxicity (gray squares) for photo-release of 6P-diNPE-GlcNAc (compound 7) Figure 8A) 1 h after photo- release. Figure 8B) 24 h after photo-release.

[0042] Figures 9A and 9B: Multiplexed cell viability (black triangles) and toxicity (gray squares) for photo-release of 6P-diNPE-ManNAc (compound 8) Figure 9A) 1 h after photo- release. Figure 9B) 24 h after photo-release.

[0043] Figure 10 shows structures of controllably-releasable glycan precursors of the present disclosure along with specific cage components; as noted, for glycans shown, R is hydroxyl, acyl, alkyne, azide, alkene, or hydrogen (H), where the alkene is C2-C8 linear or cyclic alkene, and the alkyne is C2-C8 terminal or cyclic alkyne.

[0044] Figure 11A shows Scheme 1: Synthesis of anomeric-caged sugars 1a, 1b, 2a, 2c, 3a, and 3b.

[0045] Figure 11B shows Scheme 2: Synthesis of photo-releasable glycan precursors 4, 5, and 6.

[0046] Figure 11C shows Scheme 3: Synthesis of photo-releasable glycan precursors 7, and 8.

[0047] TMSOTf = trimethylsilyl triflate. DCE = dichloroethane. CSA = camphorsulfonic acid. NaOMe = sodium methoxide⋅THF = tetrahydrofuran. DCM = dichloromethane. mCPBA = meta-chloroperoxybenzoic acid. TrtCl = trityl chloride. Ac2O = acetic anhydride. AcOH = acetic acid. Ac = acetate. NBDE = 2-nitrobenzodioxyethanol. DEAC = diethylaminocoumarin. NPE = 2- nitrophenylethanol.

[0048] Figure 12 shows Scheme IX for synthesis of compound 9.

[0049] Figure 13 shows Scheme XI for synthesis of compound 4.

[0050] Figure 14 shows Scheme XIII for synthesis of compound 5.

[0051] Figure 15 shows Scheme XV for synthesis of compound 6.

[0052] Figure 16 shows Scheme XVIII for synthesis of compound 7.

[0053] Figure 17 shows Scheme XXI for synthesis of compound 8.

[0054] Figure 18 shows synthesis of bioorthogonal “chemocage” groups for glycans.

[0055] Figure 19 shows synthesis of bis-(alcohol)-N,N-diisopropylphosphoramidites.

[0056] Figure 20 shows an example of N-caged compounds including a GlcNAc sugar moiety and a photocage moiety.

[0057] Figure 21 shows examples of N-caged hexosamine sugar caging including a GlcNAc sugar moiety linked to a cage moiety, where “cage” is a photocage or biorthogonal cage, where PG is an ester-based protecting group exemplified by an acetyl ester, ethyl ester, propyl ester, butyl ester, pentyl ester, or hexyl ester; R is an azide, C2-C8 linear or cyclic alkene, or C2-C8 terminal or cyclic alkyne; and R’ is H, C1-C6 ester, or a sugar phosphate in alpha or beta configuration.

[0058] Figure 22 shows an example of 6-caged compounds including a GlcNAc sugar moiety and a photocage moiety.

[0059] Figure 23 shows examples of sugar 6-position hexosamine sugar caging including a GlcNAc sugar moiety linked to a cage moiety, where “cage” is a photocage or biorthogonal cage, where PG is an ester-based protecting group exemplified by an acetyl ester, ethyl ester, propyl ester, butyl ester, pentyl ester, or hexyl ester; R is an azide, C2-C8 linear or cyclic alkene, or C2-C8 terminal or cyclic alkyne; and R’ is H, C1-C6 ester, or a sugar phosphate in alpha or beta configuration.

[0060] Figure 24 shows Scheme XXII demonstrating synthesis of 6Az-GlcNAc-1- diNPE-phosphate (compound 28) from compound 23, ONPE is O-nitrophenylethanol and OBMP is O-paramethoxybenzyl.

[0061] Figure 25 shows Scheme XXIII, demonstrating synthesis of 6-caged GlcNAc derivatives.

[0062] Figure 26 shows synthesis of 6-cage-GlcNAlkyne and 6-cage-GlcNAc-azide from glucosamine according to Scheme XXIV.

[0063] Figure 27 shows synthesis of GlcNAc-N-cage compound 36 from peracetylated glucosamine according to Scheme XXV. DETAILED DESCRIPTION

[0064] Scientific and technical terms used herein are intended to have the meanings commonly understood by those of ordinary skill in the art. Such terms are found defined and used in context in various standard references illustratively including J. Sambrook and D.W. Russell, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; 3rd Ed., 2001; F.M. Ausubel, Ed., Short Protocols in Molecular Biology, Current Protocols; 5th Ed., 2002; B. Alberts et al., Molecular Biology of the Cell, 4th Ed., Garland, 2002; CRISPR / Cas: A Laboratory Manual, Doudna and Mali (eds), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2016; D.L. Nelson and M.M. Cox, Lehninger Principles of Biochemistry, 4th Ed., W.H. Freeman & Company, 2004; J.-H. Fuhrhop et al. (Eds.), Organic Synthesis, Concepts and Methods, 3rdEd., Wiley-VCH Cerlag GmbH & Co. KGaA, 2003; Herdewijn, P. (Ed.), Oligonucleotide Synthesis: Methods and Applications, Methods in Molecular Biology, Humana Press, 2004; D. J. Taxman (ed.), siRNA Design, Methods and Protocols, Humana Press, 2012; Harlow, E. and Lane, D., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1988; J. D. Pound (Ed.) Immunochemical Protocols, Methods in Molecular Biology, Humana Press, 2nd ed., 1998; Chu, E. and Devita, V.T., Eds., Physicians’ Cancer Chemotherapy Drug Manual, Jones & Bartlett Publishers, 2021; J.M. Kirkwood et al., Eds., Current Cancer Therapeutics, 4th Ed., Current Medicine Group, 2001; A Adejare (Ed.), Remington: The Science and Practice of Pharmacy, Elsevier, 23rd Ed., 2021; L.V. Allen, Jr. et al., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, 11th Ed., Wolters Kluwer, 2016; and L. Brunton et al., Goodman & Gilman’s The Pharmacological Basis of Therapeutics, McGraw-Hill Education, 13th Ed., 2018.

[0065] The singular terms "a," "an," and "the" are not intended to be limiting and include plural referents unless explicitly stated otherwise or the context clearly indicates otherwise.

[0066] The terms “includes,” “comprises,” “including,” “comprising,” “has,” “having,” and grammatical variations thereof, when used in this specification, are not intended to be limiting, and specify the presence of stated features, elements, and / or components, but do notpreclude the presence or addition of one or more other features, elements, components, and / or groups thereof.

[0067] The term “about” as used herein in reference to a number is used herein to include numbers which are greater, or less than, a stated or implied value by 1%, 5%, 10%, or 20%.

[0068] Particular combinations of features are recited in the claims and / or disclosed in the specification, and these combinations of features are not intended to limit the disclosure of various aspects. Combinations of such features not specifically recited in the claims and / or disclosed in the specification. Although each dependent claim listed below may directly depend on only one claim, the disclosure of various aspects includes each dependent claim in combination with every other claim in the claim set. As used herein, a phrase referring to "at least one of" a list of items refers to any combination of those items, including single members. As an example, "at least one of: a, b, or c" is intended to cover a alone; b alone; c alone, a and b, a, b, and c, b and c, a and c, as well as any combination with multiples of the same element, such as a and a; a, a, and a; a, a, and b; a, a, and c; a, b, and b; a, c, and c; and any other combination or ordering of a, b, and c).

[0069] The terms “first,” “second,” and the like are used herein to describe various features or elements, but these features or elements are not intended to be limited by these terms, but are only used to distinguish one feature or element from another feature or element. Thus, a first feature or element could be termed a second feature or element, and vice versa, without departing from the teachings of the present disclosure.

[0070] Inventive biocompatible glycan precursors with controlled release properties and methods for their use are provided by the present disclosure.

[0071] According to aspects of the present disclosure, inventive biocompatible glycan precursors with controlled release properties are, or include, photo-releasable glycan precursors that release glycan biosynthetic precursor molecules upon light exposure.

[0072] According to aspects of the present disclosure, inventive biocompatible glycan precursors with controlled release properties are, or include, bioorthogonal chemistry- releasable glycan precursors that release glycan biosynthetic precursor molecules upon exposure to an appropriate bioorthogonal chemical reaction.

[0073] A diverse series of photo-releasable glycan precursors useful for photochemical release in cell-compatible conditions is provided according to aspects of the present disclosure. The sugars of the photo-releasable glycan precursors can be controllably releasedwith light under cell-compatible conditions, enabling a platform to track sugar-driven phenotypes with real-time chemical control.

[0074] A diverse series of bioorthogonal chemistry-releasable glycan precursors useful for bioorthogonal chemistry release in cell-compatible conditions is provided according to aspects of the present disclosure. The sugars of the bioorthogonal chemistry-releasable glycan precursors can be controllably released upon exposure to a bioorthogonal chemical reaction specific to the chemocage under cell-compatible conditions, enabling a platform to track sugar-driven phenotypes with real-time chemical control.

[0075] The term "cell-compatible conditions" is used herein interchangeably with "physiological conditions" and refers to conditions which are compatible with living cells and which do not interfere with the desired function of the glycan precursors. Cell-compatible conditions are well-known.

[0076] As noted herein, bioorthogonal chemistry-releasable glycan precursors release glycan biosynthetic precursor molecules upon exposure to an appropriate bioorthogonal chemical reaction. The term “bioorthogonal chemistry” refers to chemical reactions, which are intrinsically selective transformations not commonly found in biology that can proceed under physiological conditions without interfering with biological processes and biomolecules. The identity of an appropriate bioorthogonal chemical reaction depends on the identity of the chemocage. Appropriate pairings are readily determined, as exemplified by reactions of azides with alkynes or phosphines, tetrazines with alkenes, and other bioorthogonal ligation reactions that activate these stated motifs, such as a tetrazine and a cyclooctene.

[0077] A non-limiting example is incubation of a cell, such as a HeLa cell, with a glycan precursor covalently bonded to a chemocage, in this example a tetrazine. The HeLa cells are then incubated with a transcyclooctene to initiate release of the glycan precursor, i.e. sugar.

[0078] According to aspects of the present disclosure, the sugars of the bioorthogonal chemistry-releasable glycan precursors are releasable with bioorthogonal chemistry such as inverse electron demand Diels—Alder reactions, azide-based reactions such as alkyne cycloaddition and phosphine Staudinger reactions.

[0079] Compositions and methods provided herein are useful to provide spatiotemporal control over glycosylation pathways in living mammalian cells.

[0080] Compositions are provided according to aspects of the present disclosure which include: a photo-releasable glycan precursor, the photo-releasable glycan precursor having a photocage or chemocage component covalently bonded to a glycan precursor component.

[0081] The term “photocage” refers to a photo-labile protecting group that is released from the glycan precursor component upon exposure to a wavelength of light specific to the photocage.

[0082] The term “chemocage” refers to a bioorthogonal chemistry-labile protecting group that is released from the glycan precursor component upon exposure to a bioorthogonal chemical reaction specific to the chemocage.

[0083] Compositions are provided according to aspects of the present disclosure which include: a photo-releasable glycan precursor, the photo-releasable glycan precursor having a photocage component covalently bonded to a glycan precursor component, wherein the glycan precursor component is an N-acetyl sugar.

[0084] Compositions are provided according to aspects of the present disclosure which include: a bioorthogonal chemistry-releasable glycan precursor, the bioorthogonal chemistry- releasable glycan precursor having a chemocage component covalently bonded to a glycan precursor component, wherein the glycan precursor component is an N-acetyl sugar.

[0085] Compositions are provided according to aspects of the present disclosure which include: a photo-releasable glycan precursor, the photo-releasable glycan precursor having a photocage component covalently bonded to a glycan precursor component, wherein the glycan precursor component is selected from the group consisting of: GlcNAc, GalNAc, and ManNAc and, protected variations of any of these sugars.

[0086] Compositions are provided according to aspects of the present disclosure which include: a bioorthogonal chemistry-releasable glycan precursor, the bioorthogonal chemistry- releasable glycan precursor having a chemocage component covalently bonded to a glycan precursor component, wherein the glycan precursor component is selected from the group consisting of: GlcNAc, GalNAc, and ManNAc, and protected variations of any of these sugars.

[0087] Compositions are provided according to aspects of the present disclosure which include: a photo-releasable glycan precursor, the photo-releasable glycan precursor having a photocage component covalently bonded to a glycan precursor component, wherein the photocage component is, or includes, an o-nitrobenzyl, a p-hydroxyphenyl, a coumarinyl, a tetrazine, a cyclooctene, a nitrodibenzofuranyl, a nitrophenylbenzofuran, or a derivative of any thereof.

[0088] The term “derivative” as used herein refers to a compound having a structure derived from the structure of a reference compound wherein the derivative retains the same orsimilar activity as the reference compound. The term derivative encompasses salts, esters, amides, salts of esters, salts of amides, and N-oxides of a reference compound.

[0089] Compositions are provided according to aspects of the present disclosure which include: a photo-releasable glycan precursor, the photo-releasable glycan precursor having a photocage component covalently bonded to a glycan precursor component, wherein the glycan precursor component is an N-acetyl sugar, and wherein the photocage component is, or includes, an o-nitrobenzyl, a p-hydroxyphenyl, a coumarinyl, a tetrazine, a cyclooctene, or a nitrodibenzofuranyl, a nitrophenylbenzofuran, or a derivative of any thereof.

[0090] Compositions are provided according to aspects of the present disclosure which include: a bioorthogonal chemistry-releasable glycan precursor, the bioorthogonal chemistry- releasable glycan precursor having a chemocage component covalently bonded to a glycan precursor component, wherein the chemocage component is, or includes, an o-nitrobenzyl, a p-hydroxyphenyl, a coumarinyl, a tetrazine, a cyclooctene, or a nitrodibenzofuranyl, a nitrophenylbenzofuran, or a derivative of any thereof.

[0091] Compositions are provided according to aspects of the present disclosure which include: a bioorthogonal chemistry-releasable glycan precursor, the bioorthogonal chemistry- releasable glycan precursor having a chemocage component covalently bonded to a glycan precursor component, wherein the glycan precursor component is an N-acetyl sugar, and wherein the chemocage component is, or includes, an o-nitrobenzyl, a p-hydroxyphenyl, a coumarinyl, a tetrazine, a cyclooctene, or a nitrodibenzofuranyl, a nitrophenylbenzofuran, or a derivative of any thereof.

[0092] Compositions are provided according to aspects of the present disclosure which include: a photo-releasable glycan precursor, the photo-releasable glycan precursor having a photocage component covalently bonded to a glycan precursor component, wherein the photocage component is, or includes, nitrophenylethanol (NPE), diethylaminocoumarin (DEAC), a tetrazine, a cyclooctene, a nitrobenzodioxyethanol (NBDE), a nitrophenylbenzofuran (NPDF), or a derivative of any thereof.

[0093] Compositions are provided according to aspects of the present disclosure which include: a bioorthogonal chemistry-releasable glycan precursor, the bioorthogonal chemistry- releasable glycan precursor having a chemocage component covalently bonded to a glycan precursor component, wherein the chemocage component is, or includes, nitrophenylethanol (NPE), diethylaminocoumarin (DEAC), a tetrazine, a cyclooctene, nitrobenzodioxyethanol (NBDE) , a nitrophenylbenzofuran (NPDF), or a derivative of any thereof..

[0094] Compositions are provided according to aspects of the present disclosure which include: a photo-releasable glycan precursor, the photo-releasable glycan precursor having a photocage component covalently bonded to a glycan precursor component, wherein the glycan precursor component is an N-acetyl sugar, and wherein the photocage component is, or includes, nitrophenylethanol (NPE), diethylaminocoumarin (DEAC), nitrobenzodioxyethanol (NBDE) , a nitrophenylbenzofuran (NPDF), or a derivative of any thereof. Chemocages include tetrazine (Tz), trans-cyclooctene (TCO), or a derivative of either thereof.

[0095] Compositions are provided according to aspects of the present disclosure which include: a bioorthogonal chemistry-releasable glycan precursor, the bioorthogonal chemistry- releasable glycan precursor having a chemocage component covalently bonded to a glycan precursor component, wherein the glycan precursor component is an N-acetyl sugar, and wherein the chemocage component is, or includes, tetrazine (Tz) or trans-cyclooctene (TCO).

[0096] Compositions are provided according to aspects of the present disclosure which include: a photo-releasable glycan precursor, the photo-releasable glycan precursor having a photocage component covalently bonded to a glycan precursor component, wherein the photo-releasable glycan precursor compound is selected from the group consisting of: compound 1a, compound 2a, compound 3a, compound 1b, compound 2b, compound 3b, compound 4, compound 5, compound 6, compound 7, and compound 8 shown in Figures 2A, 2B, and 2C, or a derivative of any thereof.

[0097] Compositions are provided according to aspects of the present disclosure which include: a photo-releasable or bioorthogonal chemistry-releasable glycan precursor, the photo-releasable or bioorthogonal chemistry-releasable glycan precursor having a photocage component or chemocage component covalently bonded to a glycan precursor component; and further including one or more of: a carrier, a buffer, and a salt.

[0098] Compositions are provided according to aspects of the present disclosure which include: a photo-releasable or bioorthogonal chemistry-releasable glycan precursor, the photo-releasable or bioorthogonal chemistry-releasable glycan precursor having a photocage component or chemocage component covalently bonded to a glycan precursor component; and further including a pharmaceutically acceptable carrier.

[0099] Methods of controlled metabolic engineering are provided according to aspects of the present disclosure which include providing a photo-releasable or bioorthogonal chemistry-releasable glycan precursor of the present disclosure having a photocage component or chemocage component covalently bonded to a glycan precursor component,introducing the photo-releasable glycan precursor or bioorthogonal chemistry-releasable glycan precursor of the present disclosure into a cell, and exposing the photo-releasable or bioorthogonal chemistry-releasable glycan precursor of the present disclosure in the cell to light having a wavelength effective to release the glycan precursor from the photocage component, or a bioorthogonal chemical that can release a chemocage, thereby providing the glycan precursor to the cell. Providing a glycan precursor to a cell provides regulation of the addition of sugars onto biomolecules in the cells, each of which has the potential to alter glycan processes involved in cell morphology, signaling, and behavior.

[0100] According to aspects of the present disclosure, the cell is in vitro, in vivo, or ex vivo. According to aspects of the present disclosure, the cell is a cultured cell, including, but not limited to, a cell of a cell line, a primary cell, or a laboratory manipulated cell such as a recombinant cell.

[0101] According to aspects of the present disclosure, the cell is a human cell, a cell of a non-human mammal, or a cell of a non-human vertebrate. According to aspects of the present disclosure, the cell is an invertebrate, or a microorganism.

[0102] According to aspects of the present disclosure, the cell is a human cell.

[0103] According to aspects of the present disclosure, the cell is a human cell, including, but not limited to, a cell of a human cell line, a primary human cell, or a laboratory manipulated human cell such as a recombinant human cell.

[0104] Photo-releasable glycan precursors and bioorthogonal chemistry-releasable glycan precursors enter cells via passive diffusion or active transport. Peracetylated protected variants enhance passive diffusion.

[0105] According to aspects of the present disclosure, the wavelength is in the range of about 200 nm to about 750 nm. According to aspects of the present disclosure, the wavelength is in the range of about 250 nm to about 400 nm. According to aspects of the present disclosure, the wavelength is in the range of about 300 nm to about 400 nm. According to aspects of the present disclosure, the wavelength is in the range of about 350 nm to about 365 nm.

[0106] Any source for the light used to release the photocage can be used, such as relatively low intensity source, exemplified by a 2W flashlight, or such as a higher intensity source exemplified by a 32W photoreactor, or a source which is higher, lower, or intermediate in intensity. We tend to use the 32W photoreactor. A higher intensity source allows decaging in shorter times under 5 minutes, which is better for the cell health.

[0107] Photo-releasable or bioorthogonal chemistry-releasable glycan precursors of the present disclosure are biologically inert until decaging, thereby avoiding undesirable biological activities including metabolism and cytotoxicity. Photo-releasable glycan precursors and bioorthogonal chemistry-releasable glycan precursors of the present disclosure are cell permeable, non-toxic, and have resistance to intracellular reactivity including redox processes

[0108] Photocaged glycan biosynthetic precursor molecules according to aspects of the present disclosure are released within minutes of exposure to light having a wavelength effective to release the glycan biosynthetic precursor molecule from the photocage component.

[0109] Chemocaged glycan biosynthetic precursor molecules according to aspects of the present disclosure are released within minutes of exposure to an appropriate bioorthogonal chemical reaction effective to release the glycan biosynthetic precursor molecule from the chemocage component.

[0110] A carrier included in a composition according to aspects of the present disclosure is preferably inert with respect to a photo-releasable glycan precursors and / or bioorthogonal chemistry-releasable glycan precursors of the present disclosure. Non-limiting examples of suitable aqueous and nonaqueous carriers include water, ethanol, polyols such as propylene glycol, polyethylene glycol, glycerol, and the like, suitable mixtures of any two or more thereof.

[0111] A stabilizer is optionally included such as, for example, sucrose, EDTA, EGTA, and an antioxidant.

[0112] An included buffer is typically a biologically compatible buffers including, but not limited to, ammonium acetate, MOPS buffers, citrate buffers, HEPES buffers, carbonate buffers, Tris buffers, Tricine buffers, acetate buffers, phosphate buffers, and phosphate buffered saline.

[0113] An included salt can be any salt inert with respect to a photo-releasable glycan precursor and / or a bioorthogonal chemistry-releasable glycan precursor of the present disclosure and may be a pharmaceutically acceptable salt.

[0114] The term "pharmaceutically acceptable salt" refers to salts which are suitable for use in a subject without undue toxicity or irritation to the subject and which are effective for their intended use.

[0115] Pharmaceutically acceptable salts include pharmaceutically acceptable acid addition salts and base addition salts. Pharmaceutically acceptable salts are well-known inthe art, such as those detailed in S. M. Berge et al., J. Pharm. Sci., 66:1-19, 1977. Exemplary pharmaceutically acceptable salts are those suitable for use in a subject without undue toxicity or irritation to the subject and which are effective for their intended use which are formed with inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, phosphoric acid, sulfuric acid and sulfamic acid; organic acids such as acetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 2-acetoxybenzoic acid, butyric acid, camphoric acid, camphorsulfonic acid, cinnamic acid, citric acid, digluconic acid, ethanesulfonic acid, formic acid, fumaric acid, glutamic acid, glycolic acid, glycerophosphoric acid, hemisulfic acid, heptanoic acid, hexanoic acid, 2-hydroxyethanesulfonic acid (isethionic acid), lactic acid, maleic acid, hydroxymaleic acid, malic acid, malonic acid, mandelic acid, mesitylenesulfonic acid, methanesulfonic acid, naphthalenesulfonic acid, nicotinic acid, 2- naphthalenesulfonic acid, oxalic acid, pamoic acid, pectinic acid, phenylacetic acid, 3- phenylpropionic acid, picric acid, pivalic acid, propionic acid, pyruvic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, sulfanilic acid, tartaric acid, p-toluenesulfonic acid, trichloroacetic acid, trifluoroacetic acid and undecanoic acid; inorganic bases such as ammonia, hydroxide, carbonate, and bicarbonate of ammonium; organic bases such as primary, secondary, tertiary and quaternary amine compounds ammonium, arginine, betaine, choline, caffeine, diolamine, diethylamine, diethanolamine, 2- dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, dicyclohexylamine, dibenzylamine, N, N-dibenzylphenethylamine, 1-ephenamine, N, N'- dibenzylethylenediamine, ethanolamine, ethylamine, ethylenediamine, glucosamine, histidine, hydrabamine, isopropylamine, lH-imidazole, lysine, methylamine, N- ethylpiperidine, N-methylpiperidine, N-methylmorpholine, N, N-dimethylaniline, piperazine, trolamine, methylglucamine, purines, piperidine, pyridine, theobromine, tetramethylammonium compounds, tetraethylammonium compounds, trimethylamine, triethylamine, tripropylamine and tributylamine and metal cations such as aluminum, calcium, copper, iron, lithium, magnesium, manganese, potassium, sodium, and zinc.

[0116] The term “pharmaceutically acceptable carrier” refers to a carrier which is suitable for use in a subject without undue toxicity or irritation to the subject and which is compatible with other ingredients included in a pharmaceutical composition.

[0117] Pharmaceutically acceptable carriers, methods for making pharmaceutical compositions and various dosage forms, as well as modes of administration are well-known in the art, for example as detailed in Pharmaceutical Dosage Forms: Tablets, eds. H. A.Lieberman et al., New York: Marcel Dekker, Inc., 1989; and in L.V. Allen, Jr. et al., Ansel’s Pharmaceutical Dosage Forms and Drug Delivery Systems, 8th Ed., Philadelphia, PA: Lippincott, Williams & Wilkins, 2004; A. R. Gennaro, Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins, 21st ed., 2005, particularly chapter 89; and J. G. Hardman et al., Goodman & Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill Professional, 10th ed., 2001.

[0118] Embodiments of inventive compositions and methods are illustrated in the following examples. These examples are provided for illustrative purposes and are not considered limitations on the scope of inventive compositions and methods.

[0119] Examples

[0120] Abbreviations: CMP-Sialic Acid, cytidine monophosphate sialic acid; DEAC, diethylaminocoumarin; DMEM, Dulbecco’s Modified Eagles Medium; DMSO, dimethylsulfoxide; GalNAc, N-acetylgalactosamine; GlcNAc, N-acetylglucosamine; GlcNAc-1P, GlcNAc-1-phosphate; GlcNAc-6P, GlcNAc-6-phosphate; LED, light emitting diode; ManNAc, N-acetylmannosamine; NBDE, 2-nitrobenzodioxyethanol; NBDF, nitrodibenzofuran; NMR, nuclear magnetic resonance; NPE, nitrophenylethanol; O-GlcNAc, O-linked GlcNAc; PBS, phosphate buffered saline; PTM, post-translational modification; ROS, reactive oxygen species; UDP-GalNAc, uridine diphosphate GalNAc; UDP-GlcNAc, uridine diphosphate GlcNAc; UV, ultraviolet.

[0121] Reagents used in chemical synthesis were sourced as follows: N- acetylglucosamine (GlcNAc) (Sigma-Aldrich), acetic anhydride (Ac2O) (Sigma-Aldrich), pyridine (Acros Organics), trimethylsilyl trifluromethanesulfionate (TMSOTf) (Sigma- Aldrich), camphor sulfonic acid (CSA) (Acros Organics), m-chloro-peroxybenzoic acid (mCPBA) (Alfa Aesar), anhydrous N,N-dimethylformamide (DMF) (EMD Chemicals Inc.), phorphorous trichloride (Sigma-Aldrich), p-toluene sulfonylchloride (pTSCl) (Alfa Aesar), sodium azide (NaN3) (Fisher Scientific), 5-thioethyl-1H-tetrazole (Sigma-Aldrich), 1-(2- nitrophenyl) ethanol (NPE) (Ambeed, Inc.), 1-(6-nitro-1,3-benzodioxol-5-yl)ethanol (NBDE) (Tokyo Chemical Industry). Dichloromethane (CH2Cl2) (Sigma Aldrich) was dried over molecular sieves. Silica gel G-60 F254 aluminium TLC plates were used to monitor reaction progress. Column chromatography was performed on silica gel 60–200 mesh. Deuterated chloroform (CDCl3), deuterated methanol (CD3OD) and deuterated dioxide (D2O) were purchased from Cambridge Isotope Laboratories.

[0122] Synthesis

[0123] Synthetic Schemes

[0124] I.2-Methyl-(3,4,6-tri-O-acetyl-1,2-dideoxy-α-D-glucopyrano)-[2,1-d]-2-oxazoline (compound 11): Reported compound Ac4GlcNAc, described in Cao, Z. et al., Journal of Carbohydrate Chemistry, 34, 28-40, (2015), compound 10 (0.513 mmol, 1 equiv) was dissolved in dichloroethane (2 mL). Trimethylsilyl trifluoromethanesulfonate (0.667 mmol, 1.3 equiv) was added and the reaction was heated to 50 °C for 12 hours. The reaction mixture was cooled to room temperature and quenched with triethylamine (200 µL) and concentrated in vacuo. The crude residue was adsorbed on silica gel and purified via flash column chromatography (70:30:0.01% toluene:EtOAc:TEA) to give oxazoline compound 11 as a pale-yellow solid (150 mg, 82% yield). The analytical data matched reported values in Nakabayashi, S. et al., Carbohydrate Research, 150, c7-c10, (1986).

[0125] II.(acetylamino)-2-deoxy-1-(1-(6-nitro-1,3-benzodioxol-5-yl)ethyl)-b-D-glucopyranoside (compound 1a): Oxazoline compound 11 (110 mg, 0.334 mmol, 1 equiv) and 1-(6-nitro-1,3- benzodioxol-5-yl)ethanol (212 mg, 1.002 mmol, 3 equiv) were dissolved in dichloroethane (3 mL). A catalytic amount of camphorsulfonic acid (8 mg) was added, and the reaction mixturestirred overnight at 90 °C under inert atmosphere. The reaction mixture was quenched with saturated sodium bicarbonate (20 mL), washed with water (1×40mL), extracted with dichloromethane (2×40mL), dried over Na2SO4, and concentrated in vacuo. The crude product was adsorbed onto silica gel and purified via flash column chromatography (20% ethyl acetate in dichloromethane) to give compound 1a as a pale-yellow solid (70 mg, 39% yield) in a diastereomeric mixture.1H NMR (400 MHz, CDCl3) δ 7.43 (d, J = 1.1 Hz, 1H), 7.26 (s, 1H), 7.04 (s, 1H), 6.10 (d, J = 1.2 Hz, 1H), 5.62 (d, J = 9.0 Hz, 1H), 5.55 – 5.33 (m, 2H), 5.16 (dd, J = 10.7, 9.3 Hz, 1H), 5.10 – 5.02 (m, 1H), 4.98 (q, J = 9.5 Hz, 1H), 4.67 (d, J = 8.3 Hz, 1H), 4.31 (d, J = 8.4 Hz, 1H), 4.24 (dd, J = 12.2, 5.5 Hz, 1H), 4.11 (dd, J = 12.1, 2.5 Hz, 1H), 4.05 – 3.90 (m, 2H), 3.58 (dddd, J = 10.0, 8.0, 5.6, 2.4 Hz, 1H), 2.11 (s, 2H), 2.05 – 1.98 (m, 10H), 1.94 (s, 2H), 1.65 (s, 1H), 1.52 (d, J = 6.3 Hz, 2H), 1.46 (d, J = 6.2 Hz, 2H).13C NMR (100 MHz, CDCl3) δ 171.24, 171.21, 170.96, 170.70, 170.25, 170.07, 169.50, 152.42, 152.35, 147.41, 147.04, 137.89, 136.16, 107.78, 106.76, 105.07, 104.63, 103.17, 103.02, 100.89, 99.48, 77.48, 77.16, 76.84, 74.27, 72.57, 72.50, 72.09, 71.99, 71.95, 68.61, 62.22, 54.79, 54.68, 23.86, 23.48, 23.35, 23.13, 20.86, 20.81, 20.74, 20.67. ESI- Orbitrap HRMS: m / z calc for C23H28O13N2Na [M+Na]+563.1484, found 563.1476.

[0126] III.2-(acetylamino)-2-deoxy-1-(1-(6-nitro-1,3-benzodioxol-5-yl)ethyl)-b-D-glucopyranoside (compound 1b): compound 1a (40 mg, 0.074 mmol, 1 equiv) was dissolved in anhydrous methanol (2 mL) and anhydrous THF (0.5 mL) and cooled down to 0 °C. Freshly prepared sodium methoxide was added dropwise (200 µL) and stirred for 25 minutes. The reaction mixture was diluted with methanol (6 mL) and concentrated in vacuo. The crude product was adsorbed onto silica gel and purified via flash column chromatography (10% methanol in ethyl acetate) to give compound 1b as a pale-yellow powder (28 mg, 90% yield) in a diastereomeric mixture.1H NMR (400 MHz, CD3OD) δ 7.45 (d, J = 1.5 Hz, 1H), 7.09 (s,1H*), 6.12 (d, J = 4.6 Hz, 1H), 5.50 – 5.37 (m, 1H), 4.85 (s, 5H), 4.56 (d, J = 8.3 Hz, 1H), 4.19 (d, J = 8.4 Hz, 1H), 4.10 (d, J = 7.1 Hz, 1H*), 3.99 (d, J = 5.1 Hz, 1H*), 3.86 (dd, J = 12.0, 2.4 Hz, 1H), 3.77 – 3.66 (m, 1H), 3.62 (s, 1H*), 3.51 – 3.28 (m, 3H), 3.23 – 3.11 (m, 1H), 3.00 (s, 1H*), 2.86 (d, J = 0.7 Hz, 1H*), 2.01 (m, 3H), 1.47 (m, 3H).13C NMR (100 MHz, CD3OD) δ 172.36, 172.21, 152.26, 147.33, 146.95, 140.72, 137.36, 136.39, 106.95, 106.42, 104.01, 103.29, 103.14, 100.19, 99.69, 76.43, 76.36, 74.36, 74.06, 72.57, 71.44, 70.53, 70.48, 61.12, 56.20, 56.06, 48.23, 46.95, 22.84, 21.70, 21.60. ESI-Orbitrap HRMS: m / z calc for C17H23O10N2 [M+H]+415.1347, found 415.1343. *Less abundant diastereomer

[0127] IV.2-Methyl-(3,4-di-O-acetyl-6-azido-1,2,6-trideoxy-α-D-glucopyrano)-[2,1-d]-2-oxazoline (compound 13). Reported compound 6AzAc3GlcNAc, described in Chuh, K. N. et al., Journal of the American Chemical Society, 136, 12283-12295, (2014), compound 12 (0.806 mmol, 1 equiv) was dissolved in dichloroethane (3 mL). TMSOTf (1.129 mmol, 1.4 equiv) was added and the reaction was stirred at 50 °C for 12hrs. The reaction mixture was cooled to room temperature and quenched with triethylamine (200 µL) and concentrated in vacuo. The crude product adsorbed on silica gel and purified via flash column chromatography(70:30:0.01% toluene:EtOAc:TEA) to give oxazoline compound 13 as a colorless solid (120 mg, 48% yield).1H NMR (CDCl3, 400 MHz) δ 5.96 (d, J = 7.4Hz, 1H), 5.24 (dd, J = 1.8, 2.6Hz, 1H), 4.88 (d, J = 8.8Hz, 1H), 4.15-4.12 (m, 1H), 3.53-3.49 (m, 1H), 3.40-3.29 (m, 2H), 2.10 (s, 3H), 2.07 (d, 1.8Hz, 3H), 2.06 (s, 3H).13C NMR (CDCl3, 100 MHz) δ 169.61, 169.20, 166.72, 99.11, 69.91, 69.04, 69.01, 64.70, 52.07, 20.87, 20.83, 13.87. ESI-Orbitrap HRMS: m / z calc for C12H17O6N4[M+H]+313.1143, found 313.1137.

[0128] V.3,4-Di-O-acetyl-6-deoxy-6-azido-2-(acetylamino)-2-deoxy-1-(1-(6-nitro-1,3-benzodioxol- 5-yl)ethyl)-b-D-glucopyranoside (compound 2a): Oxazoline compound 13 (140 mg, 0.448 mmol, 1 equiv) and 1-(6-nitro-1,3-benzodioxol-5-yl)ethanol (379 mg, 0.1.794 mmol, 3 equiv) were dissolved in dichloroethane (2.5 mL). A catalytic amount of camphorsulfonic acid (11 mg) was added, and the reaction mixture stirred overnight at 90 °C under inert atmosphere. The reaction was quenched with saturated sodium bicarbonate (30mL) washed with water (1×40mL), extracted with dichloromethane (2×60mL), dried over Na2SO4, and concentrated in vacuo. The crude product was adsorbed onto silica gel and purified via flash column chromatography (20% ethyl acetate in dichloromethane) to give compound 2a as a pale- yellow solid (90 mg, 38% yield) in a diastereomeric mixture.1H NMR (400 MHz, CDCl3 δ 7.44 (d, J = 4.5 Hz, 1H), 7.21 (s, 1H), 7.06 (s, 1H*), 6.11 (dd, J = 13.1, 1.2 Hz, 1H), 6.07 (dd, J = 8.0, 1.3 Hz, 2H), 5.71 (d, J = 9.0 Hz, 1H), 5.52 (q, J = 7.9, 7.1 Hz, 1H), 5.41 (q, J = 6.2 Hz, 1H), 5.19 (dd, J = 10.7, 9.3 Hz, 1H), 5.10 (dd, J = 10.7, 9.2 Hz, 1H), 4.97 (m, 2H), 4.76 (d, J = 8.3 Hz, 1H), 4.41 (d, J = 8.4 Hz, 1H), 4.00 (dtd, J = 10.7, 8.7, 4.9 Hz, 2H), 3.58 (m, 1H), 3.38 (dd, J = 13.4, 6.8 Hz, 1H), 3.23 (dd, J = 13.4, 2.7 Hz, 1H), 3.16 (dd, J = 13.4, 6.2 Hz, 1H), 3.09 (dd, J = 13.4, 2.8 Hz, 1H), 2.03 (s, 3H), 2.01 (s, 2H), 2.00 (s, 2H), 2.00 (s, 5H), 1.99 (s, 3H), 1.94 (s, 1H), 1.53 (d, J = 6.3 Hz, 2H), 1.47 (d, J = 6.2 Hz, 3H).13C NMR (100 MHz, CDCl3) δ 171.04, 171.01, 170.16, 169.98, 169.41, 169.38, 152.43, 152.18, 147.21, 146.99, 137.55, 136.23, 107.41, 106.69, 104.57, 102.88, 100.98, 99.55, 74.61, 73.49, 73.23, 72.17, 69.36, 54.69, 54.54, 50.90, 23.30, 23.16, 23.10, 20.64, 20.59. ESI-Orbitrap HRMS: m / z calc for C21H25O11N5Na [M+Na]+546.1443, found 546.1435. *Less abundant diastereomer

[0129] VI.6-deoxy-6-azido-2-(acetylamino)-2-deoxy-1-(1-(6-nitro-1,3-benzodioxol-5-yl)ethyl)-b-D- glucopyranoside (2b): 2a (50 mg, 0.0955 mmol, 1 equiv) was dissolved in anhydrous methanol (2 mL) and anhydrous THF (1 mL) and cooled down to 0 °C. Freshly prepared sodium methoxide was added dropwise (200 µL) and stirred for 25 minutes. The reaction mixture was diluted with methanol (6 mL) and concentrated in vacuo. The crude product was adsorbed onto silica gel and purified via flash column chromatography (10% methanol in ethyl acetate) to give 2b as a pale-yellow powder (40 mg, 90% yield) in a diastereomeric mixture.1H NMR (400 MHz, CD3OD) δ 7.46 (s, 1H), 7.42 (s, 1H), 7.23 (s, 1H), 7.08 (s, 1H), 6.15 (dd, J = 8.78, 1.00 Hz, 1H), 6.09 (dd, J = 8.61, 1.07 Hz, 2H), 5.48 (s, 1H), 5.46 (d, J = 6.35 Hz, 1H*), 5.35 (q, J = 6.22 Hz, 1H), 4.56 (d, J = 8.39 Hz, 1H), 4.24 (d, J = 8.44 Hz, 1H), 3.71 (dd, J = 10.37, 8.39 Hz, 1H), 3.65 (dd, J = 10.31, 8.39 Hz, 1H), 3.52 – 3.20 (m, 11H), 2.02 (s, 3H), 1.98 (s, 2H), 1.47 (d, J = 6.36 Hz, 2H), 1.44 (d, J = 6.25 Hz, 3H).13C NMR (100 MHz, CD3OD) δ 172.38, 172.21, 152.32, 147.36, 147.01, 140.63, 137.73, 136.25, 106.95, 106.35, 104.06, 103.79, 103.30, 103.08, 101.18, 99.63, 75.58, 75.36, 74.16, 73.72, 71.60, 71.41, 71.18, 56.16, 56.03, 51.18, 51.11, 48.23, 46.95, 22.84, 22.01, 21.66, 21.57. ESI- Orbitrap HRMS: m / z calc for C17H22O9N5[M+H]+440.1412, found 440.1409. *Less abundant diastereomer

[0130] VII.3,4-Di-O-acetyl-6-deoxy-6-azido-2-(acetylamino)-2-deoxy-1-(7-diethylamino-4- hydroxymethylcoumarin)-b-D-glucopyranoside (compound 3a): Oxazoline compound 13 (60 mg, 0.192 mmol, 1 equiv) and 7-diethylamino-4-hydroxymethylcoumarin (190 mg, 0.7692 mmol, 3 equiv) were dissolved in dichloroethane (2.5 mL). A catalytic amount of camphorsulfonic acid (7 mg) was added, and the reaction mixture stirred overnight at 90 °C under inert atmosphere. Upon completion, the reaction mixture was quenched with saturated sodium bicarbonate (10mL), washed with water (1×40mL), extracted with DCM (2×40mL), dried over Na2SO4, filtered, and concentrated in vacuo. The crude product was adsorbed onto silica gel and purified via flash column chromatography (35% ethyl acetate in dichloromethane) to give compound 3a as a pale-yellow solid (53 mg, 49% yield).1H NMR (CDCl3, 400 MHz) d 7.31 (d, J = 9Hz, 1H), 6.52 (dd, J = 2.5, 9Hz, 1H), 6.47 (d, J = 8.8Hz, 1H), 6.10 (s, 1H), 5.87 (d, J = 8.8Hz, 1H), 5.29 (dd, J = 10.8, 9.2Hz, 1H), 5.00-4.95 (m, 2H), 4.83 (d, J = 8.3Hz, 1H), 4.71 (dd, J = 1.1, 14.5Hz, 1H,) 4.02-3.95 (m, 1H), 3.77-3.72 (m, 1H), 3.45 (dd, J = 7.7, 13.3Hz, 1H) 3.40 (q, J = 7.0, 4H), 3.17 (dd, J = 2.3, 13.3Hz, 1H), 2.02 (s, 6H), 1.90 (s, 3H), 1.18 (t, J = 7.0Hz, 6H).13C NMR (CDCl3, 100 MHz) d 170.77, 170.70, 170.54, 162.05, 156.33, 150.60, 125.04, 108.63, 107.14, 106.33, 99.39, 97.64, 74.03, 71.88, 69.80, 66.09, 54.63, 51.19, 44.72, 23.23, 20.63, 20.60, 12.43. ESI-Orbitrap HRMS: m / z calc for C26H33O9N5Na [M+Na]+582.2170, found 582.2167.

[0131] VIII.6-deoxy-6-azido-2-(acetylamino)-2-deoxy-1-(7-diethylamino-4- hydroxymethylcoumarin)-b-D-glucopyranoside (compound 3b): Compound 3a (20 mg, 0.0357 mmol, 1 equiv) was dissolved in anhydrous methanol (2.5 mL) and anhydrous THF (0.5 mL) and cooled down to 0 °C. Freshly prepared sodium methoxide was added dropwise (300 µL) and stirred for 15 minutes. The reaction mixture was diluted with methanol (5 mL) and concentrated in vacuo. The crude product was adsorbed onto silica gel and purified via flash column chromatography (5% methanol in ethyl acetate) to give compound 3b as a pale- yellow powder (14 mg, 82% yield).1H NMR (CD3OD, 400 MHz) δ 6.87 (d, J = 8.3Hz, 1H), 6.10 (d, J = 2.4, 1H), 6.02 (s, 1H), 5.95 (dd, J = 11, 2.5Hz, 1H), 4.68 (dd, J = 1.3, 13.2Hz, 1H), 4.52 (d, J = 8.5Hz, 1H), 4.36 (d, J = 13.2Hz, 1H), 3.89-3.81 (m, 1H), 3.68 (apt, J = 8.6Hz, 1H), 3.52-3.44 (m, 4H), 3.42-3.36 (m, 1H), 3.30 (q, J = 7Hz, 4H), 1.83 (s, 3H), 1.10 (d, J = 7.5Hz, 6H).13C NMR (CD3OD, 100 MHz) δ 175.97, 172.89, 149.26, 141.46, 130.79, 125.42, 116.27, 103.82, 100.58, 99.39, 75.80, 75.18, 71.97, 55.45, 44.09, 21.80, 11.81. ESI- Orbitrap HRMS: m / z calc for C22H30O7N5 [M+H]+476.2140, found 476.2133.

[0132] IX. Bis-(1-[2-nitrophenyl]-ethyl)-N,N-diisopropylphosphoramidite (compound 9), see Figure 12. Phosphorous trichloride (4.5 mmol, 1 equiv) and N,N-diisopropylethylamine (9.0 mmol, 2 equiv) were added to anhydrous tetrahydrofuran (9 mL) under inert atmosphere. Diisopropylamine (9.0 mmol, 2 equiv) was added dropwise and the mixture was stirred for 4 hours to yield diisopropylphosphoramidous dichloride. The reaction mixture was cooled to - 15 °C, and 2-nitrophenylethanol (9.0mmol, 2 equiv) and triethylamine (9.9 mmol, 2.2 equiv) were added. The mixture was slowly warmed to room temperature and stirred overnight. Upon completion, the reaction mixture was diluted with ethyl acetate (50 mL), washed with saturated sodium bicarbonate (2×25 mL) and brine (1×10 mL), dried over sodium sulfate, filtered, and concentrated in vacuo. The crude product was purified via flash column chromatography (5:1 hexanes:ethyl acetate) to give compound 9 as a pale yellow solid (1.2 g, 58% yield). The analytical data matched reported values in Griffiths, C. A. et al., Nature, 540, 574-578, (2016).3,4,6-Tri-O-acetyl-2-(acetylamino)-2-deoxy-D-glucopyranoside (compound 14). Known compound Ac4GlcNAc, described in Cao, Z. et al., Journal of Carbohydrate Chemistry, 34, 28-40, (2015), (8 mmol) was dissolved in anhydrous N,N-dimethylformamide (16 mL) and stirred under inert atmosphere. Hydrazine acetate (9.6 mmol, 1.2 equiv) was added and the reaction was stirred at room temperature for 8 hours. When starting material was consumed, the reaction mixture was quenched with saturated sodium bicarbonate (50 mL), extracted with ethyl acetate (2×50 mL), and concentrated in vacuo to give compound 14 (1.72 g, 62% yield) as a white solid without purification. The analytical data matched reported values in Andersen, S. M. et al., Organic Letters, 17, 944-947, (2015).

[0134] XI. 3,4,6-Tri-O-acetyl-2-(acetylamino)-2-deoxy-1-phospho(di-O-NPE)-a-D-glucopyranoside (1P-diNPE-GlcNAc, compound 4), see Figure 13. Compound 14 (0.266 mmol) was dissolved in anhydrous dichloromethane (886 µL, 0.3M) under inert atmosphere and cooled to 0 °C. 5-ethylthio-1H-tetrazole (1.33 mmol, 5.0 equiv) and compound 9 (0.665 mmol, 2 equiv) were added and the reaction mixture was stirred for 1.5 hours. When starting material was consumed, the reaction mixture was cooled to -40 °C and meta-chloroperoxybenzoic acid (0.798 mmol, 3 equiv) was added. After 30 minutes, the reaction was quenched with saturated sodium bicarbonate (5 mL) and diluted with ethyl acetate (20 mL). The organic layer was washed with bicarb (2×20 mL), and brine (1×10 mL), dried over sodium sulfate, filtered, and concentrated in vacuo. The crude residue was purified by flash column chromatography (60% ethyl acetate in hexanes) to give compound 4 (102 mg, 53% yield) as a white solid in a mixture of diastereomers due to the photocage, which was proven to be >95% pure via HPLC.1H NMR (600 MHz, CDCl3) d 7.96 (ddd, J = 9.72, 8.23, 1.25 Hz, 2H), 7.91 (ddd, J = 8.15, 2.21, 1.23 Hz, 1H), 7.83 (dt, J = 8.21, 1.36 Hz, 1H), 7.75 – 7.55 (m, 9H), 7.49 (td, J = 8.07, 1.33 Hz, 2H), 7.45 (ddp, J = 7.12, 4.85, 1.77 Hz, 1H), 7.38 (ddd, J = 8.49, 6.79, 2.10 Hz, 1H), 6.15 – 6.02 (m, 3H), 6.01 – 5.96 (m, 1H), 5.87 (dd, J = 9.17, 2.92 Hz, 1H), 5.71 (dd, J = 6.31, 3.26 Hz, H-1), 5.60 (ddd, J = 9.39, 5.93, 3.27 Hz, 1H, H-1), 5.51 (dd, J = 6.09, 3.26 Hz, 1H, H-1), 5.18 – 5.00 (m, 4H), 4.93 (dd, J = 10.96, 9.44 Hz, 1H), 4.37 – 4.26 (m, 2H), 4.18 (dd, J = 12.64, 3.84 Hz, 1H), 4.14 – 4.07 (m, 1H), 4.05 – 4.00 (m, 1H), 3.98 – 3.84 (m, 4H), 2.10 – 1.96 (m, 22H), 1.95 – 1.84 (m, 6H), 1.70 (m, 3H), 1.64 – 1.52 (m, 10H).13C NMR (150 MHz, CDCl3) d 171.02, 170.64, 170.53, 169.09, 169.06, 147.08, 146.90,146.55, 146.46, 136.87, 136.71, 136.42, 136.39, 134.06, 134.01, 133.94, 129.22, 129.15, 129.04, 128.99, 127.62, 127.39, 127.31, 127.22, 127.16, 124.75, 124.60, 96.23, 96.19, 95.92, 95.88, 73.42, 73.15, 69.90, 69.78, 67.13, 61.08, 51.78, 29.64, 24.36, 24.33, 24.08, 24.05, 24.01, 23.97, 23.79, 23.75, 22.88, 22.82, 22.78, 20.66, 20.64, 20.59, 20.52.31P NMR (243 MHz, CDCl3) d -5.57, -5.75, -6.22. ESI-Orbitrap HRMS: m / z calc for C30H36O16N3PNa [M+Na]+748.1725, found 748.1715.

[0135] XII.3,4-Di-O-acetyl-2-(acetylamino)-6-azido-2,6-dideoxy-D-glucopyranoside (compound 15). Known compound 6AzAc3GlcNAc, described in Chuh, K. N. et al., Journal of the American Chemical Society, 136, 12283-12295, (2014), (4 mmol) was dissolved in anhydrous N,N- dimethylformamide (8 mL) under inert atmosphere. Hydrazine acetate (4.8 mmol, 1.2 equiv) was added and the reaction was stirred at room temperature overnight. Upon completion, the reaction mixture was quenched with saturated sodium bicarbonate (10 mL), extracted with ethyl acetate (2×20 mL), and concentrated in vacuo to give compound 15 (1.12 g, 85% yield) as a white solid.1H NMR (600 MHz, CDCl3) δ 6.10 (d, J = 9.36 Hz, 1H), 5.28 – 5.25 (m, 1H), 5.23 (d, J = 3.54 Hz, 1H), 5.00 (t, J = 9.74 Hz, 1H), 4.24 (ddd, J = 10.80, 9.41, 3.61 Hz, 1H), 4.16 (ddd, J = 9.68, 5.22, 4.03 Hz, 1H), 3.32 – 3.28 (m, 2H), 2.01 (s, 3H), 1.99 (s, 3H), 1.94 (s, 3H).13C NMR (151 MHz, CDCl3) δ 171.38, 170.74, 169.62, 91.30, 70.84, 69.59, 68.56, 52.32, 51.21, 23.05, 20.70, 20.63.13C NMR (151 MHz, CDCl3) δ 171.38, 170.74, 169.62, 91.30, 70.84, 69.59, 68.56, 52.32, 51.21, 23.05, 20.70, 20.63. ESI-Orbitrap HRMS: m / z calc for C12H19O7N4 [M+H]+331.1248, found 331.1245.

[0136] XIII. 3,4-Di-O-acetyl-2-(acetylamino)-6-azido-2,6-dideoxy-1-phospho(di-O-NPE)-a-D- glucopyranoside (6Az-1P-diNPE-GlcNAc, compound 5), see Figure 14. Compound 15 (2.14 mmol) was dissolved in anhydrous dichloromethane (7.1 mL) under inert atmosphere and cooled to 0 °C.5-ethylthio-1H-tetrazole (10.7 mmol, 5.0 equiv) and compound 9 (2.568 mmol, 2.5 equiv) were added, and the reaction mixture was stirred for 1.5 hours. When starting material was consumed, the reaction mixture was cooled to -40 °C and meta-chloroperoxybenzoic acid (6.42 mmol, 3.0 equiv) was added. After 15 minutes, the reaction was quenched with saturated sodium bicarbonate (20mL) and diluted with dichloromethane (50 mL). The organic layer was washed with saturated sodium bicarbonate (2×30mL), and brine (1×10mL), dried over sodium sulfate, filtered, and concentrated in vacuo. The crude residue was purified by flash column chromatography (60-80% ethyl acetate in hexanes, stepwise) to give compound 5 (593 mg, 39% yield) as a white solid in a mixture of diastereomers due to the photocage, which was proven to be >95% pure via HPLC.1H NMR (600 MHz, CDCl3) d 8.01 – 7.89 (m, 5H), 7.83 (dd, J = 8.13, 1.33 Hz, 2H), 7.77 – 7.33 (m, 21H), 6.13 – 5.98 (m, 8H), 5.74 (dd, J = 6.30, 3.25 Hz, 1H), 5.64 (dd, J = 6.48, 3.25 Hz, 2H), 5.61 (dd, J = 5.84, 3.26 Hz, 1H*), 5.53 (dd, J = 6.27, 3.27 Hz, 1H*), 5.17 – 5.00 (m, 6H), 4.43 – 4.21 (m, 3H), 3.98 (ddd, J = 10.08, 5.02, 2.83 Hz, 1H), 3.94 (ddd, J = 9.91, 4.59, 3.12 Hz, 2H), 3.41 (ddd, J = 12.60, 9.64, 2.91 Hz, 1H), 3.32 – 3.24 (m, 2H), 3.23 – 3.15 (m, 4H), 2.04 (s, 3H), 2.02 (s, 8H), 2.01 (s, 5H), 1.98 (s, 3H), 1.95 (s, 5H), 1.71 (d, J = 6.31 Hz, 5H), 1.61 – 1.54 (m, 13H).13C NMR (150 MHz, CDCl3) d 171.26, 171.13, 170.68, 170.53, 169.14, 169.10, 146.57, 146.54, 136.83, 136.80, 136.60, 134.09, 134.02, 133.98, 129.03, 128.97, 128.95, 128.92, 127.43, 127.29, 127.22, 127.11, 124.65, 124.61, 124.59, 124.56, 95.90, 95.86, 73.38, 73.34, 73.24, 73.21, 73.15, 73.12, 70.83, 70.82, 69.65, 69.57, 68.29, 51.99, 51.94, 50.76, 50.52, 24.43, 24.40, 24.36, 24.32, 24.23, 24.19, 24.03, 24.00, 22.99, 22.92, 20.61, 20.60, 20.56.31P NMR (243 MHz, CDCl3) d -5.63, -5.72, -5.73, -6.20. ESI- Orbitrap HRMS: m / z calc for C28H33O14N6PNa [M+Na]+731.1685, found 731.1670. *Less abundant diastereomer

[0137] XIV.3,4,6-Tri-O-acetyl-2-(acetylamino)-2-deoxy-D-galactopyranoside (compound 16). Known compound Ac4GalNAc, described in Agarwal, K. et al., Journal of the American Chemical Society, 135, 14189-14197, (2013), (4.08 mmol) was dissolved in anhydrous N,N- dimethylformamide (8 mL) under inert atmosphere. Hydrazine acetate (4.9 mmol, 1.2 equiv) was added and stirred at room temperature. Upon completion, the reaction mixture wasquenched with saturated sodium bicarbonate, extracted with ethyl acetate (2×100mL), and concentrated in vacuo to give compound 16 (1.3 g, 94% yield) as a yellow oil without purification. The analytical data matched reported values in Probert, M. A. et al., Carbohydrate Research, 296, 149-170, (1996).

[0138] XV. 3,4,6-Tri-O-acetyl-2-(acetylamino)-2-deoxy-1-phospho(di-O-NPE)-a-D- galactopyranoside (1-PhotoGalNAc, compound 6), see Figure 15. Compound 16 (0.268 mmol) was dissolved in anhydrous dichloromethane (4 mL) at 0 °C and put under inert atmosphere. 5-ethylthio-1H-tetrazole (1.34 mmol, 5 equiv) and compound 9 (0.671 mmol) were added and the reaction mixture was stirred for 4 hours. When starting material was consumed, the reaction mixture was cooled to -40 °C and meta-chloroperoxybenzoic acid (0.805 mmol) was added and stirred. After 30 minutes, the reaction was quenched with saturated sodium bicarbonate (10 mL) and diluted with ethyl acetate (25 mL). The organic layer was washed with saturated sodium bicarbonate (2×25 mL), and brine (2×25 mL), dried over sodium sulfate, filtered, and concentrated in vacuo. The crude residue was purified by flash column chromatography (80% ethyl acetate in hexanes) to give compound 6 (43.4 mg, 22%) as a pale-yellow solid in a mixture of diastereomers due to the photocage, which was proven to be >95% pure via HPLC.1H NMR (600 MHz, CDCl3) d 7.96 (m, 2H), 7.93 – 7.89 (m, 3H), 7.85 – 7.81 (m, 2H), 7.76 – 7.68 (m, 3H), 7.68 – 7.58 (m, 9H), 7.55 (td, J = 7.96, 1.38 Hz, 1H), 7.53 – 7.48 (m, 3H), 7.48 – 7.43 (m, 3H), 7.42 – 7.35 (m, 2H), 6.25 (q, J = 5.66 Hz, 1H*), 6.12 – 5.96 (m, 7H), 5.94 (d, J = 8.95 Hz, 1H), 5.84 (d, J = 9.10 Hz, 1H), 5.77 (dd, J = 6.07, 3.28 Hz, 1H), 5.74 (d, J = 9.22 Hz, 1H), 5.67 (dd, J = 5.86, 3.24 Hz, 1H), 5.65 (dd, J = 5.53, 3.24 Hz, 1H), 5.56 (dd, J = 5.90, 3.26 Hz, 1H), 5.39 (dd, J = 3.30, 1.36 Hz, 1H), 5.35 (d, J = 3.20 Hz, 2H), 5.31 (dd, J = 3.28, 1.34 Hz, 1H), 5.04 (ddd, J = 11.48, 5.74, 3.17 Hz, 1H), 4.98 (dd, J = 11.56, 3.20 Hz, 1H), 4.90 (dd, J = 11.51, 3.21 Hz, 1H), 4.58 (m,3H), 4.31 – 4.25 (m, 1H), 4.23 (td, J = 6.75, 6.06, 1.45 Hz, 1H), 4.20 – 4.15 (m, 1H), 4.15 – 4.01 (m, 8H), 3.97 (dd, J = 11.22, 6.38 Hz, 1H), 3.87 (dd, J = 11.20, 6.11 Hz, 2H), 2.16 – 2.09 (m, 12H), 2.05 – 1.94 (m, 30H), 1.91 (s, 3H), 1.88 (s, 3H), 1.70 (m, 6H), 1.62 (d, J = 6.31 Hz, 3H), 1.61 – 1.54 (m, 12H).13C NMR (126 MHz, CDCl3) δ 170.89, 170.84, 170.82, 170.66, 170.61, 170.37, 170.33, 170.08, 162.69, 146.62, 146.56, 134.04, 133.98, 133.94, 129.31, 129.14, 129.11, 129.06, 129.00, 128.97, 128.54, 127.69, 127.47, 127.44, 127.35, 127.29, 127.24, 124.73, 124.67, 124.63, 124.60, 124.37, 97.21, 97.16, 91.37, 73.55, 73.43, 73.39, 73.14, 73.10, 73.02, 68.90, 68.78, 68.64, 67.87, 67.19, 67.09, 66.69, 66.63, 66.52, 61.31,61.18, 60.90, 60.41, 47.76, 36.55, 31.51, 30.97, 24.39, 24.35, 24.05, 23.18, 23.14, 23.05, 23.02, 22.96, 21.05, 20.70, 20.67, 20.65, 20.37, 14.20.31P NMR (243 MHz, CDCl3) d -5.27, - 5.50, -5.53, -6.03. ESI-Orbitrap HRMS: m / z calc for C30H36O16N3PNa [M+Na]+748.1725, found 748.1716. *Less abundant diastereomer

[0139] XVI.1,3,4-Tri-O-acetyl-2-(acetylamino)-6-O-(triphenylmethyl)-2-deoxy-a-D-glucopyranoside (compound 18). N-acetylglucosamine compound 17 (10 mmol, 1.0 equiv) was dissolved in pyridine (20 mL) under inert atmosphere and heated to 50 °C. Trityl chloride (20 mmol, 2.0 equiv) was added and the reaction was stirred overnight. Upon completion, the reaction was cooled to room temperature and acetic anhydride (40 mmol, 4.0 equiv) was added and the reaction was stirred at room temperature overnight. When starting material was consumed, the mixture was quenched with methanol (10 mL) and stirred for 30 minutes. The reaction was concentrated, then dissolved in ethyl acetate (50 mL) and washed with 1M HCl (3×50 mL), saturated sodium bicarbonate (2×50 mL), and brine (1×10 mL), dried over sodium sulfate, filtered, and concentrated in vacuo. The crude product was purified by flash column chromatography (50-80% ethyl acetate in hexanes, stepwise) to give compound 18 (2.4 g, 69% yield) as a white solid. The analytical data matched reported values in Horton, D., et al., The Journal of Organic Chemistry, 32, 1073-1080, (1967).

[0140] XVII.1,3,4-Tri-O-acetyl-2-(acetylamino)-2-deoxy-a-D-glucopyranoside (compound 19). Compound 18 (1.47 mmol, 1.0 equiv) was dissolved in 80% glacial acetic acid (21 mL), heated to 60 °C and stirred for 4 hours. The reaction was concentrated and co-evaporated with toluene in vacuo. The crude product was purified by flash column chromatography (80- 100% ethyl acetate in hexanes, stepwise) to give compound 19 (359 mg, 61% yield) as a white solid. The analytical data matched reported values in Filice, M. et al. Tetrahedron, 64, 9286-9292, (2008).

[0141] XVIII. 1,3,4-Tri-O-acetyl-2-(acetylamino)-2-deoxy-6-phospho(di-O-NPE)-a-D-glucopyranoside (6-PhotoGlcNAc, compound 7), see Figure 16. Compound 9 (0.96 mmol, 1.2 equiv) was dissolved in anhydrous dichloromethane (2.66 mL) under inert atmosphere and cooled to 0 °C. 5-ethylthio-1H-tetrazole (4.0 mmol, 5.0 equiv) was added and stirred for 10 minutes. Compound 19 (0.8 mmol, 1 equiv in 500 µL dichloromethane) was added dropwise and the reaction mixture was stirred for 1 hour at 0 °C. When starting material was consumed, the reaction mixture was cooled to -40 °C and meta-chloroperoxybenzoic acid (2.4 mmol, 3 equiv) was added. After 15 minutes, the reaction was quenched with saturated sodium bicarbonate (10 mL) and diluted with ethyl acetate (25 mL). The organic layer was washed with saturated sodium bicarbonate (2 × 25 mL), and brine (1×10 mL), dried over sodium sulfate, filtered, and concentrated in vacuo. The crude residue was purified by flash column chromatography (60-100% ethyl acetate in hexanes, stepwise) to give compound 7 (270 mg, 46% yield) as a white solid in a mixture of diastereomers due to the photocage, which was proven to be >95% pure by HPLC.1H NMR (600 MHz, CDCl3) d 8.04 – 7.83 (m, 5H), 7.75 – 7.58 (m, 10H), 7.55 – 7.35 (m, 5H), 6.15 – 5.94 (m, 7H), 5.65 – 5.51 (m, 2H), 5.50 (dd, J = 9.10, 2.84 Hz, 1H), 5.16 (ddd, J = 11.01, 9.42, 3.92 Hz, 1H), 5.09 (ddd, J = 11.05, 9.48, 6.06 Hz, 1H), 4.96 (t, J = 9.80 Hz, 1H), 4.87 (t, J = 9.88 Hz, 1H*), 4.60 (m, 1H), 4.33 (m, 1H), 4.22 – 4.13 (m, 1H), 4.02 – 3.86 (m, 4H), 3.79 (ddt, J = 9.89, 7.57, 2.74 Hz, 1H), 2.20 – 2.10 (m, 8H), 2.08 – 2.00 (m, 13H), 1.98 – 1.89 (m, 11H), 1.67 (m, 4H), 1.64 – 1.51 (m, 12H).13C NMR (126 MHz, CDCl3) δ 171.70170.02, 169.99, 169.95, 168.93, 168.88, 168.70168.56, 168.55, 168.54, 168.50, 146.80, 146.75, 146.70, 146.61, 146.54, 146.46, 146.41, 137.91, 137.88, 137.82, 137.79, 137.64, 137.58, 137.54, 137.49, 134.08, 134.05, 134.02, 133.97, 128.91, 128.85, 128.83, 128.79, 128.73, 128.70, 127.73, 127.60 (d, J = 1.15 Hz), 127.57, 127.51, 127.46, 124.61, 124.58, 124.56, 124.51, 124.45, 90.45, 90.42, 73.24, 73.20, 73.15, 72.87, 72.80, 72.71, 72.67, 72.63, 72.41, 70.71, 70.63, 70.46, 70.40, 70.01, 69.96, 67.21,67.13, 66.97, 66.94, 65.06, 64.98, 64.80, 64.76, 64.71, 52.75, 50.93, 50.91, 50.82, 50.79, 24.44, 24.40, 24.37, 24.32, 24.26, 24.23, 24.19, 23.15, 23.04, 20.89, 20.87, 20.71, 20.67, 20.57, 20.53, 20.42.31P NMR (243 MHz, CDCl3) d -3.80, -4.05, -4.09, -4.22. ESI-Orbitrap HRMS: m / z calc for C30H40O16N4P [M+NH4]+743.2171, found 743.2163. *Less abundant diastereomer

[0142] XIX.1,3,4-Tri-O-acetyl-2-(acetylamino)-2-deoxy-6-O-(triphenylmethyl)-a-D- mannopyranoside (compound 21). N-acetylmannosamine compound 20 (8.1 mmol, 1.0 equiv) was dissolved in pyridine (16.2 mL) under inert atmosphere and heated to 50 °C. Trityl chloride (24.3 mmol, 3.0 equiv) was added and the reaction was stirred overnight. Upon completion, the reaction was cooled to room temperature and acetic anhydride (32.4 mmol, 4.0 equiv) was added and the reaction was stirred at room temperature overnight. When starting material was consumed, the mixture was quenched with methanol (10 mL) and stirred for 30 minutes. The reaction was concentrated, then dissolved in ethyl acetate (50 mL) and washed with 1M HCl (3×50 mL), saturated sodium bicarbonate (2×50 mL), and brine (1×10 mL), dried over sodium sulfate, filtered, and concentrated in vacuo. The crude product was purified by flash column chromatography (60-100% ethyl acetate in hexanes, stepwise) to give compound 21 (291 mg, 8% yield) as a white solid. The analytical data matched reported values in Aich, U. et al. ACS Chemical Biology, 3, 230-240, (2008).

[0143] XX.1,3,4-Tri-O-acetyl-2-(acetylamino)-2-deoxy-a-D-mannopyranoside (compound 22). Compound 21 (0.85 mmol, 1.0 equiv) was dissolved in anhydrous dichloromethane (8.5 mL) under inert atmosphere. Iron (III) chloride hexahydrate (1.7 mmol, 2.0 equiv) was added and the reaction stirred at room temperature for 1 hour. The organic layer was washed with water (1×15 mL), and the aqueous layer was extracted with dichloromethane (20 mL). The combined organic layers were dried over sodium sulfate, filtered, and concentrated in vacuo. The crude product was purified by flash column chromatography (100% ethyl acetate) to give compound 22 (53 mg, 18% yield) as a white solid. The analytical data matched reported values in Aich, U. et al. ACS Chemical Biology, 3, 230-240, (2008).

[0144] XXI. 1,3,4-Tri-O-acetyl-2-(acetylamino)-2-deoxy-6-phospho(di-O-NPE)-a-D- mannopyranoside (6-PhotoManNAc, compound 8), see Figure 17. Compound 22 (0.15 mmol, 1.0 equiv) was dissolved in anhydrous dichloromethane (1 mL) under inert atmosphere and cooled to 0 °C. 5-ethylthio-1H-tetrazole (0.75 mmol, 5.0 equiv) was added and stirred for 10 minutes. Compound 9 (0.18 mmol, 1.2 equiv in 100 µL dichloromethane) was added dropwise and the reaction mixture was stirred for 1 hour at 0°C. When starting material was consumed, the reaction mixture was cooled to -40 °C and meta- chloroperoxybenzoic acid (0.45 mmol, 3 equiv) was added. After 10 minutes, the reaction was quenched with saturated sodium bicarbonate (5 mL) and diluted with ethyl acetate (15mL). The organic layer was washed with saturated sodium bicarbonate (2×10 mL), and brine (1×5 mL), dried over sodium sulfate, filtered, and concentrated in vacuo. The crude residue was purified by column chromatography (60-100% ethyl acetate in hexanes, stepwise) to give compound 8 (88 mg, 81% yield) as a white solid in a mixture of diastereomers due to the photocage, which was proven to be >95% pure by HPLC.1H NMR (600 MHz, CDCl3) δ 7.93 (ddd, J = 7.85, 6.25, 1.28 Hz, 2H), 7.91 – 7.85 (m, 3H), 7.82 (dd, J = 8.18, 1.30 Hz, 1H), 7.77 (ddd, J = 13.22, 7.33, 2.34 Hz, 4H), 7.73 – 7.60 (m, 6H), 7.59 – 7.51 (m, 5H), 7.49 – 7.32 (m, 10H), 6.07 (m, 1H), 6.03 – 5.94 (m, 5H), 5.85 – 5.79 (m, 1H), 5.75 (d, J = 1.76 Hz, 1H*), 5.74 (d, J = 1.71 Hz, 1H), 5.69 (d, J = 1.89 Hz, 1H*), 5.46 (t, J = 10.14 Hz, 1H), 5.40 (q, J = 9.90 Hz, 2H), 5.35 – 5.28 (m, 2H), 5.24 (ddd, J = 13.10, 10.26, 4.32 Hz, 1H), 4.69 – 4.64 (m, 2H), 4.55 (dddd, J = 11.47, 9.72, 4.33, 1.91 Hz, 1H), 4.25 (ddd, J = 12.58, 4.45, 1.56 Hz, 1H), 4.18 – 3.90 (m, 7H), 3.88 – 3.76 (m, 3H), 2.15 (d, J = 2.05 Hz, 6H), 2.10 (d, J = 1.61 Hz, 4H), 2.05 (s, 3H), 2.02 – 1.92 (m, 28H), 1.72 – 1.62 (m, 11H), 1.55 (d, J = 6.33 Hz, 3H), 1.52 (d, J = 6.31 Hz, 3H).13C NMR (126 MHz, CDCl3) δ 171.27, 171.24, 171.08, 170.50, 170.41, 168.96, 168.92, 168.77, 168.15, 167.98, 147.16, 146.96,146.82, 146.65, 146.44, 137.57 , 137.46, 137.24, 134.09, 133.97, 133.91, 133.88, 133.82, 133.59, 133.54, 128.84, 128.80, 128.70, 128.66, 128.58, 128.56, 127.99, 127.73, 127.70, 127.68, 127.66, 127.63, 127.23, 124.47, 124.43, 124.40, 124.26, 124.20, 124.13, 92.19, 92.10, 91.99, 91.96, 73.43, 73.39, 73.19, 73.16, 72.88, 72.71, 72.67, 72.64, 72.59, 70.99, 70.80, 69.21, 69.18, 69.05, 67.21, 67.15, 67.07, 67.01, 66.81, 64.66, 64.57, 48.60, 24.29, 24.22, 24.18, 24.09, 24.05, 23.84, 23.74, 23.70, 23.66, 23.60, 22.82, 22.76, 20.88, 20.84, 20.81, 20.68, 20.61, 20.58.31P NMR (243 MHz, CDCl3) d -4.75, -5.49, -5.82, -6.24. ESI- Orbitrap HRMS: m / z calc for C30H36O16N3PNa [M+Na]+748.1725, found 748.1709. *Less abundant diastereomer

[0145] Analytical high performance liquid chromatography (HPLC) linear gradient system was performed for purification of compounds 4, 5, 6, 7, and 8. An Agilent Poroshell 120 column packed with 2.7 µM particle size with EC C-18 was employed to load the sample. The flow rate was 1mL / minute. Solutions of 4, 5, 6, 7, and 8 in MeCN (4 mg / mL) were prepared and 5 µL was injected into the HPLC system.

[0146] 1H,13C, and31P NMR spectra were collected on a Varian 400-MR or Agilent 600 MHz DD2 instruments.

[0147] NMR Characterization of NBDE-Ac3-GlcNAc (compound 1a) and NBDE-Ac2- 6Az-GlcNAc (compound 3a) de-caging

[0148] Compound 1a (caged 1-Ac3-GlcNAc, 1mg) dissolved in 700uL of CDCl3, then placed at 1 cm distance and irradiated with 350 nm light. De-caging was observed which was confirmed1H-NMR.1H-NMR showed the disappearance of methyl group at 1.48 ppm, doublet and raising of new carbonyl methyl peak at 2.88 ppm, singlet fromnitrosoacetophenone (free photocage), indicates the successful de-caging. Similarly, light- dependent decaging experiment carried out with compound 3a using 350 nm light. Compound 3a (2mg) dissolved in 700uL of CDCl3, then placed at 1 cm distance and irradiated with 350 nm light. De-caging was observed by1H-NMR.

[0149] 31P-NMR characterization of caged sugar phosphate de-caging

[0150] Each indicated compound 4, 5, 6, 7, or 8 (2 mg) was dissolved in DMSO-D6inside a 600Hz Quartz NMR tube. D2O was added to make a 25% (v / v) D2O in DMSO-D6 mixture containing one of compounds 4, 5, 6, 7, or 8. An initial31P NMR spectrum was taken before the sample was subjected to UV exposure. The sample was then exposed to 350 nm UV light. Exposure was stopped at different time intervals to generate a31P NMR spectrum at 1 minute, 3 minutes, 5 minute, and 10 total minutes of UV exposure. The 25% DMSO was required for solubility of compound 5.

[0151] A representative31P-NMR trace for release of compound 4 (caged 1-phospho- Ac3-GlcNAc) showed increasing peak areas at ca. -4.0 to -2.4 ppm (the four caged phosphate diastereomer peaks) and 0.5 ppm (free phosphate peak) indicated compound release.

[0152] A representative31P-NMR trace for release of compound 5 (6-azido-1- phospho(caged)-Ac2-GlcNAc) showed increasing peak areas at ca. -4.0 to -2.4 ppm (the four caged phosphate diastereomer peaks) and 0.5 ppm (free phosphate peak) indicated compound release.

[0153] A representative31P-NMR trace for release of compound 6 (caged 1-phospho- Ac3-GalNAc) showed increasing peak areas at ca. -4.0 to -2.4 ppm (the four caged phosphate diastereomer peaks) and 0.5 ppm (free phosphate peak) indicated compound release.

[0154] A representative31P-NMR trace for release of compound 7 (caged 6-phospho- Ac3-GlcNAc) showed increasing peak areas at ca. -4.0 to -2.4 ppm (the four caged phosphate diastereomer peaks) and 0.5 ppm (free phosphate peak) indicated compound release.

[0155] A representative31P-NMR trace for release of compound 8 (caged 6-phospho- Ac3-ManNAc). Increasing peak areas at ca. -4.0 to -2.4 ppm (the four caged phosphate diastereomer peaks) and 0.5 ppm (free phosphate peak) indicated compound release.

[0156] High resolution mass spectra were collected on an Orbitrap Exploris 120 (ThermoFisher) in electrospray ionization (ESI)-positive mode.

[0157] High-performance liquid chromatography was performed on an Agilent 1260 Infinity II equipped with an autosampler and the traces were processed in LCOpenLAb.

[0158] Plate reads for toxicity assays were measured using a BioTek Cytation One and the data was processed in GraphPad Prism 9.

[0159] Light-driven decaging

[0160] Decaging reactions were conducted in a Rayonet RMR-600 photoreactor (Rayonet, Connecticut) equipped with eight 350 nm ultraviolet bulbs (Cat No. RMR-3500- A). Samples were placed 7.5 cm from the light sources and reactions were conducted at 32 total watts, corresponding to approximately 176 μW / cm2 intensity. For the 400–410 nm violet decaging reactions, True Violet light emitting diodes (LEDs) (Rapid LED, California) were powered by a 10 W power supply driver equipped with a dimmer switch, with samples placed 7.5 cm from the LED. All decaging experiments were conducted at room temperature in an enclosed box to prevent leakage from other sources of light.

[0161] Tracking decaging by NMR

[0162] Each of compounds 4, 5, 6, 7, and 8 (2 mg) was dissolved in DMSO-d6 (562 μL) and diluted with D2O (187.5 μL) to give a 25% v / v solution of D2O in DMSO-d6. The samples were analyzed via31P nuclear magnetic resonance (NMR) to establish baseline spectra, then exposed to 350 nm light for the indicated time intervals and analyzed by31P NMR. Exposures and measurements were repeated until the compounds had been fully decaged. The resulting spectra were processed in MestreNova.

[0163] Tracking decaging of compounds by UV–Vis

[0164] A solution of each of compounds 4, 5, 6, 7, and 8 (400 µM, 0.1% DMSO) in phosphate-buffered saline (PBS) was sonicated and vortexed until the compound was fully dissolved. The solution was divided between two 35mm culture dishes, 2 mL per dish. The UV spectrophotometer was blanked using PBS containing 0.1% DMSO. Samples were exposed to 350 nm light at 0, 1, 2, 3, 4, 5, and 10-minute intervals and the UV spectra was taken. The data was exported from Agilent ChemStation and imported into GraphPad Prism to generate curves. UV-visible traces for release of compound 4 (caged 1-phospho-Ac3- GlcNAc), compound 6 (caged 1-phospho-Ac3-GalNAc), compound 7 (caged 6-phospho-Ac3- GlcNAc, and compound 8 (caged 6-phospho-Ac3-GManNAc) were obtained and showed an increasing absorbance peak at ca.325 nm indicating release of the free photocage.

[0165] Compound 5 (6-azido-1-phospho(caged)-Ac2-GlcNAc) was insoluble in PBS or tissue culture media and was not carried forward for UV-vis release characterization.

[0166] The data was exported from ChemStation (Agilent, California) and plotted in GraphPad Prism.

[0167] Tracking decaging of compounds by high-performance liquid chromatography (HPLC)

[0168] Caged sugars were dissolved in phosphate-buffered saline (400 μM, 0.1% DMSO). After the indicated UV exposure, samples were collected and analyzed by HPLC (Agilent Technologies Infinity II, equipped with vial sampler, quat pump, and Agilent 1260 Multiwave detector.) Compounds were separated on a Poroshell 120 C18 column (2.7 μm, 4.6 mm × 50 mm). Concentration and compound identification were performed by integrating the compound peaks at 200 nm, 265 nm (caged phosphate λmax) and 310 nm (released 2-nitroacetophenone cage λmax).

[0169] Figures 5A and 5C show results of HPLC measurements to estimate photo- decaging conversion of compound 4. Figure 5A is a series of traces showing absorbance chromatograms (200 nm) for samples at increasing UV exposure time points. Figure 5B is a diagram showing a scheme for the decaging reaction. Figure 5C is a graph showing the estimated conversion from intact photocaged GlcNAc-1-phosphate (compound 4, light gray bars) to the decaged sugar, accompanied by formation of 2-nitrosoacetophenone byproduct (black bars). Data analyzed by integrating the 200 nm peaks normalized release points to 0 min (ca.100% of compound 4) and 30 min (ca.100% of 2-nitrosoacetophenone).

[0170] Cell culture conditions

[0171] HeLa cells (American Type Cell Culture #CCL-2) were cultured in Dulbecco′ s Modified Eagle′ s Medium – high glucose (4500 mg / L glucose), L-glutamine, sodium pyruvate, and sodium bicarbonate (Millipore-Sigma D6429) and incubated at 37 ◦C with 5% CO2. To seed cells, cells were released from culture dish via trypsin (0.25%, Cytiva SH30031.01). Cell counting was preformed using a Countess™ II system and trypan blue dye following the manufacturer’s procedure (ThermoFisher, Massachusetts).

[0172] Cell viability and toxicity assays

[0173] HeLa cells were seeded in 96-well plates (5,000 cells / well) and incubated with the indicated concentrations of each compound or DMSO (1% v / v) for 24 h. After treatment, cell viability was measured using Cell Proliferation Kit 1 (MTT) (Roche, Switzerland) or CellTiter Glo-2.0 (Promega, Wisconsin). Toxicity was determined using CellTox-Green (Promega, Wisconsin). The data was exported from Gen5 software and imported into GraphPad Prism to generate curves.

[0174] Multiplexed cell viability and toxicity of 1P-diNPE-GlcNAc (compound 4) when exposed to UV light

[0175] HeLa cells were seeded in 96-well plates (5,000 cells / well) and incubated with the indicated concentrations of 1P-diNPE-GlcNAc (compound 4) (24 µm, 35 µm, 53 µm, 79 µm, 178 µm, 267 µm, 400 µm) or DMSO (1% v / v) for 24 hours. Cells were then exposed to350nm light for 5minutes. CelltoxTMGreen Cytoxtoxicity Assay was used to determine toxicity, and CellTiter®-Glo 2.0 cell viability kit was used to determine viability. In brief, 0. 1uL of CelltoxTMGreen was added to each well orbital shaken for 1 minute, and then plate was allowed to equilibrate to room temperature before reading 490nmex / 525nmem. 100 uL of CellTiter®-Glo 2.0 was added to each well and after 2 minutes of orbital shaking and 10 minute room temperature equilibrium, contents were transferred to opaque white well plate for luminescence measurement. This was done 1 h after photo-release and 24 h after photo- release.

[0176] Figures 6A and 6B: Multiplexed cell viability (black triangles) and toxicity (gray squares) for photo release of 1P-diNPE-GlcNAc (compound 4) Figure 6A) 1 h after photo release. Figure 6B) 24 h after photo-release.

[0177] Multiplexed cell viability and toxicity of 1P-diNPE-GalNAc, (compound 6) when exposed to UV light

[0178] HeLa cells were seeded in 96-well plates (5,000 cells / well) and incubated with the indicated concentrations of 1P-diNPE-GalNAc (compound 6) (24 µm, 35 µm, 53 µm, 79 µm, 178 µm, 267 µm, 400 µm) or DMSO (1% v / v) for 24 hours. Cells were then exposed to 350nm light for 5minutes. CelltoxTMGreen Cytoxtoxicity Assay was used to determine toxicity, and CellTiter®-Glo 2.0 cell viability kit was used to determine viability. In brief, 0.1uL of CelltoxTMGreen was added to each well orbital shaken for 1 minute, and then plate was allowed to equilibrate to room temperature before reading 490nmex / 525nmem. 100 uL of CellTiter®-Glo 2.0 was added to each well and after 2 minutes of orbital shaking and 10 minute room temperature equilibrium, contents were transferred to opaque white well plate for luminescence measurement. This was done 1 h after photo-release and 24 h after photo- release.

[0179] Figure 7A and 7B: Multiplexed cell viability (black triangles) and toxicity (gray squares) for photo release of 1P-diNPE-GalNAc (compound 6) Figure 7A) 1 h after photo release. Figure 7B) 24 h after photo-release.

[0180] Multiplexed cell viability and toxicity of 6-P-diNPE GlcNAc, (7) when exposed to UV light

[0181] HeLa cells were seeded in 96-well plates (5,000 cells / well) and incubated with the indicated concentrations of 6-P-diNPE GlcNAc, (compound 7) (24 µm, 35 µm, 53 µm, 79 µm, 178 µm, 267 µm, 400 µm) or DMSO (1% v / v) for 24 hours. Cells were then exposed to 350nm light for 5minutes. CelltoxTMGreen Cytoxtoxicity Assay was used to determine toxicity, and CellTiter®-Glo 2.0 cell viability kit was used to determine viability. In brief, 0.1uL of CelltoxTMGreen was added to each well orbital shaken for 1 minute, and then plate was allowed to equilibrate to room temperature before reading 490nmex / 525nmem. 100 uL of CellTiter®-Glo 2.0 was added to each well and after 2 minutes of orbital shaking and 10 minute room temperature equilibrium, contents were transferred to opaque white well plate for luminescence measurement. This was done 1 h after photo-release and 24 h after photo- release.

[0182] Figures 8A and 8B: Multiplexed cell viability (black triangles) and toxicity (gray squares) for photo release of 6P-diNPE-GlcNAc (compound 7) Figure 8A) 1 h after photo release. Figure 8B) 24 h after photo-release.

[0183] Multiplexed cell viability and toxicity of 6P-diNPE-ManNAc (compound 8) when exposed to UV light

[0184] HeLa cells were seeded in 96-well plates (5,000 cells / well) and incubated with the indicated concentrations of 6P-diNPE-ManNAc (compound 8) (24 µm, 35 µm, 53 µm, 79 µm, 178 µm, 267 µm, 400 µm) or DMSO (1% v / v) for 24 hours. Cells were then exposed to 350nm light for 5minutes. CelltoxTMGreen Cytoxtoxicity Assay was used to determine toxicity, and CellTiter®-Glo 2.0 cell viability kit was used to determine viability. In brief, 0.1uL of CelltoxTMGreen was added to each well orbital shaken for 1 minute, and then plate was allowed to equilibrate to room temperature before reading 490nmex / 525nmem. 100 uL of CellTiter®-Glo 2.0 was added to each well and after 2 minutes of orbital shaking and 10 minute room temperature equilibrium, contents were transferred to opaque white well plate for luminescence measurement. This was done 1 h after photo-release and 24 h after photo- release.

[0185] Figures 9a and 9B: Multiplexed cell viability (black triangles) and toxicity (gray squares) for photo release of 6P-diNPE-ManNAc (compound 8) Figure 9A) 1 h after photo release. Figure 9B) 24 h after photo-release.

[0186] Results

[0187] Eleven photo-releasable glycan precursors were synthesized in this example and their release properties and cell compatibility were characterized for use in biological studies. These compounds cover three major glycan precursors that are typically used by cells for protein post-translational modifications: UDP-GlcNAc, UDP-GalNAc, and CMP-sialic acid. Photo-releasable glycan precursors were light-released in standard tissue culture media within 5 min. Furthermore, the phosphate caged GlcNAc-1-phosphate, GalNAc-1- phosphate, and ManNAc-6-phosphate glycan precursors showed minimal cytotoxicity.

[0188] Synthesis of photo-releasable glycan precursors

[0189] The structures of photo-releasable glycan precursors according to aspects of the present disclosure are shown in Figure 2.

[0190] Synthetic schemes for the photo-releasable glycan precursors of Fig.2 are shown in Schemes 1A, 1B, and 1C in Figures 11A, 11B, and 11C, respectively.

[0191] Synthesis of photo-releasable anomeric caged GlcNAc analogs was carried out via activation of GlcNAc to its oxazoline intermediate and subsequent glycosylation with either diethylaminocoumarin (DEAC) or 2-nitrobenzodioxyethanol (NBDE) alcohol to afford Ac3GlcNAc-NBDE 1a, 6Az-Ac2GlcNAc-NBDE 2a, and 6Az- Ac2GlcNAc-DEAC 3a. To access the unprotected sugars, 1a, 2a, and 3a were subsequently deacetylated to give GlcNAc-NBDE 1b, 6Az-GlcNAc- NBDE 2b, and 6Az-GlcNAc-DEAC 3b in 82–90% yields (Scheme 1A).

[0192] For the caged phosphosugars, low coupling efficiency of DEAC or NBDE to the phosphate was observed possibly due to steric constraints. Switching to the smaller nitrophenylethanol (NPE) photocage enabled us to access both 1-phosphate and 6-phosphate caged compounds in moderate yield. The 1-phosphate light-releasable glycan precursors 1P- diNPE-GlcNAc compound 4, 6Az-1P-diNPE-GlcNAc compound 5, and 1P-diNPE-GalNAc compound 6 were synthesized following related sugar phosphate photocage synthesis conditions8 (Scheme 1B).

[0193] Bis-(1-[2-nitrophenyl]-ethyl)-N,N-diisopropylphosphoramidite compound 9 was coupled to the anomeric alcohol of the desired sugar variants using 5-ethylthio-1H-tetrazole to give the desired compounds in 22–53% yields. This same procedure was used to form the 6-phosphate sugars, 6P-diNPE-GlcNAc compound 7 and 6P-diNPE-ManNAc compound 8, in 46% and 81% yields, respectively (Scheme 1C). Altered steric factors in the protection / deprotection of the 6-tritlyl group on mannose resulted in lower yields (8% and 18%, respectively) using conditions optimized for the β-2-OH sugars, though these conditions were not further optimized for mannose. Notably, the racemic chiral center on the NPE led to an equal mixture of 4 diastereomers for each diNPE-caged sugar phosphate. Each compound resolved to a single chiral sugar-phosphate species following light-mediated release of the NPE cages, as detailed herein.

[0194] Characterization of sugar release from photo-releasable glycan precursors

[0195] The photo-releasable glycan precursors respond to 350 nm light (NPE, NBDE) or 410 nm light (DEAC).32 To track how each compound decaged, nuclear magnetic resonance (NMR) and UV–visible light spectroscopy was performed to characterize the decaging properties of caged compounds (Fig. 3). The phosphate probes’ decaging progress wasselectively followed by31P NMR between UV-light exposure times for the photo-releasable GlcNAc-phosphate precursor compounds 4, 5, and 7 (Fig.3A and C).1H NMR was measured for non-phosphate molecules. In brief, compounds were dissolved in NMR solvent and exposed to UV 350 nm UV light for the indicated timepoints. The13CNMR spectrum were recorded post-exposure to track decaging. Though the non-azide photo-releasable glycan precursors of compounds 4, 6, 7, and 8 fully dissolved in D2O, low solubility of azide- containing compound 5 prompted the use of 25% (V / V) D2O in DMSO-d6 for its full solubility. The disappearance of the four diastereomeric caged phosphate peaks (right boxes, Fig 3A and C) and formation of decaged species (left boxes, Fig 3A and 3C) was monitored. After 10 total minutes of UV exposure the diastereomeric caged phosphate peaks collapsed to a single, upstream peak indicating the released sugar-phosphate species. The results showed that these compounds were readily decaged in under 10 min in the presence of water. However, NMR has a concentration limitation that require experiments to be done well above biological concentrations, namely 3.7 mM. As a follow-up study, UV–vis spectroscopy was used to characterize decaging in fully aqueous conditions that used μM concentrations (vs. mM) that would be more appropriate for cell experiments. Both phosphate-buffered saline (PBS) and Dulbecco’s Modified Eagles Medium (DMEM) were chosen as common buffer and media types. Because PBS and DMEM each contain high concentrations of phosphates and / or glucose that would interfere with NMR, NMR was not suitable for decaging in these media. By UV–vis, it was observed that the intact vs. released photocage provided a spectral shift from λmax = 260 nm (caged phosphate) to 325 nm (free photocage), enabling UV–vis characterization of de-caging (Fig. 3B / D). For clarity, PBS gave the best UV–vis trace, but DMEM results matched the PBS data following background subtraction. Irradiation with 350 nm light revealed that full decaging occurred within 5 min of UV exposure (vs. 10 min by NMR) for compounds 4 and 7. UV–vis de-caging profiles for sugars of compounds 6 and 8 followed the same trend. The observed accelerated decaging in PBS / DMEM may be due to a pH effect in these buffers, though this hypothesis was not confirmed. As noted during the NMR experiment, azidosugar compound 5 did not fully dissolve in PBS or DMEM, so the UV–vis decaging experiment was not possible with compound 5. However, compounds 4, 6, 7, and 8 were each rapidly decaged in fully aqueous solvents suitable for mammalian culture and carried forward for biological evaluation. To determine more exact conversions of decaging, HPLC traces were performed during decaging of compound 4. The results showed that photocaged starting material (i.e. photo-releasable glycan precursors) was ca. 95% depleted following 5 min of UV light exposure.

[0196] Photo-releasable glycan precursor cell compatibility

[0197] To evaluate effects of photo-releasable glycan precursors, cell viability studies were performed to determine the toxicity effects of these compounds on cells. HeLa cells, a well-known human cervical cancer model cell line, were used to investigate the toxicity effects of photo-releasable glycan precursors 4, 6, 7, and 8 prior to exposure with UV light. HeLa cells were treated with increasing concentrations (12.5, 25, 50, 100, 200, and 400 μM) of compounds 4, 6, 7, and 8 for 24 h (Fig. 4A). The photo-releasable glycan precursors to GlcNAc, GalNAc, and ManNAc (compounds 4, 6, 8) demonstrated low cytotoxicity when compared to the DMSO treated control. UDP-GlcNAc precursor compound 7 showed modest toxicities above 50 μM. These results indicate that compounds of the present disclosure can be incubated with HeLa cells for extended periods of time prior to decaging without disrupting the viability of the system. However, not all compounds were soluble above 500– 800 μM. Therefore, the experiments were capped at 400 μM for the direct comparison. Accordingly, accurate IC50 values for toxicity could not be measured for all compounds because the toxicity was far less than the maximum soluble concentrations for compounds 4, 5, 6, and 8. As an additional control, the toxicity of 350 nm and 410 nm light on HeLa cells was evaluated to ensure that decaging of photo-releasable glycan precursors in vitro would not affect the viability of the cells. HeLa cells were irradiated with 350 nm (6 W) or 410 nm (10 W) light for 5–60 min followed by a 24-hour incubation. While 350 nm light demonstrated low toxicity to the cells, 410 nm light was found to be toxic to cells with viability below 30% after just 2 min of irradiation (Fig. 4B). Toxicity of the 410 nm light prevented the use of coumarin-caged sugars 3a and 3b in vitro. However, 350 nm light was found to be minimally cytotoxic greater than 80% viability was maintained for at least 10 min of exposure. Notably, a 5-minute exposure to 350 nm allowed full decaging of these compounds in mammalian cell culture media (Fig.3, above), indicating that these conditions are compatible for cellular studies.

[0198] To fully determine the effects of photo-releasable glycan precursor treatment followed by UV light-release conditions on cells, a multiplexed viability and cell toxicity system was used to study both cell proliferation (viability) and cell toxicity. Cells were treated with the indicated compound 4, 6, 7, or 8 overnight to enable cellular uptake. The media was exchanged to fresh DMEM and the cells were exposed to 5 min of 350 nm light. Viability and toxicity assays were conducted 1 h and 24 h following the release of glycan precursors (Fig. 4C-D). Compounds 4, 6, and 8 (caged GlcNAc-1-phosphate, GalNAc-1- phosphate, and ManNAc-6-phosphate) showed little impact on cell viability in the 100–400μM treatment range. Some decrease in cell viability at the lower concentrations of the caged compounds was observed, which was replicated by an increase in cell toxicity observed by a CellTox-Green assay (Figs 6–9). Toxicity and loss of viability at low concentrations may be due to inefficient blocking of UV-light effects during the decaging reaction, but this observation bears further mechanistic study. The caged GlcNAc-6- phosphate compound 7 displayed significant dose-dependent cytotoxicity and loss in cell viability at both time points (Fig. 7). To confirm these results, multiplexed cell viability and toxicity studies were performed using the CellTiter-Glo and CellTox-Green systems (Fig. 4E-F). The strong toxicity response in Fig. 4F indicated that this compound was toxic to cells. The regioisomeric compound 4 showed no toxicity at the 24 h timepoint up to 400 μM and a mild decrease in viability at 400 μM, which may represent a modest reduction in signal for proliferation. However, the cell viability assay used, CellTiter-Glo 2.0, measures cellular ATP levels as its output. The CellTiter-Glo drop in signal but lack of CellTox-Green toxicity indicates that ATP levels were consumed by release of GlcNAc-1- phosphate in these cells but that toxicity did not result. These preliminary findings demonstrated controlled cellular metabolic engineering of ATP consumption following photo-release. Conversely, compound 7 showed dose-dependent toxicity (gray curve) and loss of viability (black curve) at all concentrations tested (data from all compounds found in Figs. 6–9). There was unexpected toxicity of caged GlcNAc-6- phosphate, but it was notable that toxicity occurred independently of UV release, suggesting the particular NPE-based structure was itself toxic.

[0199] Synthesis for non-acetylated, caged glycans using 6-azido-GlcNAc as example:

[0200] Compound (6): To a solution of compound 5 (390mg, 0.451mmol) in 16 mL of DCM: water (10:1) was cool down to 0oC and then DDQ was added in two portions. The resulting dark brown reaction mixture stirred for 3.5hr at 0oto 10oC. TLC indicated the complete conversion of starting material and formation of polar band. The reaction mixture quenched with saturated aq. NaHCO3 (30 mL) at 0oC and extracted with DCM (120 mL), dried over anhydrous Na2SO4, concentrated, and adsorbed on silica gel and purified by silica gel flash column chromatography using EtOAc: Hex (50:50, 75:25, and 100%), the desired product eluted with EtOAc: Acetone (95: 5%). The resulting white foamy solid compound 6 (210mg, 75% yield, diastereomeric mixture) and was stored in dry form at -80oC, until further use.1H NMR (500 MHz, CDCl3) δ 7.94 – 7.89 (m, 1H), 7.88 – 7.83 (m, 3H), 7.78 (m, 1H), 7.75 – 7.63 (m, 3H), 7.60 (m, 3H), 7.54 (m, 4H), 7.50 – 7.38 (m, 5H), 7.38 – 7.31 (m, 1H), 6.82 (dd, J = 8.0, 3.4 Hz, 1H), 6.60 (d, J = 8.1 Hz, 1H), diastereomeric anomeric proton coupling* 5.69 (dd, J = 6.2, 3.1 Hz, 0.5H*), 5.61 (dd, J = 6.2, 3.1 Hz, 1H*), 5.56 (dd, J = 5.9, 3.2 Hz, 1H*), 5.47 (dd, J = 6.1, 3.1 Hz, 1H*), 4.00 – 3.93 (m, 1H), 3.91 (ddt, J = 10.9, 8.0, 3.0 Hz, 1H), 3.80 – 3.71 (m, 1H), 3.65 (m, 1H), 3.61 – 3.56 (m, 1H), 3.52 (m, 1H), 3.50 – 3.37 (m, 5H), 3.32 (m, 4H), 2.09 – 1.93 (s, 7H), 1.84 (s, 2H), 1.65 (s, 4H), 1.57 (d, J = 6.3 Hz, 3H), 1.56 – 1.42 (merged d, 10H);13C NMR (126 MHz, CDCl3) δ 172.61, 171.21, 147.07, 146.83, 146.59, 146.54, 146.51, 146.43, 137.01, 136.97, 134.27, 134.20, 134.13, 134.07, 129.35, 129.16, 129.10, 129.03, 129.00, 127.68, 127.65, 127.54, 127.42, 127.33, 127.31, 124.58, 124.55, 124.52, 124.48, 97.17, 97.09, 96.79, 96.74, 77.30, 77.05, 76.80, 60.41, 53.60, 51.18, 50.95, 24.34, 24.29, 24.18, 24.11, 24.07, 22.96, 22.80, 22.77, 21.04, 14.19;31P NMR (202 MHz, CDCl3) δ -5.24, -5.44, -5.55, -5.80.

[0201] Synthesis of bioorthogonal “chemocage” groups for glycans, Figure 18.

[0202] Bis-(alcohol)-N,N-diisopropylphosphoramidites, see Figure 19 Phosphorous trichloride (4.5 mmol, 1 equiv) and N,N-diisopropylethylamine (9.0 mmol, 2 equiv) were added to anhydrous tetrahydrofuran (9 mL) under inert atmosphere. Diisopropylamine (9.0 mmol, 2 equiv) was added dropwise and the mixture was stirred for 4 hours to yield diisopropylphosphoramidous dichloride. The reaction mixture was cooled to - 15 °C, and the corresponding alcohol (9.0mmol, 2 equiv) and triethylamine (9.9 mmol, 2.2 equiv) were added. The mixture was slowly warmed to room temperature and stirred overnight. Upon completion, the reaction mixture was diluted with ethyl acetate (50 mL), washed with saturated sodium bicarbonate (2×25 mL) and brine (1×10 mL), dried over sodium sulfate, filtered, and concentrated in vacuo. The crude product was purified via flash column chromatography (i.e. with 5:1 hexanes:ethyl acetate) to give the product. The alcohols used are derived from published syntheses of trans-cyclooctene (reported in Davies, S. et al, Chembiochem, 20, 1541-1546, (2019)) and tetrazine (reported in Tu, J, et al Chemical Science, 11, 169-179 (2020))

[0203] Chemically caged glycan phosphates

[0204] The corresponding glycan (0.266 mmol) was dissolved in anhydrous dichloromethane (886 µL, 0.3M) under inert atmosphere and cooled to 0 °C.5-ethylthio-1H- tetrazole (1.33 mmol, 5.0 equiv) and the corresponding caged phosphine (0.665 mmol, 2 equiv) were added and the reaction mixture stirred for 1.5 hours. When starting material was consumed, the reaction mixture was cooled to -40 °C and meta-chloroperoxybenzoic acid (0.798 mmol, 3 equiv) was added. After 30 minutes, the reaction was quenched with saturated sodium bicarbonate (5 mL) and diluted with ethyl acetate (20 mL). The organic layer was washed with bicarb (2×20 mL), and brine (1×10 mL), dried over sodium sulfate, filtered, and concentrated in vacuo. The crude residue is purified by flash column chromatography (e.g. 60% ethyl acetate in hexanes) to give the product as a mixture of diastereomers due to the cage.

[0205] N-caged hexosamine sugar caging

[0206] Figure 20 shows an example of an N-caged hexosamine sugar and photocage.

[0207] Figure 21 shows examples of N-caged hexosamine sugar and photocage compounds in which the “cage” is a photocage or biorthogonal cage (chemocage); PG is an ester-based protecting group, i.e. acetyl, ethyl, propyl, butyl, pentyl, or hexyl ester; R is an azide, C2-C8 linear alkene or cyclic alkene, or C2-C8 terminal alkyne or cyclic alkyne derivative, and R’ is H, C1-C6 ester, or a sugar phosphate in alpha or beta configuration.

[0208] Sugar 6-position hexosamine sugar caging

[0209] Figure 22 shows an example of a 6-position hexosamine sugar and photocage.

[0210] Figure 23 shows examples of 6-position hexosamine sugar and photocage compounds in which the “cage” is a photocage or biorthogonal cage (chemocage); PG is an ester-based protecting group, i.e. acetyl, ethyl, propyl, butyl, pentyl, or hexyl ester; R is an azide, C2-C8 linear alkene or cyclic alkene, or C2-C8 terminal alkyne or cyclic alkyne derivative, and R’ is H, C1-C6 ester, or a sugar phosphate in alpha or beta configuration.

[0211] Scheme XXII- Synthesis of 6Az-GlcNAc-1-diNPE-phosphate (compound 28)

[0212] Figure 24 shows Scheme XXII demonstrating synthesis of 6Az-GlcNAc-1- diNPE-phosphate (compound 28) from compound 23 in 5 steps.

[0213] Scheme XXII, Compound (23): Compound 23 was synthesized from GlcNAc in two steps, by following previously reported protocols.

[0214] Scheme XXII, Compound 24 (GJ-II-030): To a solution of compound 24 (500mg, 2.26 mmol) in 9 mL of water, was cooled down to 0 ºC and then Et3N (2.84 mL, 20.35 mmol), followed by 2-Chloro-1,3-dimethylimidazolinium chloride (DMC, Shoda’s reagent) (1.147g, 6.73 mmol)) were added. The resulting brown reaction mixture stirred for 30 minutes at 0 ºC. No polarity difference was observed on TLC, the product and the starting material had same polarity. The reaction mixture was directly loaded on prepacked C18 column and eluted with 100% water (0.015% Et3N). Triethyl amine eluted first, later DMC, followed by product. The TLC fractions were pooled and frozen at -80º C and then dried using vacuum freeze centrifuge, for 24 hours resulting in a foamy light brown solid oxazoline compound 24 (450mg) in 90% yield. Compound 24 stored at -80º C, until further use.

[0215] 1H NMR (400 MHz, D2O) δ 5.98 (d, J = 7.4 Hz, 1H), 4.03 (m, 1H), 3.85 (apt, J = 3.7 Hz, 1H), 3.58 – 3.46 (m, 2H), 3.43 – 3.33 (m, 2H), 1.94 (d, J = 1.7 Hz, 3H).

[0216] Scheme XXII, Compound 25: To a solution of compound 24 (450mg, 2.03 mmol) in 8 mL of dimethylformamide (DMF), was cool down to 0º C and then sodium hydride (270mg, 6.09 mmol), was added and stirred for 10 minutes and then slowly added dropwise p-methoxybenzyl chloride (855 µL, 6.09 mmol). The resulting brown reaction mixture stirred for 16hr at 0º C to room temperature under inert atmosphere. TLC indicated the completion of starting material and formation of non-polar bands. The reaction mixture was quenched with 10 mL of ice-cold water at 0º C, washed with 50 mL of brine and extracted with EtOAc (100 mL). The organic layer dried over sodium sulfate, concentrated,and adsorbed on silica gel. The column eluted with ethylacetate / hexane (30 / 70, 50 / 50, 70 / 30) with 0.015% Et3N. The product eluted with 70 / 30, the pure fractions were pooled and concentrated, 68% yield.

[0217] 1H NMR (500 MHz, CDCl3) δ 7.24 (d, J = 8.6 Hz, 1H), 7.07 (d, J = 8.6 Hz, 1H), 6.83 (d, J = 8.7 Hz, 1H), 6.78 (d, J = 8.6 Hz, 1H), 5.89 (d, J = 7.5 Hz, 1H), 4.57 (d, J = 11.6 Hz, 1H), 4.47 (d, J = 11.6 Hz, 1H), 4.43 (d, J = 11.4 Hz, 1H), 4.15 (d, J = 11.4 Hz, 1H), 3.92 (t, J = 2.5 Hz, 1H), 3.73(s, 3H), 3.45 – 3.39 (m, 1H), 3.35 (ddd, J = 9.0, 6.0, 2.5 Hz, 1H), 3.26 (dd, J = 13.1, 2.5 Hz, 1H), 3.13 (dd, J = 13.1, 6.1 Hz, 1H), 1.97 (d, J = 1.9 Hz, 2H);13C NMR (126 MHz, CDCl3) δ 165.99, 159.54, 159.50, 129.85, 129.70, 129.69, 129.49, 113.99, 113.87, 100.12, 76.37, 75.67, 71.42, 71.14, 69.92, 65.59, 55.32, 55.29, 52.04, 14.07.

[0218] Scheme XXII, Compound 26: To a solution of compound 25 (670mg, 1.38 mmol) in 9 mL of tetrahydrofuran (THF) and 300 µL, of water, was added 250 µL of glacial acetic acid, and stirred for 36hrs at room temperature. TLC indicated the complete conversion of starting material and formation of polar band. The reaction mixture stopped and evaporated the solvents. The crude was resuspended in dichloromethane 100 mL, washed with 60mL of saturated aq. NaHCO3, separated organic layer dried over sodium sulfate, concentrated, and adsorbed on silica gel. The column eluted with ethylacetate / hexane (50 / 50, 70 / 30) and ethylacetate / chloroform (80 / 20). The pure fractions were pooled and concentrated, affording in 61% yield.

[0219] Scheme XXII, Compound 27: Compound 26 (315mg, 0.647 mmol) was dissolved in 7 mL of anhydrous dichloromethane (dry DCM) under inert atmosphere and cooled to 0º C. 5-ethylthio-1H-tetrazole (421mg, 3.23 mmol) and diNPE-phosphoramidate a (596mg, 1.29 mmol) in 3 mL of dry DCM were added and the reaction mixture was stirred for 2.5 hours at 0º C to 5º C. TLC indicated the starting material was consumed, the reaction mixture was cooled to -40 °C and meta-chloroperoxybenzoic acid (667mg, 1.94 mmol) was added. After 30 minutes, the reaction was quenched with saturated sodium bicarbonate (10 mL), diluted with DCM (20 mL) and extracted with DCM (100 mL). The organic layer was washed with bicarb (2×30 mL), and brine (1×20 mL), dried over sodium sulfate, and concentrated in vacuo. The crude residue was purified by column chromatography (70% ethyl acetate in hexanes) to give 27 (400mg) as a white foamy solid in a mixture of diastereomers in 72%

[0220] 1H NMR (400 MHz, DCCl3) δ 7.93 – 7.84 (m, 1H), 7.83 – 7.73 (m, 1H), 7.67 – 7.61 (m, 1H), 7.60 – 7.56 (m, 2H), 7.56 – 7.51 (m, 1H), 7.49 (dd, J = 8.0, 1.5 Hz, 1H), 7.45 – 7.39 (m, 1H), 7.40 – 7.32 (m, 1H), 7.25 –, 6.89 (dd, J = 8.6, 4.0 Hz, 4H), 6.03 – 5.94 (m, 1H), 5.89 (dd, J = 7.7, 6.2 Hz, 1H), 5.62 (dd, J = 5.2, 3.1 Hz, 1H), 5.60 – 5.55 (m, 1H), 5.33 – 5.19 (m, 1H), 4.92 – 4.67 (m, 3H), 4.64 – 4.46 (m, 2H), 4.27 – 4.09 (m, 1H), 3.86 (dd, J = 8.1, 4.7 Hz, 1H), 3.82 (s, 3H), 3.81 (s,3.81 (s, 3H), 3.79 (s, 3H), 3.74 – 3.67 (m, 1H), 3.67 – 3.62 (m, 1H), 3.61 – 3.56 (m, 1H), 3.51 (d, J = 2.6 Hz, 1H), 3.40 – 3.30 (m, 1H), 3.28 (dd, J = 8.6, 3.3 Hz, 1H), 1.88 (s, 3H), 1.78 (s, 3H), 1.67 (t, J = 6.7 Hz, 3H), 1.56 (d, J = 2.4 Hz, 3H), 1.55 (d, J = 2.4 Hz, 3H);31P NMR (162 MHz, cdcl3) δ -5.08, -5.69, -6.80.

[0221] Scheme XXII, Compound 28: To a solution of compound 27 (390mg, 0.451mmol) in 16 mL of DCM: water (10:1) was cool down to 0oC and then DDQ was added in two portions. The resulting dark brown reaction mixture stirred for 3.5hr at 0oC to 10oC. TLC indicated the complete conversion of starting material and formation of polar band. The reaction mixture quenched with saturated aq. NaHCO3(30 mL) at 0oC and extracted with DCM (120 mL), dried over anhydrous Na2SO4, concentrated, and adsorbed on silica gel and purified by silica gel flash column chromatography using EtOAc: Hex (50:50,75:25, and 100%), the desired product eluted with EtOAc: Acetone (95: 5%). The resulting

[0222] 1H NMR (500 MHz, CDCl3) δ 7.94 – 7.89 (m, 1H), 7.88 – 7.83 (m, 3H), 7.78 (m, 1H), 7.75 – 7.63 (m, 3H), 7.60 (m, 3H), 7.54 (m, 4H), 7.50 – 7.38 (m, 5H), 7.38 – 7.31 (m, 1H), 6.82 (dd, J = 8.0, 3.4 Hz, 1H), 6.60 (d, J = 8.1 Hz, 1H), diastereomeric anomeric proton coupling* 5.69 (dd, J = 6.2, 3.1 Hz, 0.5H*), 5.61 (dd, J = 6.2, 3.1, 5.56 (dd, J = 5.9, 3.2 Hz, 1H*), 5.47 (dd, J = 6.1, 3.1 Hz, 1H*), 4.00 – 3.93 (m, 1H), 3.91J = 10.9, 8.0, 3.0 Hz, 1H), 3.80 – 3.71 (m, 1H), 3.65 (m, 1H), 3.61 – 3.56 (m, 1H), 3.52 (m, 1H), 3.50 – 3.37 (m, 5H), 3.32 (m, 4H), 2.09 – 1.93 (s, 7H), 1.84 (s, 2H), 1.65 (s, 4H), 1.57 (d, J = 6.3 Hz, 3H), 1.56 – 1.42 (merged d, 10H);13C NMR (126 MHz, CDCl3) δ 172.61, 171.21, 147.07, 146.83, 146.59, 146.54, 146.51, 146.43, 137.01, 136.97, 134.27, 134.20, 134.13, 134.07, 129.35, 129.16, 129.10, 129.03, 129.00, 127.68, 127.65, 127.54, 127.42, 127.33, 127.31, 124.58, 124.55, 124.52, 124.48, 97.17, 97.09, 96.79, 96.74, 77.30, 77.05, 76.80, 60.41, 53.60, 51.18, 50.95, 24.34, 24.29, 24.18, 24.11, 24.07, 22.96, 22.80, 22.77, 21.04, 14.19;31P NMR (202 MHz, CDCl3) δ -5.24, -5.44, -5.55, -5.80.

[0223] Scheme XXIII: Synthesis of 6-caged GlcNAc derivatives

[0224] Figure 25 shows synthesis of 6-caged GlcNAc derivatives.

[0225] Scheme XXIII, 6-NBE-GlcNAc: N-acetyl-glucosamine (1048 mg, 4.74 mmol, 1 eq), Dibutyl tin (458 mg, 1.84 mmol, .389 eq) tertbutylamonium bromide(502 mg, 1.36 mmol, .287 eq), and 2-nitrobenzyl bromide (3948 mg, 18.3 mmol, 3.86 eq) was added into a round bottom flask under air. This was melted while stirring at 60 °C. This generated a dark yellow syrup. To this melted solution N,N-diisopropylethylamine (3.150 mL, 18.1 mmol, 3.8 eq) was added, and temperature was raised to 85°C. After 24hrs N-acetyl glucosamine was completely consumed. Reaction mixture was allowed to cool to room temperature before adding dichloromethane. Water was then used to extract product into the aqueous layer.Water was rinsed 3 times with dichloromethane. The aqueous layer was added directly on to reverse phase column. The column was rinsed with water followed by product elution using 3:1 water:methanol. Collected fractions were then frozen and speed vacuumed to obtain product. This gave 522 mg of 6-nitrobenzyl-N-acetyl-glucosamine (29% yield) as pale yellow white solid.1H NMR (500 MHz, MeOD) δ 7.95 (dd, J = 8.2, 1.3 Hz, 1H), 7.80 – 7.74 (m, 1H), 7.62 (td, J = 7.6, 1.3 Hz, 1H), 7.45 – 7.38 (m, 1H), 5.00 (d, J = 3.5 Hz, 1H), 4.88 – 4.78 (m, 2H), 3.87 (dt, J = 10.0, 3.7 Hz, 1H), 3.82 – 3.68 (m, 3H), 3.60 (dd, J = 10.7, 8.7 Hz, 1H), 3.38 – 3.23 (m, 1H), 1.88 (s, 3H).

[0226] Scheme XXIV: Synthesis of 6-cage-GlcNAkyne and 6-cage-GlcNAc-azide from glucosamine.

[0227] Figure 26 shows synthesis of 6-cage-GlcNAkyne and 6-cage-GlcNAc-azide from glucosamine according to Scheme XXIV.

[0228] Scheme XXIV, Compound 29 was synthesized according to the method described in Kakde, B. N, et al., Synthesis of Cell-Permeable N-Acetylhexosamine 1- Phosphates. The Journal of organic chemistry 2021, 86 (24), 18257–18264.

[0229] Scheme XXIV, Compound 30: To compound 29 (260mg, 1.0 mmol) in 25 mL round bottom flask, were added dibutyltin oxide (100mg, 0.40 mmol), tetrabutylammonium bromide (97mg, 0.30 mmol) and 2-nitro-benzylbromide (820mg, 3.8 mmol). The reaction mixture was heated at 60 °C for 2hr, then added diisopropylethylamine (661µL, 3.8 mmol) and continued heating at 85 °C for 16hr. Reaction mixture cooled down to room temperature and then extracted with 30 mL of CHCl3: MeOH (7:1) washed with water (20 mL). The organic layer dried over sodium sulfate, absorbed on silica gel and loaded on column. The column eluted with EtOAc / Hexane (70% / 30%) and the product isolated with EtOAc (100%). The pure fractions were pooled, concentrated resulting in a compound 30, white solid 15mg in 4.0%

[0230] 1H NMR (500 MHz, MeOD) δ 8.08 – 8.01 (m, 1H), 7.92 – 7.83 (m, 1H), 7.72 (td, J = 7.6, 1.3 Hz, 1H), 7.52 (td, J = 7.8, 1.5 Hz, 1H), 5.13 (d, J = 3.5 Hz, 1H), 4.95 (d, J = 2.5 Hz, 2H), 4.01 (ddd, J = 9.9, 4.2, 2.9 Hz, 1H), 3.90 (dd, J = 10.6, 3.5 Hz, 1H), 3.85 – 3.81 (m, 1H), 3.77 – 3.69 (m, 2H), 3.63 (q, J = 7.1 Hz, 0H), 3.46 (dd, J = 10.0, 8.8 Hz, 1H), 2.49 (d, J = 1.5 Hz, 3H).;13C NMR (126 MHz, MeOD) δ 172.96, 133.27, 133.23, 128.73, 127.86, 124.10, 91.27, 82.22, 78.13, 77.87, 77.61, 71.30, 71.16, 70.85, 70.28, 69.54, 68.78, 54.46, 48.11, 47.94, 47.89, 47.77, 47.71, 47.60, 47.43, 47.26, 47.09, 34.57, 14.23.

[0231] Scheme XXIV, compound 31: Compound 31 was synthesized according to the method described in Kakde et al., Synthesis of Cell-Permeable N-Acetylhexosamine 1- Phosphates. The Journal of organic chemistry 2021, 86 (24), 18257–18264, on different substrates.

[0232] Scheme XXIV, Compound 32: To the compound 31 (210mg, 0.826 mmol) in 25 mL round bottom flask, were added dibutyltin oxide (82mg, 0.33 mmol), tetrabutylammonium bromide (80mg, 0.247 mmol) and 2-nitro-benzylbromide (677mg, 3.13 mmol). The reaction mixture was heated at 60 °C for 2hr, then added diisopropylethylamine (545 µL, 3.13 mmol) and continued heating at 85 °C for 5hr. Reaction mixture cooled down to room temperature and then extracted with 30 mL of CHCl3: MeOH (7:1) washed with water (20 mL). The organic layer dried over sodium sulfate, absorbed on silica gel and loaded on column. The column eluted with EtOAc / Hexane (70% / 30%) and the product isolated with EtOAc (100%). The pure fractions were pooled, concentrated resulting in a compound 32 as white solid 20mg in 6.5% yield..8, .96 , J = 10.5, 3.6 Hz, 1H), 3.84 (dd, J = 3.6, 1.8 Hz, 2H), 3.74 (dd, J = 10.6, 8.8 Hz, 1H), 3.48 (dd, J = 10.0, 8.8 Hz, 1H).;13C NMR (126 MHz, MeOD) δ 168.97, 134.68, 133.22, 128.73, 127.87, 124.10, 91.13, 71.37, 71.02, 70.91, 70.22, 69.55, 54.48, 51.43.

[0234] Scheme XXV: Synthesis of GlcNAc-N-cage 36 from peracetylated glucosamine

[0235] Figure 27 shows synthesis of GlcNAc-N-cage compound 36 from peracetylated glucosamine according to Scheme XXV.

[0236] Scheme XXV, Compound 33: Peracetylated-glucosamine hydrochloride (1.60g, 4.16 mmol) in 100 mL round bottom flask, were added DIPEA (1.80mL, 10.4 mmol), and then 2-nitro-benzyl bromide (990mg, 4.58 mmol). The reaction mixture continued stirring for 72hr at room temperature. Reaction mixture diluted with 70 mL of ethyl acetate and quenched saturated sodium bicarbonate (30 mL) and washed with water (3×50 mL), dried over sodium sulfate, and concentrated and adsorbed on silica gel. The crude residue was purified by column chromatography (25% and 50% ethyl acetate in hexanes) to give 1.65g of compound 33 in 83% yield as a pale yellow solid.

[0237] 1H NMR (500 MHz, CDCl3) δ 7.97 (dd, J = 8.1, 1.3 Hz, 1H), 7.61 (td, J = 7.5, 1.3 Hz, 1H), 7.55 (d, J = 7.6 Hz,, 7.45 (ddd, J = 8.7, 7.3, 1.6 Hz, 1H), 5.63 (d, J = 8.6 Hz, 1H), 5.14 (t, J = 9.7 Hz, 1H), 5.06 (t, J = 9.6 Hz, 1H), 4.32 (dd, J = 12.4, 4.6 Hz, 1H), 4.20 – 3.99 (m, 3H), 3.81 (ddd, J = 10.1, 4.7, 2.1 Hz, 1H), 2.96 (dd, J = 10.3, 8.6 Hz, 1H), 2.17 (s, 3H), 2.09 (s, 3H), 2.08 (s, 3H), 2.04 (s, 3H);13C NMR (126 MHz, CDCl3) δ 170.86, 170.64, 169.63, 169.10, 149.00, 133.40, 131.65, 128.39, 124.89, 94.69, 73.53, 72.55, 68.37, 61.72, 60.46, 49.08, 21.04, 20.74, 20.73, 20.62.

[0238] Scheme XXV, Compound 34: Compound 33 (850mg, 1.76mmol) in 50 mL round bottom flask, was added 12 mL of pyridine: acetic anhydride (3:1) and stirred for 40hr at room temperature. TLC indicated the complete conversion of starting material, reaction mixture dropwise quenched with 20 mL of aqueous saturated. NaHCO3 (40 mL) solution and stirred for 5 minutes, until there are no effervescences and extracted with EtOAc (100 mL). The organic layer washed with 1M HCl (3 x 30 mL) and dried over sodium sulfate and concentrated and adsorbed on silica gel. The crude residue was purified by column chromatography (50% ethyl acetate in hexanes and 100% ethyl acetate) to give 847g of compound 34 in 91% yield as a white foamy solid.

[0239] 1H δ 8.10 (d, J = 8.2 Hz, 1H), 7.87 (dd, J = 8.2, 1.3 Hz, 1H), 7.57, = 7.8 Hz, 1H), 7.41 (td, J = 7.6, 1.4 Hz, 1H), 7.36 – 7.26 (m, 3H), 5.62 (d, J = 8.7 Hz, 1H), 5.46 (dd, J = 10.6, 8.8 Hz, 1H), 5.12 – 5.02 (m, 2H), 5.01 – 4.89 (m, 2H), 4.67 (d, J = 17.0 Hz, 1H), 4.26 (m, 2H), 4.15 – 4.07 (m, 2H), 3.98 (dt, J= 12.6, 2.6 Hz, 3H), 3.81 (ddd, J = 10.2, 4.3, 2.1 Hz, 1H), 2.31 (s, 4H), 2.00 (s, 4H), 1.98 (s, 3H), 1.97 (s, 3H), 1.96 (s, 7H), 1.93 (s, 3H), 1.91 (s, 4H), 1.87 (s, 4H).;13C NMR (126 MHz, CDCl3) δ 173.09, 172.63, 171.14, 170.59, 170.49, 169.75, 169.18, 167.65, 148.28, 132.82, 132.44, 129.64, 128.93, 128.38, 125.26, 90.32, 72.32, 69.93, 68.50, 61.56, 61.32, 61.20, 60.39, 40.46, 22.30, 21.06, 21.04, 20.69, 20.67, 20.62, 20.58, 20.49.

[0240] Scheme XXV, Compound 35: To a solution of compound 34 (840 mg, 1.60 mmol) in 4.5 mL of dry dimethyl formamide, was slowly added 115 µL of hydrazine hydrate (2.40mmol) stirred for 3 minutes at room temperature under argon atmosphere. Reaction mixture quenched with 500 µL of acetic acid and stirred for 3 minutes. Reaction mixture neutralized with 40 mL of aqueous saturated. NaHCO3solution and stirred for 5 minutes, until there are no effervescences and extracted with EtOAc (70 mL), dried over sodium sulfate. The organic layer concentrated and evaporated with toluene 3 mL x 2, and DCM (2 x 10 mL) and dried in vacuum, yielding white foamy solid 652 mg of hemiacetal 35 in 85% yield. The hemiacetal can be used for the next without further

[0241] 1H NMR (500 MHz, CDCl3) δ 8.17 (ddd, J = 8.2, 2.6, 1.3 Hz, 1H), 8.01 (ddd, J = 8.2, 4.0, 1.3 Hz, 0H), 7.69 (tdd, J = 7.8, 4.2, 1.3 Hz, 1H), 7.52 (td, J = 7.8, 1.5 Hz, 2H), 7.49 – 7.43 (m, 1H), 7.42 – 7.34 (m, 1H), 5.83 (s, 1H), 5.62= 11.0, 8.8 Hz, 1H), 5.36 – 5.27 (m, 1H), 5.21 – 5.08 (m, 2H), 4.98 – 4.80 (m, 1H), 4.36 – 4.22 (m, 2H), 4.23 – 4.17 (m, 1H), 4.10 – 4.00 (m, 1H), 3.77 (ddd, J = 10.2, 4.8, 2.3 Hz, 1H), 2.37 (s, 1H), 2.10 (s, 2H), 2.09 (s, 2H), 2.08 (s, 3H), 2.07 (s, 3H), 2.06 (s, 3H), 2.03 (s, 2H), 2.02 (s, 2H), 2.01 (s, 2H), 1.97 (s, 2H), 1.92 (126 MHz, CDCl3) δ 172.44, 171.22, 170.80, 170.74, 170.69,169.95, 169.85, 169.82, 169.65, 169.36, 169.32, 147.93, 147.64, 134.06, 133.86, 133.13, 132.97, 129.39, 128.78, 128.60, 127.90, 127.49, 127.29, 127.09, 125.87, 125.61, 124.94, 124.90, 93.74, 92.48, 71.66, 71.48, 70.30, 69.54, 69.44, 69.25, 68.60, 68.12, 67.41, 66.93, 63.28, 61.99, 60.42, 60.08, 22.38, 22.31, 21.04, 20.78, 20.75, 20.73, 20.64, 20.60.

[0242] Scheme XXV, Compound 36: Compound 34 (150 mg, 0.285 mmol) in 25 mL round bottom flask, was added 3 mL of dry methanol and then added potassium carbonate (60 mg, 0.42 mmol) at 0 °C and stirred for 2hr at same temperature. TLC indicated thecomplete conversion of starting material, reaction mixture quenched with 10 mL of methanol and filtered through short celite pad. Solvents were evaporated and adsorbed on silica gel. The crude residue was purified by column chromatography (100% ethyl acetate and 2.5% methanol yellow solid.

[0243] 1H NMR (500 MHz, MeOD) δ 8.12 (td, J = 8.2, 1.3 Hz, 1H), 8.01 (ddd, J = 9.3, 8.2, 1.4 Hz, 3H), 7.81 (dd, J = 8.0, 1.3 Hz, 2H), 7.76 – 7.69 (m, 1H), 7.65 (d, J = 17.0 Hz, 1H), 7.60 (ddd, J = 7.1, 5.6, 1.4 Hz, 2H), 7.58 – 7.50 (m, 1H), 7.49 – 7.44 (m, 3H), 7.43 – 7.38 (m, 2H), 5.25 (d, J = 3.0 Hz, 1H), 5.20 (d, J = 3.2 Hz, 1H), 5.17 – 5.07 (m, 2H), 4.99 – 4.91 (m, 2H), 4.78 (d, J = 7.9 Hz, 2H), 4.01 (dd, J = 10.6, 8.2 Hz, 1H), 3.98 – 3.92 (m, 2H), 3.87 (dd, J = 11.9, 2.3 Hz, 2H), 3.84 – 3.79 (m, 2H), 3.80 – 3.76 (m, 2H), 3.75 – 3.71 (m, 2H), 3.71 – 3.68 (m, 3H), 3.65 (dd, J = 10.4, 8.0 Hz, 2H), 3.49 (dd, J = 9.9, 8.2 Hz, 1H), 3.42 (dd, J = 9.8, 8.0 Hz, 2H), 3.22 (q, J = 7.3 Hz, 1H), 2.33 (s, 2H), 2.32 (s, 3H).;13C NMR (126 MHz, MeOD) δ 174.66, 174.31, 147.99, 133.28, 132.71, 132.59, 129.18, 128.05, 127.14, 126.55, 124.00, 123.94, 93.63, 92.04, 76.10, 72.05, 71.88, 71.64, 71.51, 68.92, 65.85, 61.30, 61.13, 53.40, 42.95, 21.03.

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[0289] Item List

[0290] Item 1. A composition, comprising: a controllably-releasable glycan precursor, the controllably-releasable glycan precursor having a photocage component or a chemocage component covalently bonded to a glycan precursor component.

[0291] Item 2. The composition of item 1, wherein the glycan precursor component is an N-acetyl sugar.

[0292] Item 3. The composition of item 1 or item 2, wherein the glycan precursor component is selected from the group consisting of: GlcNAc, GalNAc, ManNAc, and a derivative of any thereof.

[0293] Item 4. The composition of any of items 1, 2, or 3, wherein the photocage component or the chemocage component comprises: an o-nitrobenzyl, a p-hydroxyphenyl, a coumarinyl, a nitrodibenzofuranyl, a nitrophenylbenzofuran (NPDF), or a derivative of any thereof.

[0294] Item 5. The composition of any of items 1 to 4, wherein the photocage component comprises a member of the group consisting of: nitrophenylethanol (NPE), diethylaminocoumarin (DEAC), 2-nitrobenzodioxyethanol (NBDE), and a derivative thereof.

[0295] Item 6. The composition of any of items 1 to 5, wherein the controllably- releasable glycan precursor is a photo-releasable glycan precursor compound is selected from the group consisting of: compound 1a, compound 2a, compound 3a, compound 1b, compound 2b, compound 3b, compound 4, compound 5, compound 6, compound 7, and compound 8, or a derivative of any thereof.

[0296] Item 7. The composition of any of items 1 to 3, wherein the chemocage component comprises a member of the group consisting of: an aryl tetrazine, a trans- cyclooctene, or a derivative of any thereof.

[0297] Item 8. The composition of any of items 1 to 4, or 7, wherein the controllably- releasable glycan precursor is a bioorthogonal chemistry-releasable glycan precursor compound selected from the group consisting of: compound 28, compound 32, compound 35, compound 36, or a derivative of any thereof.

[0298] Item 9. The composition of item 1, wherein the controllably-releasable glycan precursor is 6-NBE-GlcNAc, or a derivative thereof.

[0299] Item 10. The composition of item 1, wherein the controllably-releasable glycan precursor is:where the glycan precursor component is selected from the group consisting of: GlcNAc, GalNAc, ManNAc, and a derivative of any thereof; and where R is aryl, alkyl, or H.

[0300] Item 11. The composition of item 1, wherein the controllably-releasable glycan precursor is:where the glycan precursor component is selected from the group consisting of: GlcNAc, GalNAc, ManNAc, and a derivative of any thereof.

[0301] Item 12. The composition of item 1, wherein the controllably-releasable glycan precursor is selected from the group consisting of:,where the cage is a photocage or a chemocage; where PG is an ester-based protecting group selected from the group consisting of: an acetyl ester, an ethyl ester, a propyl ester, a butyl ester, a pentyl ester, and a hexyl ester; where R is selected from the group consisting of: an azide, C2-C8 linear or cyclic alkene, and C2-C8 terminal or cyclic alkyne; and where R’ is selected from the group consisting of: H, C1-C6 ester, and a sugar phosphate in alpha or beta configuration.

[0302] Item 13. The composition of item 1, wherein the controllably-releasable glycan precursor is selected from the group consisting of:,where the cage is a photocage or a chemocage; where PG is an ester-based protecting group selected from the group consisting of: an acetyl ester, an ethyl ester, a propyl ester, a butyl ester, a pentyl ester, and a hexyl ester; where R is selected from the group consisting of: an azide, C2-C8 linear or cyclic alkene, and C2-C8 terminal or cyclic alkyne; and where R’ is selected from the group consisting of: H, C1-C6 ester, and a sugar phosphate in alpha or beta configuration.

[0303] Item 14. The composition of any of items 1 to 13, further comprising one or more of: a carrier, a buffer, and a salt.

[0304] Item 15. The composition of any of items 1 to 14, further comprising a pharmaceutically acceptable carrier.

[0305] Item 16. A method of controlled metabolic engineering in a cell, comprising: providing a controllably-releasable glycan precursor according to any of items 1 to 15; introducing the controllably-releasable glycan precursor into a cell; and exposing the controllably-releasable glycan precursor in the cell to a stimulus effective to release the glycan precursor from the photocage component or chemocage component, thereby providing the glycan precursor to the cell, and providing controlled metabolic engineering in the cell.

[0306] Item 17. The method of item 16, wherein the controllably-releasable glycan precursor comprises a photocage component covalently bonded to a glycan precursorcomponent and wherein the stimulus is light having a wavelength is in the range of about 200 nm to about 750 nm.

[0307] Item 18. The method of item 17, wherein the wavelength is in the range of about 350 nm to about 365 nm.

[0308] Item 19. The method of item 16, wherein the controllably-releasable glycan precursor comprises a chemocage component covalently bonded to a glycan precursor component and wherein the stimulus is exposure to a bioorthogonal chemical reaction effective to release the glycan precursor from the chemocage component.

[0309] Item 20. The method of any one of items 16 to 19, wherein the cell is in vitro, in vivo, or ex vivo.

[0310] Item 21. The method of any one of items 16 to 20 wherein the cell is a human cell.

[0311] Item 22. A composition, comprising: a bioorthogonal chemistry-releasable glycan precursor, the bioorthogonal chemistry-releasable glycan precursor having a chemocage component covalently bonded to a glycan precursor component substantially as shown or described herein.

[0312] Item 23. A composition, comprising: a photo-releasable glycan precursor, the photo-releasable glycan precursor having a photocage component covalently bonded to a glycan precursor component substantially as shown or described herein.

[0313] Item 24. A method of controlled metabolic engineering in a cell substantially as shown or described herein.

[0314] Any patents or publications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication is specifically and individually indicated to be incorporated by reference.

[0315] The compositions and methods described herein are presently representative of preferred embodiments, exemplary, and not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art. Such changes and other uses can be made without departing from the scope of the invention as set forth in the claims.

Claims

CLAIMS 1. A composition, comprising: a controllably-releasable glycan precursor, the controllably-releasable glycan precursor having a photocage component or a chemocage component covalently bonded to a glycan precursor component.

2. The composition of claim 1, wherein the glycan precursor component is an N- acetyl sugar.

3. The composition of claim 1 or claim 2, wherein the glycan precursor component is selected from the group consisting of: GlcNAc, GalNAc, ManNAc, and a derivative of any thereof.

4. The composition of any of claims 1, 2, or 3, wherein the photocage component or the chemocage component comprises: an o-nitrobenzyl, a p-hydroxyphenyl, a coumarinyl, a nitrodibenzofuranyl, a nitrophenylbenzofuran (NPDF), or a derivative of any thereof.

5. The composition of any of claims 1 to 4, wherein the photocage component comprises a member of the group consisting of: nitrophenylethanol (NPE), diethylaminocoumarin (DEAC), 2-nitrobenzodioxyethanol (NBDE), and a derivative thereof.

6. The composition of any of claims 1 to 5, wherein the controllably-releasable glycan precursor is a photo-releasable glycan precursor compound is selected from the group consisting of: compound 1a, compound 2a, compound 3a, compound 1b, compound 2b, compound 3b, compound 4, compound 5, compound 6, compound 7, and compound 8, or a derivative of any thereof.

7. The composition of any of claims 1 to 3, wherein the chemocage component comprises a member of the group consisting of: an aryl tetrazine, a trans-cyclooctene, or a derivative of any thereof.

8. The composition of any of claims 1 to 4, or 7, wherein the controllably- releasable glycan precursor is a bioorthogonal chemistry-releasable glycan precursorcompound selected from the group consisting of: compound 28, compound 32, compound 35, compound 36, or a derivative of any thereof.

9. The composition of claim 1, wherein the controllably-releasable glycan precursor is 6-NBE-GlcNAc, or a derivative thereof.

10. The composition of claim 1, wherein the controllably-releasable glycan precursor is:where the glycan precursor component is selected from the group consisting of: GlcNAc, GalNAc, ManNAc, and a derivative of any thereof; and where R is aryl, alkyl, or H.

11. The composition of claim 1, wherein the controllably-releasable glycan precursor is:where the glycan precursor component is selected from the group consisting of: GlcNAc, GalNAc, ManNAc, and a derivative of any thereof.

12. The composition of claim 1, wherein the controllably-releasable glycan precursor is selected from the group consisting of:where the cage is a photocage or a chemocage; where PG is an ester-based protecting group selected from the group consisting of: an acetyl ester, an ethyl ester, a propyl ester, a butyl ester, a pentyl ester, and a hexyl ester; where R is selected from the group consisting of: an azide, C2-C8 linear or cyclic alkene, and C2-C8 terminal or cyclic alkyne; and where R’ is selected from the group consisting of: H, C1-C6 ester, and a sugar phosphate in alpha or beta configuration.

13. The composition of claim 1, wherein the controllably-releasable glycan precursor is selected from the group consisting of:where the cage is a photocage or a chemocage; where PG is an ester-based protecting group selected from the group consisting of: an acetyl ester, an ethyl ester, a propyl ester, a butyl ester, a pentyl ester, and a hexyl ester; where R is selected from the group consisting of: an azide, C2-C8 linear or cyclic alkene, and C2-C8 terminal or cyclic alkyne; and where R’ is selected from the group consisting of: H, C1-C6 ester, and a sugar phosphate in alpha or beta configuration.

14. The composition of any of claims 1 to 13, further comprising one or more of: a carrier, a buffer, and a salt.

15. The composition of any of claims 1 to 14, further comprising a pharmaceutically acceptable carrier.

16. A method of controlled metabolic engineering in a cell, comprising: providing a controllably-releasable glycan precursor according to any of claims 1 to 15; introducing the controllably-releasable glycan precursor into a cell; and exposing the controllably-releasable glycan precursor in the cell to a stimulus effective to release the glycan precursor from the photocage component or chemocage component, thereby providing the glycan precursor to the cell, and providing controlled metabolic engineering in the cell.

17. The method of claim 16, wherein the controllably-releasable glycan precursor comprises a photocage component covalently bonded to a glycan precursor component and wherein the stimulus is light having a wavelength is in the range of about 200 nm to about 750 nm.

18. The method of claim 17, wherein the wavelength is in the range of about 350 nm to about 365 nm.

19. The method of claim 16, wherein the controllably-releasable glycan precursor comprises a chemocage component covalently bonded to a glycan precursor component and wherein the stimulus is exposure to a bioorthogonal chemical reaction effective to release the glycan precursor from the chemocage component.

20. The method of any one of claims 16 to 19, wherein the cell is in vitro, in vivo, or ex vivo.

21. The method of any one of claims 16 to 20 wherein the cell is a human cell.

22. A composition, comprising: a bioorthogonal chemistry-releasable glycan precursor, the bioorthogonal chemistry-releasable glycan precursor having a chemocage component covalently bonded to a glycan precursor component substantially as shown or described herein.

23. A composition, comprising: a photo-releasable glycan precursor, the photo- releasable glycan precursor having a photocage component covalently bonded to a glycan precursor component substantially as shown or described herein.

24. A method of controlled metabolic engineering in a cell substantially as shown or described herein.

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