Low immunogenic biotherapeutics

Abstract

The present disclosure provides hypoimmunogenic biotherapeutic compositions that suppress the development of a self-directed immune response in an individual. The disclosure also provides pharmaceutical compositions comprising such hypoimmunogenic biotherapeutics, methods for making such hypoimmunogenic biotherapeutics, and methods for using such hypoimmunogenic biotherapeutics as therapeutics and in research.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 136,128, filed January 11, 2021, which is incorporated by reference in its entirety.

[0002] The present invention relates to biotherapeutics that suppress a patient's own immune response. [Background technology]

[0003] Therapeutic protein and gene therapy are novel and successful drug modalities for the treatment of diseases. However, a patient's immune response to such therapeutics often results in inhibition of drug activity, accelerated drug clearance, reduced drug safety, and loss of drug efficacy. Preventing the formation of neutralizing and non-neutralizing drug-specific antibodies ("anti-drug antibodies" or "ADA") is an important unsolved problem in the field of biotherapy. Blocking the ADA response to biotherapeutics improves drug exposure, improves durability of efficacy, reduces ADA-associated toxicity, and enables favorable pharmacology for otherwise untreatable modalities (e.g., newly designed drugs, drugs based on endogenous proteins). The present invention addresses these issues. Summary of the Invention

[0004] The present disclosure provides hypoimmunogenic biotherapeutic compositions that suppress the development of a self-directed immune response in an individual. The disclosure also provides pharmaceutical compositions comprising such hypoimmunogenic biotherapeutics, methods for making such hypoimmunogenic biotherapeutics, and methods for using such hypoimmunogenic biotherapeutics as therapeutics and in research. [Brief explanation of the drawings]

[0005] [Figure 1]1 shows an embodiment of a model for biotherapeutics that engage CD22 with suppressed anti-drug antibody responses. B cell receptor-Siglec ligand coengagers (including drug-Siglec ligand conjugates) suppress or silence drug-specific B cell activation by physically recruiting the inhibitory CD22 receptor to the B cell receptor complex. [Figure 2] Another embodiment of a model for a biotherapeutic drug that engages CD22 with suppressed anti-drug antibody responses is shown: B cell receptor-Siglec ligand coengagers (including drug-Siglec ligand conjugates) suppress, silence, or delete only drug-specific B cells, while leaving those B cell clones that are not drug-specific intact. [Figure 3] Several formats of Siglec-B cell receptor co-engaging biologics with reduced immunogenicity are presented. Format 1. Siglec ligand-modified proteins: non-enzymatic or enzymatic conjugation, site-specific or non-site-specific. Format 2. Siglec ligand-modified glycans (intracellular or in vitro): biosynthetic, enzymatic glycan modification (e.g., during protein expression). Format 3. Siglec ligand-modified glycans (in vitro): post-expression, in vitro enzymatic glycan modification. Format 4. Protein / peptide fusion: CD22-binding domains or peptides are incorporated into the biologic by in-frame fusion or conjugation. [Figure 4] 1 shows an example of a conjugable CD22-binding Siglec ligand-linker structure highlighting the structural components: Siglec ligand binding moiety, Siglec ligand proximal linker structure, linker, and reactive / conjugation group. [Figure 5] Exemplary Siglec ligand structures are shown, highlighting elements that determine Siglec-2 binding affinity and species specificity. [Figure 6] 1 shows exemplary Siglec ligand structures, with structures varying with Siglec ligand valency. [Figure 7] 1 shows exemplary Siglec ligand structures showing varying linker structures, where the region adjacent to the sialic acid-based moiety consists of either a PEG-based structure or a galactose-based structure. [Figure 8] Examples of conjugated linker structures enhanced (top) or not (center) for Siglec-2 binding, as well as a negative control linker structure that does not bind Siglec-2 (bottom) are shown. Shown are an enhanced Siglec-ligand linker structure (compound no. 26288, Siglec ligand: methyl-α-9-N-(biphenyl-4-carbonyl)-amino-9-deoxy-N-glycolylneuraminic acid) (top), a Siglec ligand-linker structure containing a non-enhanced Siglec-2 binding moiety (compound no. 26614, Siglec ligand: N-glycolylneuraminic acid / Neu5Gc) (center), and a PEG-based non-Siglec-binding conjugated linker structure (compound no. 26530) (bottom). [Figure 9A-9B] Figure 9 shows exemplary purity and physicochemical characterization data for adalimumab hIgG1-Siglec ligand conjugates. Adalimumab conjugates vary in the structure of the Siglec-linker used and the ligand / linker-drug ratio ("LDR") after conjugation. Figure 9A shows capillary gel electrophoresis data for adalimumab conjugates. Figure 9B shows analytical size-exclusion chromatography profiles for adalimumab IgG and adalimumab conjugates. [Figures 10A-10B] Figure 10 shows an in vitro B cell activation assay in which mouse primary B cells are treated with either a B cell receptor-agonizing anti-IgD antibody or an anti-IgD-Siglec ligand conjugate. The B cell stimulatory activity of anti-IgD and anti-IgD-Siglec ligand conjugates is compared in a dose titration experiment with the activation readout of CD69 upregulation. CD69 levels in different treatment groups are assessed via the percentage of cells that are CD69 positive (Figure 10A) or CD69 mean fluorescence intensity (MFI) (Figure 10B). The Siglec ligands in the conjugates tested vary in linker structure ("PEG" or "Gal") and valency ("monovalent" or "bivalent"); the conjugates also vary in ligand / linker-to-drug ratio ("LDR"), or the average number of Siglec ligand structures per drug molecule. [Figures 11A-11B]Figure 11 shows an in vitro B cell activation assay in which mouse primary B cells are treated with either a B cell receptor-agonizing anti-IgD antibody or an anti-IgD-Siglec ligand conjugate. The anti-IgD antibody-Siglec ligand conjugate has a trivalent Siglec ligand structure. B cell activation is measured by upregulation of CD69 expression, as measured by cytometry. CD69 levels in different treatment groups are assessed through the percentage of cells that are CD69-positive (Figure 11A) or CD69 mean fluorescence intensity (MFI) (Figure 11B). The Siglec ligand in the conjugates tested varies in linker structure ("PEG" or "Gal"), and the conjugates also vary in ligand / linker-to-drug ratio ("LDR") or the average number of Siglec ligand structures per drug molecule. [Figures 12A-12B] Figure 12 shows an in vitro B cell activation assay in which mouse primary B cells were treated with either a B cell receptor-agonizing anti-IgD antibody or an anti-IgD-Siglec ligand conjugate. The B cell stimulatory activity of anti-IgD and anti-IgD-Siglec ligand conjugates is compared in a dose titration experiment with the activation readout of CD69 upregulation. CD69 levels in different treatment groups are assessed via the percentage of cells that are CD69-positive (Figure 12A) or CD69 mean fluorescence intensity (MFI) (Figure 12B). The Siglec ligands in the conjugates tested contain a PEG-based linker structure ("PEG") and vary in valency ("monovalent," "bivalent," and "trivalent"). The conjugates also vary in ligand / linker-to-drug ratio ("LDR"), or the average number of Siglec ligand structures per drug molecule. [Figures 13A-13B]Figure 13 shows an evaluation of Siglec ligand-anti-IgD antibody conjugate binding to mouse primary B cells compared to the parent unconjugated anti-IgD antibody. Binding was assessed by fluorescence cytometry in a competitive assay format with Alexa-647-labeled anti-IgD antibody. Figure 13A shows dose-response results for the concentration-dependent inhibition of fluorescently labeled anti-IgD binding to IgD+ B cells by unlabeled anti-IgD-Siglec ligand conjugates and unconjugated, unlabeled anti-IgD antibody. The binding IC50 is shown in nanomolar for each unlabeled test sample. Figure 13B is a schematic diagram of the binding assay system. [Figure 14] This figure shows an in vitro B cell activation assay in which mouse primary B cells were treated with either a B cell receptor-agonizing anti-IgD antibody or an anti-IgD-Siglec ligand conjugate, and the same test sample was used to treat B cells from wild-type mice. The conjugated Siglec ligands (monovalent PEG-LDR9, bivalent PEG-LDR6, trivalent PEG-LDR6, and trivalent PEG-LDR8) are enhanced due to Siglec-2 binding. The B cell stimulatory activity of anti-IgD and anti-IgD-Siglec ligand conjugates was compared with the activation readout of CD69 upregulation in a dose titration experiment. CD69 levels in different treatment groups were assessed through the percentage of CD69-positive cells. The Siglec ligands in the tested conjugates contained a PEG-based linker structure ("PEG") and differed in valency (monovalent, bivalent, and trivalent). The conjugates also vary in their ligand / linker to drug ratio ("LDR"), or the average number of Siglec ligand structures per drug molecule. [Figure 15]Figure 1 shows an evaluation of Siglec ligand-anti-IgD antibody conjugate binding to mouse primary B cells compared to the parent unconjugated anti-IgD antibody. Binding is assessed by fluorescence cytometry in a competition assay format with Alexa-647-labeled anti-IgD antibody. Figure 1 shows dose-response results for the concentration-dependent inhibition of fluorescently labeled anti-IgD binding to IgD+ B cells by unlabeled anti-IgD-Siglec ligand conjugate and unconjugated unlabeled anti-IgD antibody. The binding IC50 is shown in nanomolar for each unlabeled test sample. The test samples are the same as in Figure 15. [Figures 16A-16B] Figure 16 shows an in vitro B cell activation assay in which mouse primary B cells were treated with either a B cell receptor-agonizing anti-IgD antibody or an anti-IgD-Siglec ligand conjugate, where the Siglec ligand was either enhanced for Siglec-2 binding ("BPC-Neu5Gc monovalent PEG-LDR9" and "BPC-Neu5Gc bivalent PEG-LDR6") or not ("Neu5Gc monovalent PEG-LDR10" and "Neu5Gc bivalent PEG-LDR7"). The B cell stimulatory activity of anti-IgD and anti-IgD-Siglec ligand conjugates was compared with the activation readout of CD69 upregulation in a dose titration experiment. CD69 levels in different treatment groups were assessed via the percentage of CD69-positive cells. Dose-response curves for monovalent (Figure 16A) and bivalent (Figure 16B) Siglec ligand conjugates are shown. The conjugates also vary in their ligand / linker to drug ratio ("LDR"), or the average number of Siglec ligand structures per drug molecule. [Figures 17A-17C]This figure shows an in vitro primary mouse B cell activation assay examining the importance of CD22 engagement for Siglec ligand-conjugate-mediated inhibition of B cell receptor activation. Mouse primary B cells were treated with either a B cell receptor-agonizing anti-IgD antibody or various anti-IgD-Siglec ligand conjugates, which either had enhanced Siglec linkers ("BPC-Neu5Gc monovalent PEG-LDR9," "BPC-Neu5Gc bivalent PEG-LDR6," "BPC-Neu5Gc trivalent PEG-LDR6," and "BPC-Neu5Gc trivalent PEG-LDR8") or asialo linkers lacking Siglec binding determinants ("Asialo monovalent PEG-LDR7," "Asialo bivalent PEG-LDR8," and "Asialo trivalent PEG-LDR7"). The B cell stimulatory activity of anti-IgD and anti-IgD-Siglec ligand conjugates is compared with the activation readout of CD69 upregulation in a dose titration experiment. CD69 levels in different treatment groups are assessed through the percentage of CD69-positive cells. The Siglec ligands in the conjugates tested contain a PEG-based linker structure ("PEG") and vary in valency ("monovalent" (Figure 17A), "bivalent" (Figure 17B), and "trivalent" (Figure 17C). The conjugates also vary in their ligand / linker-to-drug ratio ("LDR"), or the average number of Siglec ligand structures per drug molecule. [Figures 18A-18C]Figure 1 shows an evaluation of Siglec ligand-anti-IgD antibody conjugate binding to B cells compared to parental anti-IgD antibodies. Anti-IgD-Siglec ligand conjugates have either enhanced Siglec linkers ("BPC-Neu5Gc monovalent PEG-LDR9," "BPC-Neu5Gc bivalent PEG-LDR6," "BPC-Neu5Gc trivalent PEG-LDR6," and "BPC-Neu5Gc trivalent PEG-LDR8") or asialo linkers lacking Siglec binding determinants ("Asialo monovalent PEG-LDR7," "Asialo bivalent PEG-LDR8," and "Asialo trivalent PEG-LDR7"). Binding was assessed by fluorescence cytometry in a competition assay format with Alexa-647-labeled anti-IgD antibodies. Figure 18 shows dose-response results for the concentration-dependent inhibition of fluorescently labeled anti-IgD binding to IgD+ B cells by unlabeled anti-IgD-Siglec ligand conjugates and unconjugated, unlabeled anti-IgD antibodies. The test samples were the same as those used in Figure 17. The Siglec ligands in the conjugates tested contained a PEG-based linker structure ("PEG") and differed in valency ("monovalent" (Figure 18A), "bivalent" (Figure 18B), and "trivalent" (Figure 18C). The conjugates also varied in their ligand / linker-to-drug ratio ("LDR"), or the average number of Siglec ligand structures per drug molecule. [Figures 19A-19B] Figure 19A shows an in vitro primary mouse B cell activation assay examining the importance of cis-B cell receptor and CD22 co-engagement for the inhibition of B cell receptor activation. Figure 19A shows a model for B cell receptor activation in which an anti-IgD BCR agonist and a Siglec-2-engaging moiety are displayed on the same or separate molecules. The B cell stimulatory activity of anti-IgD, an anti-IgD-Siglec ligand conjugate, or a mixture of 2 nM anti-IgD and various concentrations of a control antibody-Siglec ligand conjugate is compared in a dose titration experiment using CD69 upregulation as an activation readout. CD69 levels in different treatment groups are assessed via CD69 mean fluorescence intensity (MFI) (Figure 19B). The Siglec ligand in the tested conjugates contains a PEG-based linker structure ("PEG") and has a bivalent Siglec ligand structure. [Figure 20] This figure shows an in vitro primary mouse B cell activation assay for the inhibition of BCR agonism in a mixture of an agonist anti-IgD antibody and a nonagonist Siglec ligand anti-IgD conjugate. The Siglec ligand-anti-IgD conjugate is titrated in the presence or absence of 2 nM anti-IgD BCR agonist. The B cell stimulatory activity of anti-IgD, anti-IgD-Siglec ligand conjugate, or a mixture of 2 nM anti-IgD and various concentrations of anti-IgD-Siglec ligand conjugate is compared in a dose titration experiment, with CD69 upregulation as the activation readout. CD69 levels in different treatment groups are assessed via CD69 mean fluorescence intensity (MFI). The anti-IgD conjugate tested has a bivalent Siglec-2 potentiating ligand (MPB-Neu5Ac) and a PEG-based linker, with a ligand / linker-to-drug ratio ("LDR") of 6. [Figures 21A-21B] Figure 21 shows an in vitro B cell activation assay using human primary B cells and either a B cell receptor-agonizing anti-IgM antibody or a Siglec ligand conjugate with the same anti-IgM antibody. The B cell stimulatory activity of anti-IgM and anti-IgM-Siglec ligand conjugates is compared with the activation readout of CD69 upregulation in a dose titration experiment. CD69 levels in different treatment groups are assessed via the percentage of CD69-positive cells. Results are shown using conjugates using a galactose-based linker (Figure 21A) and a PEG-based linker (Figure 21B). All conjugates contain Siglec-2-potentiated MPB-Neu5Ac structures with various valencies and ligand / linker-drug ratios ("LDRs"), or an average number of Siglec ligand structures per drug molecule. [Figures 22A-22B]This shows an in vitro B cell activation assay using human primary B cells and either a B cell receptor-agonizing anti-IgM antibody or a Siglec ligand conjugate with the same anti-IgM antibody. The conjugates have Siglec ligand structures that are either enhanced for CD22 binding ("BPC-Neu5Gc monovalent PEG-LDR5-αIgM" and "BPC-Neu5Gc bivalent PEG-LDR9-αIgM") or non-enhanced ("Neu5Gc monovalent PEG-LDR6-αIgM" and "Neu5Gc bivalent PEG-LDR6-αIgM"). The B cell stimulatory activity of the anti-IgM and anti-IgM-Siglec ligand conjugates is compared with the activation readout of CD69 upregulation in a dose titration experiment. CD69 levels in different treatment groups are assessed through the percentage of CD69-positive cells. The Siglec ligands in the conjugates tested contain a PEG-based linker structure ("PEG") and differ in valency ("monovalent" (Figure 22A) or "bivalent" (Figure 22B)). [Figures 23A-23B] Figure 1 shows the evaluation of Siglec ligand-anti-IgM antibody conjugate and parent anti-IgM binding to human B cells. Anti-IgM-Siglec ligands were either enhanced for CD22 binding ("BPC-Neu5Gc monovalent PEG-LDR5-αIgM" and "BPC-Neu5Gc bivalent PEG-LDR9-αIgM") or non-enhanced ("Neu5Gc monovalent PEG-LDR6-αIgM" and "Neu5Gc bivalent PEG-LDR6-αIgM"). Binding was assessed by fluorescence cytometry in a competition assay format with Alexa-647-labeled anti-IgM antibody. Figure 1 shows dose-response results for the concentration-dependent inhibition of fluorescently labeled anti-IgM binding to IgM+ B cells by unlabeled anti-IgM-Siglec ligand conjugates and unconjugated, unlabeled anti-IgM antibody. The binding IC50 for each unlabeled test sample is shown in nanomolar. The test sample was the same as that used in the experiment of FIG. [Figures 24A-24C]Figure 24 shows the evaluation of anti-drug antibody responses in mice for adalimumab and Siglec ligand-adalimumab conjugates. The conjugates have a BPC-Neu5Gc-based Siglec ligand-linker structure that is either monovalent, bivalent, or trivalent to the Siglec ligand with a galactose-containing linker structure. Mice were administered a single 4 mg / kg intravenous dose of adalimumab or adalimumab-Siglec ligand conjugate. Serum IgG levels against adalimumab / adalimumab conjugates were measured on days 14, 21, and 28. Figures 24A, 24B, and 241C show individual animal serum IgG titers (n=5 mice) on days 14, 21, and 28, respectively. [Figures 25A-25C] Figure 25 shows the evaluation of anti-drug antibody responses in mice for adalimumab and Siglec ligand-adalimumab conjugates. The conjugates have a BPC-Neu5Gc-based Siglec ligand-linker structure that is either monovalent, bivalent, or trivalent to the Siglec ligand with a PEG-containing linker structure. Mice were administered a single 4 mg / kg intravenous dose of adalimumab or adalimumab-Siglec ligand conjugate. Serum IgG levels against adalimumab / adalimumab conjugates were measured on days 14, 21, and 28. Figures 25A, 25B, and 25C show individual animal serum IgG titers (n=5 mice) on days 14, 21, and 28, respectively. [Figures 26A-26B] Figure 26 shows serum pharmacokinetic analysis of adalimumab hIgG and Siglec ligand-adalimumab hIgG1 conjugates in mice. The conjugates have BPC-Neu5Gc-based Siglec ligand-linker structures that are either monovalent, bivalent, or trivalent to the Siglec ligand with a PEG-containing linker structure. Test samples were the same as those used in Figure 25. After intravenous administration, serum samples were analyzed for test sample concentration by anti-human IgG Fc ELISA (Figure 26A) and SPR (surface plasmon resonance, Figure 26B). [Figures 27A-27G]Figure 27A shows in vitro surface plasmon resonance (SPR) analysis of the TNFα binding activity of adalimumab hIgG and Siglec ligand-adalimumab hIgG1 conjugates. Figure 27B shows a schematic of the SPR assay setup with TNFα analyte binding to Protein A chip-immobilized adalimumab or adalimumab-Siglec ligand conjugates. The TNFα concentration is varied to assess concentration dependence, binding kinetics, and affinity. [Figure 27B] The concentration dependence of binding at the end of the association phase (RUmax) is shown in Figures 27C-G. Individual sensorgrams of adalimumab (Figure 27C), adalimumab BPC-Neu5Gc monovalent GAL LDR4 (Figure 27D), adalimumab BPC-Neu5Gc monovalent GAL LDR7 (Figure 27E), adalimumab BPC-Neu5Gc bivalent GAL LDR8.5 (Figure 27F), and adalimumab BPC-Neu5Gc trivalent GAL LDR5 (Figure 27G). [Figures 28A-28B] Figure 28 shows the evaluation of anti-drug antibody responses in mice for 1) adalimumab, 2) a Siglec ligand-adalimumab conjugate ("BPC-Neu5Gc monovalent PEG LDR10"), and 3) negative control, non-Siglec-2 binding linker conjugates ("Neg Ctrl monovalent PEG LDR7," "Neg Ctrl bivalent PEG LDR7," and "Neg Ctrl trivalent PEG LDR6"). Mice were administered a single 4 mg / kg intravenous dose of adalimumab, adalimumab-Siglec ligand conjugate, or adalimumab negative control linker conjugate. Serum IgG levels against adalimumab / adalimumab conjugates were measured on days 14 and 28. Figures 28A and 28B show individual animal serum IgG titers (n=5 mice) on days 14 and 28, respectively. [Figure 29]

[0023] Figure 1 shows the evaluation of anti-drug antibody responses in mice for 1) adalimumab, 2) an enhanced Siglec ligand-adalimumab conjugate ("BPC-Neu5Gc monovalent PEG LDR7"), and 3) non-enhanced Siglec ligand-adalimumab conjugates ("Neu5Gc monovalent PEG LDR6", "Neu5Gc bivalent PEG LDR5", and "Neu5Gc trivalent PEG LDR5"). Mice received a single 4 mg / kg intravenous dose of adalimumab IgG or conjugate. Serum IgG levels against adalimumab / adalimumab conjugates were measured on day 28. Individual animal serum IgG titers (n=5 mice) on day 28 are shown. [Figures 30A-30E] Figure 30A shows the evaluation of IgG immune responses in mice administered different combinations of 4 mg / kg adalimumab, adalimumab-Siglec ligand conjugate (adalimumab-BPC-Neu5Gc bivalent PEG LDR10), or PBS vehicle (day 0), followed by weekly administration of 200 μg hen egg white lysozyme (HEL) (days 7, 14, 21, and 28). The study plan is shown in Figure 30A. Mouse serum IgG titers were measured separately against adalimumab / adalimumab-Siglec ligand conjugate and HEL. Serum IgG levels on day 28 of anti-adalimumab / adalimumab conjugate (Figure 30B) and HEL (Figure 30D) are shown. Titers of anti-adalimumab / adalimumab conjugate (Figure 30C) and HEL (Figure 30E) are shown. [Figures 31A-31C] Figure 31 shows the evaluation of anti-drug antibody responses in mice for HEL and monovalent Siglec ligand-HEL conjugates (HEL-BPC-Neu5Gc monovalent PEG LDR1.6). The study plan is shown in Figure 31A. Mice were intravenously administered 200 µg of HEL or HEL conjugates four times weekly (days 0, 7, 14, and 21). Serum IgG levels against HEL / HEL conjugates were measured on day 27. Figure 31B shows a dilution series of IgG levels from serum samples. Figure 31C shows serum IgG titers of individual animals on day 27 (n = 5 mice). [Figure 32]Evaluation of anti-drug antibody responses in mice for recombinant asparaginase enzyme and asparaginase-Siglec ligand conjugates ("Asn'ase BPC-Neu5Gc monovalent PEG-LDR10" and "Asn'ase BPC-Neu5Gc trivalent PEG-LDR3.5") is shown. 15 μg of asparaginase or asparaginase conjugate was administered weekly (4 times) to BALB / c mice (n=5 per group). Anti-asparaginase IgG titers were measured by ELISA assay on day 28. Titers are shown for each test sample. DETAILED DESCRIPTION OF THE INVENTION

[0006] Hypoimmunogenic biotherapeutic compositions are provided that suppress the development of an autoimmune response in an individual. Also provided are pharmaceutical compositions comprising such hypoimmunogenic biotherapeutics, methods for making such hypoimmunogenic biotherapeutics, and methods for using such hypoimmunogenic biotherapeutics as therapeutics and in research. Such hypoimmunogenic biotherapeutics find particular use in the treatment of diseases that require repeated or chronic administration of the biotherapeutic to be effective. These and other objects, advantages, and features of the present invention will become more apparent to those skilled in the art upon reading the details of the compositions and methods as more fully described below.

[0007] Before the present methods and compositions are described in more detail, it is to be understood that this invention is not limited to the particular methods or compositions described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0008] Where a range of values ​​is provided, unless the context dictates otherwise, it is understood that each intervening value between the upper and lower limit of that range is also specifically disclosed, to the tenth of the unit of the lower limit. Each smaller range between any stated value or intervening value within a stated range and any other stated value or intervening value within that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded, and each range in which either, neither, or both limits are included in the smaller range is also encompassed within the invention, subject to any specifically excluded limits in the stated range. Where a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included within the invention.

[0009] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some potential and preferred methods and materials are described herein. All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials related to the cited publications. In case of conflict, it should be understood that the present disclosure supersedes any disclosure of the incorporated publication.

[0010] As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has distinct components and features which may be readily separated from or combined with the features of any of the other various embodiments without departing from the scope or spirit of the invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

[0011] It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to a "cell" includes a plurality of such cells, and a reference to a "peptide" includes a reference to one or more peptides and equivalents thereof known to those skilled in the art, e.g., polypeptides, etc.

[0012] The transitional term "comprising" is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. The transitional phrase "consisting of" excludes any unspecified element, step, or ingredient. The transitional phrase "consisting essentially of" limits the scope of the specified element to materials or steps and those that do not materially affect the basic and novel characteristics of the invention.

[0013] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0014] composition Disclosed herein are engineered biotherapeutics with reduced immunogenicity, methods for making such biotherapeutics, and methods for their use. As used herein, "biotherapeutic" refers to a composition composed of a combination of sugars, amino acids, proteins, lipids, or nucleic acids, or complexes of these substances, that is therapeutic in an individual. Non-limiting examples of biotherapeutics include protein therapeutics, e.g., antibody therapeutics, fusion protein therapeutics, enzyme therapeutics, viral therapeutics, cellular therapeutics, and nucleic acid therapeutics. "Engineered" biotherapeutics refers to biotherapeutics that have not been so engineered, i.e., biotherapeutics that have been designed and constructed to include one or more modifications relative to a parent biotherapeutic, also referred to herein as a non-engineered biotherapeutic. A "low immunogenic" composition refers to a composition that, when administered to an individual, suppresses an undesired drug-specific immune response in the individual compared to a reference composition, e.g., a corresponding non-genetically modified composition, e.g., a reduction in the immune response by 50% or more, and in some cases by 60%, 70%, 80% or more, e.g., 85%, 90%, 95% or more, and in certain cases, 98%, 99%, or 100%, compared to the reference, e.g., non-genetically modified biotherapeutic, i.e., such that the immune response is undetectable, i.e., the biotherapeutic is non-immunogenic. Thus, disclosed herein are engineered biotherapeutics that contain one or more modifications that are capable of suppressing a drug-specific immune response in an administered individual compared to the unmodified biotherapeutic, while retaining pharmacological activity. In some embodiments, the immune response is a humoral immune response, i.e., a B cell-driven response, e.g., an IgG response.

[0015] Disclosed herein are engineered, less immunogenic biotherapeutics, including biotherapeutics that have been engineered to contain a modified sialic acid-binding immunoglobulin-type lectin (Siglec) ligand profile compared to a corresponding non-engineered biotherapeutic, while retaining therapeutic activity.

[0016] "Siglec" refers to a member of a family of proteins found primarily on the surface of white blood cells that bind to sialic acid on target biologics. "Siglec ligand profile" refers to the amount and / or location of Siglec ligands covalently attached to a biotherapeutic. Often, the modification is an increase in the amount and / or location of Siglec ligands covalently attached to the biotherapeutic, which decreases the immunogenicity of the biotherapeutic in an individual compared to the corresponding unmodified biotherapeutic.

[0017] There are 14 mammalian Siglecs, which are expressed on different types of white blood cells and can exert inhibitory or activating effects depending on whether the cells in which they are expressed contain inhibitory or activating motifs. Siglecs exhibit different binding preferences for different sialic acids, and the type of linkage and base sugar also influence sialic acid recognition. (Varki, A. and Crocker, PR (2009) I-type lectins. In Essentials of Glycobiology (2nd edn) (Varki, A. et al., eds), pp. 459-474, Cold Spring Harbor Laboratory Press; Crocker, PR et al. (2007) Nat. Rev. Immunol. 7, 255-266) Together, this provides an array of alternative Siglec ligands that can be deployed to modulate immune responses to biotherapeutics. Of particular interest is the suppression of immune responses to biotherapeutics, more specifically, B cell responses to biotherapeutics. Thus, in some embodiments, the Siglec ligand is a ligand of a Siglec expressed on B lymphocytes, e.g., Siglec-2 (also known as CD22), Siglec-5 (CD170), Siglec-6, Siglec-9 (CD329), or Siglec-10 (Siglec-G). In some embodiments, the Siglec is Siglec-2. In some embodiments, the Siglec is Siglec-5. In some embodiments, the Siglec is Siglec-6. In some embodiments, the Siglec is Siglec-9. In some embodiments, the Siglec is Siglec-10. In some embodiments, the hypoimmunogenic biotherapeutic is engineered to include a sialic acid ligand for one Siglec. In other embodiments, the hypoimmunogenic biotherapeutic is engineered to include Siglec ligands for two or more Siglecs, for example, three Siglecs, or four Siglecs, and in certain cases, five Siglecs. In some cases, there are 5, 6, 7, 8, 9, 10, 11, 12, 13, or 15 Siglecs.

[0018] In some cases, the engineered, less immunogenic biotherapeutic is of formula (I): TIFF2024502850000002.tif19156, where X is a sialic acid group, L is an optional linker, Y is a biotherapeutic agent, n is an integer greater than or equal to 1, and m is an integer greater than or equal to 1. Combinations of X and L groups, i.e., [X n -L] are collectively referred to herein as Siglec ligands.

[0019] Sialic acid group X X is a sialic acid group, and the term "sialic acid" refers to an α-keto acid sugar having a nine-carbon backbone. Thus, since X is a sialic acid group, X includes sialic acid or a derivative thereof. The sialic acid or a derivative thereof can be natural or non-natural. In some cases, X includes neuraminic acid, an example of a sialic acid, or a derivative thereof.

[0020] In some embodiments, the sialic acid is a naturally occurring sialic acid. The sialic acid family includes approximately 50 naturally occurring members. The most common of these are N-acetylneuraminic acid (Neu5Ac), N-glycolylneuraminic acid (Neu5Gc, enzymatically produced from Neu5Ac by adding a single oxygen atom (i.e., hydroxylation)), 2-keto-3 deoxynonulosonic acid (Kdn), and neuraminic acid (Neu). Others are well known in the art, for example, as reviewed in Schauer (2000) Glycoconjugate J17:485-499. Thus, for example, if the target Siglec is CD22, the Siglec ligand can be a naturally occurring CD22 ligand, i.e., an α2,6-linked sialic acid, e.g., Neu5Acα2-6Galβ-4GlcNAc-6S; if the Siglec is Siglec-5, the Siglec ligand can be a naturally occurring Siglec-5 ligand, i.e., Neu5Acα8-8Neu5Ac and Neu5Acα2-6GalNAc; if the Siglec is Siglec-6, the Siglec ligand can be a naturally occurring Siglec-6 ligand, e.g., Neu5Acα8-8Neu5Ac and Neu5Acα2-6GalNAc; i.e., Neu5Acα2-6GalNAc; if the Siglec is Siglec-9, the Siglec ligand can be a naturally occurring Siglec-9 ligand, i.e., Neu5Acα2-3Galβ-4GlcNAc-6Sα-3fucose; or if the Siglec is Siglec-10 / G, the Siglec ligand can be a naturally occurring Siglec-10 ligand, i.e., α2,6-linked sialic acid or α2,3-linked sialic acid (e.g., Neu5Acα2-6Galβ-4GlcNAc) (O'Reilly, MK and Paulson, JC (2009) Trends Pharmacol. Sci. 30, 240-248). In some embodiments, the low immunogenic biotherapeutic is engineered to contain sialic acid ligands for two or more Siglecs, for example, three Siglecs, or four Siglecs, and in certain cases, five Siglecs. In some cases, there are 5, 6, 7, 8, 9, 10, 11, 12, 13, or 15 Siglecs.

[0021] In some embodiments, the sialic acid is non-naturally occurring, i.e., synthetic. "Synthetic sialic acid," also known in the art and referred to herein as "sialic acid mimic" or "SAM," refers to a non-naturally occurring sialic acid, i.e., an α-keto acid sugar derivative containing a non-natural nine-carbon backbone. Compared to Siglec ligands containing natural sialic acids, which have weak monovalent binding affinity for Siglecs (0.1-3 mM), Siglec ligands containing SAMs can be characterized by binding affinities in the nanomolar range (Crocker, P.R. et al. (2007) Nat. Rev. Immunol. 7, 255-266; Prescher, H. et al. (2014) ACS Chem. Biol. 9, 1444-1450). SAMs that find use in the compositions of the invention include those in which one or more positions of naturally occurring sialic acid, from the aglycone (C-2) through the remainder of the backbone (C-3 through C-9), have been modified to improve Siglec binding. For example, the modifications C-9-NH2 (9-NH2-Neu5Ac / Me) and C-5-FAc (Neu5FAc / Me) improved Siglec-2 binding due to increased hydrogen bonding and lipophilic interactions between the SAM and Siglec-2, and the incorporation of lipophilic groups has since been used to rationally design additional SAMs with increased binding affinity for Siglec-2 (van Rossenberg, S. M. Wet al. (2001) J. Biol. Chem. 276, 12967-12973; Kelm, S. et al. (2002) J. Exp. Med. 195, 1207-1213; Zaccai, N. R. et al. (2003) Structure 11, 557-567).Other non-limiting examples of SAMs that find use in the present application include 9-N-biphenylcarboxyl-NeuAcα2-Galβ1-4GlcNAc (6'-BPCNeuAc), NeuAcα2-6Galβ1-4GlcNAc, and NeuAcα2-6Galβ1-4(6-sulfo)GlcNAc, which SAMs are described in Bull et al. (2016) Sialic Acid Mimetics to Target the Sialic Acid-Siglec Axis. Trends Biochem Sci. 41(6):519-531 and Prescher, H. et al. (2014) Discovery of multifold modified sialosides as human CD22 / Siglec-2 ligands with nanomolar activity on B-cells. ACS Chem.Biol.9, 1444-1450, and the SAMs are those disclosed in U.S. Patent Nos. 8,357,671, 9,522,183, and 9,981,023, the disclosures of which are incorporated herein by reference in their entireties. In certain embodiments, the SAMs are those provided in Table 1 below. [Table 1] TIFF2024502850000004.tif234156TIFF2024502850000005.tif101153

[0022] As described in formula (I) above, n is an integer of 1 or greater, for example, an integer from 1 to 20, 1 to 15, 1 to 10, or 1 to 5. Optionally, n is 1, 2, 3, 4, or 5. Optionally, n is 1. Optionally, n is 2. Optionally, n is 3. Optionally, n is 4. Optionally, n is 5.

[0023] When multiple X's are present, i.e., when n is greater than 1, the X's can be the same or different from one another. When L's present, each X's is directly covalently bonded to L's, and L's is directly covalently bonded to Y's. When L's absent, each X's is directly covalently bonded to Y's. In some cases, n's is 1. In some cases, n's is an integer of 2 or greater, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0024] As noted above, m is an integer greater than or equal to 1. In some cases, m is greater than or equal to 2, greater than or equal to 3, greater than or equal to 5, or greater than or equal to 10. In some cases, m is an integer from 1 to 20, for example, from 2 to 10.

[0025] Linker L L is an optional linker. Thus, in some cases, the engineered low immunogenic biotherapeutic has a linker, and the engineered low immunogenic biotherapeutic has a linker of the formula [X n -L] m In other cases, the engineered less immunogenic biotherapeutic does not have a linker and the engineered biotherapeutic has the formula [X n ] m -Y.

[0026] Embodiments of engineered, low-immunogenic biotherapeutics can be used to treat [X n -L] m Possible configurations of -Y can be demonstrated. As noted above, X is a sialic acid group, including sialic acid or a derivative thereof. In the compound shown below, there is a single neuraminic acid group corresponding to a single X group. Thus, in the following embodiment, n is 1. The group shown to the left of neuraminic acid, having the formula (phenyl)-C(O)-phenylene, can be considered part of the X group. Although the biotherapeutic Y is not shown in the compound, the -C(O)-O-CF group can undergo a chemical reaction to form a covalent bond with the biotherapeutic Y. [ka]

[0027] In the embodiment shown below, three neuraminic acid groups are shown, indicating the presence of three X groups, and therefore n is 3. In addition, the three X groups are covalently linked to one another via a branching group comprising a derivative of a lysine residue. This branching group is part of a linker, L. Stated another way, this embodiment includes an optional linker, L, where L is a branching group that covalently links the three X groups to one another. The linker, L, also covalently links the X group to a —C(O)—O—CF group that can undergo a chemical reaction to form a covalent bond with the biotherapeutic, Y. Thus, the linker, L, also covalently links the X group to the biotherapeutic, Y. [ka]

[0028] As noted above, the combination of X and L groups is collectively referred to as a Siglec ligand. n -L] is a Siglec ligand.

[0029] In some embodiments, each sialic acid group of each X is covalently attached to the biotherapeutic agent Y via a chain of atoms that does not include a sugar group. For example, each X group includes a single sialic acid group but does not include any other sugar groups. Further, in some embodiments, if L is present, L directly covalently attaches each X to Y, and L does not include a sugar group. As used herein, the term "sugar" refers to monosaccharides and disaccharides. Thus, even if L includes a trisaccharide, such L is not within the scope of such an embodiment, since a trisaccharide includes a disaccharide unit and a monosaccharide unit. In other words, [X n The only sugar groups present in [-L] are single sialic acid groups for each X. The sialic acid groups are not directly covalently bonded to each other.

[0030] In some embodiments, each sialic acid group of each X is covalently attached to the biotherapeutic agent Y through a chain of atoms that does not include an oxygen-containing heterocyclic group. In particular, monosaccharides such as glucose and galactose are heterocyclic groups that include an oxygen atom in the ring. In some embodiments, each sialic acid group of each X is covalently attached to the biotherapeutic agent Y through a chain of atoms that is comprised of one or more chemical moieties selected from the group consisting of alkyl, alkenyl, alkynyl, polyethylene glycol, aryl, heteroaryl, sulfur-containing heterocycle, nitrogen-containing heterocycle, amino acid residue, amino, acyl, halo, hydroxy, carboxy, sulfoxy, and substituted analogs thereof.

[0031] In some embodiments, when present, the linker L is comprised of one or more chemical moieties selected from the group consisting of alkyl, alkenyl, alkynyl, polyethylene glycol, aryl, heteroaryl, sulfur-containing heterocycle, nitrogen-containing heterocycle, amino acid residue, amino, acyl, halo, hydroxy, carboxy, sulfoxy, and substituted analogs thereof. Furthermore, the cross section of X between the sialic acid and L or Y is comprised of one or more chemical moieties selected from the group consisting of alkyl, alkenyl, alkynyl, polyethylene glycol, aryl, heteroaryl, sulfur-containing heterocycle, nitrogen-containing heterocycle, amino acid residue, amino, acyl, halo, hydroxy, carboxy, sulfoxy, and substituted analogs thereof.

[0032] In some instances where L is present, L is a branched linker. In other words, L directly covalently attaches the biotherapeutic Y to two or more X groups. In some cases, the branching positions of L include an amino acid residue or derivative thereof, e.g., lysine or a derivative thereof. For example, the branching positions of L may have the formula shown below, where each position marked with an asterisk (*) is a site for directing an X or Y group. [ka]

[0033] In some cases, the branching positions of L do not include aryl or heteroaryl groups. In some cases, the branching positions of L include alkyl groups, amide groups, amino acid residues, or combinations thereof.

[0034] In some embodiments, the linker L comprises a polyethylene glycol group, a triazole group, or a combination thereof. In some embodiments, the cross section of X between the sialic acid group and L or Y comprises a polyethylene glycol group, a triazole group, or a combination thereof. In some embodiments, the triazole group is part of the covalent bond between the X group and the L group.

[0035] In some embodiments, the linker, L, can include one or more linker subunits (LS), such as 2, 3, 4, 5, 6, 7, 8, 9, or 10, or even more linker subunits (LS). For example, some embodiments of the linker can include 1 to 10 linker subunits (LS) described by formula (II): -(LS 1 ) a -(LS 2 ) b -(LS 3 ) c -(LS 4 ) d -(LS 5 ) e -(LS 6 ) f -(LS 7 ) g -(LS 8 ) h -(LS 9 ) i -(LS 10 ) j -(II) In the formula, LS 1 , L.S. 2 , L.S. 3 , L.S. 4 , L.S. 5 , L.S. 6 , L.S. 7 , L.S. 8 , L.S. 9 and LS 10are each independently a linker subunit, and a, b, c, d, e, f, g, h, i, and j are each independently 0 or 1. In some embodiments, the sum of a through j is 1 (e.g., a is 1 and b through j are each 0). In these embodiments, the linker subunit LS 1 are attached to the ends of Y and X. In some embodiments, the sum of a through j is 2 (e.g., a and b are each 1 and c through j are each 0). In these embodiments, the linker subunit LS 1 binds to Y and the linker subunit LS 2 is attached to X. In some embodiments, the sum of a through j is 3 (e.g., a through c are each 1 and d through j are each 0). In these embodiments, the linker subunit LS 1 binds to Y and the linker subunit LS 3 is attached to X. In some embodiments, the sum of a through j is 4 (e.g., a through d are each 1 and e through j are each 0). In these embodiments, the linker subunit LS 1 binds to Y and the linker subunit LS 4 is attached to X. In some embodiments, the sum of a through j is 5 (e.g., a through e are each 1 and f through j are each 0). In these embodiments, the linker subunit LS 1 binds to Y and the linker subunit LS 5 is attached to X. In some embodiments, the sum of a through j is 6 (e.g., a through f are each 1 and g through j are each 0). In these embodiments, the linker subunit LS 1 binds to Y and the linker subunit LS 6 is attached to X. In some embodiments, the sum of a through j is 7 (e.g., a through g are each 1 and h through j are each 0). In these embodiments, the linker subunit LS 1 binds to Y and the linker subunit LS 7is attached to X. In some embodiments, the sum of a through j is 8 (e.g., a through h are each 1 and i and j are each 0). In these embodiments, the linker subunit LS 1 binds to Y and the linker subunit LS 9 is attached to X. In some embodiments, the sum of a through j is 9 (e.g., a through j are each 1 and j is 0). In these embodiments, the linker subunit LS 1 binds to Y and the linker subunit LS 9 is attached to X. In some embodiments, the sum of a through j is 10 (e.g., each of a through j is 1). In these embodiments, the linker subunit LS 1 binds to Y and the linker subunit LS 10 is bonded to X.

[0036] Any convenient functional group can be used within each linker subunit (LS) within the linker. In some embodiments, the linker subunit (LS) may comprise a group selected from, but not limited to, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acylamino, alkylamido, substituted alkylamido, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

[0037] In some embodiments, the linker subunit is a covalent bond, (C1-C 12 ) alkyl, substituted (C1-C 12 ) alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, (PEG) n , and (AA) pwherein each n is independently an integer from 1 to 50, and each p is independently an integer from 1 to 20. As used herein, "PEG" refers to polyethylene glycol.

[0038] As used herein, "AA" refers to an amino acid residue. Amino acid residues include those commonly found in naturally occurring proteins (e.g., Ala or A, Cys or C, Asp or D, Glu or E, Phe or F, Gly or G, His or H, Ile or I, Lys or K, Leu or L, Met or M, Asn or N, Pro or P, Gln or Q, Arg or R, Ser or S, Thr or T, Val or V, Trp or W, Tyr or Y). In some embodiments, amino acid residues used in the linkers and linker subunits described herein also include amino acid analogs and amino acid derivatives, which are naturally occurring amino acids with modified side chains or backbones. Amino acid analogs also include amino acid analogs that have the same stereochemistry as the naturally occurring D-form, as well as L-forms of amino acid analogs. In some examples, amino acid analogs share the backbone and / or side chain structure of one or more naturally occurring amino acids, differing only in one or more modified groups within the molecule. Such modifications can include, but are not limited to, substituting an atom (e.g., N) for a related atom (e.g., S), adding a group (e.g., methyl or hydroxyl) or atom (e.g., Cl or Br), deleting a group, substituting a covalent bond (e.g., a single bond for a double bond), or attaching another group to the side chain or backbone, or a combination thereof. For example, amino acid analogs can include alpha-hydroxy acids, alpha-amino acids, and the like. In some examples, amino acid analogs or amino acid derivatives can include another group, e.g., another sialic acid moiety (X), attached to the side chain or backbone of the amino acid analog or amino acid derivative via an optional linker.

[0039] In some embodiments, the linker subunits are (C1-C 12 ) alkyl or substituted (C1-C 12In some embodiments, the alkyl group includes a functional group independently selected from (C-C 12 The alkyl group is a straight or branched chain alkyl group containing 1 to 12 carbon atoms, such as 1 to 10 carbon atoms, or 1 to 8 carbon atoms, or 1 to 6 carbon atoms, or 1 to 5 carbon atoms, or 1 to 4 carbon atoms, or 1 to 3 carbon atoms. In some examples, (C1-C 12 )Alkyl is C1-C 12 Alkyl, or C1-C 10 It can be an alkyl, such as an alkyl, or a C1-C6 alkyl, or a C1-C3 alkyl. In some examples, (C1-C 12 ) alkyl is C2 alkyl. For example, (C1-C 12 )Alkyl is C1-C 12 Alkylene, or C1-C 10 It can be an alkylene or substituted alkylene, such as alkylene, or C1-C6 alkylene, or C1-C3 alkylene. In some examples, (C1-C 12 ) alkyl is C1-alkylene (e.g., CH2). In some examples, (C1-C 12 ) Alkyl is C2-alkylene (e.g., CH2CH2).

[0040] In some embodiments, the substitution (C-C 12 The substituted (C1-C2) alkyl group is a straight or branched chain substituted alkyl group containing 1 to 12 carbon atoms, such as 1 to 10 carbon atoms, or 1 to 8 carbon atoms, or 1 to 6 carbon atoms, or 1 to 5 carbon atoms, or 1 to 4 carbon atoms, or 1 to 3 carbon atoms. In some examples, the substituted (C1-C2) alkyl group is 12 ) alkyl is a substituted C1-C 12 Alkyl or substituted C1-C 10 It can be alkyl, or substituted alkyl, such as substituted C1-C6 alkyl, or substituted C1-C3 alkyl. In some examples, substituted (C1-C 12 ) alkyl is a substituted C2-alkyl. For example, substituted (C1-C 12 ) alkyl is a substituted C1-C 12 Alkylene or substituted C-C 10It can be an alkylene, or a substituted alkyl, such as a substituted C1-C6 alkylene, or a substituted C1-C3 alkylene. In some examples, the substituted (C1-C 12 ) alkyl is a substituted C1-alkylene. In some examples, a substituted (C1-C 12 ) Alkyl is a substituted C2-alkylene.

[0041] In some embodiments, the linker subunits comprise functional groups independently selected from aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

[0042] In some examples, the linker subunits include functional groups independently selected from aryl or substituted aryl. In some examples, the linker subunits include aryl. For example, the aryl can be phenyl. In some examples, the linker subunits include substituted aryl. In some cases, the substituted aryl is a substituted phenyl. The substituted aryl can be any of (C1-C 12 ) alkyl, substituted (C1-C 12 ) can be substituted with one or more substituents selected from alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

[0043] In some examples, the linker subunits comprise functional groups independently selected from heteroaryl or substituted heteroaryl. In some examples, the linker subunits comprise heteroaryl. In some examples, the linker subunits comprise substituted heteroaryl. Substituted heteroaryl is a heteroaryl selected from (C1-C 12 ) alkyl, substituted (C1-C 12 ) can be substituted with one or more substituents selected from alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

[0044] In some examples, the linker subunits comprise a functional group independently selected from cycloalkyl or substituted cycloalkyl. In some examples, the linker subunits comprise a cycloalkyl. In some examples, the linker subunits comprise a substituted cycloalkyl. The substituted cycloalkyl is selected from (C1-C 12 ) alkyl, substituted (C1-C 12 ) can be substituted with one or more substituents selected from alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

[0045] In some examples, the linker subunits include functional groups independently selected from heterocyclyl or substituted heterocyclyl. In some examples, the linker subunits include heterocycloalkyl. For example, the linker subunits can include triazole (e.g., 1,2,3-triazole). In some examples, the linker subunits include substituted heterocycloalkyl. Substituted heterocyclyls include (C1-C 12 ) alkyl, substituted (C1-C 12 ) can be substituted with one or more substituents selected from alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

[0046] In some embodiments, the linker does not include natural sugars.

[0047] In some embodiments, the linker (L) comprises one or more tether groups adjacent to or between one or more linker subunits (LS) within the linker. The tether groups may facilitate attachment between two linker subunits, between a linker subunit and a reactive terminus for conjugation to a moiety of interest (Y), or between a linker subunit and a sialic acid moiety (X). The tether groups may include any convenient functional group that facilitates their attachment, such as, but not limited to, amino, carbonyl, amido, oxycarbonyl, carboxy, thioether, sulfonyl, sulfoxide, sulfonylamino, aminosulfonyl, thio, oxy, phospho, phosphoramidate, thiophosphoridate, and the like. In some embodiments, the tether groups each independently comprise a covalent bond, -CO-, -NR 15 -, -NR 15 (CH2) q -,-CONR 15 -, -NR 15 CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR 15 -, -NR 15 and -SO2-, and -P(O)OH-, where q is an integer from 1 to 6. In some embodiments, q is an integer from 1 to 6 (e.g., 1, 2, 3, 4, 5, or 6). In some embodiments, q is 1. In some embodiments, q is 2. In some embodiments, q is 3. In some embodiments, q is 4. In some embodiments, q is 5. In some embodiments, q is 6.

[0048] In some embodiments, each R 15is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

[0049] In some embodiments, R 15 is hydrogen. In some embodiments, each R 15 is hydrogen. In some embodiments, R 15 is alkyl or substituted alkyl, e.g., C 1-6 Alkyl or C 1-6 Substituted alkyl, or C 1-4 Alkyl or C 1-4 Substituted alkyl, or C 1-3 Alkyl or C 1-3 In some embodiments, R 15 is alkenyl or substituted alkenyl, e.g., C 2-6 Alkenyl or C 2-6 substituted alkenyl, or C 2-4 Alkenyl or C 2-4 substituted alkenyl, or C 2-3 Alkenyl or C 2-3 In some embodiments, R is a substituted alkenyl. 15 is alkynyl or substituted alkynyl. In some embodiments, R 15 is alkoxy or substituted alkoxy. In some embodiments, R 15 is amino or substituted amino. In some embodiments, R 15 is carboxyl or carboxyl ester. In some embodiments, R 15 is acyl or acyloxy. In some embodiments, R 15 is acylamino or aminoacyl. In some embodiments, R15 is an alkylamide or a substituted alkylamide. In some embodiments, R 15 is sulfonyl. In some embodiments, R 15 is thioalkoxy or substituted thioalkoxy. In some embodiments, R 15 is aryl or substituted aryl, e.g., C 5-8 Aryl or C 5-8 In some embodiments, R is a C aryl or a C substituted aryl, for example, a C aryl or a C substituted aryl. 15 is heteroaryl or substituted heteroaryl, e.g., C 5-8 Heteroaryl or C 5-8 In some embodiments, R is a substituted heteroaryl, such as a C heteroaryl or a C substituted heteroaryl, or a C heteroaryl or a C substituted heteroaryl. 15 is cycloalkyl or substituted cycloalkyl, e.g., C 3-8 Cycloalkyl or C 3-8 Substituted cycloalkyl, e.g., C 3-6 Cycloalkyl or C 3-6 substituted cycloalkyl, or C 3-5 Cycloalkyl or C 3-5 In some embodiments, R 15 is heterocyclyl or substituted heterocyclyl, for example, C 3-8 Heterocyclyl or C 3-8 Substituted heterocyclyl, e.g., C 3-6 Heterocyclyl or C 3-6 substituted heterocyclyl, or C 3-5 Heterocyclyl or C 3-5 It is a substituted heterocyclyl.

[0050] In some embodiments, the linker subunit (LS) can comprise a polymer. For example, the polymer can comprise polyalkylene glycols and derivatives thereof, including polyethylene glycol, methoxypolyethylene glycol, polyethylene glycol homopolymer, polypropylene glycol homopolymer, copolymers of ethylene glycol and propylene glycol (e.g., the homopolymers and copolymers are unsubstituted or substituted at one end with an alkyl group), polyvinyl alcohol, polyvinyl ethyl ether, polyvinylpyrrolidone, combinations thereof, and the like. In some embodiments, the polymer is a polyalkylene glycol. In some embodiments, the polymer is polyethylene glycol (PEG).

[0051] In some cases, the linker is not branched, but rather connects one X group to a Y group, and may therefore be referred to as monovalent. In some cases, the linker is divalent and is a branched linker connecting two X groups to a Y group. In certain cases, the linker is trivalent and is a branched linker connecting three X groups to a Y group. In some examples, the linker is a branched linker of higher multivalency, connecting multiple X groups to a Y group. In some cases, the linker has a linear or branched backbone of 500 atoms or less (e.g., 400 atoms or less, 300 atoms or less, 200 atoms or less, 100 atoms or less, 80 atoms or less, 60 atoms or less, 50 atoms or less, 40 atoms or less, 30 atoms or less, or even 20 atoms or less), for example, as measured between two or more moieties. The linking moiety can be a covalent bond connecting two groups or a straight or branched chain of 1 to 500 atoms, e.g., about 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20, 30, 40, 50, 100, 150, 200, 300, 400, or 500 carbon atoms in length, and the linker can be straight, branched, cyclic, or a single atom. In certain cases, one, two, three, four, five or more, ten or more, or even more carbon atoms in the linker backbone can be optionally substituted with a heteroatom, e.g., a sulfur, nitrogen, or oxygen heteroatom. In certain cases, when the linker includes a PEG group, every third atom in that segment of the linker backbone is substituted with an oxygen. The bond between the backbone atoms can be saturated or unsaturated, and typically, one, two, or no more than three unsaturated bonds are present in the linker backbone. The linker may include one or more substituents, such as an alkyl, aryl, or alkenyl group. The linker may include, but is not limited to, one or more of the following: oligo(ethylene glycol), ether, thioether, disulfide, amide, carbonate, carbamate, tertiary amine, alkyl, which may be, for example, methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), etc. The linker backbone may include a cyclic group, such as an aryl, heterocycle, cycloalkyl group, or heterocyclic group, where two or more atoms, for example, 2, 3, or 4 atoms, of the cyclic group are included in the backbone.

[0052] In some embodiments, the linker subunit (LS) can be a branched subunit. A branched subunit can be a linker subunit that is bound to two or more sialic acid moieties, either directly or through a respective linker, for each sialic acid moiety. For example, a branched subunit can be bound to two sialic acid moieties. In some embodiments, a branched subunit comprises an amino acid (AA). For example, a branched subunit can comprise an amino acid whose backbone forms part of a linker bound to a first sialic acid moiety and whose side chain is conjugated to a second sialic acid moiety directly or through a branched linker (i.e., a "branched linker"). For example, in some embodiments, a branched subunit comprises a lysine, whose backbone forms part of a linker bound to a first sialic acid moiety and whose side chain is conjugated to a second sialic acid moiety directly or through a branched linker. In some embodiments, the second sialic acid moiety is conjugated to the lysine by bonding to the terminal amine of the lysine side chain. In some embodiments, the branched linker is a linker described by formula (II) above.

[0053] Examples of linkers according to the present disclosure include, but are not limited to: LS 1 is PEG (e.g., PEG4), and LS 2 is heterocyclyl (e.g., 1,2,3-triazole), and LS 3 is a PEG (e.g., PEG2), and d to j are each 0; LS 1 is PEG (e.g., PEG4), and LS 2 is a branched subunit (e.g., lysine), and LS 3 is alkyl (e.g., C alkyl), and LS 4 is PEG (e.g., PEG2), and LS 5 is heterocyclyl (e.g., 1,2,3-triazole), and LS 6is a PEG (e.g., PEG2), g to j are each 0, and LS 2 The branched subunits are attached to a branched linker described by: LS 1 is PEG (e.g., PEG2), and LS 2 is heterocyclyl (e.g., 1,2,3-triazole), and LS 3 is a PEG (e.g., PEG2), and d to j are each 0; LS 1 is PEG (e.g., PEG4), and LS 2 is heterocyclyl (e.g., 1,2,3-triazole), and LS 3 is alkyl (e.g., C2-alkyl), and d to j are each 0; LS 1 is PEG (e.g., PEG4), and LS 2 is a branched subunit (e.g., lysine), and LS 3 is alkyl (e.g., C alkyl), and LS 4 is PEG (e.g., PEG2), and LS 5 is heterocyclyl (e.g., 1,2,3-triazole), and LS 6 is alkyl (e.g., C2-alkyl), g to j are each 0, and LS 2 The branched subunits are attached to a branched linker described by: LS 1 is PEG (e.g., PEG2), and LS 2 is heterocyclyl (e.g., 1,2,3-triazole), and LS 3 is alkyl (e.g., C2-alkyl), and d to j are each 0; LS 1 is PEG (e.g., PEG4), and LS 2 is a branched subunit (e.g., lysine), and LS 3 is a branched subunit (e.g., lysine), and LS 4 is alkyl (e.g., C3-alkyl), and LS 5 is PEG (e.g., PEG2), and LS 6is heterocyclyl (e.g., 1,2,3-triazole), and LS 7 is alkyl (e.g., C2-alkyl), h to j are each 0, and LS 2 The branched subunits are attached to a branched linker described by: LS 1 is PEG (e.g., PEG2), and LS 2 is heterocyclyl (e.g., 1,2,3-triazole), and LS 3 is alkyl (e.g., C2-alkyl), d to j are each 0, and LS 3 The branched subunits are attached to a branched linker described by: LS 1 is PEG (e.g., PEG2), and LS 2 is heterocyclyl (e.g., 1,2,3-triazole), and LS 3 is alkyl (e.g., C2-alkyl), and d to j are each 0; LS 1 is PEG (e.g., PEG4), and LS 2 is a branched subunit (e.g., lysine), and LS 3 is a branched subunit (e.g., lysine), and LS 4 is alkyl (e.g., C3-alkyl), and LS 5 is PEG (e.g., PEG2), and LS 6 is heterocyclyl (e.g., 1,2,3-triazole), and LS 7 is PEG (e.g., PEG2), h to j are each 0, and LS 2 The branched subunits are attached to a branched linker described by: LS 1 is PEG (e.g., PEG2), and LS 2 is heterocyclyl (e.g., 1,2,3-triazole), and LS 3 is a PEG (e.g., PEG2), d to j are each 0, and LS 3 The branched subunits are attached to a branched linker described by: LS 1 is PEG (e.g., PEG2), and LS 2is heterocyclyl (e.g., 1,2,3-triazole), and LS 3 is a PEG (e.g., PEG2), and d to j are each 0; LS 1 is a branched subunit (e.g., lysine), and LS 2 is a branched subunit (e.g., lysine), and LS 3 is alkyl (e.g., C3-alkyl), and LS 4 is heterocyclyl (e.g., 1,2,3-triazole), and LS 5 is a PEG (e.g., PEG2), f to j are each 0, and LS 1 The branched subunits are attached to a branched linker described by: LS 1 is heterocyclyl (e.g., 1,2,3-triazole), and LS 2 is PEG (e.g., PEG2), c to j are each 0, and LS 2 The branched subunits are attached to a branched linker described by: LS 1 is heterocyclyl (e.g., 1,2,3-triazole), and LS 2 is a PEG (e.g., PEG2), and c to j are each 0; LS 1 is PEG (e.g., PEG4), and LS 2 is a branched subunit (e.g., lysine), and LS 3 is a branched subunit (e.g., lysine), and LS 4 is alkyl (e.g., C3-alkyl), and LS 5 is heterocyclyl (e.g., 1,2,3-triazole), and LS 6 is a PEG (e.g., PEG2), g to j are each 0, and LS 2 The branched subunits are attached to a branched linker described by: LS 1 is heterocyclyl (e.g., 1,2,3-triazole), and LS 2 is PEG (e.g., PEG2), c to j are each 0, and LS 3The branched subunits are attached to a branched linker described by: LS 1 is heterocyclyl (e.g., 1,2,3-triazole), and LS 2 is a PEG (e.g., PEG2), and c to j are each 0; LS 1 is PEG (e.g., PEG4), and LS 2 is a branched subunit (e.g., lysine), and LS 3 is alkyl (e.g., C3-alkyl), and LS 4 is heterocyclyl (e.g., 1,2,3-triazole), and LS 5 is a PEG (e.g., PEG2), f to j are each 0, and LS 2 The branched subunits are attached to a branched linker described by: LS 1 is heterocyclyl (e.g., 1,2,3-triazole), and LS 3 is a PEG (for example, PEG2), and c to j are each 0.

[0054] The above-described linkers may contain one or more tether groups to facilitate attachment between two linker subunits, between a linker subunit and a reactive terminus for conjugation to a moiety of interest (Y), or between a linker subunit and a sialic acid moiety (X).

[0055] Siglec ligand [X n -L] m Expression [X n -L] m -Y, [X n

[0023] -L] represents a Siglec ligand, and m represents an integer from 1 to 25. It is contemplated that one or more Siglec ligands, e.g., 2, 3, 4, or 5 or more Siglec ligands, in some cases 6, 7, 8, 9, or 10 or more Siglec ligands, in some such cases 11, 12, 13, 14, 15 or more Siglec ligands, in some cases 16, 17, 18, 19, 20 or more Siglec ligands, and in some cases 21, 22, 23, 24, or 25 Siglec ligands, may be conjugated to the biotherapeutic, attached to either the same or different amino acids of the biotherapeutic. The Siglec ligand may be naturally occurring, i.e., a moiety comprising a naturally occurring sialic acid and a naturally occurring glycan, where the sialic acid and glycan are typically found in association with one another in nature to form the Siglec ligand. The Siglec ligand may be non-naturally occurring, e.g., a moiety comprising a natural sialic acid and a linker, a moiety comprising a non-natural sialic acid and a glycan found in nature as part of a Siglec ligand, a moiety comprising a non-natural sialic acid and a linker, or a moiety comprising a peptide with affinity for a Siglec.

[0056] Biotherapy drug Y Expression [X n -L] m In [X -Y], Y is a biotherapeutic. Y itself is referred to as an unengineered biotherapeutic, and this term is used interchangeably with the term parent biotherapeutic. However, [X n -L] m The element combinations [X -Y] are referred to as engineered hypoimmunogenic biotherapeutics. Y, by itself, has therapeutic activity. n -L] does not mediate the therapeutic activity of Y. Stated another way, the therapeutic activity of Y is n -L] is irrelevant.

[0057] Exemplary biotherapeutic Y groups include antibodies, enzymes, virus particles, nanoparticles, polypeptides, and nucleic acids.

[0058] Any protein or nucleic acid biotherapeutic can function as a biotherapeutic engineered to be a less immunogenic biotherapeutic according to the present disclosure, including, for example, proteins such as antibodies, fusion proteins, enzymes, viral particles, DNA molecules, or RNA molecules. The biotherapeutic can be naturally occurring, e.g., a native protein, native capsid, etc., delivered to a patient as a therapeutic. The biotherapeutic can be an engineered protein, e.g., an antibody therapeutic, a fusion protein, or a "chimeric" protein, i.e., a protein containing protein domains from two or more different proteins, or a completely non-naturally occurring protein, i.e., a protein having 30% or less identity to any native protein across its entire functional domain (see, e.g., Chen et al. (2020) De novo design of protein logic gates. Science 368(6486):78-84, and Polizzi et al. (2020) A defined structural unit enables de novo design of small-molecule-binding proteins Science 369(6508):1227-1233). In some embodiments, the biotherapeutic is a variant of a naturally occurring protein or a known engineered protein. By "variant" is meant a variant of a protein that has less than 100% sequence identity to the protein from which it is derived. For example, a variant protein can have 60% or more sequence identity to the full-length naturally occurring protein, e.g., 65%, 70%, 75%, or 80% or more identity to the full-length naturally occurring protein, e.g., 85%, 90%, or 95% or more identity, e.g., 98% or 99% identity to the full-length naturally occurring protein. Variants also include fragments of naturally occurring proteins, particularly those that have similar or improved activity to the naturally occurring protein. Biotherapeutic agents can be from any source, e.g., human, non-human, or engineered.

[0059] In some embodiments, the protein is an antibody or fragment thereof, such as, for example, a monoclonal antibody, a bispecific antibody, a trispecific antibody, an scFv, a Fab, a camelid nanobody, etc. Non-limiting examples of antibodies for which the engineering contemplated herein finds particular use include adalimumab and infliximab (for the treatment of autoimmune or inflammatory diseases such as rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, Crohn's disease, ulcerative colitis, psoriasis, mucositis, uveitis, or juvenile idiopathic arthritis), cetuximab (for the treatment of cancer, including metastatic colorectal cancer, metastatic non-small cell lung cancer, and head and neck cancer), natalizumab (for the treatment of multiple sclerosis), lumiti / moxetumumab daspotox (hairy cell white for the treatment of hematologic malignancies), Tecentriq / atezolizumab (for the treatment of various cancers), Opdivo / nivolumab (for the treatment of various cancers), ReoPro / abiximab (anti-GPIIb / IIIa, for the prevention of thrombosis during and after coronary artery procedures such as angioplasty), brentuximab (for the treatment of relapsed or refractory Hodgkin lymphoma (HL) and systemic anaplastic large cell lymphoma (ALCL)), certolizumab pegol (for the treatment of Crohn's disease, rheumatoid arthritis, psoriatic arthritis, and ankylosing spondylitis), elotuzumab (for the treatment of relapsed multiple myeloma) benralizumab (for treating asthma), vedolizumab (for treating ulcerative colitis and Crohn's disease), galcanezumab (for treating migraines and cluster headaches), rituximab (for treating autoimmune diseases and various cancers), alemtuzumab (for treating chronic lymphocytic leukemia (CLL) and multiple sclerosis), dupilumab (for treating allergic diseases such as eczema (atopic dermatitis), asthma and nasal polyps), golimumab (for treating inflammation), obinutuzumab (for treating lymphoid malignancies such as chronic lymphocytic leukemia and follicular lymphoma) (for the treatment of immunologically mediated inflammatory diseases), tildrakizumab (for the treatment of immunologically mediated inflammatory diseases), elenumab (for the prevention of migraines), mepolizumab (for the treatment of severe eosinophilic asthma, eosinophilic granulomatosis and hypereosinophilic syndrome (HES)), ramucirumab (for the treatment of solid tumors), ranibizumab (for the treatment of "wet" age-related macular degeneration (AMD, also ARMD), diabetic retinopathy and macular edema), ustekinumab (for the treatment of psoriasis, Crohn's disease and ulcerative colitis), reslizumab (for the treatment of asthma), ipilimumab (for the treatment of various cancers),Alirocumab (for the treatment of high cholesterol), belimumab (for the treatment of systemic lupus erythematosus (SLE)), panitumab (for the treatment of various cancers), avelumab (for the treatment of Merkel cell carcinoma, urothelial carcinoma, and renal cell carcinoma), necitumumab (for the treatment of metastatic squamous non-small cell lung cancer (NSCLC)), mogamulizumab (for the treatment of relapsed or refractory mycoses and Sézary disease, relapsed or refractory CCR4+ adult T-cell leukemia / lymphoma (ATCLL)), and relapsed or refractory CCR4+ cutaneous T-cell lymphoma (CTCL), olaratumab (for the treatment of solid tumors), brodalumumab (for the treatment of inflammatory diseases), eculizumab (for the treatment of paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), and neuromyelitis optica), pertuzumab (for the treatment of metastatic HER2-positive breast cancer), pembrolizumab (for the treatment of various cancers), and tocilizumab (for the treatment of rheumatoid arthritis (RA) and systemic juvenile idiopathic arthritis).

[0060] In some embodiments, biotherapeutic agent Y is an antibody that is not specific for a receptor selected from a B cell receptor (BCR), a receptor for the Fc region of immunoglobulin E (FcIRI), a Toll-like receptor (TLR), a T cell receptor (TCR), or a complex thereof.

[0061] As used herein, an antibody that specifically binds to a target antigen refers to an antibody that comprises a complementarity-determining region (CDR) domain that specifically recognizes and binds to the target antigen. Thus, an antibody that specifically binds to a B cell receptor or a complex thereof refers to an antibody that comprises a CDR that specifically recognizes and binds to a B cell receptor or a complex containing a B cell receptor, an antibody that specifically binds to a receptor in the Fc region of IgE refers to an antibody that comprises a CDR that specifically recognizes and binds to a receptor in the Fc region of IgE, an antibody that specifically binds to a Toll-like receptor refers to an antibody that comprises a CDR that specifically recognizes and binds to a Toll-like receptor or a complex containing a Toll-like receptor, and an antibody that specifically binds to a T cell receptor refers to an antibody that comprises a CDR that specifically recognizes and binds to a T cell receptor or a complex containing a T cell receptor.

[0062] In some embodiments, the protein is a native or naturally occurring protein. In other embodiments, the protein is an engineered protein. Examples of proteins for which the engineering contemplated herein finds particular use include erythropoietin (EPO for stimulating the production of red blood cells), thrombopoietin (TPO for stimulating the production of platelets), human growth hormone, tissue factor, IFNβ-1b (for the treatment of multiple sclerosis), IFNβ-1a (for the treatment of multiple sclerosis), IL-2 or the IL-2 mimetic aldesleukin (for the treatment of melanoma and renal cell carcinoma), exenatide (for the treatment of type 2 diabetes), albiglutide (for the treatment of type 2 diabetes), and the like. These include fluticasone (for the treatment of type 2 diabetes), alefacept (to suppress inflammation in moderate to severe psoriasis with plaque formation, and for the treatment of cutaneous T-cell lymphoma and T-cell non-Hodgkin's lymphoma), palifermin (to promote the growth of cells lining the mouth and intestinal tract after chemotherapy), belatacept (to promote graft / transplant survival), and the neutral and basic amino acid transport protein rBAT or b(0,+)-type amino acid transporter 1 (to treat cystinuria).

[0063] In some embodiments, biotherapeutic agent Y is a protein that is not ovalbumin or immunoglobulin E (IgE).

[0064] In some embodiments, the protein is an enzyme, e.g., a metabolic enzyme, lysosomal enzyme, protease, peptidase, etc. Non-limiting examples of enzymes for which the manipulations contemplated herein find particular use include enzymes from Erwinia chrysanthemi (Erwinia asparaginase from P. chrysanthemi (for the treatment of leukemia), bacterial IdeS (for immunosuppression after tissue transplantation or for the treatment of IgG antibody-driven diseases such as systemic lupus erythematosus, pemphigus vulgaris, or IgA nephropathy, in the administration of therapies to which the patient has pre-existing immunity, e.g., gene therapy), bacterial mucinase (for the treatment of MUC+ cancers, e.g., MUC1+ cancers), factor VIII (for the treatment of hemophilia A), factor IX (for the treatment of hemophilia B), factor Xa (to promote clotting), complement-degrading proteases, e.g., from bacterial or fungal pathogens (e.g., Pseudomonas elastase (PaE), Pseudomonas alkaline protease (PaAP), Streptococcus pyrogenic exotoxin B (SpeB), gingipain from Porphyromonas gingivalis, Aspergillus alkaline protease 1 (Alp1), C.and pathogen-derived aspartyl proteases such as S. albicans secreted aspartyl proteases 1 (Sap1), 2 (Sap2), and 3 (Sap3) for the treatment of complement-mediated diseases, e.g., IgA nephropathy), phenylalanine ammonia-lyase or mimetic PEGylalanine valerase (for the treatment of PKU), alpha-galactosidase A (for the treatment of Fabry disease), acid alpha-glucosidase or mimetic alglucosidase alfa (GAA, for the treatment of Pompe disease), glucocerebrosidase (GCase, for the treatment of Gaucher disease), aspartylglucosaminidase (AGA, for the treatment of aspartylglucosaminuria), asphatase (for the treatment of hypophosphatemia (HPP)), alpha-L-iduronidase (for the treatment of MPS I), idronate sulfatase or idronate sulfatase mimetic iduronidase (for the treatment of MPS II), sulfaminase (MPS IIIa), α-acetylglucosaminase (NAGLU, for the treatment of MPS IIIB), heparin acetyl-CoA:α-glucosaminide N-acetyltransferase (HGSNAT, for the treatment of MPS IIIC), N-acetylglucosamine 6-sulfatase (GNS, for the treatment of MPS IIID), N-glucosamine 3-O-sulfatase (arylsulfatase G or ARSG, for the treatment of MPS IIIE), N-acetylgalactosamine 6-sulfatase (for the treatment of MPS IVA), beta-galactosidase (for the treatment of MPS IVB), N-acetylgalactosamine 4-sulfatase (for the treatment of MPS VI), beta-glucuronidase (MPS These include: palmitoyl protein thioesterase (PPT1, for the treatment of Batten disease / CLN1); tripeptidyl peptidase (TPP1, for the treatment of Batten disease / CLN2); arginase-1 or pegdilarginase (for the treatment of arginase-1 deficiency); or cystathionine beta synthase or Aeglea product AGLE-177 (for the treatment of cystathionine beta synthase (CBS) deficiency (also known as classical homocystinuria)).

[0065] In some embodiments, biotherapeutic agent Y is an enzyme that is not Factor VIII.

[0066] In some embodiments, the protein is a viral protein or viral particle, e.g., a recombinant viral particle. A "recombinant" virus or viral particle refers to a virus / viral particle that contains a genome that includes a polynucleotide that is heterologous to the virus, i.e., a genome that is not naturally found associated with the viral capsid / envelope, where the polynucleotide encodes a gene product (RNA or protein). Recombinant viral particles find use in delivering polynucleotides that encode therapeutic gene products for purposes of gene therapy or oncolytic virotherapy. Gene therapy is an established technology. It is also well established that gene therapy is significantly hindered by the inability to re-administer the same viral therapeutic agent multiple or several times due to the fact that viral particles induce an immune response in individuals. Thus, those skilled in the art will understand that any viral particle used in gene therapy would benefit from engineering as contemplated herein.Non-limiting examples of viral particles that can function as biotherapeutics engineered to be low immunogenic biotherapeutics according to the present disclosure include recombinant adeno-associated viral (rAAV) particles, such as AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrhlO, AAVll, AAV12, or AAV13. rAAV particles comprising a capsid VP1 protein consisting of the group consisting of a VP1 protein, or a variant or pseudotyped virus thereof; recombinant human adenovirus particles, for example, rHAdV particles comprising a capsid protein consisting of the group consisting of rHAdV-A, rHAdV-B, rHAdV-C, rHAdV-D, rHAdV-E, rHAdV-F, or rHAdV-G, or a variant thereof; herpes simplex virus (rHSV) particles, rHSV1, or rHSV2, or a variant or pseudotyped virus thereof; recombinant papillomavirus (PV) particles; recombinant polyomavirus particles; recombinant vaccinia virus particles. recombinant cytomegalovirus (CMV) particles; recombinant baculovirus particles; recombinant human papillomavirus (HPV) particles; or recombinant retrovirus particles, such as recombinant lentivirus, recombinant human immunodeficiency virus (HIV) particles, simian immunodeficiency virus (SIV) particles, feline immunodeficiency virus (FIV) particles, puma lentivirus (PLV) particles, ectemia virus (EIAV) particles, bovine immunodeficiency virus (BIV) particles, caprine arthritis encephalitis virus particles, gammaretrovirus particles, and murine leukemia virus (MLV) particles, or mutants or pseudotypes thereof.

[0067] In some embodiments, biotherapeutic agent Y is not a toxin. Generally, toxins are compounds that are generally harmful to cells in a non-specific manner, i.e., toxins cause similar amounts of harm to different cells, even if such cells belong to significantly different categories. In contrast, selectively damaging compounds harm certain cells to a much greater extent than they harm other types of cells. For example, a selectively harmful compound may cause harm based on a biochemical process that is common in lung cells but rare in kidney cells, whereas a toxin may cause harm based on a biochemical process that is common to both lung cells and kidney cells. In some cases, the harm is cell death. In some cases, the toxin is Pseudomonas exotoxin A. In some embodiments, biotherapeutic agent Y is not a B cell modulator. Y does not increase or decrease the immune function of B cells. Examples of B cell modulation include promoting differentiation of B cells into biotherapy-specific mature B cells, e.g., plasma cells or memory cells, preventing B cells from producing antigen-specific antibodies, preventing upregulation of activation markers, e.g., CD69, and reducing the viability of the biotherapy-specific B cell population. In some embodiments, B cell activation is inhibited only for B cells with a B cell receptor that recognizes Y (as opposed to the entire B cell population that recognizes X).

[0068] As discussed above, unlike parent biotherapeutics that have not been engineered to contain an altered Siglec ligand profile, the hypoimmunogenic biotherapeutic compositions of the present disclosure suppress the development of an immune response to themselves. Thus, the engineered hypoimmunogenic biotherapeutics of the present disclosure can be functionally distinguished from the non-engineered, i.e., parent, biotherapeutic from which they are derived by assessing the extent to which the engineered hypoimmunogenic biotherapeutic attenuates immune cell activity. By attenuating activity, it is meant slowing the increase in activity, reducing activity, or preventing activity, e.g., silencing, inhibiting, deleting, etc., a cell or cell population. Thus, for example, attenuating the activity of a B cell or B cell population can include preventing B cells from differentiating into biotherapeutic-specific mature B cells, e.g., plasma cells or memory cells, preventing B cells from producing antigen-specific antibodies, preventing the upregulation of activation markers (e.g., CD69), or promoting reduced viability of a biotherapeutic-specific B cell population. Typically, the therapeutic activity of the non-engineered or parent biotherapeutic does not involve attenuating the activity of immune cells, and more typically, the therapeutic activity of the non-engineered or parent biotherapeutic does not involve attenuating the activity of B cells or B cell populations.

[0069] The ability of engineered biotherapeutics according to the present disclosure to suppress an immune response can be readily measured in vitro or in vivo in any number of ways. In vitro, immunosuppression can be measured, for example, as the extent to which a population of B cells is activated by the biotherapeutic, with less activation indicating greater immunosuppression. Any approach known in the art for measuring B cell activation may be used. For example, the extent to which cells of a population upregulate CD69 expression upon contact with the engineered biotherapeutic can be assessed, for example, by measuring the percent of CD69+ cells by FACS, assessing the mean fluorescence intensity (MFI) of B cells, assessing B cell activity, etc. In such analyses, the engineered biotherapeutic is expected to activate B cells at least about 2.5-fold less robustly than the non-engineered biotherapeutic, and in some instances, at least about 5-fold less robustly, at least about 7.5-fold less robustly, or at least about 10-fold less robustly, and in some instances, about 20-fold less robustly. In vivo, immunosuppression can be measured, for example, as the extent to which an engineered biotherapeutic induces "anti-drug antibodies" or ADAs in an individual, e.g., a mouse, injected intramuscularly or intravenously, with or without an immunological adjuvant such as alum, or, e.g., a human, compared to the ADAs elicited in an individual, e.g., a mouse, upon administration of the corresponding unmodified biotherapeutic, where fewer ADAs indicate greater immunosuppression. In some examples, the ADA titer for the less immunogenic biotherapeutic is reduced by 50% or more, e.g., 60%, 70%, 80% or more, and in certain cases, 85%, 90%, 95% or more, preferably 98%, 99%, or 100%, i.e., undetectable, compared to the corresponding unmodified biotherapeutic. In other words, the ADA titer induced by a low-immunogenic biotherapeutic is 50% or less of that induced by a corresponding non-engineered biotherapeutic, e.g., 40%, 30%, or 20% or less, and in certain cases, 15%, 10%, 5% or less, and preferably only 2%, 1% or less, of that induced by a corresponding non-engineered biotherapeutic.

[0070] Methods for detecting antibodies are well known in the art, including, but not limited to, enzyme-linked immunosorbent assay (ELISA), microparticle ELISA, ELISPOT, radioimmunoprecipitation assay, electrochemiluminescence immunoassay (ECLIA), DELFIA (dissociation-enhanced lanthanide fluorescence immunoassay), time-resolved fluorescence (TRF) assay, surface plasmon resonance immunoassay (SPRIA), Western blotting (immunoblotting), and the like, and any method that can be used to detect antibodies in an individual's serum to determine whether ADAs have been generated is well known in the art. ADA titer can be assessed in the serum of an individual after administration of a hypoimmunogenic biotherapeutic, where the level of ADA detected in serum collected 24 hours or more, e.g., 48 hours or 78 hours or more, and in some examples, 1, 2, 3, or 4 weeks or more, e.g., 6 weeks or 8 weeks after administration of the hypoimmunogenic biotherapeutic, is lower than the level of ADA detected in a control individual treated with the corresponding unengineered biotherapeutic for the same period of time with the same dosing regimen.

[0071] As another example, immunosuppression may be observed in vitro as a decreased leukocyte response upon exposure to an engineered biotherapeutic compared to a corresponding unengineered biotherapeutic. For example, greater activation of downstream signaling pathways (e.g., Erk phosphorylation, NFAT nuclear translocation) may be observed in CD22 Siglec-containing B cells exposed to an unengineered biotherapeutic compared to CD22 Siglec-containing B cells exposed to a biotherapeutic modified to contain more CD22 ligands. As yet another example, CD22 Siglecs (and similarly, cells expressing CD22 Siglecs) may have a higher binding affinity for a biotherapeutic engineered to contain more CD22 ligands than for a unengineered biotherapeutic.

[0072] In some embodiments, ADA to a hypoimmunogenic biotherapeutic is lower in the serum of a treated individual after administration of the subject biotherapeutic for one or more days, e.g., one or more months, six or more months, nine or more months, or one or more years, compared to the level of ADA detected in serum from a control individual treated with the same dosing regimen and compared to the level of ADA over the same period with the corresponding non-engineered biotherapeutic. In certain embodiments, ADA to a hypoimmunogenic biotherapeutic is undetectable in the serum of an individual after administration of the subject biotherapeutic for one or more days, e.g., six or more months, nine or more months, or one or more years, while ADA can be detected in the serum of a control individual treated with the same dosing regimen and the corresponding non-engineered biotherapeutic for the same period. Such administration may be daily, weekly, biweekly, monthly, quarterly, semi-annually, annually, biennially, triennially, tetraennially, quinennially, or decennially.

[0073] In some embodiments, the engineered, low-immunogenic biotherapeutics of the present disclosure can be further engineered to contain increased amounts of ligands for the asialoglycoprotein receptor (ASGPR). The asialoglycoprotein receptor is a lectin that binds asialoglycoproteins to glycoproteins, removing sialic acid from them to expose galactose residues. Ligands for ASGPR, such as galactosyl moieties (galactose, galactosamine, N-acetylgalactosamine (GalNAc)), glucosyl moieties (glucose, glucosamine, N-acetylglucosamine (GlcNAc)), and glycomimetics thereof, have been shown to induce immune tolerance to antigens that would normally elicit a T cell response when covalently linked to the antigen; see, e.g., US2017 / 0007708A1, the entire disclosure of which is incorporated herein in its entirety. In certain embodiments, the ASGPR ligand is a naturally occurring galactosyl moiety, such as galactose, galactosamine, or GalNAc. In other embodiments, the ASGPR ligand is a glycosylated moiety such as glucose, glucosamine, or GlcNAc. In other embodiments, the ASGPR ligand is a synthetic ligand, for example, a glycomimetic such as those disclosed in Mamidyala, SK et al. (2012) J. Am. Chem. Soc. 2012, 134, 4, 1978-1981.

[0074] Methods for covalently associating ASGPR ligands to biotherapeutics are well known in the art (see, e.g., US2017 / 0007708A1, the entire disclosure of which is incorporated herein by reference), any of which may be used to engineer the biotherapeutics of the present disclosure. In some embodiments, the low-immunogenic biotherapeutic contains at least 2-fold more ASGPR ligand than a corresponding unengineered biotherapeutic that induces a T cell response in an individual, e.g., 3-fold or more, 4-fold or more, 5-fold or more, 6-fold or more, 7-fold or more, 8-fold or more, 9-fold or more, 10-fold or more, 11-fold or more, 12-fold or more, 13-fold or more, 14-fold or more, 15-fold or more, 16-fold or more, 17-fold or more, 18-fold or more, 19-fold or more, or even 20-fold more ASGPR ligand than the unengineered biotherapeutic. Any approach for measuring the content of glycans on a biotherapeutic composition can be used to determine the amount of ASGPR ligand attached to the biotherapeutic, including, for example, glycoprotein LC / MS, glycan LC / MS, capillary gel electrophoresis glycan analysis, analytical ion exchange HPLC, etc.

[0075] Covalent attachment of an ASGPR ligand to a subject low-immunogenic biotherapeutic is expected to target the subject low-immunogenic biotherapeutic to antigen-presenting cells in the liver (especially hepatocytes, which specifically bind ASGPR). Specificity of binding to antigen-presenting cells in the liver can be confirmed, for example, by labeling the subject low-immunogenic biotherapeutic, including the ASGPR ligand, with a marker (such as the fluorescent marker phycoerythrin ("PE"). The subject biotherapeutic is administered to suitable experimental subjects. A control, e.g., unconjugated PE or vehicle (saline), is administered to another group of subjects. The subject biotherapeutic and control are allowed to circulate for 1-5 hours, after which the subject's spleen and liver are harvested and assayed for fluorescence. The specific cells in which fluorescence is found can then be identified. When tested in this manner, the subject ASGPR ligand-associated biotherapeutic exhibits higher levels of concentration in antigen-presenting cells in the liver compared to unconjugated PE or vehicle.

[0076] Efficacy in immune modulation can be tested by measuring the proliferation of OT-1 CD8+ cells (transplanted into host mice) in response to administration of a subject's hypoimmunogenic biotherapeutic containing an ASGPR ligand, compared to administration of the hypoimmunogenic biotherapeutic alone or simply vehicle. When tested in this manner, ASGPR ligand-associated biotherapeutics demonstrate increased OT-1 cell proliferation and increased CD8+ T cell cross-priming compared to the biotherapeutic alone or vehicle. To distinguish T cells that have expanded to a functional effector phenotype from those that have expanded and been deleted, expanded OT-1 CD8+ T cells can be phenotypically analyzed for molecular signatures of exhaustion [such as programmed death-1 (PD-1), FasL, and others], as well as annexin-V binding as a hallmark of apoptosis and therefore deletion. OT-1 CD8+ T cells can also be assessed for their responsiveness to challenge with an unmanipulated biotherapeutic plus adjuvant to demonstrate functional non-responsiveness to the biotherapeutic and therefore immune tolerance. To this end, the compositions of the present disclosure are administered to host mice, and then cells are analyzed for inflammatory signatures, followed by protein challenge.Compared to control groups, the compositions of the present disclosure when tested in this manner exhibit very low (for example, background) levels of inflammatory OT-1CD8+ T cell responses to biotherapeutic agents, thus demonstrating immune tolerance.

[0077] The efficacy of a target ASGPR ligand-associated biotherapeutic in inducing tolerance in humans can be assessed by evaluating inflammatory signatures associated with T cell responses. Typically, a biotherapeutic having an ASGPR ligand covalently attached thereto induces a T cell response in an individual administered the biotherapeutic that is 50% or less of the T cell response elicited by the corresponding biotherapeutic, e.g., 40%, 30%, or 20% or less of that elicited by the corresponding non-engineered biotherapeutic, and in certain cases, 15%, 10%, 5% or less, preferably only 2% or 1% or less. The induction of tolerance by an ASGPR-associated, low-immunogenic biotherapeutic can be readily assessed by quantifying the anti-biotherapeutic antibody titer specific to the non-engineered biotherapeutic administered several weeks after treatment with the ASGPR ligand-associated biotherapeutic. When tested in this manner, the compositions of the present disclosure demonstrate lower levels of antibody formation in response to challenge with the biotherapeutic in groups pretreated with the ASGPR ligand-associated biotherapeutic compared to groups not pretreated.

[0078] As discussed above, the engineered, hypoimmunogenic biotherapeutic agents of the present disclosure contain one or more modifications disclosed herein that enable them to suppress an immune response, while retaining pharmacological activity. By "retaining pharmacological activity," we mean that the biotherapeutic is no more than 5-fold more therapeutically active than the corresponding non-engineered biotherapeutic when administered to a naive individual (i.e., the individual receiving treatment for the first time), and in some cases, no more than 3-fold more active, preferably no more than 2-fold more active, and more preferably at least as therapeutically active as the corresponding non-engineered biotherapeutic when administered to a naive individual.

[0079] In some cases, when Y is a polypeptide, the polypeptide conjugation reactive end of the linker is a site capable of conjugating to a polypeptide via a cysteine ​​thiol or lysine amine group on the polypeptide, and thus may be a thiol-reactive group such as maleimide or dibromomaleimide, or as defined herein, or an amine-reactive group such as an active ester (e.g., perfluorophenyl ester or tetrafluorophenyl ester), or a thiol-reactive group as defined herein. In other words, the bond between Y and L or X may be the product of the reaction between a cysteine, thiol, or lysine amine group on the polypeptide and a thiol-reactive group such as maleimide or dibromomaleimide on the L or X group, or an amine-reactive group on the L or X group.

[0080] In some embodiments, the X or L group is covalently attached to an amino acid residue or the terminus of a glycan on biotherapeutic Y that is typically not sialylated, i.e., the sialic acid residue is heterologous to the amino acid residue or glycan moiety. As used herein, the term "heterologous" refers to a component of a composition that is non-native to the composition, i.e., not typically found in nature in association with the rest of the entity to which it is compared. For example, sialic acid may be covalently attached to a glycan structure such as GO, G1, G2, GOF, G1F, or G2F. In some embodiments, sialic acid is covalently attached to a structure that is typically sialylated in glycans such as G1S, G2S, G2S2, G1FS, G2FS, and G2FS2. In some embodiments, sialic acid is covalently attached to an N- or O-linked native glycan present in the corresponding unengineered biotherapeutic that lacks the sialic acid modification. In other such embodiments, sialic acid is covalently attached to a novel N-linked glycan site. In other embodiments, sialic acid is directly covalently attached to an amino acid of the biotherapeutic, e.g., a random lysine or cysteine, an engineered transglutaminase site, an engineered catalent formylglycine aldehyde site using formylglycine generating enzyme (FGE), N-terminal selective conjugation to biotherapeutics containing an N-terminal 2-hydroxyethylamine (serine) moiety (SeriMab technology), or a novel O-linked glycan site. Any approach for determining the site of sialylation or Siglec ligand conjugation on a biotherapeutic may be used to determine the location of the Siglec ligand within the biotherapeutic, including, for example, LC / MS of proteolytic products (peptide mapping LC / MS) and LC / MS of larger product fragments (e.g., antibody Fc vs. light chain, Fd').

[0081] In some embodiments, X or L is covalently attached to a naturally occurring element of biotherapeutic Y, i.e., a glycan.

[0082] Additional Aspects As discussed above, some aspects of the present disclosure provide engineered, hypoimmunogenic biotherapeutics, wherein the engineered, hypoimmunogenic biotherapeutics (hereinafter referred to as "hypoimmunogenic biotherapeutics," "modified biotherapeutics," or simply "subject biotherapeutics") are biotherapeutics that have been engineered to include a modified Siglec ligand profile.

[0083] In some embodiments, the modified Siglec ligand profile comprises an enrichment for sialic acid relative to the parent biotherapeutic. In other words, the engineered, less immunogenic biotherapeutic is enriched for sialic acid, i.e., "highly silylated." For example, an engineered, less immunogenic biotherapeutic may comprise one or more sialic acid moieties, e.g., 1, 2, 3, 4 or more sialic acid moieties, and in some examples, 5, 6, 7, 8, 9, or 10 or more sialic acid moieties, and in some such examples, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more moieties, while the parent biotherapeutic, in another example, does not comprise a sialic acid moiety, the subject biotherapeutic may comprise two or more sialic acid moieties, e.g., 2, 3, 4 or more sialic acid moieties, and in some examples, 5, 6, 7, 8, 9, or 10 or more sialic acid moieties, and in some such examples, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more sialic acid moieties, while the parent biotherapeutic comprises only one sialic acid moiety. In some embodiments, the hypoimmunogenic biotherapeutic comprises 2-fold more sialic acid than a corresponding non-engineered biotherapeutic that induces an immune response in an individual, e.g., 3-fold or more, 4-fold or more, 5-fold or more, 6-fold or more, 7-fold or more, 8-fold or more, 9-fold or more, 10-fold or more, 11-fold or more, 12-fold or more, 13-fold or more, 14-fold or more, 15-fold or more, 16-fold or more, 17-fold or more, 18-fold or more, 19-fold or more, or even 20-fold more sialic acid than the non-engineered biotherapeutic. In some embodiments, 50% or more of the glycan moieties of the engineered immunosuppressive biotherapeutic comprise sialic acid, e.g., 60%, 70%, 80%, 85%, 90%, 95%, 98%, or 100% of the glycan moieties.

[0084] In some embodiments, 50% or more of the poorly immunogenic biotherapeutics in a sample are highly silylated, e.g., 60%, 70%, 80%, 85%, 90%, 95%, 98%, or 100% of the poorly immunogenic biotherapeutics in a sample are highly silylated. For example, 50% or more of the poorly immunogenic biotherapeutics in a sample can be highly silylated to the same or greater extent, including embodiments in which the poorly immunogenic biotherapeutics contain more sialic acid than the corresponding unengineered biotherapeutic that elicits an immune response in an individual, as described above. In some embodiments, 60%, 70%, 80%, 85%, 90%, 95%, 98%, or 100% of the poorly immunogenic biotherapeutics in a sample are highly silylated to the same or greater extent.

[0085] Any approach for measuring the sialylation, i.e., sialic acid content, of a biotherapeutic composition can be used to determine the amount of Siglec ligand applied to the biotherapeutic, including, for example, glycoprotein LC / MS, glycan LC / MS, protein LC / MS, intact drug LC / MS, capillary gel electrophoresis glycan analysis, analytical ion exchange HPLC, analytical reversed-phase HPLC, analytical hydrophobic interaction chromatography HPLC, analytical mixed-mode chromatography HPLC, total sialic acid or Siglec ligand analysis by plate-based assay, UV / Vis absorbance spectroscopy, surface plasmon resonance-based Siglec ligand quantification assay, biolayer interference-based Siglec ligand quantification assay, etc.

[0086] In some embodiments, the Siglec ligand comprises a Siglec-binding fragment derived from a Siglec-specific antibody, e.g., a Siglec-specific CDR, Fab, Fab', Fv, nanobody, etc., such as a monoclonal antibody, scFv, minibody, diabody, triabody, tetrabody, darpin, camelid nanobody, affimer, fynomer, bispecific antibody, triabody, etc. In some embodiments, the Siglec ligand comprises a Siglec-binding fragment derived from a Siglec-specific chimeric antigen receptor ("CAR"). In some embodiments, the Siglec-specific antibody or Siglec-specific CAR is specific for Siglec-2. In some such embodiments, the Siglec-2-specific antibody is selected from the group consisting of epratuzumab, inotuzumab, squirasulimab, bectomomab, pinatuzumab, GTB-1550, hLL2, RFB4, JNJ-75348780, HB-22.7, m971, H10-2-4, and moxetumomab. In some such embodiments, the Siglec ligand comprises a Siglec-binding fragment derived from an engineered scFv polypeptide sequence from epratuzumab, or a peptide selected from the group consisting of PV1 (GYINPRNDYTEYNQ), PV2 (CGYRNPRNDYREYCNQ), and PV3 (RNDYTE), the chemical structures of which can be found in Table 2 (Kim, B. et al. Nanoscale 2020, 12, 11672-11683). In certain such embodiments, the Siglec-binding fragment consists essentially of an scFv polypeptide sequence engineered from epratuzumab, or a peptide selected from the group consisting of PV1 (GYINPRNDYTEYNQ), PV2 (CGYRNPRNDYREYCNQ), and PV3 (RNDYTE). [Table 2]

[0087] In some such embodiments, the Siglec ligand is synthetically derived from monoclonal antibody polypeptides, including HB-22.5, 22.7, 22.23, 22.33, 22.13, and HB22.196, as described in Pearson, et al. (International Journal of Peptide Research and Therapeutics, 14, 3, 237-246 (2008)). One such peptide is "peptide 5," which is derived from the CDR2 region of monoclonal antibody HB22-7 and has the amino acid sequence CLGIIWGDGRTDYNSALKSRC and a disulfide bond between the N-terminal and C-terminal cysteines.

[0088] In some embodiments, the Siglec ligand comprises a Siglec-binding fragment derived from a Siglec-specific aptamer. Non-limiting examples of Siglec-specific aptamers comprising Siglec-binding fragments that find use in the subject biotherapeutics include TD-05, TD-05.1, and TD-05.17.

[0089] In some such embodiments, the Siglec ligand comprises a synthetic, non-antibody-derived Siglec-binding peptide that binds to CD22 with measurable affinity and high specificity. For example, the peptide may be one described in WO2014 / 044793, such as "peptide 26," also known as "G635BVI07IM1TK," having the amino acid sequence RALLSIFGSLDHRHHHRTCNTTHYRVTTMSHPQFEKKKKLRMKMSHPQLINTTHYRGGPTMGGSPSRQV.

[0090] Thus, in some embodiments, subject engineered, low-immunogenic biotherapeutics comprise biotherapeutics conjugated to one or more naturally occurring Siglec ligands, i.e., moieties comprising naturally occurring sialic acid and naturally occurring glycans, where the sialic acid and glycans are typically found in nature in association with one another to form Siglec ligands. In other embodiments, subject engineered, low-immunogenic biotherapeutics comprise biotherapeutics conjugated to one or more non-naturally occurring Siglec ligands, e.g., moieties comprising naturally occurring sialic acid and a linker, moieties comprising non-naturally occurring sialic acid and a glycan found in nature as part of a Siglec ligand, moieties comprising non-naturally occurring sialic acid and a linker, moieties comprising peptides with affinity for Siglecs, etc. For example, in some embodiments of the engineered, low-immunogenic biotherapeutics, the Siglec ligand is a non-naturally occurring Siglec ligand. In some embodiments, the non-naturally occurring Siglec ligand comprises naturally occurring sialic acid and a non-naturally occurring linker. In some embodiments, the non-naturally occurring Siglec ligand consists essentially of naturally occurring sialic acid and a non-naturally occurring linker. In some embodiments, the non-naturally occurring Siglec ligand comprises a non-naturally occurring sialic acid. In some embodiments, the non-naturally occurring Siglec ligand comprises a non-naturally occurring linker. In some embodiments, the non-naturally occurring Siglec ligand consists essentially of a non-naturally occurring sialic acid and a non-naturally occurring linker.

[0091] Manufacturing method As discussed above, a less immunogenic biotherapeutic is a biotherapeutic that has been modified to include a heterologous Siglec ligand (whether heterologous to the biotherapeutic or to the amino acids to which they are added) and / or an increased amount of native Siglec ligand on the biotherapeutic. Typically, the modification is not simply by associating the Siglec ligand with the biotherapeutic via a formulation, e.g., a liposomal formulation. Rather, the modification is a covalent attachment of the Siglec ligand to the biotherapeutic.

[0092] Methods for covalently attaching sialic acid to biotherapeutics are well understood in the art, and any of these can be deployed to modify a selected biotherapeutic to become an engineered, less immunogenic biotherapeutic of the present disclosure. For example, the modification can be performed by an engineered biotherapeutic. "Biosynthesis" refers to a synthetic process mediated by a cell. For example, in the Golgi apparatus, a subset of 20 known sialyltransferases attach sialic acid to a basic monosaccharide, such as galactose, via three different types of linkages (α2,3, α2,6, and α2,8). By engineered biosynthesis, it refers to a synthetic process mediated by a cell that has been engineered to perform the process, in some instances de novo, and in other cases, in a modified manner. Thus, for example, the producer cell line may contain one or more sialyltransferases, such as sialyltransferase (EC 2.4.99), beta-galactosamide α-2,6-sialyltransferase (EC 2.4.99.1), α-N-acetylgalactosaminide α-2,6-sialyltransferase (EC 2.4.99.3), beta-galactoside α-2,3-sialyltransferase (EC 2.4.99.4), N-acetyllactosaminide α-2,3-sialyltransferase (EC 2.4.99.6), α-N-acetyl-neuraminic acid α-2,8-sialyltransferase (EC 2.4.99.7), α-N-acetyl-neuraminic acid α-2,8-sialyltransferase (EC 2.4.99.8), α-N-acetyl-neuraminic acid α-2,8-sialyltransferase (EC 2.4.99.9), α-N-acetyl-neuraminic acid α-2,8-sialyltransferase (EC 2.4.99.1 ...2), α-N-acetyl-neuraminic acid α-2,8-sialyltransferase (EC 2.4.99.3), α-N-acetyl-neuraminic acid α-2,8-sialyltransferase (EC 2.4.99.4), α-N-acetyl-neuraminic acid α-2,8-sialyltransferase (EC 2.4.99.1), α-N-acetyl-neuraminic acid (EC 2.4.99.8); lactosylceramide α-2,3-sialyltransferase (EC 2.4.99.9) or other enzymes in the enzymatic pathway, such as CMP-Neu5Ac hydroxylase, sialate 4-O-acetyltransferase, sialate 4-O-acetylesterase, sialate 7(9)-O-acetyltransferase, sialate 8-O-methyltransferase, sialate 9-)-acetyltransferase, etc., to covalently attach specific sialic acids to biotherapeutics or to target specific novel amino acid residues for covalent modification with sialic acids. As another example, producer cell lines can be supplied with precursor substrates, which are incorporated by the producer strain into the manufactured biotherapeutics as specific Siglec ligands.Any production cell that finds use in expressing proteins for use as therapeutic biotherapeutics may be used in this process, for example, mammalian cells (such as CHO, HEK), insect cells (such as SF9), bacteria, protozoa (such as Leishmania), etc., as disclosed in WO2017 / 093291, WO2019 / 002512, WO2019 / 234021, the disclosures of which are incorporated herein by reference in their entireties.

[0093] As another example, modification may be achieved by chemical conjugation. "Chemical conjugation" refers to a process that is exogenous to the cell. Thus, for example, a Siglec ligand may be enzymatically or chemically linked to the biotherapeutic after biosynthesis from the production strain. Non-limiting examples of such in vitro processes are disclosed in U.S. Patent Nos. 7,220,555, 6,376,475B, and 5,409,817, the entire disclosures of which are incorporated herein by reference. In some such embodiments, a linker may be developed to covalently attach sialic acid to the biotherapeutic. Many examples of linkers exist in the art, any of which may be used to chemically conjugate sialic acid to the biotherapeutic to arrive at the low-immunogenic biotherapeutic of the present disclosure.

[0094] As a third example, specifically related to embodiments in which the Siglec ligand is a peptide or polypeptide sequence, e.g., an scFv or peptide derived from epratuzumab, e.g., PV1, PV2, or PV3, modification may be achieved by genetic engineering of the biotherapeutic to include the peptide / polypeptide sequence within the biotherapeutic. For example, a polynucleotide used to manufacture the biotherapeutic can be modified by standard molecular biology cloning techniques to include a polynucleotide sequence encoding the peptide / polypeptide in the same translational reading frame ("in frame"), such that upon transcription and translation of the biotherapeutic in a producing cell, the biotherapeutic will include the peptide / polypeptide sequence covalently linked to the amino acids that make up the biotherapeutic, resulting in a less immunogenic biotherapeutic. Preferably, the peptide / polypeptide sequence is genetically engineered into a domain of the biotherapeutic that is not involved in the therapeutic effect of the biotherapeutic, such as an enzymatic domain of an enzyme, a Fab, or more specifically, a CDR domain of an antibody. When modifying viral particles, the peptide / polypeptide sequence is preferably engineered into the capsid or envelope protein so that it is exposed on the outside of the viral particle, e.g., in an exposed loop of the viral capsid protein, a surface-exposed tegument protein, etc. Such structural features are well understood by those skilled in the art of viral therapy.

[0095] How to use In some aspects of the present invention, methods for the treatment of individuals in need of medical intervention are provided. The terms "treatment," "treating," and the like are used herein to generally mean obtaining a desired pharmacological and / or physiological effect. The effect can be preventative, in that a disease or its symptoms are completely or partially prevented, and / or therapeutic, in that a disease and / or side effects resulting from the disease are partially or completely cured. As used herein, "treatment" encompasses any treatment of a mammalian disease, including (a) preventing the disease from occurring in a subject who may be susceptible to the disease but has not yet been diagnosed with it; (b) suppressing the disease, i.e., arresting its development; or (c) relieving the disease, i.e., causing regression of the disease. Therapeutic agents can be administered before, during, or after the onset of a disease or injury. Treatment of ongoing disease, where treatment stabilizes or reduces undesirable clinical symptoms in the patient, is of particular interest. Such treatment is desirably administered before complete loss of function in affected tissues. The subject therapies are desirably administered during, and in some cases after, the symptomatic stage of the disease.

[0096] The terms "individual," "subject," "host," and "patient" are used interchangeably herein and refer to any mammalian subject, particularly humans, for whom diagnosis, treatment, or therapy is desired.

[0097] The hypoimmunogenic compositions of the present disclosure are particularly useful in the treatment of diseases that require repeated or chronic administration of a therapeutic agent to be effective. There are many examples of such conditions, some non-limiting examples of which are provided below and elsewhere. It is expected that one of skill in the art will be able to extrapolate from these examples to other indications and biotherapeutics known in the art.

[0098] For example, the individual may be suffering from a chronic autoimmune or inflammatory disease, such as rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, Crohn's disease, ulcerative colitis, psoriasis, mucositis, uveitis, and juvenile idiopathic arthritis. In such cases, the method may comprise administering to the individual a hypoimmunogenic TNFα-specific antibody, such as hypoimmunogenic adalimumab engineered from adalimumab or hypoimmunogenic infliximab engineered from infliximab, in an amount effective to treat the chronic immune disease.

[0099] As another example, the individual may be suffering from leukemia, e.g., ALL. In such cases, the method may include administering to the individual an engineered, hypoimmunogenic asparaginase from Erwinia chrysanthemi in an amount effective to treat the leukemia.

[0100] As another example, the individual may be suffering from colorectal cancer, non-small cell lung cancer, or head and neck cancer, in which case the method may include administering to the individual an engineered, hypoimmunogenic cetuximab in an amount effective to treat the colorectal cancer, non-small cell lung cancer, or head and neck cancer.

[0101] As another example, the individual may have multiple sclerosis. In such a case, the method may include administering to the individual an engineered, less immunogenic natalizumab, an engineered, less immunogenic IFNβ-1b, or an engineered, less immunogenic IFNβ-1a in an amount effective to treat multiple sclerosis.

[0102] As another example, the individual may be an organ transplant recipient and require an immunosuppressant to protect the transplanted tissue from rejection by the individual's immune system. In such cases, the method may include administering to the individual an engineered, hypoimmunogenic IdeS in an amount effective to prevent an antibody response against the transplanted tissue. In some embodiments, the transplanted organ is an allograft. In some embodiments, the transplanted organ is a xenograft. In some embodiments, the organ is selected from kidney, heart, lung, liver, pancreas, trachea, vascular tissue, skin, bone, cartilage, adrenal tissue, fetal thymus, and cornea.

[0103] As another example, the individual may have type 2 diabetes. In such a case, the method includes administering to the individual an engineered, less immunogenic exenatide or an engineered, less immunogenic albiglutide in an amount effective to treat diabetes.

[0104] As another example, the individual may be suffering from a complement-mediated disease. In such cases, the method would involve administering to the individual an engineered, hypoimmunogenic complement-degrading protease derived from a pathogen, such as a bacterial or fungal pathogen (e.g., Pseudomonas elastase (PaE), Pseudomonas alkaline protease (PaAP), Streptococcus pyrogenic exotoxin B (SpeB), gingipain from Porphyromonas gingivalis, Aspergillus alkaline protease 1 (Alp1), C. albicans secreted aspartyl proteinases 1 (Sap1), 2 (Sap2), and 3 (Sap3)), in an amount effective to degrade complement and treat the disease.

[0105] As another example, the individual may be suffering from an enzyme deficiency, in which case the method comprises administering to the individual an engineered, less immunogenic enzyme in an amount effective to treat the deficiency.Non-limiting examples of such enzyme deficiencies include PKU, in which a low immunogenic phenylalanine ammonia lyase is administered; Fabry disease, in which a low immunogenic alpha-galactosidase A is administered; Pompe disease, in which a low immunogenic acid alpha-glucosidase (GAA) is administered; Gaucher disease, in which a low immunogenic glucocerebrosidase (GCase) is administered; aspartylglucosaminuria, in which a low immunogenic aspartylglucosaminidase (AGA) is administered; hypophosphatasia (HPP), in which a low immunogenic asphaltase is administered; MPS I, in which a low immunogenic alpha-L-iduronidase is administered; MPS II, in which a low immunogenic iduronate sulfatase is administered; MPS IIIa, in which a low immunogenic sulfaminase is administered; and MPS IIIb, in which a low immunogenic alpha-N-acetylglucosaminidase (NAGLU) is administered. MPS IIIB, in which low-immunogenic heparin acetyl-CoA:α-glucosaminide N-acetyltransferase (HGSNAT) is administered; MPS IIIC, in which low-immunogenic N-acetylglucosamine 6-sulfatase (GNS) is administered; MPS IIID, in which low-immunogenic N-glucosamine 3-O-sulfatase (arylsulfatase G or ARSG) is administered; MPS IIIE, in which low-immunogenic N-acetylgalactosamine 6-sulfatase is administered; MPS IVA, in which low-immunogenic beta-galactosidase is administered; MPS IVB, in which low-immunogenic N-acetylgalactosamine 4-sulfatase is administered; MPS VI, in which low-immunogenic beta-glucuronidase is administered. Hemophilia VI, in which hypoimmunogenic factor VIII is administered, Hemophilia B in which hypoimmunogenic factor IX is administered, CLN1 form of Batten disease in which hypoimmunogenic palmitoyl protein thioesterase (PPT1) is administered, CLN2 form of Batten disease in which hypoimmunogenic tripeptidyl peptidase (TPP1) is administered, Arginase-1 deficiency in which hypoimmunogenic arginase-1 or pegdilarginase is administered, and Cystathionine beta synthase (CBS) deficiency (also known as Classical Homocystinuria) in which hypoimmunogenic cystathionine beta synthase or the Aeglea product AGLE-177 is administered.

[0106] As another example, an individual may be suffering from a disease that would benefit from gene therapy, e.g., a genetic disease or a complex disease (i.e., not limited to being associated with a specific genetic etiology) in which chronic expression of a therapeutic RNA or protein treats the condition. In such cases, the method involves administering to the individual an engineered, hypoimmunogenic viral particle containing a polynucleotide sequence (a "transgene") encoding a therapeutic gene product of interest in an amount effective to treat the disease. Non-limiting examples of suitable transgenes or gene products that can be delivered via the subject immunosuppressive viral particle include those associated with muscular dystrophy, cystic fibrosis, familial hypercholesterolemia, and rare or orphan diseases. Examples of such rare diseases may include, among others, spinal muscular atrophy (SMA), Huntington's disease, Rett syndrome (e.g., methyl-CpG binding protein 2 (MeCP2), UniProtKB-P51608), amyotrophic lateral sclerosis (ALS), Duchenne muscular dystrophy, Friedreich's ataxia (e.g., frataxin), spinocerebellar ataxia-associated ATXN2 type 2 (SCA2) / ALS; ALS-associated TDP-43, progranulin (PRGN) (associated with non-Alzheimer's brain degeneration, including frontotemporal dementia (FTD), progressive non-fluent aphasia (PNFA), and semantic dementia). See, e.g., www.orpha.net / consor / cgi-bin / Disease_Search_List.php; rarediseases.info.nih.gov / diseases.

[0107] Other useful therapeutic gene products that may be encoded by the transgene also include hormones and growth and differentiation factors, including, but not limited to, insulin, glucagon, glucagon-like peptide-1 (GLP-1), growth hormone (GH), parathyroid hormone (PTH), growth hormone-releasing factor (GRF), follicle-stimulating hormone (FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG), vascular endothelial growth factor (VEGF), angiopoietin, angiostatin, granulocyte colony-stimulating factor (GCSF), erythropoietin (EPO), connective tissue growth factor (CTGF), basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), epidermal growth factor (EGF), transforming growth factor α (TGFα), platelet-derived growth factor (PDGF), insulin growth factor I, and the like. and II (IGF-I and IGF-II), any one of the transforming growth factor b superfamily (TGFb, activin, inhibin), or any of the bone morphogenetic proteins (BMP) BMP1-15, any one of the heregluin / neuregulin / ARIA / neu differentiation factor (NDF) family of growth factors, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophins (NT-3 and NT-4 / 5), ciliary neurotrophic factor (CNTF), glial cell line-derived neurotrophic factor (GDNF), neurturin, agrin, any one of the semaphorin / collapsin family, netrin-1 and netrin-2, hepatocyte growth factor (HGF), ephrin, noggin, sonic hedgehog, and tyrosine hydroxylase.

[0108] Other useful transgenes include those encoding proteins that regulate the immune system, including, but not limited to, cytokines and lymphokines such as thrombopoietin (TPO), interleukins (IL), IL-1 through IL-25 (e.g., IL-2, IL-4, IL-12, and IL-18), monocyte chemotactic proteins, leukemia inhibitory factor, granulocyte-macrophage colony-stimulating factor, Fas ligand, tumor necrosis factors α and β, interferons a, b, and g, stem cell factor, and Hk-2 / flt3 ligand. Gene products produced by the immune system are also useful in the present invention. These include, but are not limited to, immunoglobulins IgG, IgM, IgA, IgD, and IgE, chimeric immunoglobulins, humanized antibodies, single-chain antibodies, T cell receptors, chimeric T cell receptors, single-chain T cell receptors, class I and class II MHC molecules, and engineered immunoglobulins and MHC molecules. Useful gene products also include complement regulatory proteins such as complement regulatory proteins, membrane cofactor protein (MCP), decay accelerating factor (DAF), CR1, CF2, and CD59.

[0109] Still other useful gene products include those that encode gene products for any one of the receptors for hormones, growth factors, cytokines, lymphokines, regulatory proteins, and immune system proteins.

[0110] Still other useful transgenes include those encoding receptors for cholesterol regulation, including the low density lipoprotein (LDL) receptor, the high density lipoprotein (HDL) receptor, the very low density lipoprotein (VLDL) receptor, and scavenger receptors. The present invention also encompasses gene products such as members of the steroid hormone receptor superfamily, including glucocorticoid and estrogen receptors, vitamin D receptors, and other nuclear receptors. Additionally, useful gene products include transcription factors such as jun, fos, max, mad, serum response factor (SRF), AP-1, AP2, myb, MyoD, and myogenin, ETS box containing proteins, TFE3, E2F, ATF1, ATF2, ATF3, ATF4, ZF5, NFAT, CREB, HNF-4, C / EBP, SP1, CCAAT box binding proteins, interferon regulatory factor (IRF-1), Wilms tumor protein, ETS binding proteins, STATs, GATA box binding proteins, e.g., GATA-3, and the forkhead family of winged helix proteins.

[0111] Other useful gene products include carbamoyl synthetase I, ornithine transcarbamylase, argininosuccinate synthetase, argininosuccinate lyase, arginase, fumarylacetoacetate hydrolase, phenylalanine hydroxylase, alpha-1 antitrypsin, glucose-e-phosphatase, porphobilinogen deaminase, Factor VIII, Factor IX, cystathione beta synthase, branched-chain keto acid decarboxylase, albumin, isovaleryl CoA dehydrogenase, propionyl CoA carboxylase, methylmalonyl CoA mutase, glutaryl CoA dehydrogenase, insulin, beta-glucosidase, pyruvate carboxylate, hepatic phosphorylase, phosphorylase kinase, glycine decarboxylase, H-protein, T-protein, cystic fibrosis transmembrane conductance regulator (CFTR) sequence, and dystrophin sequence, or functional fragments thereof. Still other useful gene products include enzymes that may be useful in enzyme replacement therapy, which is useful in various conditions caused by insufficient enzyme activity. For example, enzymes containing mannose-6-phosphate may be used in the therapy of lysosomal storage diseases (e.g., suitable genes include those encoding β-glucuronidase (GUSB)). In another example, the gene product is ubiquitin protein ligase E3A (UBE3A). Further useful gene products include UDP glucuronosyltransferase family 1 member A1 (UGT1A1).

[0112] In some embodiments, the gene product is not Factor VIII.

[0113] Other useful gene products include non-naturally occurring polypeptides, such as chimeric or hybrid polypeptides with non-naturally occurring amino acid sequences containing insertions, deletions, or amino acid substitutions. For example, single-chain engineered immunoglobulins may be useful in certain immunodeficient patients. Other types of non-naturally occurring gene sequences include antisense molecules and catalytic nucleic acids, such as ribozymes, which can be used to reduce overexpression of targets.

[0114] Reducing and / or modulating gene expression is particularly desirable for treating hyperproliferative conditions characterized by hyperproliferative cells, such as cancer and psoriasis. Target polypeptides include polypeptides produced exclusively or at higher levels in hyperproliferative cells compared to normal cells. Target antigens include polypeptides encoded by oncogenes such as myb, myc, and fyn, as well as translocation genes bcr / abl, ras, src, p53, neu, trk, and EGRF. In addition to oncogene products as target antigens, target polypeptides for anti-cancer therapeutic and protective regimens include variable regions of antibodies produced by B-cell lymphomas and variable regions of T-cell receptors in T-cell lymphomas, and in some embodiments are also used as target antigens for autoimmune diseases. Other tumor-associated polypeptides can be used as target polypeptides, such as polypeptides found at higher levels in tumor cells, including polypeptides recognized by monoclonal antibody 17-1A and folate-binding polypeptides.

[0115] Other suitable transgenes include those encoding therapeutic agents that may be useful for treating individuals suffering from autoimmune diseases and disorders by conferring a broad protective immune response against targets associated with autoimmunity, including cellular receptors and cells that produce "self"-directed antibodies. T cell-mediated autoimmune diseases include rheumatoid arthritis (RA), multiple sclerosis (MS), Sjögren's syndrome, sarcoidosis, insulin-dependent diabetes mellitus (IDDM), autoimmune thyroiditis, reactive arthritis, ankylosing spondylitis, scleroderma, polymyositis, dermatomyositis, psoriasis, vasculitis, Wegener's granulomatosis, Crohn's disease, and ulcerative colitis. Each of these diseases is characterized by a T cell receptor (TCR) that binds to an endogenous antigen and initiates the inflammatory cascade associated with the autoimmune disease.

[0116] Still other useful gene products include those used to treat hemophilia, including hemophilia B (including factor IX) and hemophilia A (including factor VIII and its variants, e.g., heterodimers and B-deleted domain light and heavy chains; U.S. Patent Nos. 6,200,560 and 6,221,349). In some embodiments, the minigene contains the first 57 base pairs of the factor VIII heavy chain encoding a 10-amino acid signal sequence and the human growth hormone (hGH) polyadenylation sequence. In alternative embodiments, the minigene further contains the A1 and A2 domains, and 5 amino acids from the N-terminus of the B domain and / or 85 amino acids from the C-terminus of the B domain, as well as the A3, Cl, and C2 domains. In yet other embodiments, nucleic acids encoding the factor VIII heavy and light chains are provided in a single minigene separated by 42 nucleic acids encoding the 14 amino acids of the B domain [U.S. Patent No. 6,200,560].

[0117] Additional exemplary genes that can be delivered via low-immunogenic viral particles include, but are not limited to, glucose-6-phosphatase, associated with glycogen storage disease or type 1A deficiency (GSD1), phosphoenolpyruvate-carboxykinase (PEPCK), associated with PEPCK deficiency, cyclin-dependent kinase-like 5 (CDKL5), also known as serine / threonine kinase 9 (STK9), associated with seizures and severe neurodevelopmental disorders, galactose-1-phosphate uridyltransferase, associated with galactosemia, phenylketone kinase, and phosphoenolpyruvate-carboxykinase (PGK). Gene products associated with primary hyperoxaluria type 1, including phenylalanine hydroxylase (PAH), hydroxyacid oxidase 1 (GO / HAOl), and AGXT, associated with oxaluria type 1 (PKU), branched-chain α-ketoacid dehydrogenases (including BCKDH, BCKDH-E2, BAKDH-Ela, and BAKDH-Elb) associated with maple syrup urine disease, fumarylacetoacetate hydrolase associated with tyrosinemia type 1, methylmalonyl-CoA mutase associated with methylmalonic acidemia, and medium-chain acyl-CoA deficiency associated with medium-chain acetyl-CoA deficiency. Dehydrogenase, ornithine transcarbamylase (OTC) associated with ornithine transcarbamylase deficiency, argininosuccinate synthetase (ASS1) associated with citrullinemia, lecithin-cholesterol acyltransferase (LCAT) deficiency, methylmalonic acidemia (MMA), NPC1 associated with Niemann-Pick disease (type C1), propionibacterium academia (PA), transthyretin (TTR) associated with TTR-related hereditary amyloidosis, and low-density lipoprotein (LDL) associated with familial hypercholesterolemia (FH). Protein receptor (LDLR) proteins, LDLR variants described in WO2015 / 164778, PCSK9, ApoE and ApoC proteins associated with dementia, UDP-glucourosyltransferase associated with Crigler-Najjar disease, adenosine deaminase associated with severe combined immunodeficiency, hypoxanthine guanine phosphoribosyltransferase associated with gout and Lesch-Naird syndrome, biotimidase associated with biotimidase deficiency, alpha-galactosidase A (α-GalA) associated with Fabry disease,Beta-galactosidase (GLB1) associated with GM1 gangliosidosis, ATP7B associated with Wilson's disease, β-glucocerebrosidase associated with Gaucher disease types 2 and 3, peroxisomal membrane protein 70 kDa associated with Zellweger syndrome, arylsulfatase A (ARSA) enzyme associated with metachromatic leukodystrophy, galactocerebrosidase (GALC) enzyme associated with Krabbe disease, α-glucosidase (GAA) associated with Pompe disease, sphingomyelinase (S) associated with Niemann-Pick disease type A. SMPD1 gene, argininosuccinate synthase associated with adult-onset type II citrullinemia (CTLN2), carbamoyl phosphate synthase 1 (CPS1) associated with urea cycle disorders, survival motor neuron (SMN) protein associated with spinal muscular atrophy, ceramidase associated with Farber lipogranulomatosis, b-hexosaminidase associated with GM2 gangliosidosis and Tay-Sachs and Sandhoff disease, aspartylglucosaminidase associated with aspartyl-glucosaminuria, and a-fucosidosis associated with fucosidosis. α-mannosidase, α-mannosidase associated with α-mannosidosis, porphobilinogen deaminase associated with acute intermittent porphyria (AIP), α-1 antitrypsin for the treatment of α-1 antitrypsin deficiency (emphysema), erythropoietin for the treatment of anemia due to thalassemia or renal failure, vascular endothelial growth factor, angiopoietin-1, and fibroblast growth factor for the treatment of ischemic disease, such as thrombomodulin and tissue factor for the treatment of blocked blood vessels found in atherosclerosis, thrombosis, or embolism. inhibitors of the mitochondrial pathway, aromatic amino acid decarboxylase (AADC) and tyrosine hydroxylase (TH) for the treatment of Parkinson's disease, beta-adrenergic receptors for the treatment of congestive heart failure, antisense to phospholamban or its mutants, sarco(endo)plasmic reticulum adenosine triphosphatase-2 (SERCA2), and cardiac adenylate cyclase, tumor suppressor genes such as p53 for the treatment of various cancers, cytokines such as one of the various interleukins for the treatment of inflammatory and immune disorders and cancer,Dystrophin or mini-dystrophin and utrophin or mini-utrophin for the treatment of muscular dystrophy, and insulin or GLP-1 for the treatment of diabetes.

[0118] Pharmaceutical Compositions In methods of treating an individual with a subject hypoimmunogenic biotherapeutic, the patient is typically administered a pharmaceutical composition comprising the subject hypoimmunogenic biotherapeutic. By pharmaceutical composition is meant the engineered hypoimmunogenic biotherapeutic of the present disclosure formulated in a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier, diluent, or excipient" includes, but is not limited to, any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier approved by the U.S. Food and Drug Administration as acceptable for use in humans or veterinary medicine.

[0119] The pharmaceutical compositions of the present disclosure are administered at a therapeutically effective dose, e.g., a dose sufficient to provide treatment for the aforementioned disease states. Administration of the compounds of the present disclosure or their pharmaceutically acceptable salts can be via any of the accepted modes of administration for agents providing similar utilities. While human dosage levels have not been optimized for the compounds of the present disclosure, they can be readily extrapolated from doses administered to relevant animal models, e.g., mice, that result in treatment of the disease or disorder in the animal model. Generally, individual human doses range from about 0.01 to 2.0 mg / kg body weight, preferably about 0.1 to 1.5 mg / kg body weight, and most preferably about 0.3 to 1.0 mg / kg body weight. Treatment can be administered for one or several days and can be repeated at intervals of several days, one or several weeks, or one or several months. Administration can be as a single dose (e.g., as a bolus) or as an initial bolus, followed by continuous infusion of the remainder of the complete dose over time, e.g., for one to seven days. The amount of active compound administered will depend on any or all of the following: the subject and disease state being treated, the severity of the affliction, the manner and schedule of administration, and the judgment of the prescribing physician, etc. It will also be understood that the amount administered will depend on the molecular weight of the biotherapeutic, the amount of covalently attached Siglec ligand, and the size of the linker.

[0120] While all typical routes of administration (e.g., oral, topical, transdermal, injection (intramuscular, intravenous, or intraarterial)) are contemplated, it is currently preferred to provide a liquid dosage form suitable for injection. Generally, depending on the intended mode of administration, a pharmaceutically acceptable composition will contain from about 0.1% to 95% by weight, preferably from about 0.5% to 50% by weight, of the subject low immunogenic biotherapeutic of the present disclosure, with the remainder being suitable pharmaceutical excipients, carriers, etc. Dosage forms or compositions can be prepared containing from 0.005% to 95% of the active ingredient, with the remainder consisting of a non-toxic carrier.

[0121] The subject pharmaceutical compositions can be administered alone or in combination with other pharmaceutical agents. These compositions can include other active agents, pharmaceutical agents, carriers, etc., including, but not limited to, other active agents that can act as immunomodulators and, more specifically, have inhibitory effects on B cells, including antifolates, immunosuppressants, cytostatics, antimitotics, and antimetabolites, or combinations thereof.

[0122] Liquid pharmaceutically administrable compositions can be prepared, for example, by dissolving, dispersing, etc., the active composition of the present disclosure (e.g., lyophilized powder) and optional pharmaceutical adjuvants in a carrier, such as water (water for injection), saline, dextrose water, glycerol, glycol, ethanol (excluding galactose), etc., thereby forming a solution or suspension. If desired, the administered pharmaceutical composition can also contain small amounts of non-toxic auxiliary substances such as wetting agents, emulsifiers, stabilizers, solubilizers, pH buffers, e.g., sodium acetate, sodium citrate, cyclodextrin derivatives, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, etc., osmolytes, amino acids, sugars and carbohydrates, proteins and polymers, salts, surfactants, chelating agents and antioxidants, preservatives, and specific ligands. Actual methods for preparing such dosage forms are known or will be apparent to those skilled in the art; see, for example, Remington: The Science and Practice of Pharmacy, 22nd Edition, 2012. The composition or formulation to be administered will, in any event, contain a quantity of the active compound(s) in an amount effective to treat the condition of the subject being treated. [Example]

[0123] The following examples are presented to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent that the following experiments are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. "Average" means the arithmetic mean. Standard abbreviations may be used, such as bp, base pairs; kb, kilobases; pl, picoliters; s or sec, seconds; min, minutes; h or hr, hours; aa, amino acids; kb, kilobases; bp, base pairs; nt, nucleotides; im, intramuscular; ip, intraperitoneal; sc, subcutaneous, etc.

[0124] General synthetic procedure All synthetic chemistry was performed in standard laboratory glassware unless otherwise indicated in the examples. Commercially available reagents were used as received. Microwave reactions were performed on an Anton Paar Monowave 400 using instrument software to control heating time and pressure. Analytical LC / MS was performed on either a Waters Acquity UPLC Instrument equipped with a PDA and a single quadrupole detector (alternating positive and negative ion scans) using Masslynx software or a Shimadzu LCMS-2020 using LabSolutions software. Retention times were determined from extracted 214 and / or 254 nm UV chromatograms. Preparative HPLC was performed on a Waters Autopurification system consisting of a fractionation module 2767, pump 2545, and 2998 PDA detector with Masslynx software, an Agilent 1260 Infinity Autopurification system with a DAD detector, or on a Gilson system with a 215 liquid handler, 333 and 334 pumps, a UV / VIS-155 detector, and Trilution lc software. 1 H NMR was performed on either a Bruker Avance 400 MHz or a Bruker Fourier 300 MHz using Topspin software. Analytical thin layer chromatography was performed on silica (Sigma Aldrich TLC Silica Gel 60 F 254 TLC was performed on aluminum or glass plates (silica gel coated with the fluorescent indicator F254) and visualized under UV light. Silica gel chromatography was performed manually or using a Teledyne ISCO CombiFlash NextGen300+ automated chromatograph for gradient elution.

[0125] Many general references are available that provide generally known chemical synthetic schemes and conditions useful for synthesizing the disclosed compounds (see, for example, Smith and March, March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, Fifth Edition, Wiley-Interscience, 2001, or Vogel, A Textbook of Practical Organic Chemistry, Including Qualitative Organic Analysis, Fourth Edition, New York: Longman, 1978).

[0126] During any of the processes for the preparation of the compound of interest, it may be necessary and / or desirable to protect any sensitive or reactive groups on the molecule in question. This can be achieved by following a variety of methods, including those described in J.F.W.M. Comie, "Protective Groups in Organic Chemistry," Plenum Press, London and New York, 1973; T.W. Greene and P.G.W. Muts, "Protective Groups in Organic Synthesis," Third Edition, Wiley, New York, 1999; "The Peptides"; Volume 3 (editors: E. Gross and J. Meienhofer), Academic Press, London and New York, 1981; "Methoden der organischen Chemie," Houben-Weyl, 4 thedition, Vol. 15 / 1, Georg Thieme Verlag, Stuttgart 1974; H.-D. Jakubke and H. Jescheit, "Aminosauren, Peptide, Protein," Verlag Chemie, Weinheim, Deerfield Beach, and Basel 1982; and / or Jochen Lehmann, "Chemie der Kohlenhydrate: Monosaccharide and Derivate," Georg Thieme Verlag, Stuttgart 1974. The protecting groups may be removed at a convenient subsequent stage using methods known in the art.

[0127] The compounds of the present invention can be synthesized via a variety of different synthetic routes using commercially available starting materials and / or starting materials prepared by conventional synthetic methods. Various examples of synthetic routes that can be used to synthesize the compounds disclosed herein are illustrated in the following schemes.

[0128] Example 1: Concept of a class of biotherapeutics involving CD22 that are suppressed due to anti-drug antibody responses B cells and their clonotypic B cell receptors are at the center of the antibody-based immune response to foreign drugs. Anti-drug antibodies are a ubiquitous challenge to drug exposure for many biotherapeutic classes, including monoclonal antibodies, bispecific and multispecific antibodies, enzyme replacement therapies, recombinant microbial enzymes, protein-Fc fusion proteins, intracellular delivery constructs, and gene therapy vectors.

[0129] A novel class of biomolecules and biotherapeutics with reduced or ablated humoral immunogenicity has been designed based on the principle that B cell clones with the potential to differentiate into anti-drug antibody-secreting plasma cells can be inhibited by recruiting Siglec inhibitory receptors to clonotypic anti-drug B cell receptors. Siglecs are a class of sialic acid-binding lectin proteins expressed on most or all types of hematopoietic cells.

[0130] Figure 1 shows one embodiment of a model for biotherapeutics that engage CD22 with suppressed anti-drug antibody responses. B cell receptor-Siglec ligand coengagers (including drug-Siglec ligand conjugates) suppress or silence drug-specific B cell activation by physically recruiting the inhibitory CD22 receptor to the B cell receptor complex.

[0131] Figure 2 shows another embodiment of a model for a biotherapeutic drug that engages CD22 with a suppressed anti-drug antibody response. B cell receptor-Siglec ligand coengagers (including drug-Siglec ligand conjugates) suppress, silence, or delete only drug-specific B cells, while leaving those B cell clones that are not drug-specific intact.

[0132] Example 2: Different formats of low or non-immunogenic biotherapeutics involving Siglec-2 / CD22 Four formats of biotherapeutics that engage Siglec-2 / CD22 may be engineered to be low or non-immunogenic. Figure 3 illustrates representative structures for each format. While antibodies are depicted to illustrate such biotherapeutics, other biological modalities (e.g., bispecific and multispecific antibodies, enzyme replacement therapies, recombinant microbial enzymes, protein-Fc fusion proteins, intracellular delivery constructs, gene therapy vectors) can be similarly engineered using the four formats presented. Formats 1, 2, and 3 use chemically incorporated, synthetic, small molecule Siglec ligands to confer Siglec-2 binding activity to a given drug. Such synthetic Siglec-2 ligand structures are described in Example 3.

[0133] "Format 1" is a biotherapeutic covalently modified on its polypeptide chain with one or more conjugable Siglec-2 ligand-linker structures. Conjugation of the Siglec-2 ligand-linker structure can be achieved by site-specific or non-site-specific methodologies.

[0134] "Format 2" refers to biotherapeutics modified on natural or engineered glycans bearing Siglec-2 ligand structures, where incorporation of the Siglec-2 ligand occurs biosynthetically during drug expression in cells. This approach includes providing cells with a Siglec-2 ligand-based substrate during drug expression to enable biosynthetic incorporation into the drug glycan. Incorporation of the Siglec ligand into the glycan can also be achieved by treatment with a Siglec-2 ligand-based enzyme substrate in an in vitro protein translation system.

[0135] "Format 3" is a biotherapeutic covalently modified on a native or engineered glycan with a Siglec-2 ligand structure. Glycan modification with a terminal Siglec-2 ligand structure is achieved by chemical and / or chemoenzymatic conjugation after purification of the biologic.

[0136] "Format 4" for engineering hypoimmune or non-immunogenic biologics relies on incorporating protein or peptide-based CD22 binding agents into the polypeptide chain of the biotherapeutic. Examples of such CD22 binding agents include: 1) immunoglobulin-based binding agents, such as Fab domains, single-chain Fv (scFv) fragments, diabodies, and single-chain antibody fragments (Camelidae V) H H or Shark V NAR), 2) non-immunoglobulin-based binding domains, e.g., affibodies, finomers, monobodies, DARPins, Knottins, variable lymphocyte receptors (VLRs), and affimers; 3) CD22-binding peptides, e.g., peptide aptamers; and 4) oligonucleotide-based Siglec binders, e.g., oligonucleotide aptamers.

[0137] All four exemplified formats enable CD22 recruitment to anti-drug, clonotypic B cell receptors on drug-specific B cells, resulting in the suppression of B cell activation, proliferation, and differentiation, ultimately blocking anti-drug antibody production.

[0138] Example 3: A set of conjugated linker compounds was designed and synthesized to establish the importance of drug-Siglec-2 binding and enhanced versus non-enhanced Siglec-2 binding for the suppression of B cell activation in vitro and anti-drug antibody responses in vivo. Figure 4 shows examples of conjugable CD22-binding Siglec ligand-linker structures, highlighting the structural components: Siglec ligand binding moiety, Siglec ligand proximal linker structure, linker, and reactive / conjugation group. Figure 5 shows exemplary Siglec ligand structures, highlighting elements that determine Siglec-2 binding affinity and species specificity. Figure 6 shows exemplary Siglec-2 ligand structures, illustrating structures with varying ligand valency. Figure 7 shows exemplary Siglec ligand structures, illustrating structures with varying linker structures, where the region proximal to the sialic acid-based moiety is composed of either a PEG-based structure or a galactose-based structure. Figure 8 shows examples of asialo negative control linker structures that are enhanced for Siglec-2 binding (top), not enhanced for Siglec-2 binding (middle), and not bound to Siglec-2 (bottom). Shown are an enhanced Siglec-ligand linker structure (compound no. 26288, Siglec ligand: methyl-α-9-N-(biphenyl-4-carbonyl)-amino-9-deoxy-N-glycolylneuraminic acid) (top), a Siglec ligand-linker structure containing a non-enhanced Siglec-2 binding moiety (compound no. 26614, Siglec ligand: N-glycolylneuraminic acid / Neu5Gc) (middle), and a PEG-based non-Siglec binding conjugated linker structure (compound no. 26530) (bottom). These compounds were synthesized to high purity and conjugated to different biotherapeutics and proteins, as shown in Example 5.

[0139] All synthetic chemistry is carried out in standard laboratory glassware unless otherwise indicated in the examples. Commercially available reagents are used as received. Microwave reactions are performed on an Anton Paar Monowave 400 using instrument software to control heating time and pressure. Analytical LC / MS is performed on either a Waters Acquity UPLC Instrument equipped with a PDA and single quadrupole detector (alternating positive and negative ion scans) using Masslynx software or a Shimadzu LCMS-2020 using LabSolutions software. Retention times are determined from extracted 214 and / or 254 nm UV chromatograms. Preparative HPLC is performed on a Waters Autopurification system consisting of a fractionation module 2767, pump 2545, and 2998 PDA detector with Masslynx software, an Agilent 1260 Infinity Autopurification system with a DAD detector, or on a Gilson system with a 215 liquid handler, 333 and 334 pumps, a UV / VIS-155 detector, and Trilution lc software. 1H NMR is performed on either a Bruker Avance 400 MHz or a Bruker Fourier 300 MHz using Topspin software. Analytical thin-layer chromatography is performed on silica (Sigma Aldrich TLC Silica Gel 60 F 254 TLC is performed on aluminum or glass plates (silica gel coated with the fluorescent indicator F254) and visualized under UV light. Silica gel chromatography is performed manually or using a Teledyne ISCO CombiFlash NextGen300+ automated chromatograph for gradient elution.

[0140] Example 4: Synthesis of conjugable Siglec ligands (2R,3S,4S,5R,6R)-2-(acetoxymethyl)-6-(but-3-yn-1-yloxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (Compound No. 26499) and (2R,3R,4S,5S,6R)-2-(but-3-yn-1-yloxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triyl tribenzoate (Example 1) [ka]

[0141] Synthesis of (2R,3S,4S,5R,6R)-2-(acetoxymethyl)-6-(but-3-yn-1-yloxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (3) Under an argon atmosphere, (2S,3R,4S,5S,6R)-6-(acetoxymethyl)tetrahydro-2H-pyran-2,3,4,5-tetrayltetraacetate (1, 10.0 g, 25.62 mmol) and but-3-yn-1-ol (2, 2.69 g, 38.43 mmol) were dissolved in anhydrous dichloromethane (100.0 mL) with stirring. To this solution, activated powdered 4 Å molecular sieves (10.0 g, 100% w / w) were added. The reaction mixture was stirred at room temperature for 30 min, then cooled to 0 °C, followed by the dropwise addition of boron trifluoride diethyl etherate (22.32 mL, 76.86 mmol) over 30 min. The mixture was stirred at room temperature for 12 h. Upon completion, the reaction mixture was quenched with triethylamine to neutral pH, filtered over Celite, and washed with dichloromethane (50 mL). Aqueous sodium bicarbonate (100 mL) was added to the filtrate with stirring. After 10 min, the organic layer was separated, washed with water (2 × 50 mL), dried over anhydrous sodium sulfate, and concentrated on a rotary evaporator to give the crude residue. The crude residue was purified by column chromatography (60–90% ethyl acetate in hexanes) to give (2R,3S,4S,5R,6R)-2-(acetoxymethyl)-6-(but-3-yn-1-yloxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (3) as a white solid. Yield: 8.50 g, 82.87%, ELSD-MS (ESI) m / z 401.38 [M+1] + .

[0142] (2R,3S,4S,5R,6R)-2-(acetoxymethyl)-6-(but-3-yn-1-yloxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (compound number 26499) To a stirred solution of (2R,3R,4S,5R,6R)-2(but-3-yn-1-yloxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol (3, 8.5.0 g, 21.23 mmol) in methanol (80 mL) was added sodium methoxide (25% in methanol) solution (0.44 mL, 2.12 mmol) dropwise at 0° C. The reaction mixture was stirred at room temperature for 1 hour. Upon completion, the reaction mixture was cooled to 0° C. and quenched with DOWEX hydrogen foam to maintain a pH of 6. The mixture was filtered through Celite and concentrated under reduced pressure to give a solid, which was then triturated with diethyl ether and filtered to give (2R,3R,4S,5R,6R)-2(but-3-yn-1-yloxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol (compound number 26499) as an off-white solid. Yield: 4.0 g, 81.13%, ELSD-MS (ESI) m / z 250.0 [M+18]. + .

[0143] Synthesis of (2R,3R,4S,5R,6R)-2-(but-3-yn-1-yloxy)-6-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydro-2H-pyran-3,4,5-triol (4) To a stirred solution of (2R,3R,4S,5R,6R)-2-(but-3-yn-1-yloxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol (26499, 1.0 gm, 4.31 mmol) in dry pyridine (10.0 ml) was added tert-butyl(chloro)dimethylsilane (0.779 gm, 5.17 mmol) at 0 °C. The reaction mixture was stirred at room temperature for 16 h. Upon completion, the reaction mixture was directly concentrated under reduced pressure to give a crude residue, which was then purified by column chromatography (70-95% ethyl acetate in hexanes) to give (2R,3R,4S,5R,6R)-2-(but-3-yn-1-yloxy)-6-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydro-2H-pyran-3,4,5-triol (4) as a colorless viscous liquid. Yield: 0.80 g, 53.62%. LCMS m / z 347.0 [M+1] + .

[0144] Synthesis of (2R,3R,4S,5S,6R)-2-(but-3-yn-1-yloxy)-6-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydro-2H-pyran-3,4,5-triyl tribenzoate (5) To a stirred solution of (2R,3R,4S,5R,6R)-2-(but-3-yn-1-yloxy)-6-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydro-2H-pyran-3,4,5-triol (4, 0.75 g, 2.16 mmol) in pyridine (7.5 mL) was added benzoyl chloride (2.01 mL, 17.3 mmol) at 0 °C. The mixture was stirred at room temperature overnight. After completion, the reaction mixture was directly concentrated on a rotary evaporator to give a crude residue, which was then purified by column chromatography (10–30% ethyl acetate in hexanes) to give (2R,3R,4S,5S,6R)-2-(but-3-yn-1-yloxy)-6-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydro-2H-pyran-3,4,5-triyl tribenzoate (5) as a colorless solid. Yield: 1.25g, 87.66%, LCMS m / z 659.44[M+1] + .

[0145] Synthesis of (2R,3R,4S,5S,6R)-2-(but-3-yn-1-yloxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triyl tribenzoate (Example 1) To a stirred solution of (2R,3R,4S,5S,6R)-2(but-3-yn-1-yloxy)-6-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydro-2H-pyran-3,4,5-triyltribenzoate (5, 3.0 g, 4.55 mmol) in oxolane (30.0 ml) was added tetrabutylazanium fluoride (6.83 mL, 6.83 mmol) at 0° C. The reaction mixture was stirred at 0° C. for 2.5 hours. After completion, the reaction mixture was quenched with water and extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate and evaporated under reduced pressure to give a crude residue, which was purified by column chromatography (30-40% ethyl acetate in hexanes) to give (2R,3S,4S,5R,6R)-4,5-bis(acetyloxy)-6-(but-3-yn-1-yloxy)-2-(hydroxymethyl)oxan-3-yl acetate (Example 1) as a white solid. Yield: 1.2 g, 48.39%, ELSD-MS (ESI) m / z 250.0 [M+18]. + . 1 H NMR (400MHz, methanol-d4) δ4.25 (d, J=7.2Hz, 1H), 3.98~3.92 (m, 3H), 3.81 (d, J=2.8Hz, 1H), 3.78~3.66 (m, 3H), 3.53~3.43 (m, 3H), 2.50 (dt, J=7.2 and 2.8Hz, 2H), 2.25 (t, J=2.4Hz, 1H).

[0146] (1R,2R)-1-((2R,3R,4S,6R)-3-acetamido-4-acetoxy-6-(methoxycarbonyl)-6-(p-tolylthio)tetrahydro-2H-pyran-2-yl)-3-(3-phenoxybenzamido)propane-1,2-diyl diacetate (Example 3) [ka] TIFF2024502850000012.tif135163

[0147] Synthesis of methyl (2R,4S,5R,6R)-5-acetamido-2,4-dihydroxy-6-((1R,2R)-1,2,3-trihydroxypropyl)tetrahydro-2H-pyran-2-carboxylate (2) To a stirred suspension of (2R,4S,5R,6R)-5-acetamido-2,4-dihydroxy-6-((1R,2R)-1,2,3-trihydroxypropyl)tetrahydro-2H-pyran-2-carboxylic acid (1, 100.0 g, 323.3 mmol) in anhydrous methanol (2500 mL) was added Amberlite IR-120 (H + ) resin (80.0 g) was added at room temperature under an argon atmosphere. The reaction mixture was stirred under an inert atmosphere until the suspension became a clear solution. The resin was removed by filtration, and the filtrate was concentrated under reduced pressure to give a residue. The residue was triturated with diethyl ether and filtered to give methyl (2R,4S,5R,6R)-5-acetamido-2,4-dihydroxy-6-((1R,2R)-1,2,3-trihydroxypropyl)tetrahydro-2H-pyran-2-carboxylate (2) as a pale pink solid. Yield: 104.0 g, 99.49%, LCMS (ESI) m / z 324.2 [M+1] + .

[0148] Synthesis of (1S,2R)-1-((2R,3R,4S,6S)-3-acetamido-4,6-diacetoxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)propane-1,2,3-triyl triacetate (3) In a 2000 mL round-bottom flask, methyl (2R,4S,5R,6R)-5-acetamido-2,4-dihydroxy-6-((1R,2R)-1,2,3-trihydroxypropyl)tetrahydro-2H-pyran-2-carboxylate (2, 102.0 g, 315 mmol) was dissolved in pyridine (600 mL) with stirring under an argon atmosphere. To this solution, acetic anhydride (298 mL, 3.15 mmol) was added dropwise with stirring over 30 minutes at 0 °C. The mixture was stirred overnight from 0 °C to room temperature. Upon completion, the reaction mixture was directly concentrated under reduced pressure on a rotary evaporator. The resulting thick syrup was then poured into a separatory funnel with ethyl acetate (500 mL) and washed with 1 N aqueous HCl (200 mL), followed by saturated sodium bicarbonate (200 mL) solution and DM water (2 × 200 mL). The organic layer was separated, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give a thick syrup. The syrup was triturated with diethyl ether and filtered to give (1S,2R)-1-((2R,3R,4S,6S)-3-acetamido-4,6-diacetoxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)propane-1,2,3-triyl triacetate (3) as a white solid. Yield: 130.0 g, 71.83%, LCMS (ESI) m / z 534.2 [M+1] + .

[0149] Synthesis of (1S,2R)-1-((2R,3R,4S,6R)-3-acetamido-4-acetoxy-6-(methoxycarbonyl)-6-(p-tolylthio)tetrahydro-2H-pyran-2-yl)propane-1,2,3-triyl triacetate (4) Under an argon atmosphere, (1S,2R)-1-((2R,3R,4S,6S)-3-acetamido-4,6-diacetoxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)propane-1,2,3-triyl triacetate (3, 130.0 g, 243.68 mmol) was dissolved in anhydrous dichloromethane (1300.0 mL) with stirring. To this solution, activated powdered 4 Å molecular sieves (40.0 g) were added. The reaction mixture was stirred at room temperature for 30 minutes and cooled to 0 °C. Subsequently, boron trifluoride diethyl etherate (111.0 mL, 365.5 mmol) was added dropwise over 30 minutes. The mixture was stirred at room temperature. Upon completion, the reaction mixture was quenched with triethylamine to neutral pH, filtered over Celite, and washed with dichloromethane (100 mL). Aqueous sodium bicarbonate (300 mL) was added to the filtrate with stirring. After 10 min, the organic layer was separated, washed with water (2 × 300 mL), dried over anhydrous sodium sulfate, and concentrated on a rotary evaporator to give a crude residue. The crude residue was purified by column chromatography (60–90% ethyl acetate in hexanes) to give (1S,2R)-1-((2R,3R,4S,6R)-3-acetamido-4-acetoxy-6-(methoxycarbonyl)-6-(p-tolylthio)tetrahydro-2H-pyran-2-yl)propane-1,2,3-triyl triacetate (4) as a white solid. Yield: 125.0 g, 85.83%, LCMS (ESI) m / z 598.32 [M+1] + .

[0150] Synthesis of methyl (2R,4S,5R,6R)-5-acetamido-4-hydroxy-2-(p-tolylthio)-6-((1R,2R)-1,2,3-trihydroxypropyl)tetrahydro-2H-pyran-2-carboxylate (5) To a stirred solution of (1S,2R)-1-((2R,3R,4S,6R)-3-acetamido-4-acetoxy-6-(methoxycarbonyl)-6-(p-tolylthio)tetrahydro-2H-pyran-2-yl)propane-1,2,3-triyl triacetate (4, 100.0 g, 167 mmol) in methanol (800 mL) was added sodium methoxide (25% in MeOH) solution (3.58 mL, 16.7 mmol) slowly at 0° C. The reaction mixture was stirred at room temperature for 2 hours. Upon completion, the reaction mixture was cooled to 0° C. and quenched with DOWEX hydrogen form to maintain pH 6. The mixture was filtered through Celite and concentrated under reduced pressure to give a solid, which was then triturated with diethyl ether and filtered to give methyl (2R,4S,5R,6R)-5-acetamido-4-hydroxy-2-(p-tolylthio)-6-((1R,2R)-1,2,3-trihydroxypropyl)tetrahydro-2H-pyran-2-carboxylate (5) as an off-white solid. Yield: 71.0 g, 98.80%, LCMS (ESI) m / z 430.10 [M+1] + .

[0151] Synthesis of methyl (2R,4S,5R,6R)-5-acetamido-6-((1R,2R)-1,2-dihydroxy-3-(tosyloxy)propyl)-4-hydroxy-2-(p-tolylthio)tetrahydro-2H-pyran-2-carboxylate (6) To a stirred solution of methyl (2R,4S,5R,6R)-5-acetamido-4-hydroxy-2-(p-tolylthio)-6-((1R,2R)-1,2,3-trihydroxypropyl)tetrahydro-2H-pyran-2-carboxylate (5, 40.0 g, 93.1 mmol) in pyridine (300 mL) was added dropwise a solution of 4-methylbenzene-1-sulfonyl chloride (30.2 g, 158 mmol) in pyridine (100 mL) at 0° C. The resulting reaction solution was stirred overnight. Upon completion, the reaction mixture was directly concentrated under reduced pressure to a thick syrup. The thick syrup was purified by flash column chromatography (80-95% ethyl acetate in hexanes) to give methyl (2R,4S,5R,6R)-5-acetamido-6-((1R,2R)-1,2-dihydroxy-3-(tosyloxy)propyl)-4-hydroxy-2-(p-tolylthio)tetrahydro-2H-pyran-2-carboxylate (6) as a white solid. Yield: 21.2 g, 39.0%. LC-MS (ESI) m / z 584.05 [M+1] + .

[0152] Synthesis of methyl (2R,4S,5R,6R)-5-acetamido-6-((1R,2R)-3-azido-1,2-dihydroxypropyl)-4-hydroxy-2-(p-tolylthio)tetrahydro-2H-pyran-2-carboxylate (7) In an inert atmosphere, methyl (2R,4S,5R,6R)-5-acetamido-6-((1R,2R)-1,2-dihydroxy-3-(tosyloxy)propyl)-4-hydroxy-2-(p-tolylthio)tetrahydro-2H-pyran-2-carboxylate (6, 21.0 g, 35.98 mmol) was dissolved in anhydrous N,N-dimethylformamide (210.0 mL) with stirring. To this solution, sodium azide (7.80 g, 120 mmol) was added at room temperature. The resulting reaction mixture was stirred at 60° C. for 16 hours. After completion, the reaction mixture was directly concentrated on a rotary evaporator to give a crude solid. The resulting crude solid was purified by column chromatography (80-95% ethyl acetate in hexanes) to give methyl (2R,4S,5R,6R)-5-acetamido-6-((1R,2R)-3-azido-1,2-dihydroxypropyl)-4-hydroxy-2-(p-tolylthio)tetrahydro-2H-pyran-2-carboxylate (7) as an off-white solid. Yield: 11.7 g, 71.55%, LCMS (ESI) m / z 453.19 [M-1]. - .

[0153] Synthesis of methyl (2R,4S,5R,6R)-5-acetamido-6-((1R,2R)-3-amino-1,2-dihydroxypropyl)-4-hydroxy-2-(p-tolylthio)tetrahydro-2H-pyran-2-carboxylate (8) To a stirred solution of methyl (2R,4S,5R,6R)-5-acetamido-6-((1R,2R)-3-amino-1,2-dihydroxypropyl)-4-hydroxy-2-(p-tolylthio)tetrahydro-2H-pyran-2-carboxylate (7, 10.0 g, 22.0 mmol) in tetrahydrofuran (100 mL) was added 10% Pd / C (10.0 g, 100% w / w) at room temperature. The reaction was then hydrogenated using balloon pressure of H gas for 12 hours. Upon completion, the reaction was filtered through Celite and the filtrate was concentrated. The residue was dried under vacuum to give crude methyl (2R,4S,5R,6R)-5-acetamido-6-((1R,2R)-3-amino-1,2-dihydroxypropyl)-4-hydroxy-2-(p-tolylthio)tetrahydro-2H-pyran-2-carboxylate (8) as a thick syrup. Yield 9.9 g, 105.3%, LCMS m / z 428.16 [M+1] + .

[0154] Synthesis of methyl (2R,4S,5R,6R)-5-acetamido-6-((1R,2R)-1,2-dihydroxy-3-(3-phenoxybenzamido)propyl)-4-hydroxy-2-(p-tolylthio)tetrahydro-2H-pyran-2-carboxylate (10) Methyl (2R,4S,5R,6R)-5-acetamido-6-((1R,2R)-3-amino-1,2-dihydroxypropyl)-4-hydroxy-2-(p-tolylthio)tetrahydro-2H-pyran-2-carboxylate (8, 9.90 g, 21.78 mmol) and 2,5-dioxopyrrolidin-1-yl 3-phenoxybenzoate (9, 7.91 g, 25.41 mmol) were dissolved in tetrahydrofuran (90.0 mL) with stirring under an argon atmosphere. To this solution was added ethylbis(propan-2-yl)amine (12.07 mL, 69.31 mmol) at 0° C. The resulting reaction mixture was stirred at room temperature for 12 hours. After completion, the reaction mixture was concentrated under reduced pressure to give the crude residue, which was then purified by column chromatography (80-90% ethyl acetate in hexanes) to give methyl (2R,4S,5R,6R)-5-acetamido-6-((1R,2R)-1,2-dihydroxy-3-(3-phenoxybenzamido)propyl)-4-hydroxy-2-(p-tolylthio)tetrahydro-2H-pyran-2-carboxylate (10) as a white solid. Yield: 8.10 g, 58.93% (over two steps), LCMS (ESI) m / z 625.23 [M+1]. + .

[0155] Synthesis of (1R,2R)-1-((2R,3R,4S,6R)-3-acetamido-4-acetoxy-6-(methoxycarbonyl)-6-(p-tolylthio)tetrahydro-2H-pyran-2-yl)-3-(3-phenoxybenzamido)propane-1,2-diyl diacetate (Example 3) To a stirred solution of methyl (2R,4S,5R,6R)-5-acetamido-6-((1R,2R)-1,2-dihydroxy-3-(3-phenoxybenzamido)propyl)-4-hydroxy-2-(p-tolylthio)tetrahydro-2H-pyran-2-carboxylate (10, 8.0 g, 12.81 mmol) in pyridine (80.0 mL) was added acetic anhydride (5.45 mL, 57.63 mmol) dropwise over 30 minutes at 0 °C. The mixture was stirred overnight from 0 °C to room temperature. Upon completion, the reaction mixture was directly concentrated under vacuum. The resulting thick syrup was poured into a separatory funnel with ethyl acetate (80.0 mL) and washed with 1 N HCl solution (50 mL), followed by saturated sodium sulfate solution (50 mL) and DM water (2 × 100 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give the crude residue. The residue was purified by column chromatography (55-70% ethyl acetate in hexanes) to give (1R,2R)-1-((2R,3R,4S,6R)-3-acetamido-4-acetoxy-6-(methoxycarbonyl)-6-(p-tolylthio)tetrahydro-2H-pyran-2-yl)-3-(3-phenoxybenzamido)propane-1,2-diyl diacetate (Example 3) as a white solid. Yield: 4.80 g, 49.92%, LCMS (ESI) m / z 751.25 [M+1] + . 1 H NMR (400 MHz, methanol-d4) δ 8.20 (d, J = 9.6 Hz, 1H), 7.82 (t, J = 5.6 Hz, 1H), 7.57-7.35 (m, 7H), 7.17-7.01 (m, 6H), 5.44-5.42 (m, 1H), 5.35 (dt, J = 10.4 and 4.8 Hz, 1H), 4.75 (dd, J=10.4 and 1.8Hz, 1H), 4.03(d, J=10.4Hz, 2H), 3.85~3.74(m, 1H), 3.28~4.26(m , 1H), 2.61 (dd, J=14.0 and 4.8Hz, 1H), 2.19 (s, 3H), 2.08~1.96 (m, 7H), 1.85 (s, 3H).

[0156] (1R,2R)-3-(2-([1,1′-biphenyl]-4-yl)acetamido)-1-((2R,3R,4S,6R)-4-acetoxy-3-(2-acetoxyacetamido)-6-(methoxycarbonyl)-6-(p-tolylthio)tetrahydro-2H-pyran-2-yl)propane-1,2-diyl diacetate (Example 4) [ka]

[0157] Synthesis of methyl (2R,4S,5R,6R)-5-amino-6-((1R,2R)-3-azido-1,2-dihydroxypropyl)-4-hydroxy-2-(p-tolylthio)tetrahydro-2H-pyran-2-carboxylate (2) To a stirred solution of methyl (2R,4S,5R,6R)-5-acetamido-6-((1R,2R)-3-azido-1,2-dihydroxypropyl)-4-hydroxy-2-(p-tolylthio)tetrahydro-2H-pyran-2-carboxylate (1, 21.0 g, 46.20 mmol) in methanol (210.0 mL) was added methanesulfonic acid (18.0 mL, 277.2 mmol) dropwise at 0° C. The resulting reaction mixture was stirred at 63° C. for 30 hours. Upon completion, the reaction mixture was cooled to 0° C. and quenched with triethylamine (approximately 15.0 mL, pH 7). The mixture was concentrated under reduced pressure to give crude methyl (2R,4S,5R,6R)-5-amino-6-((1R,2R)-3-azido-1,2-dihydroxypropyl)-4-hydroxy-2-(p-tolylthio)tetrahydro-2H-pyran-2-carboxylate (2) as a light brown gel. Yield: 19.0 g, 99.68%, LC-MS (ESI) m / z 413.57 [M+1] + .

[0158] Synthesis of methyl (2R,4S,5R,6R)-5-(2-acetoxyacetamido)-6-((1R,2R)-3-azido-1,2-dihydroxypropyl)-4-hydroxy-2-(p-tolylthio)tetrahydro-2H-pyran-2-carboxylate (3) In an inert atmosphere, crude methyl (2R,4S,5R,6R)-5-amino-6-((1R,2R)-3-azido-1,2-dihydroxypropyl)-4-hydroxy-2-(p-tolylthio)tetrahydro-2H-pyran-2-carboxylate (2, 19.0 g, 46.06 mmol) was dissolved in dry tetrahydrofuran (200.0 mL) with stirring and cooled to 0° C. To this solution was added triethylamine (17.70 mL, 138.2 mmol) followed by 2-chloro-2-oxoethyl acetate (4.95 mL, 46.06 mmol) slowly at 0° C. The reaction was stirred at 0° C. until completion. The mixture was concentrated under reduced pressure to give a crude residue, which was then purified by column chromatography (60-75% ethyl acetate in hexanes) to give methyl (2R,4S,5R,6R)-5-(2-acetoxyacetamido)-6-((1R,2R)-3-azido-1,2-dihydroxypropyl)-4-hydroxy-2-(p-tolylthio)tetrahydro-2H-pyran-2-carboxylate (3) as a white solid. Yield: 15.2 g, 64.38%, LCMS (ESI) m / z 513.42 [M+1] + .

[0159] Synthesis of methyl (2R,4S,5R,6R)-6-((1R,2R)-3-azido-1,2-dihydroxypropyl)-4-hydroxy-5-(2-hydroxyacetamido)-2-(p-tolylthio)tetrahydro-2H-pyran-2-carboxylate (4) To a stirred solution of methyl (2R,4S,5R,6R)-5-(2-acetoxyacetamido)-6-((1R,2R)-3-azido-1,2-dihydroxypropyl)-4-hydroxy-2-(p-tolylthio)tetrahydro-2H-pyran-2-carboxylate (3, 15.0 g, 29.27 mmol) in methanol (150.0 mL) was added sodium methoxide solution (25% in methanol, 0.061 ml, 2.93 mmol) slowly at 0° C. The reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was cooled to 0° C. and quenched with DOWEX hydrogen form to maintain a pH of 6. The mixture was filtered through Celite and concentrated under reduced pressure to give a solid, which was triturated with diethyl ether and filtered on a centered funnel to give methyl (2R,4S,5R,6R)-6-((1R,2R)-3-azido-1,2-dihydroxypropyl)-4-hydroxy-5-(2-hydroxyacetamido)-2-(p-tolylthio)tetrahydro-2H-pyran-2-carboxylate (4) as an off-white solid. Yield: 13.0 g, 94.41%, LCMS (ESI) m / z 471.15 [M+1] + .

[0160] Synthesis of methyl (2R,4S,5R,6R)-6-((1R,2R)-3-amino-1,2-dihydroxypropyl)-4-hydroxy-5-(2-hydroxyacetamido)-2-(p-tolylthio)tetrahydro-2H-pyran-2-carboxylate (5) To a stirred solution of methyl (2R,4S,5R,6R)-6-((1R,2R)-3-azido-1,2-dihydroxypropyl)-4-hydroxy-5-(2-hydroxyacetamido)-2-(p-tolylthio)tetrahydro-2H-pyran-2-carboxylate (4, 13.0 g, 27.63 mmol) in methanol (130 mL) was added 10% Pd / C (13.0 g, 100% w / w) at room temperature. The reaction was then hydrogenated using balloon pressure of H gas for 12 hours. Upon completion, the reaction was filtered through Celite and the filtrate was concentrated. The resulting residue was then dried under high vacuum to give crude methyl (2R,4S,5R,6R)-6-((1R,2R)-3-amino-1,2-dihydroxypropyl)-4-hydroxy-5-(2-hydroxyacetamido)-2-(p-tolylthio)tetrahydro-2H-pyran-2-carboxylate (5) as a thick syrup. Yield: 12.2 g, 99.41%, LCMS (ESI) m / z 445.16 [M+1] + .

[0161] Synthesis of methyl (2R,4S,5R,6R)-6-((1R,2R)-3-(2-([1,1'-biphenyl]-4-yl)acetamido)-1,2-dihydroxypropyl)-4-hydroxy-5-(2-hydroxyacetamido)-2-(p-tolylthio)tetrahydro-2H-pyran-2-carboxylate (7) To a stirred solution of methyl (2R,4S,5R,6R)-6-((1R,2R)-3-amino-1,2-dihydroxypropyl)-4-hydroxy-5-(2-hydroxyacetamido)-2-(p-tolylthio)tetrahydro-2H-pyran-2-carboxylate (5, 12.2 g, 27.45 mmol) and 2,5-dioxocyclopentyl 2-([1,1′-biphenyl]-4-yl)acetate (6, 10.19 g, 32.94 mmol) in tetrahydrofuran (40.0 mL) was added ethyl bis(propan-2-yl)amine (22.4 mL, 137.23 mmol) at 0° C. The resulting reaction mixture was stirred at room temperature for 12 hours. After completion, the mixture was concentrated under reduced pressure to provide a crude residue. The crude residue was purified by column chromatography (80-90% ethyl acetate in hexanes) to give methyl (2R,4S,5R,6R)-6-((1R,2R)-3-(2-([1,1'-biphenyl]-4-yl)acetamido)-1,2-dihydroxypropyl)-4-hydroxy-5-(2-hydroxyacetamido)-2-(p-tolylthio)tetrahydro-2H-pyran-2-carboxylate (7) as a white solid. Yield: 8.0 g, 45.63%, LCMS (ESI) m / z 639.23 [M+1] + .

[0162] Synthesis of (1R,2R)-3-(2-([1,1'-biphenyl]-4-yl)acetamido)-1-((2R,3R,4S,6R)-4-acetoxy-3-(2-acetoxyacetamido)-6-(methoxycarbonyl)-6-(p-tolylthio)tetrahydro-2H-pyran-2-yl)propane-1,2-diyl diacetate (Example 4) To a stirred solution of methyl (2R,4S,5R,6R)-6-((1R,2R)-3-(2-([1,1'-biphenyl]-4-yl)acetamido)-1,2-dihydroxypropyl)-4-hydroxy-5-(2-hydroxyacetamido)-2-(p-tolylthio)tetrahydro-2H-pyran-2-carboxylate (7, 8.0 g, 12.52 mmol) in pyridine (80.0 mL) was added acetic anhydride (11.61 mL, 125.2 mmol) dropwise over 30 minutes at 0 °C. The reaction mixture was stirred from 0 °C to room temperature overnight. Upon completion, the volatiles were removed under vacuum to give a crude thick syrup. The crude thick syrup was then poured into a separatory funnel with ethyl acetate (240.0 mL) and washed with 1 N HCl solution, followed by saturated sodium sulfate solution. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give a crude thick syrup. The crude thick syrup was purified by column chromatography (60-70% ethyl acetate in hexanes) to give (1R,2R)-3-(2-([1,1'-biphenyl]-4-yl)acetamido)-1-((2R,3R,4S,6R)-4-acetoxy-3-(2-acetoxyacetamido)-6-(methoxycarbonyl)-6-(p-tolylthio)tetrahydro-2H-pyran-2-yl)propane-1,2-diyl diacetate (Example 4) as a white solid. Yield: 5.40 g, 53.43%, LCMS (ESI) m / z 807.2 [M+1] + . 1 H NMR (400MHz, methanol-d4) δ7.27(d, J=8.4Hz, 2H), 7.14(d, J=8.4Hz, 2H), 4.03(t, J=8. 4Hz, 1H), 3.77(d, J=7.6Hz, 2H), 3.51~3.47(m, 2H), 3.20(t, J=6.4Hz, 2H), 2.91(dd, J=9.6, 14.4Hz, 1H), 2.82(dd, J=6, 14Hz, 1H), 2.24~2.20(m, 3H), 2.07(d, J=9.6Hz, 1 H), 1.75(d, J=12.8Hz, 2H), 1.68~1.62(m, 2H), 1.60~1.57(m, 2H), 1.56~1.47(m, 1H).

[0163] (2R,4S,5R,6R)-5-acetamido-6-((1R,2R)-1,2-dihydroxy-3-(3-phenoxybenzamido)propyl)-4-hydroxy-2-(2-(2-((1-(15-oxo-15-(perfluorophenoxy)-3,6,9,12-tetraoxapentadecyl)-1H-1,2,3-triazol-4-yl)methoxy)ethoxy)ethoxy)tetrahydro-2H-pyran-2-carboxylic acid (compound number 24836) [ka]

[0164] Synthesis of (2R,4S,5R,6R)-5-acetamido-6-((1R,2R)-1,2-dihydroxy-3-(3-phenoxybenzamido)propyl)-4-hydroxy-2-(2-(2-((1-(15-oxo-15-(perfluorophenoxy)-3,6,9,12-tetraoxapentadecyl)-1H-1,2,3-triazol-4-yl)methoxy)ethoxy)ethoxy)tetrahydro-2H-pyran-2-carboxylic acid (compound number 24836) To a stirred solution of (2R,4S,5R,6R)-5-acetamido-6-((1R,2R)-1,2-dihydroxy-3-(3-phenoxybenzamido)propyl)-4-hydroxy-2-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)tetrahydro-2H-pyran-2-carboxylic acid (26334, 0.025 g, 0.039 mmol) and perfluorophenyl 1-azido-3,6,9,12-tetraoxapentadecan-15-oate (1, 0.020 g, 0.043 mmol) in dimethyl sulfoxide (2.0 mL) was added tetrakis(acetonitrile)copper(I) hexafluorophosphate (0.041 g, 0.111 mmol). The resulting reaction mixture was stirred at room temperature for 30 minutes. Acetic acid (0.5 mL) was then added, and the reaction mixture was diluted with acetonitrile and purified by preparative HPLC (20–42% acetonitrile in water with 0.1% TFA). Fractions containing the desired product were combined and lyophilized to give (2R,4S,5R,6R)-5-acetamido-6-((1R,2R)-1,2-dihydroxy-3-(3-phenoxybenzamido)propyl)-4-hydroxy-2-(2-(2-((1-(15-oxo-15-(perfluorophenoxy)-3,6,9,12-tetraoxapentadecyl)-1H-1,2,3-triazol-4-yl)methoxy)ethoxy)ethoxy)tetrahydro-2H-pyran-2-carboxylic acid (compound no. 24836) as an amorphous solid. Yield: 0.018 g, 41.73%, LCMS, m / z 1088.53 [M+1] + ; 1 H NMR (400MHz, DMSO-d6 with D2O exchange) δ8.00 (d, J=3.2Hz, 1H), 7.59 (d, J=7.2Hz, 1H) , 7.46~7.36(m, 4H), 7.16~7.12(m, 2H), 6.99(d, J=8.0Hz, 2H), 4.48~4.45(m, 4 H), 3.76~3.72(m, 6H), 3.62~3.57(m, 2H), 3.56~3.43(m, 23H), 3.24~3.20(m, 2 H), 2.95(t, J=6.0Hz, 2H), 2.42~2.39(m, 1H), 1.84(s, 3H), 1.53~1.47(m, 1H).

[0165] (2S,4S,5R,6R)-5-Acetamido-6-((1R,2R)-1,2-dihydroxy-3-(3-phenoxybenzamido)propyl)-4-hydroxy-2-(2-(2-((1-(15-oxo-15-(perfluorophenoxy)-3,6,9,12-tetraoxapentadecyl)-1H-1,2,3-triazol-4-yl)methoxy)ethoxy)ethoxy)tetrahydro-2H-pyran-2-carboxylic acid (compound number 24838) [ka] TIFF2024502850000016.tif116159 Room temperature

[0166] Synthesis of (2R,4S,5R,6R)-5-acetamido-6-((1R,2R)-1,2-dihydroxy-3-(3-phenoxybenzamido)propyl)-4-hydroxy-2-(2-(2-((1-(15-oxo-15-(perfluorophenoxy)-3,6,9,12-tetraoxapentadecyl)-1H-1,2,3-triazol-4-yl)methoxy)ethoxy)ethoxy)tetrahydro-2H-pyran-2-carboxylic acid (compound number 24838) To a stirred solution of (2S,4S,5R,6R)-5-acetamido-6-((1R,2R)-1,2-dihydroxy-3-(3-phenoxybenzamido)propyl)-4-hydroxy-2-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)tetrahydro-2H-pyran-2-carboxylic acid (26335, 0.035 g, 0.055 mmol) and perfluorophenyl 1-azido-3,6,9,12-tetraoxapentadecan-15-oate (1, 0.028 g, 0.061 mmol) in dimethyl sulfoxide (2.0 mL) was added tetrakis(acetonitrile)copper(I) hexafluorophosphate (0.057 g, 0.155 mmol). The resulting reaction mixture was stirred at room temperature for 30 minutes. Acetic acid (0.5 mL) was added, and the reaction mixture was diluted with acetonitrile and purified by preparative HPLC (18–40% acetonitrile in water with 0.1% TFA). Fractions containing the desired product were combined and lyophilized to give (2R,4S,5R,6R)-5-acetamido-6-((1R,2R)-1,2-dihydroxy-3-(3-phenoxybenzamido)propyl)-4-hydroxy-2-(2-(2-((1-(15-oxo-15-(perfluorophenoxy)-3,6,9,12-tetraoxapentadecyl)-1H-1,2,3-triazol-4-yl)methoxy)ethoxy)ethoxy)tetrahydro-2H-pyran-2-carboxylic acid (compound no. 24838) as an amorphous solid. Yield: 0.017 g, 28.15%, LCMS, m / z 1088.56 [M+1] + ; 1 H NMR (400MHz, DMSO-d6 with D2O exchange) δ7.98(s, 1H), 7.57(d, J=7.6Hz, 1H), 7.47~7.36(m , 4H), 7.16~7.12(m, 2H), 6.98(d, J=8.0Hz, 2H), 4.74~4.44(m, 4H), 3.76~3.55(m, 9 H), 3.50~3.42(m, 17H), 3.30~3.17(m, 3H), 3.04(dd, J=14.8 and 7.6Hz, 2H), 2.93(t, J=6.0Hz, 2H), 2.16~2.11(m, 1H), 1.84(s, 3H), 1.53~1.47(m, 1H) 1.19~1.13(m, 3H).

[0167] (2R,4S,5R,6R)-5-Acetamido-6-((1R,2R)-1,2-dihydroxy-3-(3-phenoxybenzamido)propyl)-4-hydroxy-2-(2-(2-((1-(15-oxo-3,6,9,12-tetraoxa-16-azaoctadecyl)-1H-1,2,3-triazol-4-yl)methoxy)ethoxy)ethoxy)tetrahydro-2H-pyran-2-carboxylic acid (compound number 24839) [ka]

[0168] Synthesis of (2R,4S,5R,6R)-5-acetamido-6-((1R,2R)-1,2-dihydroxy-3-(3-phenoxybenzamido)propyl)-4-hydroxy-2-(2-(2-((1-(15-oxo-3,6,9,12-tetraoxa-16-azaoctadecyl)-1H-1,2,3-triazol-4-yl)methoxy)ethoxy)ethoxy)tetrahydro-2H-pyran-2-carboxylic acid (compound number 24839) To a stirred solution of (2R,4S,5R,6R)-5-acetamido-6-((1R,2R)-1,2-dihydroxy-3-(3-phenoxybenzamido)propyl)-4-hydroxy-2-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)tetrahydro-2H-pyran-2-carboxylic acid (26334, 0.025 g, 0.039 mmol) and 1-azido-N-ethyl-3,6,9,12-tetraoxapentadecan-15-amide (1, 0.014 g, 0.043 mmol) in dimethyl sulfoxide (2.0 mL) was added tetrakis(acetonitrile)copper(I) hexafluorophosphate (0.041 g, 0.111 mmol). The resulting reaction mixture was stirred at room temperature for 30 minutes. Acetic acid (0.5 mL) was then added, and the reaction mixture was diluted with acetonitrile and purified by preparative HPLC (20–42% acetonitrile in water with 0.1% TFA). Fractions containing the desired product were combined and lyophilized to give (2R,4S,5R,6R)-5-acetamido-6-((1R,2R)-1,2-dihydroxy-3-(3-phenoxybenzamido)propyl)-4-hydroxy-2-(2-(2-((1-(15-oxo-3,6,9,12-tetraoxa-16-azaoctadecyl)-1H-1,2,3-triazol-4-yl)methoxy)ethoxy)ethoxy)tetrahydro-2H-pyran-2-carboxylic acid (compound number 24839) as an off-white solid. Yield: 0.026 g, 60.28%, LCMS, m / z 475.62 [M / 2+1]. + ; 1H NMR (400MHz, DMSO-d6) δ13.84(bs, 1H), 8.31(d, J=4.8Hz, 1H), 8.04(s, 1H), 7.97(d, J=6.8Hz, 1H), 7.80~7.77(m, 1H), 7.64(d, J=7.6Hz, 1H), 7.48~7.38(m, 4H), 7.18~7.14(m, 2H), 7.02(d, J=8.0Hz, 2H), 4.51~4.48(m, 4H), 3.81~3.72(m, 4H), 3.64~3.46(m, 27H), 3.26~3.16(m, 1H), 3 .07~3.02(m, 2H), 2.65(t, J=6.0Hz, 2H), 1.86(s, 3H), 1.55~1.49(m, 1H) 0.98(t, J=7.2Hz, 3H).

[0169] (2S,4S,5R,6R)-5-Acetamido-6-((1R,2R)-1,2-dihydroxy-3-(3-phenoxybenzamido)propyl)-4-hydroxy-2-(2-(2-((1-(15-oxo-3,6,9,12-tetraoxa-16-azaoctadecyl)-1H-1,2,3-triazol-4-yl)methoxy)ethoxy)ethoxy)tetrahydro-2H-pyran-2-carboxylic acid (compound number 24840) [ka]

[0170] Synthesis of (2S,4S,5R,6R)-5-acetamido-6-((1R,2R)-1,2-dihydroxy-3-(3-phenoxybenzamido)propyl)-4-hydroxy-2-(2-(2-((1-(15-oxo-3,6,9,12-tetraoxa-16-azaoctadecyl)-1H-1,2,3-triazol-4-yl)methoxy)ethoxy)ethoxy)tetrahydro-2H-pyran-2-carboxylic acid (compound number 24840) To a stirred solution of (2S,4S,5R,6R)-5-acetamido-6-((1R,2R)-1,2-dihydroxy-3-(3-phenoxybenzamido)propyl)-4-hydroxy-2-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)tetrahydro-2H-pyran-2-carboxylic acid (26335, 0.020 g, 0.032 mmol) and 1-azido-N-ethyl-3,6,9,12-tetraoxapentadecan-15-amide (1, 0.011 g, 0.034 mmol) in dimethyl sulfoxide (2.0 mL) was added tetrakis(acetonitrile)copper(I) hexafluorophosphate (0.033 g, 0.088 mmol). The resulting reaction mixture was stirred at room temperature for 30 minutes. Acetic acid (0.5 mL) was then added, and the reaction mixture was diluted with acetonitrile and purified by preparative HPLC (18–40% acetonitrile in water with 0.1% TFA). Fractions containing the desired product were combined and lyophilized to give (2S,4S,5R,6R)-5-acetamido-6-((1R,2R)-1,2-dihydroxy-3-(3-phenoxybenzamido)propyl)-4-hydroxy-2-(2-(2-((1-(15-oxo-3,6,9,12-tetraoxa-16-azaoctadecyl)-1H-1,2,3-triazol-4-yl)methoxy)ethoxy)ethoxy)tetrahydro-2H-pyran-2-carboxylic acid (compound number 24840) as an off-white solid. Yield: 0.005 g, 14.49%, LCMS, m / z 949.62 [M / 2+1]. + ; 1 H NMR (400MHz, DMSO-d6 with D2O exchange) δ8.00(s, 1H), 7.59(d, J=7.6Hz, 1H), 7.48~7.37(m , 4H), 7.17~7.13(m, 2H), 7.00(d, J=8.0Hz, 2H), 4.49~4.46(m, 4H), 3.84~3.73(m, 5 H), 3.62~3.42(m, 23H), 3.24~3.16(m, 3H), 3.02(dd, J=14.4 and 7.2Hz, 2H), 2.25(t, J=6.4Hz, 2H), 2.17~2.12(m, 1H), 1.84(s, 3H), 1.51~1.45(m, 1H) 0.98~0.94(m, 3H).

[0171] (2R,4S,5R,6R)-5-acetamido-6-((1R,2R)-1,2-dihydroxy-3-(3-phenoxybenzamido)propyl)-4-hydroxy-2-(((2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(2-(1-(15-oxo-15-(perfluorophenoxy)-3,6,9,12-tetraoxapentadecyl)-1H-1,2,3-triazol-4-yl)ethoxy)tetrahydro-2H-pyran-2-yl)methoxy)tetrahydro-2H-pyran-2-carboxylic acid (compound number 24906) [ka] TIFF2024502850000020.tif83155

[0172] Synthesis of (2R,4S,5R,6R)-5-acetamido-6-((1R,2R)-1,2-dihydroxy-3-(3-phenoxybenzamido)propyl)-4-hydroxy-2-(((2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(2-(1-(15-oxo-15-(perfluorophenoxy)-3,6,9,12-tetraoxapentadecyl)-1H-1,2,3-triazol-4-yl)ethoxy)tetrahydro-2H-pyran-2-yl)methoxy)tetrahydro-2H-pyran-2-carboxylic acid (Compound No. 24906) To a stirred solution of perfluorophenyl 1-azido-3,6,9,12-tetraoxapentadecan-15-oate (1, 0.156 g, 0.342 mmol) and (2R,4S,5R,6R)-5-acetamido-2-(((2R,3R,4S,5R,6R)-6-(but-3-yn-1-yloxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)methoxy)-6-((1R,2R)-1,2-dihydroxy-3-(3-phenoxybenzamido)propyl)-4-hydroxytetrahydro-2H-pyran-2-carboxylic acid (26409, 0.087 g, 0.310 mmol) in dimethyl sulfoxide (3 mL) was added tetrakis(acetonitrile)copper(I) hexafluorophosphate (0.324 g, 0.86 9 mmol) was added. The resulting reaction mixture was stirred at room temperature for 30 minutes. After that, acetic acid (0.5 mL) was added, and the reaction mixture was diluted with acetonitrile and purified by preparative HPLC (23-41% acetonitrile in water with 0.1% TFA). Fractions containing the desired product were combined and lyophilized to give (2-((2R,3S,4R,5S,6R)-3,4,5-trihydroxy-6-((1-(15-oxo-15-(perfluorophenoxy)-3,6,9,12-tetraoxapentadecyl)-1H-1,2,3-triazol-4-yl)methyl)tetrahydro-2H-pyran-2-yl)ethyl)phosphonic acid (compound number 24906) as an off-white solid. Yield: 0.101 g, 44.11%, LCMS (ESI) m / z 738.20 [M+1] + ; 1 H NMR (400MHz, DMSO-d6 with D2O exchange) δ4.44 (t, J=5.2Hz, 2H), 3.89~3.86 (m, 1H), 3.77~3.73 (m, 4H), 3.60~3.56 (m, 2H), 3.53~3.4 6(m, 13H), 3.29~3.28(m, 2H), 2.97(t, J=5.6Hz, 2H), 2.86(d, J=7.2Hz, 2H), 1.82(bs, 1H), 1.57(bs, 1H), 1.46~1.31(m, 2H).

[0173] (2R,4S,5R,6R)-5-acetamido-6-((1R,2R)-1,2-dihydroxy-3-(3-phenoxybenzamido)propyl)-4-hydroxy-2-(((2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(2-(1-(15-oxo-3,6,9,12-tetraoxa-16-azaoctadecyl)-1H-1,2,3-triazol-4-yl)ethoxy)tetrahydro-2H-pyran-2-yl)methoxy)tetrahydro-2H-pyran-2-carboxylic acid (compound number 24924) [ka]

[0174] Synthesis of (2R,4S,5R,6R)-5-acetamido-6-((1R,2R)-1,2-dihydroxy-3-(3-phenoxybenzamido)propyl)-4-hydroxy-2-(((2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(2-(1-(15-oxo-3,6,9,12-tetraoxa-16-azaoctadecyl)-1H-1,2,3-triazol-4-yl)ethoxy)tetrahydro-2H-pyran-2-yl)methoxy)tetrahydro-2H-pyran-2-carboxylic acid (Compound No. 24924) (2R,4S,5R,6R)-5-acetamido-2-(((2R,3R,4S,5R,6R)-6-(but-3-yn-1-yloxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)methoxy)-6-((1R,2R)-1,2-dihydroxy-3-(3-phenoxybenzamido)propyl)-4-hydrogen in dimethyl sulfoxide (2.0 mL). To a stirred solution of 2H-pyran-2-hydroxytetrahydro-carboxylic acid (26409, 0.023 g, 0.032 mmol) and 1-azido-N-ethyl-3,6,9,12-tetraoxapentadecan-15-amide (1, 0.011 g, 0.032 mmol), tetrakis(acetonitrile)copper(I) hexafluorophosphate (0.029 g, 0.080 mmol) was added. The resulting reaction mixture was stirred at room temperature for 30 min. Acetic acid (0.3 mL) was added, and the reaction mixture was diluted with acetonitrile and purified by preparative HPLC (15-35% acetonitrile in water with 0.1% TFA). Fractions containing the desired product were combined and lyophilized to give (2R,4S,5R,6R)-5-acetamido-6-((1R,2R)-1,2-dihydroxy-3-(3-phenoxybenzamido)propyl)-4-hydroxy-2-((2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(2-(1-(15-oxo-3,6,9,12-tetraoxa-16-azaoctadecyl)-1H-1,2,3-triazol-4-yl)ethoxy)tetrahydro-2H-pyran-2-yl)methoxy)tetrahydro-2H-pyran-2-carboxylic acid (compound number 24924) as a white solid as the TFA salt. Yield: 0.006 g, 18.08%; LCMS (ESI) m / z 1037.75 [M+1] + ; 1H NMR (400MHz, DMSO-d6 with D2O exchange) δ7.86(s, 1H), 7.59(d, J=8.0Hz, 1H), 7.46~7.37(m, 4H), 7.16~7.11(m, 2H), 6.99(d, J=7.6Hz, 1H), 4.42(t, J=5.0Hz, 2H), 4.13(d, J=6.8Hz, 1H), 3.91~3.85(m, 1H), 3.79~3.73( m, 6H), 3.63~3.60(m, 2H), 3.56~3.52(m, 5H), 3.49~3.43(m, 15H), 3.27~3.21(m, 4H), 3.02(ABq, J=7.6H) z, 2H), 2.83~2.80(m, 2H), 2.25(t, J=6.4Hz, 1H), 1.83(s, 3H), 1.57~1.51(m, 1H), 0.96(t, J=7.2Hz, 3H).

[0175] (2R,4S,5R,6R)-5-acetamido-6-((1R,2R)-1,2-dihydroxy-3-(3-phenoxybenzamido)propyl)-4-hydroxy-2-(((2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(2-(1-(15-oxo-15-(perfluorophenoxy)-3,6,9,12-tetraoxapentadecyl)-1H-1,2,3-triazol-4-yl)ethoxy)tetrahydro-2H-pyran-2-yl)methoxy)tetrahydro-2H-pyran-2-carboxylic acid (compound number 26288) [ka]

[0176] Synthesis of (2R,4S,5R,6R)-6-((1R,2R)-3-(2-([1,1'-biphenyl]-4-yl)acetamido)-1,2-dihydroxypropyl)-4-hydroxy-5-(2-hydroxyacetamido)-2-(2-(2-((1-(15-oxo-15-(perfluorophenoxy)-3,6,9,12-tetraoxapentadecyl)-1H-1,2,3-triazol-4-yl)methoxy)ethoxy)ethoxy)tetrahydro-2H-pyran-2-carboxylic acid (compound number 26288) To a stirred solution of (2R,4S,5R,6R)-6-((1R,2R)-3-(2-([1,1'-biphenyl]-4-yl)acetamido)-1,2-dihydroxypropyl)-4-hydroxy-5-(2-hydroxyacetamido)-2-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)tetrahydro-2H-pyran-2-carboxylic acid (26463, 0.035 g, 0.054 mmol) and perfluorophenyl 1-azido-3,6,9,12-tetraoxapentadecan-15-oate (1, 0.025 g, 0.054 mmol) in dimethyl sulfoxide (0.5 mL) was added tetrakis(acetonitrile)copper(I) hexafluorophosphate (0.050 g, 0.135 mmol). The resulting reaction mixture was stirred at room temperature for 30 minutes. Upon completion, acetic acid (0.3 mL) was added. The resulting solution was diluted with acetonitrile and purified by preparative HPLC (19-35% acetonitrile in water with 0.1% TFA). Fractions containing the desired product were combined and lyophilized to give (2R,4S,5R,6R)-6-((1R,2R)-3-(2-([1,1'-biphenyl]-4-yl)acetamido)-1,2-dihydroxypropyl)-4-hydroxy-5-(2-hydroxyacetamido)-2-(2-(2-((1-(15-oxo-15-(perfluorophenoxy)-3,6,9,12-tetraoxapentadecyl)-1H-1,2,3-triazol-4-yl)methoxy)ethoxy)ethoxy)tetrahydro-2H-pyran-2-carboxylic acid (compound number 26288) as a white solid as the TFA salt. Yield: 0.022 g, 36.77%; LCMS (ESI) m / z 1102.72 [M+1] + ; 1 H NMR (400MHz, DMSO-d6 with D2O exchange) δ7.98 (d, J=3.6Hz, 2H), 7.59 (d, J=7.2Hz, 2H), 7.56~7.52 (m, 2H), 7.42 (t, J=8.0Hz, 2H), 7.33~7.31 (m, 3 H), 4.46(s, 4H), 4.11(m, 2H), 3.55~3.40(m, 22H), 3.19(d, J=9.2Hz, 2H), 2.99~2.92(m, 5H), 2.45~2.43(m, 6H), 2.50(m, 1H), 1.20(s, 1H).

[0177] (2R,2'R,4S,4'S,5R,5'R,6R,6'R)-2,2'-(((((((S)-9,14,22-trioxo-16-((15-oxo-15-(perfluorophenoxy)-3,6,9,12-tetraoxapentadecyl)carbamoyl)-3,6,25,28-tetraoxa-10,15,21-triazatriacontane-1,30-diyl) Bis(1H-1,2,3-triazole-1,4-diyl))bis(methylene))bis(oxy))bis(ethane-2,1-diyl))bis(oxy))bis(5-acetamido-6-((1R,2R)-1,2-dihydroxy-3-(3-phenoxybenzamido)propyl)-4-hydroxytetrahydro-2H-pyran-2carboxylic acid) (Compound No. 26289) [ka]

[0178] (2R,2'R,4S,4'S,5R,5'R,6R,6'R)-2,2'-(((((((S)-9,14,22-trioxo-16-((15-oxo-15-(perfluorophenoxy)-3,6,9,12-tetraoxapentadecyl)carbamoyl)-3,6,25,28-tetraoxa-10,15,21-triazatriacontane-1,30-diyl)bi Synthesis of bis(1H-1,2,3-triazole-1,4-diyl))bis(methylene))bis(oxy))bis(ethane-2,1-diyl))bis(oxy))bis(5-acetamido-6-((1R,2R)-1,2-dihydroxy-3-(3-phenoxybenzamido)propyl)-4-hydroxytetrahydro-2H-pyran-2carboxylic acid) (Compound No. 26289) (2R,4S,5R,6R)-5-acetamido-6-((1R,2R)-1,2-dihydroxy-3-(3-phenoxybenzamido)propyl)-4-hydroxy-2-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)tetrahydro-2H-pyran-2-carboxylic acid (26334, 0.025 g, 0.039 mmol) and perfluorophenyl (S)-1-azide in dimethyl sulfoxide (0.5 mL). To a stirred solution of do-16-(4-(3-(2-(2-azidoethoxy)ethoxy)propanamido)butyl)-9,14,17-trioxo-3,6,21,24,27,30-hexaoxa-10,15,18-triazatritriacontan-33-oate (26332, 0.020 g, 0.019 mmol) was added tetrakis(acetonitrile)copper(I) hexafluorophosphate (0.036 g, 0.099 mmol). The resulting reaction mixture was stirred at room temperature for 30 min. The reaction progress was monitored by LC-MS, and upon completion, acetic acid (0.3 mL) was added. The resulting solution was diluted with acetonitrile and purified by preparative HPLC (25–44% acetonitrile in water with 0.1% TFA). Fractions containing the desired product were combined, lyophilized, and purified to give (2R,2'R,4S,4'S,5R,5'R,6R,6'R)-2,2'-(((((((S)-9,14,22-trioxo-16-((15-oxo-15-(perfluorophenoxy)-3,6,9,12-tetraoxapentadecyl)carbamoyl)-3,6,25,28-tetraoxa-10,15,21-triazatriacontane-1,30-di (1R,2R)-1,2-dihydroxy-3-(3-phenoxybenzamido)propyl)-4-hydroxytetrahydro-2H-pyran-2carboxylic acid) (compound number 26289) was obtained as a white solid as the TFA salt. Yield: 0.012 g, 27.67%; LCMS (ESI) m / z 738.20 [M+1]. + ; 1H NMR (400MHz, DMSO-d6 with D2O exchange) δ7.98(s, 2H), 7.58(d, J=8.0Hz, 2H), 7.46~7.36(m, 9H), 7.16 ~7.12(m, 4H), 6.98(d, J=7.6Hz, 4H), 4.46~4.43(m, 9H), 4.11(m, 2H), 3.53~3.35(m, 56H), 3.23 ~3.21(m, 5H), 3.14(bs, 2H), 2.97~2.93(m, 9H), 2.25~2.22(m, 6H), 2.08(bs, 4H), 1.83(s, 6H), 1.56~1.44(m, 8H), 1.49~1.44(m, 2H), 1.33~1.27(m, 4H), 1.20(bs, 5H), 0.98(d, J=6.8Hz, 2H).

[0179] Perfluorophenyl (S)-1-azido-16-(4-(3-(2-(2-azidoethoxy)ethoxy)propanamido)butyl)-9,14,17-trioxo-3,6,21,24,27,30-hexaoxa-10,15,18-triazatritriacontan-33-oate (Compound No. 26332) [ka]

[0180] Synthesis of perfluorophenyl (S)-1-azido-16-(4-(3-(2-(2-azidoethoxy)ethoxy)propanamido)butyl)-9,14,17-trioxo-3,6,21,24,27,30-hexaoxa-10,15,18-triazatritriacontan-33-oate (compound number 26332) To a stirred solution of (S)-1-azido-16-(4-(3-(2-(2-azidoethoxy)ethoxy)propanamido)butyl)-9,14,17-trioxo-3,6,21,24,27,30-hexaoxa-10,15,18-triazatritriacontan-33-oic acid (26337, 1.0 g, 1.18 mmol) in tetrahydrofuran (10.0 mL) was added 2,3,4,5,6-pentafluorophenol (1, 0.434 g, 2.36 mmol) and N,N'-diisopropylcarbodiimide (0.461 mL, 2.94 mmol) at 0°C. The resulting reaction mixture was stirred at room temperature for 24 hours. Upon completion, the solvent was concentrated and purified by preparative HPLC (30-75% acetonitrile in water with 0.1% TFA). Fractions containing the desired product were combined and lyophilized to give perfluorophenyl (S)-1-azido-16-(4-(3-(2-(2-azidoethoxy)ethoxy)propanamido)butyl)-9,14,17-trioxo-3,6,21,24,27,30-hexaoxa-10,15,18-triazatritriacontan-33-oate (compound no. 26332) as an off-white solid. Yield: 0.291 g, 24%; ELSD (ESI) m / z 1015.6 [M+1]. + ; 1 H NMR (400MHz, DMSO-d6) δ7.92(t, J=11.6Hz, 1H), 7.85~7.80(m, 2H), 7.78(t, J=11.2Hz, 1H), 4.19~4.14(m, 1H), 3.78(t, J=12). 0Hz, 2H), 3.61~3.49(m, 29H), 3.38~3.35(m, 5H), 3.25~3.13(m, 3H), 3.07~2.99(m, 6H), 2.32~2.26(m, 4H), 1.62~1.20(m, 9H).

[0181] Perfluorophenyl(16S,19S)-1-azido-16,19-bis(4-(3-(2-(2-azidoethoxy)ethoxy)propanamido)butyl)-9,14,17,20-tetraoxo-3,6,24,27,30,33-hexaoxa-10,15,18,21-tetraazahexatriacontan-36-oate (compound number 26333) [ka] TIFF2024502850000026.tif50155

[0182] Synthesis of perfluorophenyl(16S,19S)-1-azido-16,19-bis(4-(3-(2-(2-azidoethoxy)ethoxy)propanamido)butyl)-9,14,17,20-tetraoxo-3,6,24,27,30,33-hexaoxa-10,15,18,21-tetraazahexatriacontan-36-oate (compound number 26333) To a stirred solution of (16S,19S)-1-azido-16,19-bis(4-(3-(2-(2-azidoethoxy)ethoxy)propanamido)butyl)-9,14,17,20-tetraoxo-3,6,24,27,30,33-hexaoxa-10,15,18,21-tetraazahexatriacontan-36-oic acid (26338, 1.5 g, 1.29 mmol) in tetrahydrofuran (20.0 mL) was added 2,3,4,5,6-pentafluorophenol (1, 0.475 g, 2.58 mmol) and N,N'-diisopropylcarbodiimide (0.472 mL, 3.22 mmol) at 0°C. The resulting reaction mixture was stirred at room temperature for 24 hours. Upon completion, the solvent was concentrated to give a residue that was then purified by preparative HPLC (20-55% acetonitrile in water with 0.1% TFA). The desired fractions were combined and lyophilized to give perfluorophenyl (16S,19S)-1-azido-16,19-bis(4-(3-(2-(2-azidoethoxy)ethoxy)propanamido)butyl)-9,14,17,20-tetraoxo-3,6,24,27,30,33-hexaoxa-10,15,18,21-tetraazahexatriacontan-36-oate (compound no. 26333) as an off-white sticky solid. Yield: 0.556 g, 32.25%; LC-MS (ESI) m / z 1329.09 [M+1]. + 1H NMR (400MHz, DMSO-d6) δ7.98~7.68(m, 6H), 4.16(bs, 2H), 3.78(t, J=11.6Hz, 2H), 3.59~3.49(m, 35H), 3.38~3.33(m, 9H), 3.21~3.17(m, 4H), 3.03~2.99(m, 9H), 2.30~2.27(m, 6H), 2.14(t, J=14.4Hz, 2H), 1.60~1.57(m, 4H), 1.48~1.23(m, 10H).

[0183] (2R,4S,5R,6R)-5-acetamido-6-((1R,2R)-1,2-dihydroxy-3-(3-phenoxybenzamido)propyl)-4-hydroxy-2-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)tetrahydro-2H-pyran-2-carboxylic acid (compound number 26334) and (2S,4S,5R,6R)-5-acetamido-6-((1R,2R)-1,2-dihydroxy-3-(3-phenoxybenzamido)propyl)-4-hydroxy-2-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)tetrahydro-2H-pyran-2-carboxylic acid (compound number 26335) [ka]

[0184] Synthesis of (1R,2R)-1-((2R,3R,4S,6R)-3-acetamido-4-acetoxy-6-(methoxycarbonyl)-6-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)tetrahydro-2H-pyran-2-yl)-3-(3-phenoxybenzamido)propane-1,2-diyl diacetate (3) To a stirred solution of (1R,2R)-1-((2R,3R,4S,6S)-3-acetamido-4-acetoxy-6-(methoxycarbonyl)-6-(p-tolylthio)tetrahydro-2H-pyran-2-yl)-3-(3-phenoxybenzamido)propane-1,2-diyl diacetate (1, 1.0 g, 1.33 mmol) and 2-(2-(prop-2-yn-1-yloxy)ethoxy)ethan-1-ol (2, 0.288 g, 2.0 mmol) in dry dichloromethane (20.0 mL) was added 4 Å molecular sieves (1.0 g, 100% w / w). The resulting reaction mixture was stirred under a nitrogen atmosphere for 12 hours. Then, 1-iodopyrrolidine-2,5-dione (0.576 g, 2.56 mmol) and trifluoromethanesulfonic acid (0.094 mL, 1.066 mmol) were added at −45° C. The resulting reaction mixture was stirred at −45° C. for 1 h. Upon completion, the reaction mixture was quenched with trimethylamine to neutral pH, filtered through Celite, diluted with dichloromethane, and washed with aqueous sodium bicarbonate, followed by DM water. The organic layer was concentrated to give the crude residue, which was then purified by flash column chromatography (5-7.5% methanol in ethyl acetate). The desired fractions were concentrated and dried to give (1R,2R)-1-((2R,3R,4S,6R)-3-acetamido-4-acetoxy-6-(methoxycarbonyl)-6-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)tetrahydro-2H-pyran-2-yl)-3-(3-phenoxybenzamido)propane-1,2-diyl diacetate (3) as a white solid as a mixture of anomers. Yield: 1.0 g, 97.41%; LCMS (ESI) m / z 771.79.

[0185] Synthesis of (2R,4S,5R,6R)-5-acetamido-6-((1R,2R)-1,2-dihydroxy-3-(3-phenoxybenzamido)propyl)-4-hydroxy-2-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)tetrahydro-2H-pyran-2-carboxylic acid (compound number 26334) and (2S,4S,5R,6R)-5-acetamido-6-((1R,2R)-1,2-dihydroxy-3-(3-phenoxybenzamido)propyl)-4-hydroxy-2-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)tetrahydro-2H-pyran-2-carboxylic acid (compound number 26335) To a stirred solution of (1R,2R)-1-((2R,3R,4S,6R)-3-acetamido-4-acetoxy-6-(methoxycarbonyl)-6-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)tetrahydro-2H-pyran-2-yl)-3-(3-phenoxybenzamido)propane-1,2-diyl diacetate (3, 1.0 g, 1.302 mmol) in ethanol (10.0 mL) was added lithium hydroxide (0.186 g, 7.78 mmol) at 0 °C. The resulting reaction mixture was stirred at room temperature for 4 h. After completion, Dowex® hydrogen carbonate was added to pH 6 and the reaction mass was filtered. The filtrate was concentrated and dried to give a crude residue, which was then diluted with acetonitrile and purified by preparative HPLC (22-38% acetonitrile in water with 0.1% TFA). Fractions containing the desired product were separately combined and lyophilized to give (2R,4S,5R,6R)-5-acetamido-6-((1R,2R)-1,2-dihydroxy-3-(3-phenoxybenzamido)propyl)-4-hydroxy-2-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)tetrahydro-2H-pyran-2-carboxylic acid (compound number 26334) as the TFA salt as a thick syrup. Yield: 0.24 g, 29.33%; LCMS (ESI) m / z 631.55 [M+1] + ; 1H NMR (400MHz, DMSO-d6) δ8.36(t, J=5.4Hz, 1H), 8.06(d, J=7.6Hz, 1H), 7.65(d, J=8.0Hz, 1H), 7.50~7.39(m, 4H), 7.17~7.15(m, 2H), 7.02(d, J=8.0Hz, 2H), 4.83(bs, 2H), 4.13(d J = 2.0 Hz, 1H), 3.76–3.71 (m, 4H), 3.59–3.14 (m, 14H), 2.17 (dd, J = 12.8 and 4.8 Hz, 1H), 1.87 (s, 3H), 1.49 (t, J = 12.0 Hz, 1H); (2S,4S,5R,6R)-5-acetamido-6-((1R,2R)-1,2-dihydroxy-3-(3-phenoxybenzamido)propyl)-4-hydroxy-2-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)tetrahydro-2H-pyran-2-carboxylic acid (compound number 26335) was obtained as a white solid as the TFA salt. Yield: 0.23g, 28.11%; LCMS (ESI) m / z 631.55[M+1] + ; 1 H NMR (400MHz, DMSO-d6) δ8.32(t, J=5.2Hz, 1H), 7.99(d, J=7.2Hz, 1H), 7.64(d, J=7.6Hz, 1H), 7.48~7.39(m, 4H), 7.18~7.14(m, 2H), 7.30(d, J=6.8Hz, 2H), 4.33(bs, 1H), 4.11(d J=2.0Hz, 1H), 3.82~3.73(m, 2H), 3.66~3.24(m, 11H), 3.25~3.21(m, 1H), 1.88(s, 3H), 1.52(t, J=12.0Hz, 1H).

[0186] (S)-1-Azido-16-(4-(3-(2-(2-azidoethoxy)ethoxy)propanamido)butyl)-9,14,17-trioxo-3,6,21,24,27,30-hexaoxa-10,15,18-triazatritriacontan-33-oic acid (compound number 26337) [ka] TIFF2024502850000029.tif74154

[0187] Synthesis of methyl N6-((benzyloxy)carbonyl)-N2-(4-(((benzyloxy)carbonyl)amino)butanoyl)-L-lysinate (3). To a solution of methyl N6-((benzyloxy)carbonyl)-L-lysinate hydrochloride (1, 5.00 g, 19.5 mmol), 4-(((benzyloxy)carbonyl)amino)butanoic acid (2, 4.63 g, 17.0 mmol), [(dimethylamino)({3H-[1,2,3]triazolo[4,5-b]pyridin-3-yloxy})methylidene]dimethylazanium; hexafluoro-λ 5 To the resulting mixture was added methyl N-((benzyloxy)carbonyl)-N-(4-((benzyloxy)carbonyl)amino)butanoyl)-L-lysinate (3) as an off-white solid. Yield: 7.0 g, 90.25%, LC-MS (ESI) m / z 514.2 [M+1]. + .

[0188] Synthesis of N6-((benzyloxy)carbonyl)-N2-(4-(((benzyloxy)carbonyl)amino)butanoyl)-L-lysine (4) To a solution of methyl N-((benzyloxy)carbonyl)-N-(4-(((benzyloxy)carbonyl)amino)butanoyl)-L-lysinate (3, 7.0 g, 13.6 mmol) in methanol (25.0 mL), tetrahydrofuran (25.0 mL), and water (5.0 mL) was added lithium hydroxide (0.979 g, 40.9 mmol) at room temperature. The resulting mixture was stirred at −40° C. for 4 hours. Upon completion, the reaction mixture was acidified with 1N HCl solution (pH 4) and extracted with ethyl acetate. The organic layer was dried over sodium sulfate, filtered, and concentrated under high vacuum to give crude N-((benzyloxy)carbonyl)-N-(4-(((benzyloxy)carbonyl)amino)butanoyl)-L-lysine (4) as an off-white solid. Yield: 6.0g, 94.25%, LCMS (ESI) m / z 500.2[M+1] + .

[0189] Synthesis of tert-butyl (S)-10-(4-(((benzyloxy)carbonyl)amino)butyl)-3,8,11-trioxo-1-phenyl-2,15,18,21,24-pentaoxa-4,9,12-triazaheptacosane-27-oate (6) To a solution of N-((benzyloxy)carbonyl)-N-(4-((benzyloxy)carbonyl)amino)butanoyl)-L-lysine (4, 3.0 g, 6.01 mmol) in tetrahydrofuran (30.0 mL) was added 2,3,4,5,6-pentafluorophenol (2.21 g, 12.0 mmol) and N,N'-diisopropylcarbodiimide (1.91 mL, 15.0 mmol) at 0 °C. The resulting reaction mixture was stirred at room temperature for 4 h. tert-Butyl 1-amino-3,6,9,12-tetraoxapentadecan-15-oate (5, 2.32 g, 7.21 mmol) was added at room temperature, and the reaction mixture was stirred for 12 h. After completion, the solvent was concentrated under high vacuum to give the crude residue, which was then purified by flash column chromatography (70-100% ethyl acetate in hexanes). The desired fractions were concentrated under reduced pressure to give tert-butyl (S)-10-(4-(((benzyloxy)carbonyl)amino)butyl)-3,8,11-trioxo-1-phenyl-2,15,18,21,24-pentaoxa-4,9,12-triazaheptacosane-27-oate (6) as a pale yellow sticky substance, yield: 4.0 g, 84.25%, LC-MS(ESI) m / z 803.41 [M+1]. + .

[0190] Synthesis of tert-butyl (S)-23-amino-18-(4-aminobutyl)-17,20-dioxo-4,7,10,13-tetraoxa-16,19-diazatricosanoate (7) To a stirred solution of tert-butyl (S)-10-(4-(((benzyloxy)carbonyl)amino)butyl)-3,8,11-trioxo-1-phenyl-2,15,18,21,24-pentaoxa-4,9,12-triazaheptacosane-27-oate (6, 4.0 g, 4.98 mmol) in methanol (50 mL) was added 10% palladium on carbon (4.0 g, 100% w / w) at room temperature under nitrogen. The resulting mixture was stirred at room temperature under hydrogen gas pressure for 12 hours. The reaction mixture was filtered through Celite and washed with methanol. The filtrate was concentrated in vacuo to give tert-butyl (S)-23-amino-18-(4-aminobutyl)-17,20-dioxo-4,7,10,13-tetraoxa-16,19-diazatricosanoate (7) as an off-white solid. Yield: 2.0 g, 75%, ELSD (ESI) m / z 803.4 [M+1] + .

[0191] Synthesis of tert-butyl (S)-1-azido-16-(4-(3-(2-(2-azidoethoxy)ethoxy)propanamido)butyl)-9,14,17-trioxo-3,6,21,24,27,30-hexaoxa-10,15,18-triazatritriacontan-33-oate (9) To a solution of tert-butyl (S)-23-amino-18-(4-aminobutyl)-17,20-dioxo-4,7,10,13-tetraoxa-16,19-diazatricosanoate (7, 2.0 g, 3.74 mmol) in tetrahydrofuran (40.0 mL) was added 2,5-dioxopyrrolidin-1-yl 3-(2-(2-azidoethoxy)ethoxy)propanoate (2.25 g, 7.48 mmol) at 0 °C. The resulting reaction mixture was stirred at room temperature for 4 h. After completion, the solvent was concentrated under high vacuum to give the crude residue, which was then purified by flash column chromatography (0-7.5% methanol in dichloromethane). The desired fractions were concentrated under reduced pressure to give tert-butyl (S)-1-azido-16-(4-(3-(2-(2-azidoethoxy)ethoxy)propanamido)butyl)-9,14,17-trioxo-3,6,21,24,27,30-hexaoxa-10,15,18-triazatritriacontan-33-oate (9) as a pale yellow viscous liquid, yield: 1.10 g, 32.25%, ELSD (ESI) m / z 905.4 [M+1]. + .

[0192] Synthesis of (S)-1-azido-16-(4-(3-(2-(2-azidoethoxy)ethoxy)propanamido)butyl)-9,14,17-trioxo-3,6,21,24,27,30-hexaoxa-10,15,18-triazatritriacontan-33-oic acid (compound number 26337) To a solution of tert-butyl (S)-1-azido-16-(4-(3-(2-(2-azidoethoxy)ethoxy)propanamido)butyl)-9,14,17-trioxo-3,6,21,24,27,30-hexaoxa-10,15,18-triazatritriacontan-33-oate (9, 1.1 g, 1.21 mmol) in dichloromethane (10 mL) was added trifluoroacetic acid (5.0 mL) at 0° C. The resulting mixture was stirred at room temperature under nitrogen for 4 hours. Upon completion, the reaction mixture was concentrated, washed with diethyl ether, and dried to give (S)-1-azido-16-(4-(3-(2-(2-azidoethoxy)ethoxy)propanamido)butyl)-9,14,17-trioxo-3,6,21,24,27,30-hexaoxa-10,15,18-triazatritriacontan-33-oic acid (compound number 26337) as a pale yellow viscous liquid. Yield: 1.0 g, 99.25%, ELSD (ESI) m / z 849.6 [M+1]. + . 1 H NMR (400MHz, DMSO-d6) δ12.0(bs, 1H), 7.92(t, J=11.6Hz, 1H), 7.85~7.80(m, 2H), 7.78(t, J=11.2Hz, 1H), 4.20~4.15(m, 1H), 3.63~3.49(m, 31 H), 3.40~3.36(m, 5H), 3.25~3.17(m, 2H), 3.15~3.07(m, 4H), 2.45~2.4 2(m, 2H), 2.32~2.26(m, 4H), 2.13(t, J=14.8Hz, 2H), 1.62~1.14(m, 8H).

[0193] (16S,19S)-1-Azido-16,19-bis(4-(3-(2-(2-azidoethoxy)ethoxy)propanamido)butyl)-9,14,17,20-tetraoxo-3,6,24,27,30,33-hexaoxa-10,15,18,21-tetraazahexatriacontan-36-oic acid (compound number 26338) [ka]

[0194] Synthesis of methyl N6-((benzyloxy)carbonyl)-N2-(N6-((benzyloxy)carbonyl)-N2-(4-(((benzyloxy)carbonyl)amino)butanoyl)-L-lysyl)-L-lysinate (3) To a stirred solution of N6-((benzyloxy)carbonyl)-N2-(4-(((benzyloxy)carbonyl)amino)butanoyl)-L-lysine (1, 6.0 g, 12.0 mmol) in N,N-dimethylformamide (50.0 mL) was added methyl N6-((benzyloxy)carbonyl)-L-lysinate (2, 4.24 g, 14.4 mmol), followed by [(dimethylamino)({3H-[1,2,3]triazolo[4,5-b]pyridin-3-yloxy})methylidene]dimethylazanium;hexafluoro-λ 5 N-phosphanide (9.59 g, 25.2 mmol) and ethyl bis(propan-2-yl)amine (7.32 mL, 42.0 mmol) were added at 0 °C. The resulting reaction mixture was stirred at room temperature for 12 h. Upon completion, the reaction mixture was diluted with saturated sodium bicarbonate solution and extracted with ethyl acetate. The organic layer was dried over sodium sulfate, filtered, and concentrated under high vacuum to give the crude residue. The crude residue was purified by flash column chromatography (50-70% ethyl acetate in hexanes) to give methyl N-((benzyloxy)carbonyl)-N-(N-((benzyloxy)carbonyl)-N-(4-(((benzyloxy)carbonyl)amino)butanoyl)-L-lysyl)-L-lysinate (3) as an off-white solid. Yield: 9.0 g, 96.25%, LCMS (ESI) m / z 776.54 [M+1] + .

[0195] Synthesis of N6-((benzyloxy)carbonyl)-N2-(N6-((benzyloxy)carbonyl)-N2-(4-(((benzyloxy)carbonyl)amino)butanoyl)-L-lysyl)-L-lysine (4) To a stirred solution of methyl N6-((benzyloxy)carbonyl)-N2-(N6-((benzyloxy)carbonyl)-N2-(4-(((benzyloxy)carbonyl)amino)butanoyl)-L-lysyl)-L-lysinate (3, 9.0 g, 11.6 mmol) in methanol (30.0 mL) and tetrahydrofuran (15.0 mL) was added lithium hydroxide (0.833 g, 34.8 mmol) dissolved in water (1.0 mL). The resulting mixture was stirred at −40° C. for 16 hours. Upon completion, the reaction mixture was concentrated and the crude residue was dissolved in water and acidified to pH 4 with 1 N hydrochloric acid solution, followed by extraction with ethyl acetate. The organic layer was separated, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give N-((benzyloxy)carbonyl)-N-(N-((benzyloxy)carbonyl)-N-(4-(((benzyloxy)carbonyl)amino)butanoyl)-L-lysyl)-L-lysine (4) as an off-white solid. Yield: 8.0 g, 90%, LCMS (ESI) m / z 762.43 [M+1] + .

[0196] Synthesis of tert-butyl (10S,13S)-10,13-bis(4-(((benzyloxy)carbonyl)amino)butyl)-3,8,11,14-tetraoxo-1-phenyl-2,18,21,24,27-pentaoxa-4,9,12,15-tetraazatriacontan-30-oate (6) To a stirred solution of N-((benzyloxy)carbonyl)-N-(N-((benzyloxy)carbonyl)-N-(4-(((benzyloxy)carbonyl)amino)butanoyl)-L-lysyl)-L-lysine (4, 4.0 g, 5.25 mmol) in tetrahydrofuran (40.0 mL) was added 2,3,4,5,6-pentafluorophenol (1.93 g, 12.0 mmol) and N,N'-diisopropylcarbodiimide (1.67 mL, 13.1 mmol) at 0°C. The resulting reaction mixture was stirred at room temperature for 4 hours. To the reaction mixture was added tert-butyl 1-amino-3,6,9,12-tetraoxapentadecan-15-oate (5, 2.03 g, 6.30 mmol). The resulting reaction mixture was stirred at room temperature for 12 hours. After completion, the solvent was concentrated under high vacuum to give the crude residue, which was then purified by flash column chromatography (70-100% ethyl acetate in hexanes). The desired fractions were concentrated under reduced pressure to give tert-butyl (10S,13S)-10,13-bis(4-(((benzyloxy)carbonyl)amino)butyl)-3,8,11,14-tetraoxo-1-phenyl-2,18,21,24,27-pentaoxa-4,9,12,15-tetraazatriacontan-30-oate (6) as a pale yellow semi-solid. Yield: 5.0 g, 89.25%, LC-MS (ESI) m / z 1065.8 [M+1]. + .

[0197] Synthesis of tert-butyl (18S,21S)-26-amino-18,21-bis(4-aminobutyl)-17,20,23-trioxo-4,7,10,13-tetraoxa-16,19,22-triazahexacosanoate (7) To a stirred solution of tert-butyl (10S,13S)-10,13-bis(4-(((benzyloxy)carbonyl)amino)butyl)-3,8,11,14-tetraoxo-1-phenyl-2,18,21,24,27-pentaoxa-4,9,12,15-tetraazatriacontan-30-oate (6, 5.0 g, 4.69 mmol) in methanol (40.0 mL) was added 10% palladium on carbon (5.0 g, 100% w / w) at room temperature under nitrogen. The resulting mixture was stirred at room temperature under hydrogen gas pressure for 12 hours. The reaction mixture was filtered through Celite and washed with methanol. The filtrate was concentrated in vacuo to give tert-butyl (18S,21S)-26-amino-18,21-bis(4-aminobutyl)-17,20,23-trioxo-4,7,10,13-tetraoxa-16,19,22-triazahexacosanoate (7) as a pale yellow viscous liquid. Yield: 3.0 g, 96.23%, ELSD (ESI) m / z 663.5 [M+1] + .

[0198] Synthesis of tert-butyl (16S,19S)-1-azido-16,19-bis(4-(3-(2-(2-azidoethoxy)ethoxy)propanamido)butyl)-9,14,17,20-tetraoxo-3,6,24,27,30,33-hexaoxa-10,15,18,21-tetraazahexatriacontan-36-oate (9) To a stirred solution of tert-butyl (18S,21S)-26-amino-18,21-bis(4-aminobutyl)-17,20,23-trioxo-4,7,10,13-tetraoxa-16,19,22-triazahexacosanoate (7, 3.0 g, 4.53 mmol) in tetrahydrofuran (30.0 mL) was added 2,5-dioxopyrrolidin-1-yl 3-(2-(2-azidoethoxy)ethoxy)propanoate (8, 4.08 g, 13.6 mmol) at 0 °C. The resulting reaction mixture was stirred at room temperature for 4 h. After completion, the solvent was concentrated under high vacuum to give the crude residue, which was then purified by flash column chromatography (0-7.5% methanol in dichloromethane). The desired fractions were concentrated under reduced pressure to give tert-butyl (16S,19S)-1-azido-16,19-bis(4-(3-(2-(2-azidoethoxy)ethoxy)propanamido)butyl)-9,14,17,20-tetraoxo-3,6,24,27,30,33-hexaoxa-10,15,18,21-tetraazahexatriacontan-36-oate (9) as a pale yellow viscous liquid, yield: 2.2 g, 39.30%, ELSD (ESI) m / z 1217.6 [M+1]. + .

[0199] Synthesis of (16S,19S)-1-azido-16,19-bis(4-(3-(2-(2-azidoethoxy)ethoxy)propanamido)butyl)-9,14,17,20-tetraoxo-3,6,24,27,30,33-hexaoxa-10,15,18,21-tetraazahexatriacontan-36-oic acid (compound number 26338) To a stirred solution of tert-butyl (16S,19S)-1-azido-16,19-bis(4-(3-(2-(2-azidoethoxy)ethoxy)propanamido)butyl)-9,14,17,20-tetraoxo-3,6,24,27,30,33-hexaoxa-10,15,18,21-tetraazahexatriacontan-36-oate (9, 2.2 g, 1.80 mmol) in dichloromethane (20 mL) was added trifluoroacetic acid (5.0 mL) at 0° C. The resulting reaction mixture was stirred at room temperature under nitrogen for 4 hours. Upon completion, the reaction mixture was concentrated, washed with diethyl ether, and dried to give (16S,19S)-1-azido-16,19-bis(4-(3-(2-(2-azidoethoxy)ethoxy)propanamido)butyl)-9,14,17,20-tetraoxo-3,6,24,27,30,33-hexaoxa-10,15,18,21-tetraazahexatriacontan-36-oic acid (compound No. 26338) as an off-white viscous liquid. Yield: 1.4 g, 70.35%, ELSD (ESI) m / z 1161.4 [M-1]. - . 1 H NMR (400MHz, DMSO-d6) δ12.0(bs, 1H), 7.97~7.77(m, 5H), 4.16(bs, 2H), 3.60~3.57(m, 14H), 3.53~3.48(m, 24H), 3.38~3.19(m, 14H), 2.94(bs, 9H), 2.43~2.42(m, 8H), 2.30~2.27(m, 5H), 2.14(t, J=15.2Hz, 2H), 1.62~1.59(m, 4H), 1.49~1.48(m, 2H), 1.37~1.23(m, 8H).

[0200] (2R,4S,5R,6R)-6-((1R,2R)-3-(2-([1,1'-biphenyl]-4-yl)acetamido)-1,2-dihydroxypropyl)-4-acetoxy-2-(((2R,3R,4S,5R,6R)-6-(but-3-yn-1-yloxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)methoxy)-5-(2-hydroxyacetamido)tetrahydro-2H-pyran-2-carboxylic acid (compound number 26339) and (2S,4S,5R,6R)-6-((1R,2R)-3-(2-([1,1'-biphenyl]-4-yl)acetamido)-1,2-dihydroxypropyl)-4-acetoxy-2-(((2R,3R,4S,5R,6R)-6-(but-3-yn-1-yloxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)methoxy)-5-(2-hydroxyacetamido)tetrahydro-2H-pyran-2-carboxylic acid (compound number 26411). [ka]

[0201] Synthesis of (2R,3S,4S,5R,6R)-2-((((2R,4S,5R,6R)-6-((1R,2R)-3-(2-([1,1'-biphenyl]-4-yl)acetamido)-1,2-diacetoxypropyl)-4-acetoxy-5-(2-hydroxyacetamido)-2-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy)methyl)-6-((but-3-yn-1-yloxy)tetrahydro-2H-pyran-3,4,5-triyl tribenzoate (3) To a stirred solution of (1R,2R)-3-(2-([1,1'-biphenyl]-4-yl)acetamido)-1-((2R,3R,4S,6S)-4-acetoxy-3-(2-acetoxyacetamido)-6-(methoxycarbonyl)-6-(p-tolylthio)tetrahydro-2H-pyran-2-yl)propane-1,2-diyl diacetate (1, 1.50 g, 1.86 mmol) and (2R,3R,4S,5S,6R)-2(but-3-yn-1-yloxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triyl tribenzoate (2, 2.53 g, 4.65 mmol) in dry dichloromethane (30 mL) was added 4 Å molecular sieves (1.50 g, 100% w / w). The resulting reaction mixture was stirred under a nitrogen atmosphere for 12 hours. After cooling the reaction mixture to -45°C, 1-iodopyrrolidine-2,5-dione (1.05 g, 4.65 mmol) was added dropwise, followed by trifluoromethanesulfonic acid (0.164 mL, 1.86 mmol). The reaction mixture was stirred at -45°C for 30 minutes. Upon completion, the reaction mixture was quenched with trimethylamine to neutral pH, filtered through Celite, diluted with dichloromethane, and washed with aqueous sodium bicarbonate, followed by DM water. The organic layer was concentrated to give the crude residue, which was then purified by flash column chromatography (5-7.5% methanol in ethyl acetate). The desired fractions were concentrated and dried to give (2R,3S,4S,5R,6R)-2-(((((2R,4S,5R,6R)-6-((1R,2R)-3-(2-([1,1'-biphenyl]-4-yl)acetamido)-1,2-diacetoxypropyl)-4-acetoxy-5-(2-acetoxyacetamido)-2-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy)methyl)-6-(but-3-yn-1-yloxy)tetrahydro-2H-pyran-3,4,5-triylenezoate (3) as a white solid as an inseparable mixture of anomers. Yield: 1.40 g, 63.54%, LCMS (ESI) m / z 1186.20 [M+1]. + .

[0202] (2R,4S,5R,6R)-6-((1R,2R)-3-(2-([1,1'-biphenyl]-4-yl)acetamido)-1,2-dihydroxypropyl)-4-acetoxy-2-(((2R,3R,4S,5R,6R)-6-(but-3-yn-1-yloxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)methoxy)-5-(2-hydroxyacetamido)tetrahydro-2H-pyran-2-carboxylic acid (compound number 26339) and Synthesis of (2S,4S,5R,6R)-6-((1R,2R)-3-(2-([1,1'-biphenyl]-4-yl)acetamido)-1,2-dihydroxypropyl)-4-acetoxy-2-(((2R,3R,4S,5R,6R)-6-(but-3-yn-1-yloxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)methoxy)-5-(2-hydroxyacetamido)tetrahydro-2H-pyran-2-carboxylic acid (Compound No. 26411) (2R,3S,4S,5R,6R)-2-(((((2R,4S,5R,6R)-6-((1R,2R)-3-(2-([1,1'-biphenyl]-4-yl)acetamido)-1,2-diacetoxypropyl)-4-acetoxy-5-(2-acetoxyacetamido)-2-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy)methyl) in ethanol (5.0 mL) To a stirred solution of 1.40 g (1.18 mmol) of 6-(but-3-yn-1-yloxy)tetrahydro-2H-pyran-3,4,5-triylenezoate (3), a solution of lithium hydroxide (0.071 g, 2.95 mmol) in water (1.0 mL) was added at 0° C. The reaction mixture was stirred at room temperature for 4 hours. After completion, Dowex® hydrogen chloride was added until pH 6 and the reaction mass was filtered. The filtrate was concentrated. The crude residue was concentrated and dried, diluted with acetonitrile, and purified by preparative HPLC (17-35% acetonitrile in water with 0.1% TFA). Fractions containing the desired product were separately combined and lyophilized to afford (2R,4S,5R,6R)-6-((1R,2R)-3-(2-([1,1'-biphenyl]-4-yl)acetamido)-1,2-dihydroxypropyl)-4-acetoxy-2-(((2R,3R,4S,5R,6R)-6-(but-3-yn-1-yloxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)methoxy)-5-(2-hydroxyacetamido)tetrahydro-2H-pyran-2-carboxylic acid (compound no. 26339) as the TFA salt as a white solid. Yield: 0.12 g; 13.11%; LCMS (ESI) m / z 733.18[M+1] + ; 1H NMR (400 MHz, methanol-d4) δ 7.99-7.97 (m, 1H), 7.88 (d, J = 7.2 Hz, 1H), 7.61-7.57 (m, 4H), 7.43-7.37 (m, 4H), 7.31 (t, J = 7.6 Hz, 1H), 4.25 (d, J = 7.2 Hz, 1H), 4.01 (s, 2H). 3.94~3.88(m, 3H), 3.86~3.82(m, 3H), 3.79~3.63(m, 5H), 3.60(s, 2H), 3.52~3.44( m, 2H), 3.38 (d, J=8.4Hz, 1H), 2.70 (dd, J=13.2 and 3.6Hz, 1H), 2.49 (dt, J=7.6 and 2.8 2H), 2.25 (t, J = 2.8 Hz, 1H), 1.75 (t, J = 13.2 Hz, 1H); (2S,4S,5R,6R)-6-((1R,2R)-3-(2-([1,1'-biphenyl]-4-yl)acetamido)-1,2-dihydroxypropyl)-4-acetoxy-2-(((2R,3R,4S,5R,6R)-6-(but-3-yn-1-yloxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)methoxy)-5-(2-hydroxyacetamido)tetrahydro-2H-pyran-2-carboxylic acid (compound number 26411) was obtained as a white solid as the TFA salt. Yield: 0.230 g, 25.13%; LCMS (ESI) m / z 733.24 [M+1] + ; 1 H NMR (400MHz, methanol-d4) δ8.06~8.04 (m, 1H), 7.89 (d, J=9.2Hz, 1H), 7.61~7. 57(m, 4H), 7.43~7.36(m, 4H), 7.31(t, J=7.6Hz, 1H), 4.26(d, J=7.2Hz, 1H), 4 .16~4.13(m, 1H), 4.04~4.02(m, 3H), 3.94~3.83(m, 5H), 3.71~3.64(m, 3H), 3.60(s, 2H), 3.53~3.44(m, 3H), 3.39(d, J=9.2Hz, 1H), 2.50(dt, J=7.2 and 2.4 2H), 2.40 (dd, J=12.8 and 4.4Hz, 1H), 2.25 (t, J=2.4Hz, 1H), 1.66 (t, J=12.8Hz, 1H).

[0203] (2S,4S,5R,6R)-6-((1R,2R)-3-(2-([1,1'-biphenyl]-4-yl)acetamido)-1,2-dihydroxypropyl)-4-hydroxy-5-(2-hydroxyacetamido)-2-((2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(2-(1-(15-oxo-15-(perfluorophenoxy)-3,6,9,12-tetraoxapentadecyl)-1H-1,2,3-triazol-4-yl)ethoxy)tetrahydro-2H-pyran-2-yl)methoxy)tetrahydro-2H-lan-2-carboxylic acid (Compound No. 26373) [ka]

[0204] Synthesis of (2S,4S,5R,6R)-6-((1R,2R)-3-(2-([1,1'-biphenyl]-4-yl)acetamido)-1,2-dihydroxypropyl)-4-hydroxy-5-(2-hydroxyacetamido)-2-((2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(2-(1-(15-oxo-15-(perfluorophenoxy)-3,6,9,12-tetraoxapentadecyl)-1H-1,2,3-triazol-4-yl)ethoxy)tetrahydro-2H-pyran-2-yl)methoxy)tetrahydro-2H-lan-2-carboxylic acid (Compound No. 26373) (2S,4S,5R,6R)-6-((1R,2R)-3-(2-([1,1'-biphenyl]-4-yl)acetamido)-1,2-dihydroxypropyl)-2-(((2R,3R,4S,5R,6R)-6-(but-3-yn-1-yloxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)methoxy)-4-hydroxy-5-(2-hydroxyacetamido)tetrahydrofuran-2-yl)methoxy) in NMP (0.3 mL) in a 1-dram vial with a stir bar To a solution of hydro-2H-pyran-2-carboxylic acid (26411, 1.00 equiv., 19.5 mg, 0.0266 mmol) was added a solution of perfluorophenyl 1-azido-3,6,9,12-tetraoxapentadecan-15-oate (1, 1.10 equiv., 13.4 mg, 0.0293 mmol) in NMP (0.3 mL), followed by tetrakis(acetonitrile)copper(I) hexafluorophosphate (2.50 equiv., 24.8 mg, 0.0665 mmol). The resulting clear green solution was capped and stirred at room temperature for 10 min. The reaction was diluted with acetic acid, filtered, and purified by preparative HPLC (20-100% acetonitrile in water with 0.1% TFA). Fractions containing the desired product were combined and lyophilized to give (2S,4S,5R,6R)-6-((1R,2R)-3-(2-([1,1′-biphenyl]-4-yl)acetamido)-1,2-dihydroxypropyl)-4-hydroxy-5-(2-hydroxyacetamido)-2-((2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(2-(1-(15-oxo-15-(perfluorophenoxy)-3,6,9,12-tetraoxapentadecyl)-1H-1,2,3-triazol-4-yl)ethoxy)tetrahydro-2H-pyran-2-yl)methoxy)tetrahydro-2H-lan-2-carboxylic acid (compound number 26373) as a white solid. Yield: 19.4 mg, 61%; LCMS m / z 1190.8 [M+1]+; 1H NMR (300MHz, DMSO with D2O) δ7.87(s, 1H), 7.75(d, J=8.8Hz, 1H), 7.64~7.52(m, 4H), 7.47~7.39(m, 2H), 7.36~7.28(m, 3H), 4.42(t, J=5 .0Hz, 2H), 4.20~4.11(m, 1H), 4.07~3.58(m, 9H), 3.57~3.38(m, 16H), 3.36~2.81(m, 14H), 2.19~2.08(m, 1H), 1.48(t, J=12.1Hz, 1H).

[0205] (2R,4S,5R,6R)-5-acetamido-2-(((2R,3R,4S,5R,6R)-6-(but-3-yn-1-yloxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)methoxy)-6-((1R,2R)-1,2-dihydroxy-3-(3-phenoxybenzamido)propyl)-4-hydroxytetrahydro-2H-pyran-2-carboxylic acid (compound number 26409) and (2S,4S,5R,6R)-5-acetamido-2-(((2R,3R,4S,5R,6R)-6-(but-3-yn-1-yloxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)methoxy)-6-((1R,2R)-1,2-dihydroxy-3-(3-phenoxybenzamido)propyl)-4-hydroxytetrahydro-2H-pyran-2-carboxylic acid (compound number 26410). [ka]

[0206] Synthesis of (2R,3S,4S,5R,6R)-2-((((2R,4S,5R,6R)-5-acetamido-4-acetoxy-6-((1R,2R)-1,2-diacetoxy-3-(3-phenoxybenzamido)propyl)-2-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy)methyl)-6-(but-3-yn-1-yloxy)tetrahydro-2H-pyran-3,4,5,triyltribenzoate (3) To a stirred solution of (1R,2R)-1-((2R,3R,4S,6S)-3-acetamido-4-acetoxy-6-(methoxycarbonyl)-6-(p-tolylthio)tetrahydro-2H-pyran-2-yl)-3-(3-phenoxybenzamido)propane-1,2-diyl diacetate (1, 0.50 g, 0.665 mmol) and (2R,3R,4S,5S,6R)-2(but-3-yn-1-yloxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triyl tribenzoate (2, 0.543 g, 0.998 mmol) in dry dichloromethane (20 mL), 4 Å molecular sieves (0.50 g, 100% w / w) were added. The resulting reaction mixture was stirred under a nitrogen atmosphere for 12 h. Then, 1-iodopyrrolidine-2,5-dione (0.288 g, 1.28 mmol) and trifluoromethanesulfonic acid (0.047 mL, 0.533 mmol) were added at −45° C. The resulting reaction mixture was stirred at −45° C. for 30 minutes. Upon completion, the reaction mixture was quenched with trimethylamine to neutral pH, filtered through Celite, diluted with dichloromethane, and washed with aqueous sodium bicarbonate, followed by DM water. The organic layer was concentrated to give the crude residue, which was then purified by flash column chromatography (5-7.5% methanol in ethyl acetate). The desired fractions were concentrated and dried to give (2R,3S,4S,5R,6R)-2-((((2R,4S,5R,6R)-5-acetamido-4-acetoxy-6-((1R,2R)-1,2-diacetoxy-3-(3-phenoxybenzamido)propyl)-2-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy)methyl)-6-(but-3-yn-1-yloxy)tetrahydro-2H-pyran-3,4,5,triyl tribenzoate (3) as a yellow amorphous solid as a mixture of anomers. Yield: 0.60 g, 76.93%, LCMS (ESI) m / z 1172.17 [M+1] + .

[0207] (2R,4S,5R,6R)-5-acetamido-2-(((2R,3R,4S,5R,6R)-6-(but-3-yn-1-yloxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)methoxy)-6-((1R,2R)-1,2-dihydroxy-3-(3-phenoxybenzamido)propyl)-4-hydroxytetrahydro-2H-pyran-2-carboxylic acid (compound number 26409) and ( Synthesis of 2S,4S,5R,6R)-5-acetamido-2-(((2R,3R,4S,5R,6R)-6-(but-3-yn-1-yloxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)methoxy)-6-((1R,2R)-1,2-dihydroxy-3-(3-phenoxybenzamido)propyl)-4-hydroxytetrahydro-2H-pyran-2-carboxylic acid (compound number 26410) To a stirred solution of (2R,3S,4S,5R,6R)-2-((((2R,4S,5R,6R)-5-acetamido-4-acetoxy-6-((1R,2R)-1,2-diacetoxy-3-(3-phenoxybenzamido)propyl)-2-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy)methyl)-6-(but-3-yn-1-yloxy)tetrahydro-2H-pyran-3,4,5,triyl tribenzoate (3, 0.60 g, 0.512 mmol) in ethanol (5.0 mL) was added lithium hydroxide (0.129 g, 3.07 mmol) at 0° C. The reaction mixture was stirred at room temperature for 4 hours. After completion, Dowex® hydrogen carbonate was added until pH 6 and the reaction mass was filtered. The filtrate was concentrated and dried to give a crude residue, which was then diluted with acetonitrile and purified by preparative HPLC (15-35% acetonitrile in water with 0.1% TFA). Fractions containing the desired product were separately combined and lyophilized to give (2R,4S,5R,6R)-5-acetamido-2-(((2R,3R,4S,5R,6R)-6-(but-3-yn-1-yloxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)methoxy)-6-((1R,2R)-1,2-dihydroxy-3-(3-phenoxybenzamido)propyl)-4-hydroxytetrahydro-2H-pyran-2-carboxylic acid (compound no. 26409) as a white solid as the TFA salt. Yield: 0.045 g, 12%; LCMS (ESI) m / z 719.57 [M+1]. + ; 1H NMR (400MHz, methanol-d4) δ8.28~8.26 (m, 1H), 8.15~8.12 (m, 1H), 7.58 (d, J=7. 6Hz, 1H), 7.48~7.35(m, 4H), 7.15~7.11(m, 2H), 7.01(d, J=8.0Hz, 2H), 4.21( t, J=3.6Hz, 1H), 4.04~4.00(m, 2H), 3.91~3.77(m, 2H), 3.72~3.57(m, 6H), 3. 49~3.45(m, 3H), 3.39(d, J=8.8Hz, 1H), 2.83~2.80(m, 1H), 2.45~2.41(m, 2H) , 2.21 (t, J = 2.8 Hz, 1H), 1.97 (s, 3H), 1.61 (t, J = 11.6 Hz, 1H); (2S,4S,5R,6R)-5-acetamido-2-(((2R,3R,4S,5R,6R)-6-(but-3-yn-1-yloxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)methoxy)-6-((1R,2R)-1,2-dihydroxy-3-(3-phenoxybenzamido)propyl)-4-hydroxytetrahydro-2H-pyran-2-carboxylic acid (compound number 26410) was obtained as a white solid as the TFA salt. Yield: 0.045 g, 12%; LCMS (ESI) m / z 719.57 [M+1] + ; 1 H NMR (400MHz, methanol-d4) δ7.59 (d, J=7.2Hz, 1H), 7.49~7.35 (m, 4H), 7.15~7.1 3(m, 2H), 7.00(d, J=7.6Hz, 2H), 4.25(t, J=6.8Hz, 1H), 3.99~3.85(m, 6H), 3.8 1~3.63(m, 4H), 3.54~3.44(m, 4H), 3.36(d, J=8.8Hz, 1H), 2.49~2.45(m, 2H), 2 .42~2.37(m, 1H), 2.23(t, J=2.8Hz, 1H), 1.95(s, 3H), 1.60(t, J=12.4Hz, 1H).

[0208] (2S,2'S,4S,4'S,5R,5'R,6R,6'R)-2,2'-(((2R,2'R,3R,3'R,4S,4'S,5R,5'R,6R,6'R)-(((((RS)-9,14,22-trioxo-16-((15-oxo-15-(perfluorophenoxy)-3,6,9,12-tetraoxapentadecyl)carbamoyl)-3,6,25,28-tetraoxa-10,15,21-triazatriacontane-1,30-diyl)bis(1H-1,2,3 ... riazole-1,4-diyl))bis(ethane-2,1-diyl))bis(oxy))bis(3,4,5-trihydroxytetrahydro-2H-pyran-6,2-diyl))bis(methylene))bis(oxy))bis(6-((1R,2R)-3-(2-([1,1'-biphenyl]-4-yl)acetamido)-1,2-dihydroxypropyl)-4-hydroxy-5-(2-hydroxyacetamido)tetrahydro-2H-pyran-2-carboxylic acid) (Compound No. 26412) [ka]

[0209] (2S,2'S,4S,4'S,5R,5'R,6R,6'R)-2,2'-(((2R,2'R,3R,3'R,4S,4'S,5R,5'R,6R,6'R)-(((((RS)-9,14,22-trioxo-16-((15-oxo-15-(perfluorophenoxy)-3,6,9,12-tetraoxapentadecyl)carbamoyl)-3,6,25,28-tetraoxa-10,15,21-triazatriacontane-1,30-diyl)bis(1H-1,2,3 ... Synthesis of))azole-1,4-diyl)bis(ethane-2,1-diyl))bis(oxy))bis(3,4,5-trihydroxytetrahydro-2H-pyran-6,2-diyl))bis(methylene))bis(oxy))bis(6-((1R,2R)-3-(2-([1,1'-biphenyl]-4-yl)acetamido)-1,2-dihydroxypropyl)-4-hydroxy-5-(2-hydroxyacetamido)tetrahydro-2H-pyran-2-carboxylic acid) (Compound No. 26412) Perfluorophenyl (R)-1-azido-16-(4-(3-(2-(2-azidoethoxy)ethoxy)propanamido)butyl)-9,14,17-trioxo-3,6,21,24,27,30-hexaoxa-10,15,18-triazatriacontan-33-oate (26332, 1.00 equiv., 14.3 mg, 0.0141 mmol) and (2S,4S,5R,6R)-6-((1R,2R)-3-(2-([1,1'-biphenyl]-4-yl)acetamido)-1,4-diol in NMP (0.6 mL) in a 1-dram vial with a stir bar. To a solution of (2R,3R,4S,5R,6R)-6-(but-3-yn-1-yloxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)methoxy)-4-hydroxy-5-(2-hydroxyacetamido)tetrahydro-2H-pyran-2-carboxylic acid (26411, 2.10 equiv., 21.7 mg, 0.0296 mmol), tetrakis(acetonitrile)copper(I) hexafluorophosphate (5.00 equiv., 26.3 mg, 0.0704 mmol) was added. The resulting clear green solution was capped and stirred at room temperature for 10 min. The reaction was diluted with acetic acid, filtered, and purified by preparative HPLC (20-80% acetonitrile in water with 0.1% TFA).Fractions containing the desired product were combined, lyophilized, and purified to give (2S,2'S,4S,4'S,5R,5'R,6R,6'R)-2,2'-(((2R,2'R,3R,3'R,4S,4'S,5R,5'R,6R,6'R)-(((((RS)-9,14,22-trioxo-16-((15-oxo-15-(perfluorophenoxy)-3,6,9,12-tetraoxapentadecyl)carbamoyl)-3,6,25,28-tetraoxa-10,15,21-triazatriacontane-1,30-diyl)bis(1H- 1,2,3-Triazole-1,4-diyl))bis(ethane-2,1-diyl))bis(oxy))bis(3,4,5-trihydroxytetrahydro-2H-pyran-6,2-diyl))bis(methylene))bis(oxy))bis(6-((1R,2R)-3-(2-([1,1'-biphenyl]-4-yl)acetamido)-1,2-dihydroxypropyl)-4-hydroxy-5-(2-hydroxyacetamido)tetrahydro-2H-pyran-2-carboxylic acid) (compound number 26412) was obtained as a white solid. Yield: 22.8 mg, 65%; LCMS m / z 1240.7 [M / 2+1]+; 1 H NMR (300MHz, DMSO with D2O) δ7.86(s, 2H), 7.75(d, J=8.8Hz, 2H), 7.63~7.51(m, 8H), 7.42(t, J=7.5Hz, 4H), 7.35~7.27(m, 6H), 4.46~ 4.34(m, 4H), 4.19~3.58(m, 19H), 3.57~2.79(m, 66H), 2.30~2.19(m, 4H), 2.18~2.03(m, 4H), 1.64~1.39(m, 6H), 1.37~1.07(m, 4H).

[0210] (2S,2'S,4S,4'S,5R,5'R,6R,6'R)-2,2'((((((((RS)-9,14,22-trioxo-16-((15-oxo-15-(perfluorophenoxy)-3,6,9,12-tetraoxapentadecyl)carbamoyl)-3,6,25,28-tetraoxa-10,15,21-triazatriacontane-1,30-diyl)bis(1H-1,2,3 -triazole-1,4-diyl))bis(methylene))bis(oxy))bis(ethane-2,1-diyl))bis(oxy))bis(ethane-2,1-diyl))bis(oxy))bis(5-acetamido-6-((1R,2R)-1,2-dihydroxy-3-(3-phenoxybenzamido)propyl)-4-hydroxytetrahydro-2H-pyran-2carboxylic acid) (compound number 26414) [ka]

[0211] (2S,2'S,4S,4'S,5R,5'R,6R,6'R)-2,2'((((((((RS)-9,14,22-trioxo-16-((15-oxo-15-(perfluorophenoxy)-3,6,9,12-tetraoxapentadecyl)carbamoyl)-3,6,25,28-tetraoxa-10,15,21-triazatriacontane-1,30-diyl)bis(1H-1,2,3- Synthesis of triazole-1,4-diyl))bis(methylene))bis(oxy))bis(ethane-2,1-diyl))bis(oxy))bis(ethane-2,1-diyl))bis(oxy))bis(5-acetamido-6-((1R,2R)-1,2-dihydroxy-3-(3-phenoxybenzamido)propyl)-4-hydroxytetrahydro-2H-pyran-2carboxylic acid) (Compound No. 26414) Perfluorophenyl (R)-1-azido-16-(4-(3-(2-(2-azidoethoxy)ethoxy)propanamido)butyl)-9,14,17-trioxo-3,6,21,24,27,30-hexaoxa-10,15,18-triazatriacontan-33-oate (26332, 1.00 equiv., 22.5 mg, 0.0222 mmol) and (2S,4S,5R,6R)-5-acetamido in NMP (0.6 mL) in a 1-dram vial with a stir bar. To a solution of do-6-((1R,2R)-1,2-dihydroxy-3-(3-phenoxybenzamido)propyl)-4-hydroxy-2-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)tetrahydro-2H-pyran-2-carboxylic acid (26335, 2.10 equiv., 29.4 mg, 0.0466 mmol), tetrakis(acetonitrile)copper(I) hexafluorophosphate (5.00 equiv., 41.3 mg, 0.111 mmol) was added. The resulting clear, pale yellow solution was capped and stirred at room temperature for 10 min (slowly turning green). The reaction was diluted with acetic acid, filtered, and purified by preparative HPLC (20-90% acetonitrile in water with 0.1% TFA). Fractions containing the desired product were combined, lyophilized, and purified to give (2S,2'S,4S,4'S,5R,5'R,6R,6'R)-2,2'((((((((RS)-9,14,22-trioxo-16-((15-oxo-15-(perfluorophenoxy)-3,6,9,12-tetraoxapentadecyl)carbamoyl)-3,6,25,28-tetraoxa-10,15,21-triazatriacontane-1,30-diyl)bis(1 H-1,2,3-triazole-1,4-diyl))bis(methylene))bis(oxy))bis(ethane-2,1-diyl))bis(oxy))bis(ethane-2,1-diyl))bis(oxy))bis(5-acetamido-6-((1R,2R)-1,2-dihydroxy-3-(3-phenoxybenzamido)propyl)-4-hydroxytetrahydro-2H-pyran-2carboxylic acid) (compound number 26414) was obtained as a white solid. Yield: 32.9 mg, 65%; LCMS m / z 1138.8 [M / 2+1]+; 1H NMR (300MHz, DMSO with D2O) δ7.96(s, 2H), 7.55(d, J=7.7Hz, 2H), 7.48~7.32(m, 8H), 7.13(t, J=7.4Hz, 4H), 6.97(d, J=8.0Hz, 4H), 4.51~4.38(m, 8H), 3.89~2.87(m, 71H), 2.29~2.19(m, 4H), 2.18~2.03(m, 4H), 1.82(s, 6H), 1.62~1.39(m, 6H), 1.36~1.06(m, 4H).

[0212] (2R,4S,5R,6R)-6-((1R,2R)-3-(2-([1,1'-biphenyl]-4-yl)acetamido)-1,2-dihydroxypropyl)-4-hydroxy-5-(2-hydroxyacetamido)-2-((2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(2-(1-(15-oxo-15-(perfluorophenoxy)-3,6,9,12-tetraoxapentadecyl)-1H-1,2,3-triazol-4-yl)ethoxy)tetrahydro-2H-pyran-2-yl)methoxy)tetrahydro-2H-pyran-2-carboxylic acid (compound number 26415) [ka]

[0213] Synthesis of (2R,4S,5R,6R)-6-((1R,2R)-3-(2-([1,1'-biphenyl]-4-yl)acetamido)-1,2-dihydroxypropyl)-4-hydroxy-5-(2-hydroxyacetamido)-2-((2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(2-(1-(15-oxo-15-(perfluorophenoxy)-3,6,9,12-tetraoxapentadecyl)-1H-1,2,3-triazol-4-yl)ethoxy)tetrahydro-2H-pyran-2-yl)methoxy)tetrahydro-2H-pyran-2-carboxylic acid (Compound No. 26415) (2R,4S,5R,6R)-6-((1R,2R)-3-(2-([1,1'-biphenyl]-4-yl)acetamido)-1,2-dihydroxypropyl)-2-(((2R,3R,4S,5R,6R)-6-(but-3-yn-1-yloxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)methoxy)-4-hydroxy-5-(2-hydroxyacetamido)tetrahydrofuran-2-yl)methoxy) in NMP (0.3 mL) in a 1-dram vial with a stir bar To a solution of hydro-2H-pyran-2-carboxylic acid (26339, 1.00 equiv., 26.2 mg, 0.0358 mmol) in NMP (0.3 mL) was added a solution of perfluorophenyl 1-azido-3,6,9,12-tetraoxapentadecan-15-oate (1, 1.10 equiv., 18.0 mg, 0.0393 mmol), followed by tetrakis(acetonitrile)copper(I) hexafluorophosphate (2.50 equiv., 33.3 mg, 0.0894 mmol). The resulting clear green solution was capped and stirred at room temperature for 10 min. The reaction was diluted with acetic acid, filtered, and purified by preparative HPLC (20-70% acetonitrile in water with 0.1% TFA). Fractions containing the desired product were combined and lyophilized to give (2R,4S,5R,6R)-6-((1R,2R)-3-(2-([1,1′-biphenyl]-4-yl)acetamido)-1,2-dihydroxypropyl)-4-hydroxy-5-(2-hydroxyacetamido)-2-((2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(2-(1-(15-oxo-15-(perfluorophenoxy)-3,6,9,12-tetraoxapentadecyl)-1H-1,2,3-triazol-4-yl)ethoxy)tetrahydro-2H-pyran-2-yl)methoxy)tetrahydro-2H-puran-2-carboxylic acid (compound number 26415) as a white solid. Yield: 26.5 mg, 62%; LCMS m / z 1190.7[M+1]+; 1H NMR (300MHz, DMSO with D2O) δ7.83(s, 1H), 7.79(d, J=7.4Hz, 1H), 7.62~7.50(m, 4H), 7.42(t, J=7.6Hz, 2H), 7.34~7.26 (m, 3H), 4.39(t, J=4.8Hz, 2H), 4.15(d, J=6.6Hz, 1H), 3.96~2.79(m, 38H), 2.47~2.40(m, 1H), 1.55(t, J=12.2Hz, 1H).

[0214] (2R,2'R,4S,4'S,5R,5'R,6R,6'R)-2,2'-(((2R,2'R,3R,3'R,4S,4'S,5R,5'R,6R,6'R)-(((((RS)-9,14,22-trioxo-16-((15-oxo-15-(perfluorophenoxy)-3,6,9,12-tetraoxapentadecyl)carbamoyl)-3,6,25,28-tetraoxa-10,15,21-triazatriacontane-1,30-diyl)bis(1H-1,2,3-trioxo-16-((15-oxo-15-(perfluorophenoxy)-3,6,9,12-tetraoxapentadecyl)carbamoyl) riazole-1,4-diyl))bis(ethane-2,1-diyl))bis(oxy))bis(3,4,5-trihydroxytetrahydro-2H-pyran-6,2-diyl))bis(methylene))bis(oxy))bis(6-((1R,2R)-3-(2-([1,1'-biphenyl]-4-yl)acetamido)-1,2-dihydroxypropyl)-4-hydroxy-5-(2-hydroxyacetamido)tetrahydro-2H-pyran-2-carboxylic acid) (Compound No. 26416) [ka]

[0215] (2R,2'R,4S,4'S,5R,5'R,6R,6'R)-2,2'-(((2R,2'R,3R,3'R,4S,4'S,5R,5'R,6R,6'R)-(((((RS)-9,14,22-trioxo-16-((15-oxo-15-(perfluorophenoxy)-3,6,9,12-tetraoxapentadecyl)carbamoyl)-3,6,25,28-tetraoxa-10,15,21-triazatriacontane-1,30-diyl)bis(1H-1,2,3 ... Synthesis of))azole-1,4-diyl)bis(ethane-2,1-diyl))bis(oxy))bis(3,4,5-trihydroxytetrahydro-2H-pyran-6,2-diyl))bis(methylene))bis(oxy))bis(6-((1R,2R)-3-(2-([1,1'-biphenyl]-4-yl)acetamido)-1,2-dihydroxypropyl)-4-hydroxy-5-(2-hydroxyacetamido)tetrahydro-2H-pyran-2-carboxylic acid) (Compound No. 26416) Perfluorophenyl (R)-1-azido-16-(4-(3-(2-(2-azidoethoxy)ethoxy)propanamido)butyl)-9,14,17-trioxo-3,6,21,24,27,30-hexaoxa-10,15,18-triazatriacontan-33-oate (26332, 1.00 equiv., 20.7 mg, 0.0204 mmol) and (2R,4S,5R,6R)-6-((1R,2R)-3-(2-([1,1'-biphenyl]-4-yl)acetamido)-1,4-diol in NMP (0.6 mL) in a 1-dram vial with a stir bar. To a solution of (2R,3R,4S,5R,6R)-6-(but-3-yn-1-yloxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)methoxy)-4-hydroxy-5-(2-hydroxyacetamido)tetrahydro-2H-pyran-2-carboxylic acid (26339, 2.10 equiv., 31.4 mg, 0.0428 mmol), tetrakis(acetonitrile)copper(I) hexafluorophosphate (5.00 equiv., 38.0 mg, 0.102 mmol) was added. The resulting clear green solution was capped and stirred at room temperature for 10 min. The reaction was diluted with acetic acid, filtered, and purified by preparative HPLC (20-65% acetonitrile in water with 0.1% TFA).Fractions containing the desired product were combined, lyophilized, and purified to give (2R,2'R,4S,4'S,5R,5'R,6R,6'R)-2,2'-(((2R,2'R,3R,3'R,4S,4'S,5R,5'R,6R,6'R)-(((((RS)-9,14,22-trioxo-16-((15-oxo-15-(perfluorophenoxy)-3,6,9,12-tetraoxapentadecyl)carbamoyl)-3,6,25,28-tetraoxa-10,15,21-triazatriacontane-1,30-diyl)bis(1H- 1,2,3-Triazole-1,4-diyl))bis(ethane-2,1-diyl))bis(oxy))bis(3,4,5-trihydroxytetrahydro-2H-pyran-6,2-diyl))bis(methylene))bis(oxy))bis(6-((1R,2R)-3-(2-([1,1'-biphenyl]-4-yl)acetamido)-1,2-dihydroxypropyl)-4-hydroxy-5-(2-hydroxyacetamido)tetrahydro-2H-pyran-2-carboxylic acid) (compound number 26416) was obtained as a white solid. Yield: 27.2 mg, 54%; LCMS m / z 1240.8 [M / 2+1]+; 1 H NMR (300MHz, DMSO with D2O) δ7.84~7.75(m, 4H), 7.61~7.49(m, 8H), 7.41(t, J=7.5Hz, 4H), 7.34~7.25(m, 6H), 4.42~4.32(m, 4 H), 3.94~2.76(m, 85H), 2.47~2.39(m, 2H), 2.30~2.17(m, 4H), 2.08(t, J=7.5Hz, 2H), 1.64~1.37(m, 6H), 1.35~1.07(m, 4H).

[0216] Compound number 26417 [ka]

[0217] Synthesis of compound No. 26417 Perfluorophenyl(16R,19R)-1-azido-16,19-bis(4-(3-(2-(2-azidoethoxy)ethoxy)propanamido)butyl)-9,14,17,20-tetraoxo-3,6,24,27,30,33-hexaoxa-10,15,18,21-tetraazahexatriacontan-36-oate (26333, 1.00 equiv., 19.1 mg, 0.0144 mmol) and (2R,4S,5R,6R)-6-((1R,2R)-3-(2-([1,1'-biphenyl] To a solution of ((2R,3R,4S,5R,6R)-6-(but-3-yn-1-yloxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)methoxy)-4-hydroxy-5-(2-hydroxyacetamido)tetrahydro-2H-pyran-2-carboxylic acid (26339, 3.10 equiv., 32.7 mg, 0.0446 mmol) was added tetrakis(acetonitrile)copper(I) hexafluorophosphate (7.50 equiv., 40.2 mg, 0.108 mmol). The resulting clear green solution was capped and stirred at room temperature for 10 min. The reaction was diluted with a mixture of 70% acetic acid in NMP, filtered, and purified by preparative HPLC (15–65% acetonitrile in water with 0.1% TFA). Fractions containing the desired product were combined and lyophilized to give compound no. 26417 as a white solid. Yield: 22.8 mg, 45%; LCMS m / z [M+1]+; 1 H NMR (300MHz, DMSO with D2O) δ7.83~7.73(m, 6H), 7.60~7.48(m, 12H), 7.40(t, J=7.5Hz, 6H), 7.33~7.25(m, 9H), 4.40~4.32(m , 6H), 3.93~2.77(m, 120H), 2.47~2.40(m, 2H), 2.30~2.18(m, 6H), 2.13~2.03(m, 2H), 1.66~1.38(m, 8H), 1.36~1.06(m, 8H).

[0218] Compound No. 26418 [ka] TIFF2024502850000040.tif113156

[0219] Synthesis of compound No. 26418 Perfluorophenyl(16R,19R)-1-azido-16,19-bis(4-(3-(2-(2-azidoethoxy)ethoxy)propanamido)butyl)-9,14,17,20-tetraoxo-3,6,24,27,30,33-hexaoxa-10,15,18,21-tetraazahexatriacontan-36-oate (26333, 1.00 equiv., 18.3 mg, 0.0138 mmol) and (2S,4S,5R,6R)-6-((1R,2R)-3-(2-([1,1'-biphenyl] To a solution of ((2R,3R,4S,5R,6R)-6-(but-3-yn-1-yloxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)methoxy)-4-hydroxy-5-(2-hydroxyacetamido)tetrahydro-2H-pyran-2-carboxylic acid (26411, 3.10 equiv., 31.3 mg, 0.0427 mmol) was added tetrakis(acetonitrile)copper(I) hexafluorophosphate (7.50 equiv., 38.5 mg, 0.103 mmol). The resulting clear green solution was capped and stirred at room temperature for 10 min. The reaction was diluted with a mixture of 70% acetic acid in NMP, filtered, and purified by preparative HPLC (15–65% acetonitrile in water with 0.1% TFA). Fractions containing the desired product were combined and lyophilized to give compound no. 26418 as a white solid. Yield: 32.7 mg, 67%; LCMS m / z 1763.7 [M / 2+1]+; 1H NMR (300MHz, DMSO with D2O) δ7.83(s, 3H), 7.76(d, J=8.9Hz, 3H), 7.61~7.49(m, 12H), 7.40(t, J=7.5Hz, 6H), 7.34~ 7.26(m, 9H), 4.43~4.34(m, 6H), 4.02~2.80(m, 120H), 2.30~2.04(m, 10H), 1.64~1.40(m, 8H), 1.35~1.08(m, 8H).

[0220] (2R,4S,5R,6R)-6-((1R,2R)-3-(2-([1,1'-biphenyl]-4-yl)acetamido)-1,2-dihydroxypropyl)-4-hydroxy-5-(2-hydroxyacetamido)-2-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)tetrahydro-2H-pyran-2-carboxylic acid (compound number 26463) and (2S,4S,5R,6R)-6-((1R,2R)-3-(2-([1,1'-biphenyl]-4-yl)acetamido)-1,2-dihydroxypropyl)-4-hydroxy-5-(2-hydroxyacetamido)-2-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)tetrahydro-2H-pyran-2-carboxylic acid (compound number 26464) [ka] TIFF2024502850000042.tif81156

[0221] Synthesis of (1R,2R)-3-(2-([1,1'-biphenyl]-4-yl)acetamido)-1-((2R,3R,4S)-4-acetoxy-3-(2-acetoxyacetamido)-6-(methoxycarbonyl)-6-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)tetrahydro-2H-pyran-2-yl)propane-1,2-diyl diacetate (3) To a stirred solution of (1R,2R)-3-(2-([1,1'-biphenyl]-4-yl)acetamido)-1-((2R,3R,4S,6S)-4-acetoxy-3-(2-acetoxyacetamido)-6-(methoxycarbonyl)-6-(p-tolylthio)tetrahydro-2H-pyran-2-yl)propane-1,2-diyl diacetate (1, 1.0 g, 1.240 mmol) in anhydrous dichloromethane (20.0 mL) was added 2-(2-(prop-2-yn-1-yloxy)ethoxy)ethan-1-ol (2, 0.893 g, 6.20 mmol) and activated 4 Å powdered molecular sieves (1.0 g, 100% w / w). The resulting reaction solution was stirred at room temperature under nitrogen for 15 hours. Then, 1-iodopyrrolidine-2,5-dione (0.697 g, 3.10 mmol) and trifluoromethanesulfonic acid (0.109 mL, 1.240 mmol) were added at −40° C. The resulting reaction solution was stirred at −40° C. for 1 hour. Upon completion, the reaction mixture was quenched with triethylamine (0.5 mL) and allowed to warm to room temperature. The reaction mixture was filtered through a sintered funnel and washed with dichloromethane. The filtrate was washed with saturated sodium bicarbonate (aq), dried over sodium sulfate, filtered, and concentrated under reduced pressure to provide a crude residue. The residue was purified by column chromatography (60-80% ethyl acetate in hexanes) to give (1R,2R)-3-(2-([1,1'-biphenyl]-4-yl)acetamido)-1-((2R,3R,4S)-4-acetoxy-3-(2-acetoxyacetamido)-6-(methoxycarbonyl)-6-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)tetrahydro-2H-pyran-2-yl)propane-1,2-diyl diacetate (3) as a white solid as an anomeric mixture. Yield: 0.80 g, 78.07%, LCMS (ESI) m / z 827.30 [M+1] + .

[0222] (2R,4S,5R,6R)-6-((1R,2R)-3-(2-([1,1'-biphenyl]-4-yl)acetamido)-1,2-dihydroxypropyl)-4-hydroxy-5-(2-hydroxyacetamido)-2-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)tetrahydro-2H-pyran-2-carboxylic acid (compound number 26463) and Synthesis of (2S,4S,5R,6R)-6-((1R,2R)-3-(2-([1,1'-biphenyl]-4-yl)acetamido)-1,2-dihydroxypropyl)-4-hydroxy-5-(2-hydroxyacetamido)-2-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)tetrahydro-2H-pyran-2-carboxylic acid (compound number 26464) To a stirred solution of (1R,2R)-3-(2-([1,1'-biphenyl]-4-yl)acetamido)-1-((2R,3R,4S,6R)-4-acetoxy-3-(2-acetoxyacetamido)-6-(methoxycarbonyl)-6-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)tetrahydro-2H-pyran-2-yl)propane-1,2-diyl diacetate (3, 0.450 g, 0.544 mmol) in methanol (5.0 mL) was added a solution of lithium hydroxide monohydrate (0.137 g, 3.27 mmol) in water (0.50 mL). The resulting reaction mixture was stirred at room temperature for 6 hours. Upon completion, the reaction mixture was treated with Dowex 50 (H+) to a pH of approximately 6, and the suspension was filtered and washed with methanol. The filtrate was concentrated under reduced pressure to give a crude residue. The residue was purified by preparative HPLC to give (2R,4S,5R,6R)-6-((1R,2R)-3-(2-([1,1'-biphenyl]-4-yl)acetamido)-1,2-dihydroxypropyl)-4-hydroxy-5-(2-hydroxyacetamido)-2-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)tetrahydro-2H-pyran-2-carboxylic acid (compound number 26463) as a white solid. Yield: 0.316 g, 90%, LCMS (ESI) m / z 645.45 [M+1] + . 1H NMR (400MHz, methanol-d4) δ7.87(d, J=7.6Hz, 1H), 7.61~7.56(m, 4H), 7.43~7.37(m, 4H), 7.31(t, J=7.2Hz, 1H), 4.17(d, J=2.4 Hz, 2H), 3.90(s, 2H), 3.89~3.81(m, 4H), 3.74~3.59(m, 11H), 3.39~3.25(m, 2H), 2.82(t, J=0.8Hz, 1H), 2.71(dd, J=8.8 and 4. 0 Hz, 1H), 1.75 (t, J = 12.4 Hz, 1H); (2S,4S,5R,6R)-6-((1R,2R)-3-(2-([1,1'-biphenyl]-4-yl)acetamido)-1,2-dihydroxypropyl)-4-hydroxy-5-(2-hydroxyacetamido)-2-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)tetrahydro-2H-pyran-2-carboxylic acid (compound number 26464) was obtained as a white solid. Yield: 0.192 g, 80.25%, LCMS (ESI) m / z 645.42 [M+1] + . 1 H NMR (400MHz, methanol-d4) δ7.82 (d, J=8.8Hz, 1H), 7.61~7.57 (m, 4H), 7.44~7.36 (m, 4H), 7. 31(t, J=7.2Hz, 1H), 4.17(d, J=2.4Hz, 2H), 4.15~4.119(m, 1H), 4.03~3.96(m, 3H), 3.94~3 .86(m, 1H), 3.86~3.78(m, 2H), 3.36~3.62(m, 2H) 3.59(s, 2H), 3.51~3.46(m, 1H), 3.42~3 .35(m, 2H), 2.84(t, J=2.4Hz, 1H), 2.38(dd, J=12.8 and 3.8Hz, 1H), 1.66(t, J=11.6Hz, 1H).

[0223] (2S,4S,5R,6R)-5-acetamido-6-((1R,2R)-1,2-dihydroxy-3-(3-phenoxybenzamido)propyl)-4-hydroxy-2-(((2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(2-(1-(15-oxo-15-(perfluorophenoxy)-3,6,9,12-tetraoxapentadecyl)-1H-1,2,3-triazol-4-yl)ethoxy)tetrahydro-2H-pyran-2-yl)methoxy)tetrahydro-2H-pyran-2-carboxylic acid (compound number 26465) [ka]

[0224] Synthesis of (2S,4S,5R,6R)-5-acetamido-6-((1R,2R)-1,2-dihydroxy-3-(3-phenoxybenzamido)propyl)-4-hydroxy-2-(((2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(2-(1-(15-oxo-15-(perfluorophenoxy)-3,6,9,12-tetraoxapentadecyl)-1H-1,2,3-triazol-4-yl)ethoxy)tetrahydro-2H-pyran-2-yl)methoxy)tetrahydro-2H-pyran-2-carboxylic acid (Compound No. 26465) (2S,4S,5R,6R)-5-acetamido-2-(((2R,3R,4S,5R,6R)-6-(but-3-yn-1-yloxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)methoxy)-6-((1R,2R)-1,2-dihydroxy-3-(3-phenoxybenzamido)propyl)-4-hydroxytetrahydro-2H-pyran-2 in NMP (0.3 mL) in a 1-dram vial with a stir bar To a solution of 2-carboxylic acid (26410, 1.00 equiv., 25.8 mg, 0.0359 mmol) was added a solution of perfluorophenyl 1-azido-3,6,9,12-tetraoxapentadecan-15-oate (1, 1.10 equiv., 18.1 mg, 0.0395 mmol) in NMP (0.3 mL), followed by tetrakis(acetonitrile)copper(I) hexafluorophosphate (2.50 equiv., 33.5 mg, 0.0898 mmol). The resulting clear green solution was capped and stirred at room temperature for 10 min. The reaction was diluted with acetic acid, filtered, and purified by preparative HPLC (15-65% acetonitrile in water with 0.1% TFA). Fractions containing the desired product were combined and lyophilized to give (2S,4S,5R,6R)-5-acetamido-6-((1R,2R)-1,2-dihydroxy-3-(3-phenoxybenzamido)propyl)-4-hydroxy-2-(((2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(2-(1-(15-oxo-15-(perfluorophenoxy)-3,6,9,12-tetraoxapentadecyl)-1H-1,2,3-triazol-4-yl)ethoxy)tetrahydro-2H-pyran-2-yl)methoxy)tetrahydro-2H-pyran-2-carboxylic acid (compound no. 26465) as a white solid. Yield: 23.5 mg, 56%; LCMS m / z 1176.8 [M+1]+; 1H NMR (300MHz, D2O with DMSO-d6) δ7.93 (d, J=7.5Hz, 1H), 7.84 (s, 1H), 7.55 (d, J =7.7Hz, 1H), 7.45(t, J=7.9Hz, 1H), 7.41~7.32(m, 3H), 7.14(t, J=7.1Hz, 2H) , 6.97(d, J=8.0Hz, 2H), 4.44~4.34(m, 2H), 3.95~3.78(m, 2H), 3.78~2.95(m, 30H), 2.95~2.81(m, 4H), 2.18~2.06(m, 1H), 1.83(s, 3H), 1.54~1.40(m, 1H).

[0225] Compound number 26466

change

[0226] Synthesis of compound number 26466 Perfluorophenyl (16R,19R)-1-azido-16,19-bis(4-(3-(2-(2-azidoethoxy)ethoxy)propanamido)butyl)-9,14,17,20-tetraoxo-3,6,24,27,30,33-hexaoxa-10,15,18,21-tetraazahexatriacontan-36-oate (26333, 1.00 equiv., 23.1 mg, 0.0160 mmol) and (2R,4R,5S, To a solution of 6S)-5-acetamido-6-((1S,2S)-1,2-dihydroxy-3-(3-phenoxybenzamido)propyl)-4-hydroxy-2-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)tetrahydro-2H-pyran-2-carboxylic acid (26335, 3.10 equiv., 36.9 mg, 0.0495 mmol), tetrakis(acetonitrile)copper(I) hexafluorophosphate (7.50 equiv., 44.7 mg, 0.120 mmol) was added. The resulting clear, pale yellow solution was capped and stirred at room temperature for 10 min (slowly turning green). The reaction was diluted with a mixture of 70% acetic acid in NMP, filtered, and purified by preparative HPLC (15-65% acetonitrile in water with 0.1% TFA). Fractions containing the desired product were combined and lyophilized to give compound no. 26466 as a white solid. Yield: 35.4 mg, 69%; LCMS m / z 1610.7 [M / 2+1]+; 1 H NMR (300MHz, DMSO-d6 with D2O) δ7.95(s, 3H), 7.54(d, J=7.8Hz, 3H), 7.47~7.30(m, 12H), 7.12(t, J=7.4Hz, 6H), 6.96(d, J=8.0Hz, 6H) , 4.52~4.37(m, 12H), 3.90~2.78(m, 99H), 2.29~2.18(m, 6H), 2.18~2.02(m, 4H), 1.82(s, 9H), 1.68~1.36(m, 8H), 1.36~1.05(m, 8H).

[0227] Compound No. 26467 [ka] TIFF2024502850000046.tif57155

[0228] Synthesis of compound No. 26467 Perfluorophenyl (16R,19R)-1-azido-16,19-bis(4-(3-(2-(2-azidoethoxy)ethoxy)propanamido)butyl)-9,14,17,20-tetraoxo-3,6,24,27,30,33-hexaoxa-10,15,18,21-tetraazahexatriacontan-36-oate (26333, 1.00 equiv., 23.7 mg, 0.0164 mmol) and (2R,4S,5R, To a solution of 6R)-5-acetamido-6-((1R,2R)-1,2-dihydroxy-3-(3-phenoxybenzamido)propyl)-4-hydroxy-2-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)tetrahydro-2H-pyran-2-carboxylic acid (26334, 3.10 equiv., 37.9 mg, 0.0508 mmol), tetrakis(acetonitrile)copper(I) hexafluorophosphate (7.50 equiv., 45.8 mg, 0.123 mmol) was added. The resulting clear, pale yellow solution was capped and stirred at room temperature for 10 min (slowly turning green). The reaction was diluted with a mixture of 70% acetic acid in NMP, filtered, and purified by preparative HPLC (15-65% acetonitrile in water with 0.1% TFA). Fractions containing the desired product were combined and lyophilized to give compound no. 26467 as a white solid. Yield: 36.8 mg, 70%; LCMS m / z 1610.6 [M / 2+1]+; 1 H NMR (300MHz, DMSO-d6 with D2O) δ7.93(s, 3H), 7.51(d, J=7.5Hz, 3H), 7.46~7.27(m, 12H), 7.11(t, J=8.0Hz, 6H), 6.94(d, J=7.9Hz, 6H), 4.48~4. 35(m, 12H), 4.10~2.82(m, 99H), 2.45~2.39(m, 2H), 2.29~2.17(m, 6H), 2.13~2.02(m, 2H), 1.82(s, 9H), 1.66~1.36(m, 8H), 1.36~1.05(m, 8H).

[0229] (2RS,2'RS,4SR,4'SR,5RS,5'RS,6RS,6'RS)-2,2'-((((2R,2'R,3R,3'R,4S,4'S,5R,5'R,6R,6'R)-(((((SR)-9,14,22-trioxo-16-((15-oxo-15-(perfluorophenoxy)-3,6,9,12-tetraoxapentadecyl)carbamoyl)-3,6,25,28-tetraoxa-10,15,21-triazatriacontane-1,30-di yl)bis(1H-1,2,3-triazole-1,4-diyl))bis(ethane-2,1-diyl))bis(oxy))bis(3,4,5-trihydroxytetrahydro-2H-pyran-6,2-diyl))bis(methylene))bis(oxy))bis(5-acetamido-6-((1RS,2RS)-1,2-dihydroxy-3-(3-phenoxybenzamido)propyl)-4-hydroxytetrahydro-2H-pyran-2carboxylic acid) (Compound No. 26468) [ka]

[0230] (2RS,2'RS,4SR,4'SR,5RS,5'RS,6RS,6'RS)-2,2'-((((2R,2'R,3R,3'R,4S,4'S,5R,5'R,6R,6'R)-(((((SR)-9,14,22-trioxo-16-((15-oxo-15-(perfluorophenoxy)-3,6,9,12-tetraoxapentadecyl)carbamoyl)-3,6,25,28-tetraoxa-10,15,21-triazatriacontane-1,30-diyl) Synthesis of)bis(1H-1,2,3-triazole-1,4-diyl))bis(ethane-2,1-diyl))bis(oxy))bis(3,4,5-trihydroxytetrahydro-2H-pyran-6,2-diyl))bis(methylene))bis(oxy))bis(5-acetamido-6-((1RS,2RS)-1,2-dihydroxy-3-(3-phenoxybenzamido)propyl)-4-hydroxytetrahydro-2H-pyran-2carboxylic acid) (Compound No. 26468) Perfluorophenyl (R)-1-azido-16-(4-(3-(2-(2-azidoethoxy)ethoxy)propanamido)butyl)-9,14,17-trioxo-3,6,21,24,27,30-hexaoxa-10,15,18-triazatriacontan-33-oate (26332, 1.00 equiv., 18.4 mg, 0.0163 mmol) and (2RS,4SR,5RS,6RS)-5-acetamido-2-(((2R,3R,4S,5R,6 To a solution of (R)-6-(but-3-yn-1-yloxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)methoxy)-6-((1RS,2RS)-1,2-dihydroxy-3-(3-phenoxybenzamido)propyl)-4-hydroxytetrahydro-2H-pyran-2-carboxylic acid (26409, 2.10 equiv., 24.6 mg, 0.0342 mmol), tetrakis(acetonitrile)copper(I) hexafluorophosphate (5.00 equiv., 30.4 mg, 0.0815 mmol) was added. The resulting clear solution was capped and stirred at room temperature for 10 min (slowly turning green). The reaction was diluted with a mixture of 70% acetic acid in NMP, filtered, and purified by preparative HPLC (15-65% acetonitrile in water with 0.1% TFA).Fractions containing the desired product were combined and lyophilized to give (2RS,2'RS,4SR,4'SR,5RS,5'RS,6RS,6'RS)-2,2'-((((2R,2'R,3R,3'R,4S,4'S,5R,5'R,6R,6'R)-(((((SR)-9,14,22-trioxo-16-((15-oxo-15-(perfluorophenoxy)-3,6,9,12-tetraoxapentadecyl)carbamoyl)-3,6,25,28-tetraoxa-10,15,21-triazatriacontane (1RS,2RS)-1,2-dihydroxy-3-(3-phenoxybenzamido)propyl)-4-hydroxytetrahydro-2H-pyran-2carboxylic acid) (compound number 26468) was obtained as a white solid. Yield: 28.2 mg, 71%; LCMS m / z 1226.8 [M / 2+1]+. 1 H NMR (300MHz, DMSO-d6 with D2O) δ7.79(s, 2H), 7.51(d, J=7.8Hz, 2H), 7.45~7.30(m, 8H), 7.16~7.05(m, 4H), 6.94(d, J=8.0Hz, 4H), 4.42~4.3 3(m, 4H), 4.15~2.69(m, 77H), 2.44~2.39(m, 2H), 2.30~2.18(m, 4H), 2 .14~2.02(m, 2H), 1.82(s, 6H), 1.64~1.38(m, 6H), 1.38~1.07(m, 4H).

[0231] (2RS,2'RS,4RS,4'RS,5SR,5'SR,6SR,6'SR)-2,2'-((((2R,2'R,3R,3'R,4S,4'S,5R,5'R,6R,6'R)-(((((RS)-9,14,22-trioxo-16-((15-oxo-15-(perfluorophenoxy)-3,6,9,12-tetraoxapentadecyl)carbamoyl)-3,6,25,28-tetraoxa-10,15,21-triazatriacontane-1,30-di yl)bis(1H-1,2,3-triazole-1,4-diyl))bis(ethane-2,1-diyl))bis(oxy))bis(3,4,5-trihydroxytetrahydro-2H-pyran-6,2-diyl))bis(methylene))bis(oxy))bis(5-acetamido-6-((1SR,2SR)-1,2-dihydroxy-3-(3-phenoxybenzamido)propyl)-4-hydroxytetrahydro-2H-pyran-2carboxylic acid) (Compound No. 26469) [ka]

[0232] (2RS,2'RS,4RS,4'RS,5SR,5'SR,6SR,6'SR)-2,2'-((((2R,2'R,3R,3'R,4S,4'S,5R,5'R,6R,6'R)-(((((RS)-9,14,22-trioxo-16-((15-oxo-15-(perfluorophenoxy)-3,6,9,12-tetraoxapentadecyl)carbamoyl)-3,6,25,28-tetraoxa-10,15,21-triazatriacontane-1,30-diyl) Synthesis of)bis(1H-1,2,3-triazole-1,4-diyl))bis(ethane-2,1-diyl))bis(oxy))bis(3,4,5-trihydroxytetrahydro-2H-pyran-6,2-diyl))bis(methylene))bis(oxy))bis(5-acetamido-6-((1SR,2SR)-1,2-dihydroxy-3-(3-phenoxybenzamido)propyl)-4-hydroxytetrahydro-2H-pyran-2carboxylic acid) (Compound No. 26469) Perfluorophenyl (R)-1-azido-16-(4-(3-(2-(2-azidoethoxy)ethoxy)propanamido)butyl)-9,14,17-trioxo-3,6,21,24,27,30-hexaoxa-10,15,18-triazatriacontan-33-oate (26332, 1.00 equiv., 19.3 mg, 0.0171 mmol) and (2RS,4RS,5SR,6SR)-5-acetamido-2-(((2R,3R,4S,5R,6 To a solution of (R)-6-(but-3-yn-1-yloxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)methoxy)-6-((1SR,2SR)-1,2-dihydroxy-3-(3-phenoxybenzamido)propyl)-4-hydroxytetrahydro-2H-pyran-2-carboxylic acid (26410, 2.10 equiv., 25.8 mg, 0.0359 mmol), tetrakis(acetonitrile)copper(I) hexafluorophosphate (5.00 equiv., 31.8 mg, 0.0854 mmol) was added. The resulting clear solution was capped and stirred at room temperature for 10 min (slowly turning green). The reaction was diluted with a mixture of 70% acetic acid in NMP, filtered, and purified by preparative HPLC (15-65% acetonitrile in water with 0.1% TFA).Fractions containing the desired product were combined and lyophilized to give (2RS,2'RS,4RS,4'RS,5SR,5'SR,6SR,6'SR)-2,2'-((((2R,2'R,3R,3'R,4S,4'S,5R,5'R,6R,6'R)-(((((RS)-9,14,22-trioxo-16-((15-oxo-15-(perfluorophenoxy)-3,6,9,12-tetraoxapentadecyl)carbamoyl)-3,6,25,28-tetraoxa-10,15,21-triazatriacontane (1,30-diyl)bis(1H-1,2,3-triazole-1,4-diyl))bis(ethane-2,1-diyl))bis(oxy))bis(3,4,5-trihydroxytetrahydro-2H-pyran-6,2-diyl))bis(methylene))bis(oxy))bis(5-acetamido-6-((1SR,2SR)-1,2-dihydroxy-3-(3-phenoxybenzamido)propyl)-4-hydroxytetrahydro-2H-pyran-2carboxylic acid) (compound number 26469) was obtained as a white solid. Yield: 26.0 mg, 62%; LCMS m / z 1226.8 [M / 2+1]+; 1 H NMR (300MHz, DMSO-d6 with D2O) δ7.92(bs, 2H), 7.83(s, 2H), 7.54(d, J=7.6Hz, 2H), 7.44(t, J=7.8Hz, 2H), 7.41~7.31(m, 6H), 7.13(t, J=7.2H) z, 4H), 6.96(d, J=8.0Hz, 4H), 4.45~4.32(m, 4H), 3.95~2.70(m, 77H), 2.31~2.02(m, 8H), 1.82(s, 6H), 1.63~1.37(m, 6H), 1.37~1.07(m, 4H).

[0233] Compound No. 26470 [ka] TIFF2024502850000050.tif60156

[0234] Compound No. 26470 Perfluorophenyl (16R,19R)-1-azido-16,19-bis(4-(3-(2-(2-azidoethoxy)ethoxy)propanamido)butyl)-9,14,17,20-tetraoxo-3,6,24,27,30,33-hexaoxa-10,15,18,21-tetraazahexatriacontan-36-oate (26333, 1.00 equiv., 21.6 mg, 0.0149 mmol) and (2RS,4RS,5SR,6SR)-5-acetamido-2-(((2R To a solution of (3R,4S,5R,6R)-6-(but-3-yn-1-yloxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)methoxy)-6-((1SR,2SR)-1,2-dihydroxy-3-(3-phenoxybenzamido)propyl)-4-hydroxytetrahydro-2H-pyran-2-carboxylic acid (26410, 3.10 equiv., 33.3 mg, 0.0463 mmol) was added tetrakis(acetonitrile)copper(I) hexafluorophosphate (7.50 equiv., 41.8 mg, 0.112 mmol). The resulting clear yellow solution was capped and stirred at room temperature for 10 min (slowly turning green). The reaction was diluted with a mixture of 70% acetic acid in NMP, filtered, and purified by preparative HPLC (15-65% acetonitrile in water with 0.1% TFA). Fractions containing the desired product were combined and lyophilized to give compound no. 26470 as a white solid. Yield: 27.3 mg, 52%; LCMS m / z 1742.4 [M / 2+1]+; 1 H NMR (300MHz, DMSO-d6 with D2O) δ7.92(bs, 3H), 7.82(s, 3H), 7.52(d, J=7.7Hz, 3H), 7.43(t, J=7.9Hz, 3H), 7.39~7.30(m, 9H), 7.12(t, J=7.2Hz) , 6H), 6.95(d, J=7.9Hz, 6H), 4.42~4.32(m, 6H), 4.17~2.70(m, 108H), 2 .31~2.02(m, 10H), 1.82(s, 9H), 1.66~1.38(m, 8H), 1.38~1.05(m, 8H). Yield: 12.7 mg, 24%; 1H NMR (300MHz, D2OをbandうDMSO-d6) δ7.92 (bs, 3H), 7.83 (s, 3H), 7.54 (d, J=7.7Hz, 3H), 7.45 (t, J=7.9Hz, 3H), 7.40~7.31 (m, 9H), 7.13 (t, J=7.0Hz , 6H), 6.96 (d, J=8.0Hz, 6H), 4.42~4.33(m, 6H), 3.96~2.70(m, 108H), 2 .32~2.03(m, 10H), 1.82(s, 9H), 1.66~1.39(m, 8H), 1.39~1.07(m, 8H).

[0235] Compound number 26471

change

[0236] Compound number 26471 perfluorophenyl(16R,19R)-1-azido-16,19-bis(4-(3-(2-(2-azidoethoxy)ethoxy)propanamido)butyl)-9,14,17,20-tetraoxo-3,6,24,27,30,33-hexaoxa-10,15,18,21-tetraazahexatriacontan-36-oate (26333, 1.00 equiv., 17.1 mg, 0.0119 mmol) and (2RS,4SR,5RS,6RS)-5-acetamido-2-(((2R To a solution of (3R,4S,5R,6R)-6-(but-3-yn-1-yloxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)methoxy)-6-((1RS,2RS)-1,2-dihydroxy-3-(3-phenoxybenzamido)propyl)-4-hydroxytetrahydro-2H-pyran-2-carboxylic acid (26409, 3.10 equiv., 26.4 mg, 0.0368 mmol), tetrakis(acetonitrile)copper(I) hexafluorophosphate (7.50 equiv., 33.1 mg, 0.0889 mmol) was added. The resulting clear yellow solution was capped and stirred at room temperature for 10 min (slowly turning green). The reaction was diluted with a mixture of 70% acetic acid in NMP, filtered, and purified by preparative HPLC (15-65% acetonitrile in water with 0.1% TFA). Fractions containing the desired product were combined and lyophilized to give compound no. 26471 as a white solid. Yield: 19.7 mg, 48%; LCMS m / z 1742.5 [M / 2+1]+; 1 H NMR (300 MHz, DMSO-d6 with DO) δ 7.94 (bs, 3H), 7.81 (s, 3H), 7.53 (d, J = 7.7 Hz, 3H), 7.47–7.31 (m, 12H), 7.17–7.05 (m, 6H), 6.95 (d, J = 8.0 Hz, 6H), 4.44–4.30 ( m, 6H), 4.16~4.09(m, 2H), 3.90~2.69(m, 106H), 2.45~2.37(m, 2H), 2.31~2.16 (m, 6H), 2.14~2.01(m, 2H), 1.82(s, 9H), 1.65~1.38(m, 8H), 1.38~1.04(m, 8H). Yield: 8.1 mg, 20%; 1H NMR (300MHz, DMSO-d6 with D2O) δ7.93(bs, 3H), 7.78(s, 3H), 7.50(d, J=7.6Hz, 3H), 7.45~7.29(m, 12H), 7.16~7.03(m, 6H), 6.94(d, J=8.0H) z, 6H), 4.41~4.32(m, 6H), 4.15~2.70(m, 108H), 2.32~2.18(m, 8H), 2.14~2.04(m, 2H), 1.82(s, 9H), 1.66~1.38(m, 8H), 1.38~1.05(m, 8H).

[0237] (2S,4S,5R,6R)-5-acetamido-6-((1R,2R)-1,2-dihydroxy-3-(3-phenoxybenzamido)propyl)-2,4-dihydroxytetrahydro-2H-pyran-2-carboxylic acid (compound number 26473) [ka]

[0238] Synthesis of (1R,2R)-1-((2R,3R,4S,6S)-3-acetamido-4-acetoxy-6-(benzyloxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)-3-(3-phenoxybenzamido)propane-1,2-diyl diacetate (3) A suspension of (1R,2R)-1-((2R,3R,4S,6R)-3-acetamido-4-acetoxy-6-(methoxycarbonyl)-6-(p-tolylthio)tetrahydro-2H-pyran-2-yl)-3-(3-phenoxybenzamido)propane-1,2-diyl diacetate (1, 0.50 g, 0.66 mmol), phenylmethanol (2, 0.203 mL, 2.0 mmol), and activated 4 Å powdered molecular sieves (0.50 g, 100% w / w) in anhydrous dichloromethane (10.0 mL) was stirred at room temperature under a nitrogen atmosphere for 15 hours. The reaction mixture was cooled to -40°C, followed by the addition of 1-iodopyrrolidine-2,5-dione (0.374 g, 1.66 mmol) and trifluoromethanesulfonic acid (0.058 mL, 0.66 mmol). The reaction was stirred at -40°C for 1 hour, and progress was monitored by TLC and LCMS. Upon completion, the reaction mixture was quenched with triethylamine (0.1 mL, neutral pH) and allowed to warm to room temperature. The reaction mixture was filtered through a sintered funnel and washed with dichloromethane. The filtrate was washed with a saturated solution of sodium bicarbonate, dried over sodium sulfate, filtered, and concentrated under reduced pressure to give the crude residue. The residue was purified by column chromatography (45-60% ethyl acetate in hexanes) to give (1R,2R)-1-((2R,3R,4S,6S)-3-acetamido-4-acetoxy-6-(benzyloxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)-3-(3-phenoxybenzamido)propane-1,2-diyl diacetate (3) as an off-white solid. Yield: 0.30 g, 61.31%, LCMS (ESI) m / z 7354.22 [M+1] + .

[0239] Synthesis of (2R,4S,5R,6R)-5-acetamido-2-(benzyloxy)-6-((1R,2R)-1,2-dihydroxy-3-(3-phenoxybenzamido)propyl)-4-hydroxytetrahydro-2H-pyran-2-carboxylic acid (4) To a stirred solution of (1R,2R)-1-((2R,3R,4S,6S)-3-acetamido-4-acetoxy-6-(benzyloxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)-3-(3-phenoxybenzamido)propane-1,2-diyl diacetate (3, 0.30 g, 0.408 mmol) in methanol (10.0 mL) was added lithium hydroxide (0.048 g, 2.04 mmol) at 0° C. The mixture was stirred and allowed to warm to room temperature. Upon completion, Dowex® hydrogen form was added to the reaction mass to pH 6. The reaction mixture was filtered through a sintered funnel and the filtrate was concentrated on a rotary evaporator to give (2S,4S,5R,6R)-5-acetamido-2-(benzyloxy)-6-((1R,2R)-1,2-dihydroxy-3-(3-phenoxybenzamido)propyl)-4-hydroxytetrahydro-2H-pyran-2-carboxylic acid (4) as a white solid. Yield: 0.24 g, crude, LCMS (ESI) m / z 595.22 [M+1] + .

[0240] Synthesis of (2S,4S,5R,6R)-5-acetamido-6-((1R,2R)-1,2-dihydroxy-3-(3-phenoxybenzamido)propyl)-2,4-dihydroxytetrahydro-2H-pyran-2-carboxylic acid (compound number 26473) To a stirred solution of (2S,4S,5R,6R)-5-acetamido-2-(benzyloxy)-6-((1R,2R)-1,2-dihydroxy-3-(3-phenoxybenzamido)propyl)-4-hydroxytetrahydro-2H-pyran-2-carboxylic acid (4, 0.24 g, 0.403 mmol) in methanol (3.0 mL) was added 10% Pd / C (0.12 g, 50% w / w) at room temperature. The reaction was hydrogenated using balloon pressure of H gas for 12 h. The reaction was monitored by LC-MS and TLC. Upon completion, the reaction was filtered through Celite, and the filtrate was concentrated. The residue was purified by preparative HPLC (17–35% acetonitrile in water with 0.1% TFA). Fractions containing the desired product were combined and lyophilized to give (2S,4S,5R,6R)-5-acetamido-6-((1R,2R)-1,2-dihydroxy-3-(3-phenoxybenzamido)propyl)-2,4-dihydroxytetrahydro-2H-pyran-2-carboxylic acid (compound number 26473) as a white solid as the TFA salt. Yield: 0.078 g, 38.31%; LCMS (ESI) m / z 505.35 [M+1] + ; 1 H NMR (400MHz, methanol-d4) δ8.42~8.40(m, 1H), 8.09(d, J=8.4Hz, 1H), 7.56(d, J=7.6Hz, 1H), 7.46~7.42(m, 2H), 7.39~7.35(m, 2H), 7.16~7.12(m, 2H) , 7.02~7.00(m, 2H), 4.06~4.00(m, 2H), 3.88~3.75(m, 3H), 3.51~3.42(m, 2H), 2.21(dd, J=12.4 and 4.8Hz, 1H), 1.97(s, 3H), 1.83(t, J=12.4Hz, 1H).

[0241] (2R,4R,5S,6S)-6-((1S,2S)-3-(2-([1,1'-biphenyl]-4-yl)acetamido)-1,2-dihydroxypropyl)-4-hydroxy-5-(2-hydroxyacetamido)-2-(2-(2-(1-(15-oxo-15-(perfluorophenoxy)-3,6,9,12-tetraoxapentadecyl)-1H-1,2,3-triazol-4-yl)methoxy)ethoxy)ethoxy)tetrahydro-2H-pyran-2-carboxylic acid (compound number 26474) [ka]

[0242] Synthesis of (2R,4R,5S,6S)-6-((1S,2S)-3-(2-([1,1'-biphenyl]-4-yl)acetamido)-1,2-dihydroxypropyl)-4-hydroxy-5-(2-hydroxyacetamido)-2-(2-(2-(1-(15-oxo-15-(perfluorophenoxy)-3,6,9,12-tetraoxapentadecyl)-1H-1,2,3-triazol-4-yl)methoxy)ethoxy)ethoxy)tetrahydro-2H-pyran-2-carboxylic acid (compound number 26474) (2R,4R,5S,6S)-6-((1S,2S)-3-(2-([1,1'-biphenyl]-4-yl)acetamido)-1,2-dihydroxypropyl)-4-hydroxy-5-(2-hydroxyacetamido)-2-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)tetrahydro-2H-pyran-2-carboxylic acid (26 To a solution of 464 (1.00 equiv., 28.0 mg, 0.0434 mmol) in NMP (0.3 mL) was added a solution of perfluorophenyl 1-azido-3,6,9,12-tetraoxapentadecan-15-oate (1, 1.10 equiv., 21.9 mg, 0.0478 mmol), followed by tetrakis(acetonitrile)copper(I) hexafluorophosphate (2.50 equiv., 40.5 mg, 0.109 mmol). The resulting clear green solution was capped and stirred at room temperature for 10 min. The reaction was diluted with acetic acid, filtered, and purified by preparative HPLC (15-65% acetonitrile in water with 0.1% TFA). Fractions containing the desired product were combined and lyophilized to give (2R,4R,5S,6S)-6-((1S,2S)-3-(2-([1,1'-biphenyl]-4-yl)acetamido)-1,2-dihydroxypropyl)-4-hydroxy-5-(2-hydroxyacetamido)-2-(2-(2-(1-(15-oxo-15-(perfluorophenoxy)-3,6,9,12-tetraoxapentadecyl)-1H-1,2,3-triazol-4-yl)methoxy)ethoxy)ethoxy)tetrahydro-2H-pyran-2-carboxylic acid (compound no. 26474) as a white solid. Yield: 10.7 mg, 22%; LCMS m / z 1102.7 [M+1]+; 1 H NMR (300MHz, DMSO-d6 with D2O) δ7.96(s, 1H), 7.69(d, J=8.8Hz, 1H), 7.61~7.48(m, 4H), 7.41(t, J=7.5Hz , 2H), 7.34~7.24(m, 3H), 4.52~4.38(m, 3H), 4.02~2.69(m, 38H), 2.31~2.07(m, 1H), 1.54~1.38(m, 1H).

[0243] (2R,2'R,4S,4'S,5R,5'R,6R,6'R)-2,2'-((((((((S)-9,14,22-trioxo-16-((15-oxo-15-(perfluorophenoxy)-3,6,9,12-tetraoxapentadecyl)carbamoyl)-3,6,25,28-tetraoxa-10,15,21-triazatriacontane-1,30-diyl)bis(1H-1,2,3-triazole-1,4- diyl))bis(methylene))bis(oxy))bis(ethane-2,1-diyl))bis(oxy))bis(ethane-2,1-diyl))bis(oxy))bis(6-((1R,2R)-3-(2-([1,1'-biphenyl]-4-yl)acetamido)-1,2-dihydroxypropyl)-4-hydroxy-5-(2-hydroxyacetamido)tetrahydro-2H-pyran-2-carboxylic acid) (Compound No. 26475) [ka]

[0244] (2R,2'R,4S,4'S,5R,5'R,6R,6'R)-2,2'-((((((((S)-9,14,22-trioxo-16-((15-oxo-15-(perfluorophenoxy)-3,6,9,12-tetraoxapentadecyl)carbamoyl)-3,6,25,28-tetraoxa-10,15,21-triazatriacontane-1,30-diyl)bis(1H-1,2,3-triazole-1,4-diyl) Synthesis of 6-((1R,2R)-3-(2-([1,1'-biphenyl]-4-yl)acetamido)-1,2-dihydroxypropyl)-4-hydroxy-5-(2-hydroxyacetamido)tetrahydro-2H-pyran-2-carboxylic acid) (Compound No. 26475) Perfluorophenyl (R)-1-azido-16-(4-(3-(2-(2-azidoethoxy)ethoxy)propanamido)butyl)-9,14,17-trioxo-3,6,21,24,27,30-hexaoxa-10,15,18-triazatriacontan-33-oate (26332, 1.00 equiv., 21.2 mg, 0.0188 mmol) and (2R,4S,5R,6R)-6-((1R,2R)-3-(2-([1 To a solution of [5-(2-hydroxyacetamido)-2-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)tetrahydro-2H-pyran-2-carboxylic acid (26463, 2.10 equiv., 25.4 mg, 0.0394 mmol) was added tetrakis(acetonitrile)copper(I) hexafluorophosphate (5.00 equiv., 35.0 mg, 0.0939 mmol). The resulting clear yellow solution was capped and stirred at room temperature for 10 min (slowly turning green). The reaction was diluted with a mixture of 70% acetic acid in NMP, filtered, and purified by preparative HPLC (15-60% acetonitrile in water with 0.1% TFA). Fractions containing the desired product were combined and lyophilized to give (2R,2'R,4S,4'S,5R,5'R,6R,6'R)-2,2'-((((((((S)-9,14,22-trioxo-16-((15-oxo-15-(perfluorophenoxy)-3,6,9,12-tetraoxapentadecyl)carbamoyl)-3,6,25,28-tetraoxa-10,15,21-triazatriacontane-1,30-diyl)bis(1H-1,2,3-triazolium) (6-((1R,2R)-3-(2-([1,1'-biphenyl]-4-yl)acetamido)-1,2-dihydroxypropyl)-4-hydroxy-5-(2-hydroxyacetamido)tetrahydro-2H-pyran-2-carboxylic acid) (compound number 26475) was obtained as a white solid. Yield: 30.2 mg, 70%. 1H NMR (300MHz, DMSO-d6 with D2O) δ7.96(s, 2H), 7.80(d, J=7.1Hz, 2H), 7.62~7.50(m, 8H), 7.41(t, J=7.5Hz, 4H), 7.35~7.26(m, 6 H), 4.50~4.38(m, 8H), 4.13~2.87(m, 79H), 2.47~2.40(m, 2H), 2.30~2.19(m, 4H), 2.08(t, J=7.6Hz, 2H), 1.62~1.08(m, 10H).

[0245] (2R,2'R,4R,4'R,5S,5'S,6S,6'S)-2,2'-((((((((R)-9,14,22-trioxo-16-((15-oxo-15-(perfluorophenoxy)-3,6,9,12-tetraoxapentadecyl)carbamoyl)-3,6,25,28-tetraoxa-10,15,21-triazatriacontane-1,30-diyl)bis(1H-1,2,3-triazole-1,4- diyl))bis(methylene))bis(oxy))bis(ethane-2,1-diyl))bis(oxy))bis(ethane-2,1-diyl))bis(oxy))bis(6-((1S,2S)-3-(2-([1,1'-biphenyl]-4-yl)acetamido)-1,2-dihydroxypropyl)-4-hydroxy-5-(2-hydroxyacetamido)tetrahydro-2H-pyran-2-carboxylic acid) (Compound No. 26476) [ka]

[0246] (2R,2'R,4R,4'R,5S,5'S,6S,6'S)-2,2'-((((((((R)-9,14,22-trioxo-16-((15-oxo-15-(perfluorophenoxy)-3,6,9,12-tetraoxapentadecyl)carbamoyl)-3,6,25,28-tetraoxa-10,15,21-triazatriacontane-1,30-diyl)bis(1H-1,2,3-triazole-1,4-diyl) Synthesis of (6-((1S,2S)-3-(2-([1,1'-biphenyl]-4-yl)acetamido)-1,2-dihydroxypropyl)-4-hydroxy-5-(2-hydroxyacetamido)tetrahydro-2H-pyran-2-carboxylic acid) (Compound No. 26476) Perfluorophenyl (R)-1-azido-16-(4-(3-(2-(2-azidoethoxy)ethoxy)propanamido)butyl)-9,14,17-trioxo-3,6,21,24,27,30-hexaoxa-10,15,18-triazatriacontan-33-oate (26332, 1.00 equiv., 21.9 mg, 0.0194 mmol) and (2R,4R,5S,6S)-6-((1S,2S)-3-(2-([1 To a solution of [5-(2-hydroxyacetamido, 1'-biphenyl]-4-yl)acetamido)-1,2-dihydroxypropyl)-4-hydroxy-5-(2-hydroxyacetamido)-2-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)tetrahydro-2H-pyran-2-carboxylic acid (26464, 2.10 equiv., 26.3 mg, 0.0407 mmol), tetrakis(acetonitrile)copper(I) hexafluorophosphate (5.00 equiv., 36.1 mg, 0.0970 mmol) was added. The resulting clear yellow solution was capped and stirred at room temperature for 10 min (slowly turning green). The reaction was diluted with a mixture of 70% acetic acid in NMP, filtered, and purified by preparative HPLC (15-65% acetonitrile in water with 0.1% TFA). Fractions containing the desired product were combined and lyophilized to give (2R,2'R,4R,4'R,5S,5'S,6S,6'S)-2,2'-((((((((R)-9,14,22-trioxo-16-((15-oxo-15-(perfluorophenoxy)-3,6,9,12-tetraoxapentadecyl)carbamoyl)-3,6,25,28-tetraoxa-10,15,21-triazatriacontane-1,30-diyl)bis(1H-1,2,3-triazolium)

[0123] To obtain bis(6-((1S,2S)-3-(2-([1,1'-biphenyl]-4-yl)acetamido)-1,2-dihydroxypropyl)-4-hydroxy-5-(2-hydroxyacetamido)tetrahydro-2H-pyran-2-carboxylic acid) (compound number 26476) as a white solid.Yield: 14.6 mg, 33%; LCMS m / z 1152.7 [M / 2+1]+;. 1 H NMR (300MHz, D2O を with DMSO-d6) δ7.95 (s, 2H), 7.74 (d, J=8.9Hz, 2H), 7.62~7.48 (m, 8H), 7.41 (t, J=7.6Hz, 4H), 7.3 5~7.24(m, 6H), 4.51~4.39(m, 7H), 4.02~2.70(m, 80H), 2.30~2.03(m, 8H), 1.62~1.37(m, 6H), 1.37~1.08(m, 4H).

[0247] Compound number 26477

change

[0248] Compound number 26477 Perfluorophenyl (16R,19R)-1-azido-16,19-bis(4-(3-(2-(2-azidoethoxy)ethoxy)propanamido)butyl)-9,14,17,20-tetraoxo-3,6,24,27,30,33-hexaoxa-10,15,18,21-tetraazahexatriacontan-36-oate (26333, 1.00 equiv., 22.5 mg, 0.0156 mmol) and (2R,4S,5R,6R)-6-((1R,2 To a solution of (R)-3-(2-([1,1'-biphenyl]-4-yl)acetamido)-1,2-dihydroxypropyl)-4-hydroxy-5-(2-hydroxyacetamido)-2-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)tetrahydro-2H-pyran-2-carboxylic acid (26463, 3.10 equiv., 31.2 mg, 0.0484 mmol), tetrakis(acetonitrile)copper(I) hexafluorophosphate (7.50 equiv., 43.6 mg, 0.117 mmol) was added. The resulting clear yellow solution was capped and stirred at room temperature for 10 min (slowly turning green). The reaction was diluted with a mixture of 70% acetic acid in NMP, filtered, and purified by preparative HPLC (15-65% acetonitrile in water with 0.1% TFA). Fractions containing the desired product were combined and lyophilized to give compound No. 26477 as a white solid. Yield: 23.4 mg, 46%; 1 H NMR (300MHz, DMSO-d6 with D2O) δ7.94(s, 3H), 7.77(bs, 3H), 7.60~7.46(m, 12H), 7.40(t, J=7.6Hz, 6H), 7.34~7. 24(m, 9H), 4.49~4.36(m, 11H), 3.90~2.84(m, 114H), 2.31~2.03(m, 8H), 1.65~1.37(m, 8H), 1.37~1.06(m, 8H).

[0249] Compound No. 26478 [ka] TIFF2024502850000058.tif116155

[0250] Compound No. 26478 Perfluorophenyl (16R,19R)-1-azido-16,19-bis(4-(3-(2-(2-azidoethoxy)ethoxy)propanamido)butyl)-9,14,17,20-tetraoxo-3,6,24,27,30,33-hexaoxa-10,15,18,21-tetraazahexatriacontan-36-oate (26333, 1.00 equiv., 24.3 mg, 0.0168 mmol) and (2R,4R,5S,6S)-6-((1S,2 To a solution of (S)-3-(2-([1,1'-biphenyl]-4-yl)acetamido)-1,2-dihydroxypropyl)-4-hydroxy-5-(2-hydroxyacetamido)-2-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)tetrahydro-2H-pyran-2-carboxylic acid (26464, 3.10 equiv., 33.7 mg, 0.0522 mmol), tetrakis(acetonitrile)copper(I) hexafluorophosphate (7.50 equiv., 47.1 mg, 0.126 mmol) was added. The resulting clear yellow solution was capped and stirred at room temperature for 10 min (slowly turning green). The reaction was diluted with a mixture of 70% acetic acid in NMP, filtered, and purified by preparative HPLC (15-65% acetonitrile in water with 0.1% TFA). Fractions containing the desired product were combined and lyophilized to give compound no. 26478 as a white solid. Yield: 23.4 mg, 43%; LCMS m / z 1631.8 [M / 2+1]+; 1 H NMR (300MHz, DMSO-d6 with D2O) δ7.96(s, 3H), 7.74(d, J=8.9Hz, 3H), 7.62~7.48(m, 12H), 7.41(t, J=7.6Hz, 6H), 7.35 ~7.25(m, 9H), 4.51~4.38(m, 11H), 3.90~2.70(m, 110H), 2.32~2.03(m, 12H), 1.65~1.37(m, 8H), 1.37~1.06(m, 8H).

[0251] Perfluorophenyl 1-(4-(13-hydroxy-2,5,8,11-tetraoxatridecyl)-1H-1,2,3-triazol-1-yl)-3,6,9,12-tetraoxapentadecan-15-oate (compound number 26530) [ka]

[0252] Perfluorophenyl 1-(4-(13-hydroxy-2,5,8,11-tetraoxatridecyl)-1H-1,2,3-triazol-1-yl)-3,6,9,12-tetraoxapentadecan-15-oate (compound number 26530) To 3,6,9,12-tetraoxapentadec-14-yn-1-ol (1, 1.00 equiv., 14.4 mg, 0.0620 mmol) in a 1-dram vial with a stir bar was added a solution of perfluorophenyl 1-azido-3,6,9,12-tetraoxapentadecan-15-oate (2, 1.10 equiv., 31.2 mg, 0.0682 mmol) in NMP (0.5 mL), followed by tetrakis(acetonitrile)copper(I) hexafluorophosphate (2.50 equiv., 57.8 mg, 0.155 mmol). The resulting clear solution was capped and stirred at room temperature for 10 min (slowly turning green). The reaction was diluted with acetic acid, filtered, and purified by preparative HPLC (15-65% acetonitrile in water with 0.1% TFA). Fractions containing the desired product were combined and lyophilized to give perfluorophenyl 1-(4-(13-hydroxy-2,5,8,11-tetraoxatridecyl)-1H-1,2,3-triazol-1-yl)-3,6,9,12-tetraoxapentadecan-15-oate (compound number 26530) as a colorless liquid. Yield: 16.2 mg, 38%; LCMS m / z 690.5 [M+1]+; 1 H NMR (300 MHz, chloroform-d) δ 7.97 (s, 1H), 4.81–4.72 (m, 2H), 4.64–4.54 (m, 2H), 3.96–3.82 (m, 4H), 3.80–3.56 (m, 29H), 2.94 (t, J = 6.1 Hz, 2H).

[0253] (RS)-9,14,17-trioxo-1-(4-(2-(((2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethyl)-1H-1,2,3-triazol-1-yl)-16-(4-(3-(2-(2-(4-(2-(((2R,3R,4S,5R,6R) -3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)propanamido)butyl)-3,6,21,24,27,30-hexaoxa-10,15,18-triazatriacontan-33-oate (compound number 26531) [ka] TIFF2024502850000061.tif51155

[0254] (RS)-9,14,17-trioxo-1-(4-(2-(((2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethyl)-1H-1,2,3-triazol-1-yl)-16-(4-(3-(2-(2-(4-(2-(((2R,3R,4S,5R,6R)-3 Synthesis of 4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)propanamido)butyl)-3,6,21,24,27,30-hexaoxa-10,15,18-triazatriacontan-33-oate (compound number 26531) Dissolve perfluorophenyl (R)-1-azido-16-(4-(3-(2-(2-azidoethoxy)ethoxy)propanamido)butyl)-9,14,17-trioxo-3,6,21,24,27,30-hexaoxa-10,15,18-triazatriacontan-33-oate (26332, 1.00 equiv., 32.4 mg, 0.0287 mmol) and (2R,3R,4S,5R,6R)-2(buta-3 To a solution of (-yn-1-yloxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol (compound (26499, 2.10 equiv., 14.0 mg, 0.0603 mmol) was added tetrakis(acetonitrile)copper(I) hexafluorophosphate (5.00 equiv., 53.5 mg, 0.143 mmol). The resulting clear yellow solution was capped and stirred at room temperature for 10 minutes (slowly turning green). The reaction was diluted with a mixture of 70% acetic acid in NMP. The mixture was diluted with hexanes, filtered, and purified by preparative HPLC (15-60% acetonitrile in water with 0.1% TFA). Fractions containing the desired product were combined, lyophilized, and purified to give (RS)-9,14,17-trioxo-1-(4-(2-(((2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethyl)-1H-1,2,3-triazol-1-yl)-16-(4-(3-(2-(2-( 4-(2-(((2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)propanamido)butyl)-3,6,21,24,27,30-hexaoxa-10,15,18-triazatriacontan-33-oate (compound number 26531) was obtained as a white solid. Yield: 28.1 mg, 66%; LCMS m / z 1480.0 [M+1]+; 1H NMR (300MHz, DMSO-d6 with D2O) δ7.85(s, 2H), 4.45~4.35(m, 3H), 4.21~4.10(m, 2H), 3.98~2. 70(m, 62H), 2.31~2.19(m, 4H), 2.09(t, J=7.5Hz, 2H), 1.64~1.37(m, 4H), 1.37~1.07(m, 4H).

[0255] Perfluorophenyl (R)-1-(4-(13-hydroxy-2,5,8,11-tetraoxatridecyl)-1H-1,2,3-triazol-1-yl)-16-(4-(3-(2-(2-(4-(13-hydroxy-2,5,8,11-tetraoxatridecyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)propanamido)butyl)-9,14,17-trioxo-3,6,21,24,27,30-hexaoxa-10,15,18-triazatritriacontan-33-oate (Compound No. 26532) [ka]

[0256] Synthesis of perfluorophenyl (R)-1-(4-(13-hydroxy-2,5,8,11-tetraoxatridecyl)-1H-1,2,3-triazol-1-yl)-16-(4-(3-(2-(2-(4-(13-hydroxy-2,5,8,11-tetraoxatridecyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)propanamido)butyl)-9,14,17-trioxo-3,6,21,24,27,30-hexaoxa-10,15,18-triazatritriacontan-33-oate (compound number 26532) To a solution of perfluorophenyl (R)-1-azido-16-(4-(3-(2-(2-azidoethoxy)ethoxy)propanamido)butyl)-9,14,17-trioxo-3,6,21,24,27,30-hexaoxa-10,15,18-triazatriacontan-33-oate (26332, 1.00 equiv., 30.2 mg, 0.0267 mmol) and 3,6,9,12-tetraoxapentadec-14-yn-1-ol (1, 2.10 equiv., 13.0 mg, 0.0562 mmol) in NMP (0.5 mL) in a 1-dram vial with a stir bar was added tetrakis(acetonitrile)copper(I) hexafluorophosphate (5.00 equiv., 49.8 mg, 0.134 mmol). The resulting clear yellow solution was capped and stirred at room temperature for 10 minutes (it slowly turned green). The reaction was diluted with a mixture of 70% acetic acid in NMP, filtered, and purified by preparative HPLC (15-60% acetonitrile in water with 0.1% TFA). Fractions containing the desired product were combined and lyophilized to give perfluorophenyl (R)-1-(4-(13-hydroxy-2,5,8,11-tetraoxatridecyl)-1H-1,2,3-triazol-1-yl)-16-(4-(3-(2-(2-(4-(13-hydroxy-2,5,8,11-tetraoxatridecyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)propanamido)butyl)-9,14,17-trioxo-3,6,21,24,27,30-hexaoxa-10,15,18-triazatritriacontan-33-oate (compound no. 26532) as a clear yellow liquid. Yield: 17.0 mg, 43%; LCMS m / z 1480.1 [M+1]+; 1 H NMR (300MHz, DMSO-d6 with D2O) δ7.98(s, 2H), 7.95~7.82(m, 3H), 4.50~4.41(m, 8H), 3.80~2. 72(m, 73H), 2.31~2.21(m, 4H), 2.10(t, J=7.4Hz, 2H), 1.65~1.38(m, 4H), 1.38~1.08(m, 4H).

[0257] Perfluorophenyl(16RS,19RS)-9,14,17,20-tetraoxo-1-(4-(2-(((2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethyl)-1H-1,2,3-triazol-1-yl)-16,19-bis(4-(3-(2-(2-(4-(2-(((2R,3R, 4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)propanamido)butyl)-3,6,24,27,30,33-hexaoxa-10,15,18,21-tetraazahexatriacontan-36-oate (Compound No. 26533) [ka] TIFF2024502850000064.tif74153

[0258] Perfluorophenyl(16RS,19RS)-9,14,17,20-tetraoxo-1-(4-(2-(((2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethyl)-1H-1,2,3-triazol-1-yl)-16,19-bis(4-(3-(2-(2-(4-(2-(((2R,3R,4 Synthesis of S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)propanamido)butyl)-3,6,24,27,30,33-hexaoxa-10,15,18,21-tetraazahexatriacontan-36-oate (compound number 26533) Perfluorophenyl (16R,19R)-1-azido-16,19-bis(4-(3-(2-(2-azidoethoxy)ethoxy)propanamido)butyl)-9,14,17,20-tetraoxo-3,6,24,27,30,33-hexaoxa-10,15,18,21-tetraazahexatriacontan-36-oate (26333, 1.00 equiv., 36.9 mg, 0.0256 mmol) and (2R,3R,4S,5R,6R To a solution of 2-(but-3-yn-1-yloxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol (compound 26499, 3.10 equiv., 18.4 mg, 0.0793 mmol), tetrakis(acetonitrile)copper(I) hexafluorophosphate (7.50 equiv., 71.5 mg, 0.192 mmol) was added. The resulting clear yellow solution was capped and stirred at room temperature for 10 minutes (slowly turning green). The reaction was diluted with a mixture of 70% acetic acid in NMP. The resulting mixture was filtered and purified by preparative HPLC (15-50% acetonitrile in water with 0.1% TFA). Fractions containing the desired product were combined, lyophilized, and purified to give perfluorophenyl(16RS,19RS)-9,14,17,20-tetraoxo-1-(4-(2-(((2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethyl)-1H-1,2,3-triazol-1-yl)-16,19-bis(4-(3- (2-(2-(4-(2-(((2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)propanamido)butyl)-3,6,24,27,30,33-hexaoxa-10,15,18,21-tetraazahexatriacontan-36-oate (compound number 26533) was obtained as a white solid. Yield: 38.1 mg, 74%; LCMS m / z 1013.8 [M / 2+1]+; 1H NMR (300 MHz, DMSO-d with DO) δ 7.85 (s, 3H), 4.45–4.36 (m, 6H), 4.18–2.65 (m, 8H), 2.31–2.16 (m, 6H), 2.16–2.04 (m, 2H), 1.66–1.38 (m, 6H), 1.38–1.06 (m, 10H).

[0259] Perfluorophenyl(16R,19R)-1-(4-(13-hydroxy-2,5,8,11-tetraoxatridecyl)-1H-1,2,3-triazol-1-yl)-16,19-bis(4-(3-(2-(2-(4-(13-hydroxy-2,5,8,11-tetraoxatridecyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)propanamido)butyl)-9,14,17,20-tetraoxo-3,6,24,27,30,33-hexaoxa-10,15,18,21-tetraazahexatriacontan-36-oate (Compound No. 26534) [ka] TIFF2024502850000066.tif106155

[0260] Synthesis of perfluorophenyl(16R,19R)-1-(4-(13-hydroxy-2,5,8,11-tetraoxatridecyl)-1H-1,2,3-triazol-1-yl)-16,19-bis(4-(3-(2-(2-(4-(13-hydroxy-2,5,8,11-tetraoxatridecyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)propanamido)butyl)-9,14,17,20-tetraoxo-3,6,24,27,30,33-hexaoxa-10,15,18,21-tetraazahexatriacontan-36-oate (compound number 26534) perfluorophenyl(16R,19R)-1-azido-16,19-bis(4-(3-(2-(2-azidoethoxy)ethoxy)propanamido)butyl)-9,14,17,20-tetraoxo-3,6,24,27,30,33-hexaoxa-10,15,18,21-tetraazahexatriacontane in NMP (0.4 mL) in a 1-dram vial with a stir bar To a solution of 2,6-dihydroxybenzoate (26333, 1.00 equiv., 35.8 mg, 0.0248 mmol) and 3,6,9,12-tetraoxapentadec-14-yn-1-ol (1, 3.10 equiv., 17.9 mg, 0.0769 mmol) was added tetrakis(acetonitrile)copper(I) hexafluorophosphate (7.50 equiv., 69.4 mg, 0.186 mmol). The resulting clear green solution was capped and stirred at room temperature for 10 min. The reaction was diluted with a mixture of 70% acetic acid in NMP, filtered, and purified by preparative HPLC (15-50% acetonitrile in water with 0.1% TFA). Fractions containing the desired product were combined and lyophilized to give perfluorophenyl(16R,19R)-1-(4-(13-hydroxy-2,5,8,11-tetraoxatridecyl)-1H-1,2,3-triazol-1-yl)-16,19-bis(4-(3-(2-(2-(4-(13-hydroxy-2,5,8,11-tetraoxatridecyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)propanamido)butyl)-9,14,17,20-tetraoxo-3,6,24,27,30,33-hexaoxa-10,15,18,21-tetraazahexatriacontan-36-oate (Compound No. 26534) as a clear pink liquid. Yield: 22.8 mg, 45%; LCMS m / z 1013.8 [M / 2+1]+; 1 H NMR (300MHz, DMSO-d6 with D2O) δ7.98(s, 3H), 7.95~7.83(m, 2H), 4.51~4.39(m, 12H), 3.80~2 .69(m, 100H), 2.31~2.19(m, 6H), 2.15~2.04(m, 2H), 1.67~1.38(m, 6H), 1.38~1.07(m, 8H).

[0261] Perfluorophenyl 1-(4-(2-(((2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethyl)-1H-1,2,3-triazol-1-yl)-3,6,9,12-tetraoxapentadecan-15-oate (compound number 26535) [ka]

[0262] Synthesis of perfluorophenyl 1-(4-(2-(((2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethyl)-1H-1,2,3-triazol-1-yl)-3,6,9,12-tetraoxapentadecan-15-oate (compound number 26535) To (2R,3R,4S,5R,6R)-2(but-3-yn-1-yloxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol (26499, 1.00 equiv., 13.6 mg, 0.0586 mmol) in a 1-dram vial with a stir bar, a solution of perfluorophenyl 1-azido-3,6,9,12-tetraoxapentadecan-15-oate (1, 1.10 equiv., 29.5 mg, 0.0645 mmol) in NMP (0.5 mL) was added, followed by tetrakis(acetonitrile)copper(I) hexafluorophosphate (2.50 equiv., 54.6 mg, 0.147 mmol). The resulting clear yellow solution was capped and stirred at room temperature for 10 min (slowly turning green). The reaction was diluted with acetic acid, filtered, and purified by preparative HPLC (15-65% acetonitrile in water with 0.1% TFA). Fractions containing the desired product were combined and lyophilized to give perfluorophenyl 1-(4-(2-(((2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethyl)-1H-1,2,3-triazol-1-yl)-3,6,9,12-tetraoxapentadecan-15-oate (compound no. 26535) as a colorless semi-solid. Yield: 29.0 mg, 72%; LCMS m / z [M+1]+; LCMS m / z 690.5 [M+1]+; 1 H NMR (300MHz, DMSO-d6 with DO) δ7.88(s, 1H), 4.47~4.37(m, 2H), 4.16~4.09(m, 1H), 3.80~3.21(m, 24H), 2.95(t, J=5.9Hz, 2H), 2.86(t, J=6.9Hz, 2H).

[0263] (1R,2R)-1-((2R,3R,4S,6R)-3-acetamido-4-acetoxy-6-hydroxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)-3-(3-phenoxybenzamido)propane-1,2-diyl diacetate (compound number 26565) [ka] TIFF2024502850000069.tif83155

[0264] Synthesis of (1R,2R)-3-(2-([1,1'-biphenyl]-4-yl)acetamido)-1-((2R,3R,4S,6R)-3-acetamido-4-acetoxy-6-(benzyloxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)propane-1,2-diyl diacetate (3) To a stirred solution of (1R,2R)-3-(2-([1,1'-biphenyl]-4-yl)acetamido)-1-((2R,3R,4S,6S)-3-acetamido-4-acetoxy-6-(methoxycarbonyl)-6-(p-tolylthio)tetrahydro-2H-pyran-2-yl)propane-1,2-diyl diacetate (1, 1.0 g, 1.24 mmol) in anhydrous dichloromethane (14.0 mL) was added phenylmethanol (0.386 mL, 3.72 mmol) and activated 4 Å powdered molecular sieves (1.0 g, 100% w / w). The resulting reaction mixture was stirred under a nitrogen atmosphere for 15 h. The reaction mixture was cooled to -40°C, followed by the addition of 1-iodopyrrolidine-2,5-dione (0.697 g, 3.10 mmol) and trifluoromethanesulfonic acid (0.109 mL, 1.24 mmol) at -40°C. The reaction was stirred at -40°C for 1 hour. Upon completion, the reaction mixture was quenched with triethylamine (0.1 mL, neutral pH) and allowed to warm to room temperature. The reaction mixture was filtered and washed with dichloromethane. The filtrate was washed with a saturated solution of sodium bicarbonate, dried over sodium sulfate, filtered, and concentrated under reduced pressure to provide the crude residue. The crude residue was purified by column chromatography (45-60% ethyl acetate in hexanes) to give (1R,2R)-3-(2-([1,1'-biphenyl]-4-yl)acetamido)-1-((2R,3R,4S,6R)-3-acetamido-4-acetoxy-6-(benzyloxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)propane-1,2-diyl diacetate (3) as an off-white solid. Yield: 0.80 g, 81.30%, LCMS (ESI) m / z 791.82 [M+1] + .

[0265] Synthesis of (2R,4S,5R,6R)-6-((1R,2R)-3-(2-([1,1'-biphenyl]-4-yl)acetamido)-1,2-dihydroxypropyl)-5-acetamido-2-(benzyloxy)-4-hydroxytetrahydro-2H-pyran-2-carboxylic acid (4) To a stirred solution of (1R,2R)-3-(2-([1,1'-biphenyl]-4-yl)acetamido)-1-((2R,3R,4S,6R)-3-acetamido-4-acetoxy-6-(benzyloxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)propane-1,2-diyl diacetate (3, 0.80 g, 1.01 mmol) in methanol (10.0 mL) was added lithium hydroxide (0.121 g, 5.06 mmol) at 0°C. The reaction mixture was allowed to warm to room temperature. After completion, Dowex-hydrogen was added to the reaction mass to pH 6. The reaction mixture was filtered, and the resulting filtrate was concentrated on a rotary evaporator to give crude (2R,4S,5R,6R)-6-((1R,2R)-3-(2-([1,1'-biphenyl]-4-yl)acetamido)-1,2-dihydroxypropyl)-5-acetamido-2-(benzyloxy)-4-hydroxytetrahydro-2H-pyran-2-carboxylic acid (4) as a white solid. Yield: 0.60 g, 95.25%, LCMS (ESI) m / z 623.67 [M+1] + .

[0266] Synthesis of (2R,4S,5R,6R)-6-((1R,2R)-3-(2-([1,1'-biphenyl]-4-yl)acetamido)-1,2-dihydroxypropyl)-2,4-dihydroxy-5-(2-hydroxyacetamido)tetrahydro-2H-pyran-2-carboxylic acid (compound number 26565) To a stirred solution of crude (2R,4S,5R,6R)-6-((1R,2R)-3-(2-([1,1'-biphenyl]-4-yl)acetamido)-1,2-diacetoxypropyl)-5-acetamido-4-acetoxy-2-(benzyloxy)tetrahydro-2H-pyran-2-carboxylic acid (4, 0.60 g, 0.985 mmol) in methanol (10.0 mL) was added 10% Pd / C (0.30 g, 50% w / w) at room temperature. The reaction was then hydrogenated using balloon pressure of H gas for 12 h. Upon completion, the reaction was filtered through Celite, and the filtrate was concentrated. The resulting residue was purified by preparative HPLC (19–38% acetonitrile in water with 0.1% TFA). Fractions containing the desired product were combined and lyophilized to give (2R,4S,5R,6R)-6-((1R,2R)-3-(2-([1,1'-biphenyl]-4-yl)acetamido)-1,2-dihydroxypropyl)-2,4-dihydroxy-5-(2-hydroxyacetamido)tetrahydro-2H-pyran-2-carboxylic acid (compound number 26565) as a white solid as the TFA salt. Yield: 0.088 g, 17.22%; LCMS (ESI) m / z 517.34 [M+1] + ; 1 H NMR (400MHz, methanol-d4) δ7.60~7.56(m, 4H), 7.43~7.36(m, 4H), 7.33~7.29(m, 1H), 4.16~4.09(m, 2H), 4.02(s, 2H), 3.90(t, J=10.4H) z, 1H), 3.77~3.73(m, 1H)3.68~3.64(m, 1H), 3.59(m, 2H), 3.39(d, J=9.2, 1H), 3.27~3.22(m, 1H), 2.21(dd, J=12.8 and 4.8Hz, 1H), 1.84 1.83(t, J=12.8Hz, 1H).

[0267] (2S,4S,5R,6R)-6-((1R,2R)-3-(2-([1,1'-biphenyl]-4-yl)acetamido)-1,2-dihydroxypropyl)-4-hydroxy-5-(2-hydroxyacetamido)-2-((2-(2-(prop-2-yn-1-yloxy)ethoxy)ethyl)thio)tetrahydro-2H-pyran-2-carboxylic acid (compound number 26566) [ka]

[0268] Synthesis of (1R,2R)-3-(2-([1,1'-biphenyl]-4-yl)acetamido)-1-((2R,3R,4S,6R)-4-acetoxy-3-(2-acetoxyacetamido)-6-hydroxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)propane-1,2-diyl diacetate (2) A stirred solution of (1R,2R)-3-(2-([1,1'-biphenyl]-4-yl)acetamido)-1-((2R,3R,4S,6S)-4-acetoxy-3-(2-acetoxyacetamido)-6-(methoxycarbonyl)-6-(p-tolylthio)tetrahydro-2H-pyran-2-yl)propane-1,2-diyl diacetate (1, 1.0 g, 1.24 mmol) in acetone:water (9:1, 20.0 mL) was cooled to 0°C. N-iodosuccinimide (0.35 g, 3.10 mmol) was added to the solution at 0°C. The resulting reaction solution was stirred at 0°C for 3 hours. Upon completion, a saturated aqueous solution of sodium metabisulfide (10.0 mL) and ethyl acetate (20.0 mL) was added. The reaction mixture was stirred for 10 minutes and transferred to a separatory funnel. The organic layer was separated, and the aqueous phase was extracted with ethyl acetate (10.0 mL). The combined organic layers were washed successively with saturated sodium bicarbonate solution and DM water. The organic layer was dried over anhydrous sodium sulfate and concentrated on a rotary evaporator to give a thick syrup. The thick syrup was purified by column chromatography (60-75% ethyl acetate in hexane) to give (1R,2R)-3-(2-([1,1'-biphenyl]-4-yl)acetamido)-1-((2R,3R,4S,6R)-4-acetoxy-3-(2-acetoxyacetamido)-6-hydroxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)propane-1,2-diyl diacetate (2) as a white solid. Yield: 0.76 g, 87.52%, LCMS (ESI) m / z 701.59 [M+1] + .

[0269] Synthesis of (1R,2R)-3-(2-([1,1'-biphenyl]-4-yl)acetamido)-1-((2R,3R,4S,6R)-4-acetoxy-3-(2-acetoxyacetamido)-6-chloro-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)propane-1,2-diyl diacetate (3) To a stirred solution of (1R,2R)-3-(2-([1,1'-biphenyl]-4-yl)acetamido)-1-((2R,3R,4S,6R)-4-acetoxy-3-(2-acetoxyacetamido)-6-hydroxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)propane-1,2-diyl diacetate (2, 0.76 g, 1.08 mmol) in acetyl chloride (30.0 mL) was added anhydrous methanol (0.3 mL) at 0° C. The resulting reaction mixture was stirred at room temperature for 24 hours. Upon completion, the reaction mixture was concentrated under reduced pressure to give crude (1R,2R)-3-(2-([1,1'-biphenyl]-4-yl)acetamido)-1-((2R,3R,4S,6R)-4-acetoxy-3-(2-acetoxyacetamido)-6-chloro-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)propane-1,2-diyl diacetate (3) as a light brown gel. Yield: 0.75 g, 97%, LC-MS (ESI) m / z 719.19 [M+1] + .

[0270] Synthesis of (1R,2R)-3-(2-([1,1'-biphenyl]-4-yl)acetamido)-1-((2R,3R,4S,6S)-4-acetoxy-3-(2-acetoxyacetamido)-6-(acetylthio)-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)propane-1,2-diyl diacetate (5) In an inert atmosphere, crude (1R,2R)-3-(2-([1,1'-biphenyl]-4-yl)acetamido)-1-((2R,3R,4S,6R)-4-acetoxy-3-(2-acetoxyacetamido)-6-chloro-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)propane-1,2-diyl diacetate (3, 0.75 g, 1.04 mmol, crude) was dissolved in dry acetone (10.0 mL) and stirred at 0°C. To this solution was added potassium thioacetate (4, 0.357 g, 3.13 mmol) pinchwise at 0°C. The reaction was stirred at 0°C for 3 hours. Upon completion, the mixture was concentrated under reduced pressure to give the crude residue. The crude residue was dissolved in ethyl acetate, and the resulting solution was washed with 1N HCl followed by DM water. The organic layer was separated, dried over anhydrous sodium sulfate, and concentrated on a rotary evaporator to give a thick residue. The residue was purified by column chromatography (60-70% ethyl acetate in hexanes) to give (1R,2R)-3-(2-([1,1'-biphenyl]-4-yl)acetamido)-1-((2R,3R,4S,6S)-4-acetoxy-3-(2-acetoxyacetamido)-6-(acetylthio)-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)propane-1,2-diyl diacetate (5) as a white solid. Yield: 0.530 g, 66.97%, LCMS (ESI) m / z 759.29 [M+1] + .

[0271] Synthesis of (1R,2R)-3-(2-([1,1'-biphenyl]-4-yl)acetamido)-1-((2R,3R,4S,6S)-4-acetoxy-3-(2-acetoxyacetamido)-6-(methoxycarbonyl)-6-((2-(2-(prop-2-yn-1-yloxy)ethoxy)ethyl)thio)tetrahydro-2H-pyran-2-yl)propane-1,2-diyl diacetate (Compound No. 26566) To a stirred solution of (1R,2R)-3-(2-([1,1'-biphenyl]-4-yl)acetamido)-1-((2R,3R,4S,6S)-4-acetoxy-3-(2-acetoxyacetamido)-6-(acetylthio)-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)propane-1,2-diyl diacetate (5, 0.53 g, 0.698 mmol) in ethanol (10.0 mL), sodium thiomethoxide (0.058 g, 0.838 mmol) was added at 0°C. The resulting reaction solution was stirred for 1 hour. To the reaction solution was added 1-iodo-2-[2-(prop-2-yn-1-yloxy)ethoxy]ethane (0.354 g, 1.40 mmol) at 0°C. The resulting reaction mixture was stirred at room temperature for 1 hour. After completion, lithium hydroxide (0.026 g, 1.12 mmol) was added at room temperature. The resulting reaction mixture was stirred at room temperature for 6 hours. After completion, Dowex Hydrogen Form was added until pH 6, and the reaction mass was filtered through a sintered funnel. The filtrate was concentrated on a rotary evaporator to give a thick residue, which was then purified by preparative HPLC (15-40% acetonitrile in water with 0.1% TFA). Fractions containing the desired product were combined and lyophilized to give (2S,4S,5R,6R)-6-((1R,2R)-3-(2-([1,1'-biphenyl]-4-yl)acetamido)-1,2-dihydroxypropyl)-4-hydroxy-5-(2-hydroxyacetamido)-2-((2-(2-(prop-2-yn-1-yloxy)ethoxy)ethyl)thio)tetrahydro-2H-pyran-2-carboxylic acid (compound number 26566) as a white solid as the TFA salt. Yield: 0.170 g, 46.04%, LC-MS (ESI) m / z 661.17 [M+1]. + . 1 H NMR (400MHz, methanol-d4) δ7.61~7.57 (m, 4H), 7.44~7.37 (, 4H), 7.33~7.30 (m, 1H), 4.15 (d, J=2.0Hz, 1H), 4.00 (s, 2H), 3.86~3. 81(m, 3H), 3.69~3.54(m, 11H), 3.37~3.35(m, 1H), 3.25~3.22(m, 1H), 2.95~2.89(m, 1H), 2.86~2.76(m, 3H), 1.85~1.75(m, 1H).

[0272] (2R,4S,5R,6R)-6-((1R,2R)-3-(2-([1,1'-biphenyl]-4-yl)acetamido)-1,2-dihydroxypropyl)-4-hydroxy-5-(2-hydroxyacetamido)-2-((2-(2-(prop-2-yn-1-yloxy)ethoxy)ethyl)thio)tetrahydro-2H-pyran-2-carboxylic acid (compound number 26567) [ka] TIFF2024502850000072.tif93156

[0273] Synthesis of (2S,4S,5R,6R)-6-((1R,2R)-3-(2-([1,1'-biphenyl]-4-yl)acetamido)-1,2-diacetoxypropyl)-5-(2-acetoxyacetamido)-2-(methoxycarbonyl)tetrahydro-2H-pyran-2,4-diyl diacetate (2) To a stirred solution of (1R,2R)-3-(2-([1,1'-biphenyl]-4-yl)acetamido)-1-((2R,3R,4S,6R)-4-acetoxy-3-(2-acetoxyacetamido)-6-hydroxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)propane-1,2-diyl diacetate (1, 0.70 g, 0.99 mmol) in pyridine (10.0 mL) was added acetic anhydride (0.188 mL, 2.0 mmol) dropwise at 0°C. The reaction mixture was warmed to room temperature and stirred overnight. Upon completion, the volatiles were removed under vacuum to give a crude thick syrup. The thick syrup was poured into a separatory funnel with ethyl acetate (20.0 mL) and washed with 1 N HCl solution, followed by saturated sodium sulfate solution and DM water. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give a crude thick syrup. The thick syrup was purified by column chromatography (55-70% ethyl acetate in hexanes) to give (2S,4S,5R,6R)-6-((1R,2R)-3-(2-([1,1'-biphenyl]-4-yl)acetamido)-1,2-diacetoxypropyl)-5-(2-acetoxyacetamido)-2-(methoxycarbonyl)tetrahydro-2H-pyran-2,4-diyl diacetate (2) as a white solid. Yield: 0.50 g, 67.39%, LCMS (ESI) m / z 743.27 [M+1] + .

[0274] Synthesis of (1R,2R)-3-(2-([1,1'-biphenyl]-4-yl)acetamido)-1-((2R,3R,4S,6R)-4-acetoxy-3-(2-acetoxyacetamido)-6-(methoxycarbonyl)-6-((2-(2-(prop-2-yn-1-yloxy)ethoxy)ethyl)thio)tetrahydro-2H-pyran-2-yl)propane-1,2-diyl diacetate (4) In an inert atmosphere, (2S,4S,5R,6R)-6-((1R,2R)-3-(2-([1,1'-biphenyl]-4-yl)acetamido)-1,2-diacetoxypropyl)-5-(2-acetoxyacetamido)-2-(methoxycarbonyl)tetrahydro-2H-pyran-2,4-diyl diacetate (2, 0.50 g, 0.673 mmol) and 2-(2-(prop-2-yn-1-yloxy)ethoxy)ethane-1-thiol (3, 0.215 g, 1.35 mmol) were dissolved in dry dichloromethane (10.0 mL) with stirring at room temperature. Activated powdered 4 Å molecular sieves (0.50 g, 100% w / w) were added to the resulting reaction solution at room temperature. The resulting reaction mixture was stirred for 30 minutes. The reaction mixture was cooled to 0 °C, and BF3·Et2O (0.586 mL, 2.02 mmol) was added dropwise over 5 min at 0 °C. The resulting reaction mixture was stirred at room temperature for 16 h. Upon completion, the reaction mixture was quenched with triethylamine to neutral pH. The reaction mixture was filtered and washed with saturated aqueous sodium bicarbonate. The organic layer was separated, dried over anhydrous sodium sulfate, and concentrated on a rotary evaporator to give a crude thick syrup. The thick syrup was purified by column chromatography (60-70% ethyl acetate in hexanes) to give (1R,2R)-3-(2-([1,1'-biphenyl]-4-yl)acetamido)-1-((2R,3R,4S,6R)-4-acetoxy-3-(2-acetoxyacetamido)-6-(methoxycarbonyl)-6-((2-(2-(prop-2-yn-1-yloxy)ethoxy)ethyl)thio)tetrahydro-2H-pyran-2-yl)propane-1,2-diyl diacetate (4) as a white solid. Yield: 0.30 g, 52.86%, LCMS (ESI) m / z 843.91 [M+1] + .

[0275] Synthesis of (2R,4S,5R,6R)-6-((1R,2R)-3-(2-([1,1'-biphenyl]-4-yl)acetamido)-1,2-dihydroxypropyl)-4-hydroxy-5-(2-hydroxyacetamido)-2-((2-(2-(prop-2-yn-1-yloxy)ethoxy)ethyl)thio)tetrahydro-2H-pyran-2-carboxylic acid (Compound No. 26567) To a stirred solution of (1R,2R)-3-(2-([1,1'-biphenyl]-4-yl)acetamido)-1-((2R,3R,4S,6R)-4-acetoxy-3-(2-acetoxyacetamido)-6-(methoxycarbonyl)-6-((2-(2-(prop-2-yn-1-yloxy)ethoxy)ethyl)thio)tetrahydro-2H-pyran-2-yl)propane-1,2-diyl diacetate (4, 0.30 g, 0.355 mmol) in methanol (10.0 mL) was added lithium hydroxide (0.051 g, 2.14 mmol) at 0°C. The reaction mixture was warmed to room temperature and stirred for 6 hours. After completion, Dowex-hydrogen was added to neutral pH and the reaction mixture was filtered through a sintered funnel. The filtrate was removed under vacuum to give a crude thick syrup, which was purified by preparative HPLC (22-48% acetonitrile in water with 0.1% TFA). Fractions containing the desired product were combined and lyophilized to give (2R,4S,5R,6R)-6-((1R,2R)-3-(2-([1,1'-biphenyl]-4-yl)acetamido)-1,2-dihydroxypropyl)-4-hydroxy-5-(2-hydroxyacetamido)-2-((2-(2-(prop-2-yn-1-yloxy)ethoxy)ethyl)thio)tetrahydro-2H-pyran-2-carboxylic acid (compound no. 26567) as an amorphous solid. Yield: 0.113 g, 48.05%, LCMS (ESI) m / z 661.36 [M+1]. + . 1 H NMR (400MHz, methanol-d4) δ7.88(d, J=8.8Hz, 1H), 7.61~7.57(m, 4H), 7.44~7.29(m, 5H), 4.26(d, J=10.4Hz, 1H), 4.17~4.15(m, 3H), 4.03(s , 2H), 3.90~3.83(m, 2H), 3.63~3.56(m, 9H), 3.41~3.34(m, 2H), 2.85~2.81(m, 3H), 2.47(dd, J=14.0, 4.8Hz, 1H), 1.95(t, J=14.0Hz, 1H).

[0276] (2R,4S,5R,6R)-4-Hydroxy-5-(2-hydroxyacetamido)-2-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)-6-((1R,2R)-1,2,3-trihydroxypropyl)tetrahydro-2H-pyran-2-carboxylic acid (compound number 26591) and (2S,4S,5R,6R)-4-Hydroxy-5-(2-hydroxyacetamido)-2-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)-6-((1R,2R)-1,2,3-trihydroxypropyl)tetrahydro-2H-pyran-2-carboxylic acid (compound number 26568) [ka] TIFF2024502850000074.tif71155

[0277] Synthesis of (1S,2R)-1-((2R,3R,4S)-4-acetoxy-3-(2-acetoxyacetamido)-6-(methoxycarbonyl)-6-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)tetrahydro-2H-pyran-2-yl)propane-1,2,3-triyl triacetate (3) To a stirred solution of (1S,2R)-1-((2R,3R,4S,6R)-3-acetamido-4-acetoxy-6-(methoxycarbonyl)-6-(p-tolylthio)tetrahydro-2H-pyran-2-yl)propane-1,2,3-triyl triacetate (1, 2.0 g, 3.34 mmol) in dichloromethane (32.0 mL) was added 2-(2-(prop-2-yn-1-yloxy)ethoxy)ethan-1-ol (2, 1.206 g, 8.36 mmol) and 4Å MS (2.0 g, 100% w / w). The resulting suspension was stirred under a nitrogen atmosphere for 12 hours. The reaction mixture was cooled to −40° C., followed by the successive dropwise addition of N-iodosuccinimide (1.882 g, 8.36 mmol) and trifluoromethanesulfonic acid (0.294 mL, 3.34 mmol) at −40° C. The resulting reaction mixture was stirred at −40° C. for 1 hour. Upon completion, triethylamine was added to neutral pH, and the reaction mixture was filtered through Celite. The filtrate was concentrated under reduced pressure to give a crude residue, which was then purified by column chromatography (40-60% ethyl acetate in hexanes) to give (1S,2R)-1-((2R,3R,4S)-4-acetoxy-3-(2-acetoxyacetamido)-6-(methoxycarbonyl)-6-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)tetrahydro-2...

Claims

1. 1. Engineered, less immunogenic biotherapeutics, including biotherapeutics that have been engineered to contain a modified sialic acid-binding immunoglobulin-type lectin (Siglec) ligand profile compared to a corresponding non-engineered biotherapeutic while retaining therapeutic activity; The poorly immunogenic biotherapeutic, wherein the Siglec ligand profile comprises an increased amount of one or more Siglec ligands covalently attached to the engineered poorly immunogenic biotherapeutic as compared to the corresponding non-engineered biotherapeutic.

2. The engineered low immunogenic biotherapeutic is represented by the formula (I) 2. The engineered, less immunogenic biotherapeutic of claim 1 , wherein X is a sialic acid group, L is an optional linker, Y is a biotherapeutic, n is an integer greater than or equal to 1, and m is an integer greater than or equal to 1.

3. 3. The engineered, hypoimmunogenic biotherapeutic of claim 1 or 2, wherein the Siglec ligand comprises a non-naturally occurring Siglec ligand and a non-naturally occurring linker L, the linker being free of saccharides.

4. 4. The engineered, hypoimmunogenic biotherapeutic of any one of claims 1 to 3, wherein the Siglec ligand comprises two sialic acids and a linker, the linker being a branched linker, and the two sialic acids are attached to the linker.

5. 4. The engineered, hypoimmunogenic biotherapeutic of any one of claims 1 to 3, wherein the Siglec ligand comprises three sialic acids and a linker, the linker being a branched linker, and the three sialic acids are attached to the linker.

6. 6. The engineered, hypoimmunogenic biotherapeutic of any one of claims 1 to 5, wherein the Siglec ligand comprises a linker L, said L comprising a polyethylene glycol group.

7. One or more Siglec ligands 2. The engineered, less immunogenic biotherapeutic of claim 1, comprising a non-naturally occurring sialic acid selected from the group consisting of:

8. The Siglec ligand is and a linker selected from the group consisting of The engineered, less immunogenic biotherapeutic of claim 1 , wherein the linker attaches the sialic acid to the biotherapeutic.

9. The non-natural Siglec ligand, 7. The engineered, hypoimmunogenic biotherapeutic of any one of claims 1 to 6, comprising:

10. 10. The engineered, hypoimmunogenic biotherapeutic of any one of claims 1 to 9, wherein the biotherapeutic is a protein.

11. 11. The engineered, hypoimmunogenic biotherapeutic of claim 10, wherein the protein is selected from the group consisting of an antibody, an enzyme, a chimeric protein, and a viral particle.

12. 12. The engineered, hypoimmunogenic biotherapeutic of claim 11, wherein the antibody is selected from the group consisting of a monoclonal antibody, a bispecific antibody, a scFv, a Fab, a camelid, or a nanobody.

13. The antibody is selected from the group consisting of adalimumab, infliximab, cetuximab, natalizumab, moxetumomab pasudotox, atezolizumab, nivolumab, abciximab, brentuximab, certolizumab pegol, elotuzumab, benralizumab, vedolizumab, galcanezumab, rituximab, alemtuzumab, dupilumab, golimumab, obinutuzumab, tildrakizumab, elenu 11. The engineered, less immunogenic biotherapeutic of claim 10, selected from the group consisting of: mab, mepolizumab, tamsilumab, ranibizumab, ustekinumab, reslizumab, ipilimumab, alirocumab, belimumab, panitumumab, avelumab, necitumumab, mogamulizumab, olaratumab, brodalumab, eculizumab, pertuzumab, pembrolizumab, and tocilizumab.

14. The protein is an enzyme, and the enzyme is selected from the group consisting of asparaginase Erwinia chrysanthemi, phenylalanine ammonia lyase, alpha-galactosidase A, acid alpha-glucosidase (GAA), glucocerebrosidase (GCase), aspartylglucosaminidase (AGA), alpha-L-iduronidase, iduronate sulfatase, sulfaminase, alpha-N-acetylglucosaminidase (NAGLU), heparin acetyl CoA: alpha-glucosamide N-acetyltransferase (HGSNAT), N-acetylglucosamine 6-sulfatase (GNS), N-glucosamine 6-sulfatase (GNS), N-acetylglucosaminidase (NAGLU), N-acetylglucosamine 6-sulfatase (N ...

11. The engineered, hypoimmunogenic biotherapeutic of claim 10, wherein the engineered, hypoimmunogenic biotherapeutic agent is selected from the group consisting of: factor 3-O-sulfatase (arylsulfatase G or ARSG), N-acetylgalactosamine 6-sulfatase, beta-galactosidase, N-acetylgalactosamine 4-sulfatase, beta-glucuronidase, factor VIII, factor IX, palmitoyl protein thioesterase (PPT1), tripeptidyl peptidase (TPP1), Pseudomonas elastase (PaE), Pseudomonas alkaline protease (PaAP), and streptococcal pyrogenic exotoxin B (SpeB).

15. An engineered, hypoimmunogenic biotherapeutic according to any one of claims 1 to 14; and a pharmaceutical excipient.

16. 16. A pharmaceutical composition according to claim 15 for treating an individual suffering from a disorder or disease that can be treated by administration of a biotherapeutic agent, comprising: The pharmaceutical composition elicits reduced anti-drug antibody titers as compared to the non-engineered biotherapeutic.

17. 17. The pharmaceutical composition of claim 16, wherein the disease is a chronic immune disease selected from the group consisting of rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, Crohn's disease, ulcerative colitis, psoriasis, hidradenitis suppurativa, uveitis, and juvenile idiopathic arthritis, and the engineered, hypoimmunogenic biotherapeutic comprises a TNFα-specific antibody selected from adalimumab and infliximab.

18. 17. The pharmaceutical composition of claim 16, wherein the disease is leukemia and the engineered, hypoimmunogenic biotherapeutic comprises asparaginase from Erwinia chrysanthemi.

19. 17. The pharmaceutical composition of claim 16, wherein the disease is multiple sclerosis and the engineered, hypoimmunogenic biotherapeutic comprises natalizumab, IFN beta-1b, or IFN beta-1a in an amount effective to treat the multiple sclerosis.

20. 17. The pharmaceutical composition of claim 16, wherein the disorder is an antibody response to transplanted tissue and the engineered, hypoimmunogenic biotherapeutic comprises IdeS.

21. 17. The pharmaceutical composition of claim 16, wherein the disorder is type 2 diabetes and the engineered, hypoimmunogenic biotherapeutic comprises exenatide or albiglutide.

22. The disorder is an enzyme deficiency, the enzyme deficiency being an enzyme deficiency of phenylalanine ammonia-lyase (PKU), alpha-galactosidase A (in Fabry), acid alpha-glucosidase (GAA, in Pompe), glucocerebrosidase (GCase, in Gaucher), aspartylglucosaminidase (AGA, in aspartylglucosaminuria), alpha-L-iduronidase (in MPS I), iduronate sulfatase (in MPS II), sulfaminase (MPS IIIa), alpha-N-acetylglucosaminidase (NAGLU, in MPS IIIB), heparin acetyl-CoA:alpha-glucosamide N-acetyltransferase (HGSNAT, in MPS IIIC), N-acetylglucosamine 6-sulfatase (GNS, in MPS 17. The pharmaceutical composition of claim 16, wherein the cause of the deficiency is an enzyme deficiency selected from the group consisting of: N-glucosamine 3-O-sulfatase (arylsulfatase G or ARSG, MPS IIID), N-acetylgalactosamine 6-sulfatase (in MPS IVA), beta-galactosidase (in MPS IVB), N-acetylgalactosamine 4-sulfatase (in MPS VI), beta-glucuronidase (in MPS VI), Factor VIII (in hemophilia A), Factor IX (in hemophilia B), palmitoyl protein thioesterase (PPT1, CLN1), tripeptidyl peptidase (TPP1, CLN2), and cystathionine beta synthase (CBS) deficiency.

23. 17. The pharmaceutical composition of claim 16, wherein the disorder is a monogenic disease and the engineered, hypoimmunogenic biotherapeutic comprises a viral particle comprising a transgene encoding a therapeutic product.

24. The pharmaceutical composition of claim 16, wherein the disorder is an antibody response against a transplanted tissue, the engineered low immunogenic biotherapeutic comprises IdeS, the transplanted tissue is an allograft or a xenograft, and the tissue is selected from kidney, heart, lung, liver, pancreas, trachea, vascular tissue, skin, bone, cartilage, adrenal tissue, fetal thymus, and cornea.

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