Bioorthogonally degradable tough hydrogels

Abstract

Disclosed are degradable tough hydrogels using enamine N-oxide based crosslinkers. Also disclosed are methods of making and using the degradable tough hydrogels.

Description

Attorney Docket No. 046094-798001 WO Date of Deposit: December 19, 2025BIOORTHOGONALLY DEGRADABLE TOUGH HYDROGELSRELATED APPLICATIONS

[0001] This application claims the benefit of priority' under 35 U. S C § 119(e) to U. S. Provisional Application No: 63 / 737,022, filed December 20, 2024, which is incorporated herein by reference in its entirely.GOVERNMENT SUPPORT

[0002] This invention was made with government support under grant number 2238040 awarded by The National Science Foundation. The government has certain rights in the invention.BACKGROUND OF THE DISCLOSURE

[0003] Hydrogels, hydrophilic polymer networks that can absorb and retain high volumes of water (X. Li el al., Nat. Rev Mater., 2024, 9:380), have been identified as ideal biomaterials due to their high-water content, tunable physical and chemical properties (X. Li et al., Front. Chem., 2018, 6:499; H Cao et al., Signal Transduct Target. Then, 2021, 6:426), and biocompatibility. As a result, hydrogels have seen many uses in biomedical applications such as biomedical devices (E. Calo et al., J, Eur. Polym., 2015, 65:252; R. D, Kasai et al., Int. J. Polym. Mater. Polym. Biomater., 2023, 72:1059), drug delivery systems (N. Joshi et cd., Nat. Commun., 2018, 9:1275; R. Zhong et al., Nat. Mater.. 2023, 22:818; J. Li, D. J. Mooney, Nat. Rev. Mater., 2016, 1:16071), and tissue engineering (K. Y. Lee et al., Chem. Rev., 2001, 101:1869; F. Xu et al.. Front. Bioeng. Biotechnol., 2022, 10:849831; O. Chaudhuri et al.. Nat. Mater., 2016, 15:326). Beyond tailoring the hydrogel's properties for its intended applications, development of degradable chemical motifs that can be integrated within the polymeric backbone (P Shieh et al., Chem. Rev., 2021, 121:7059; R. A Smith et al., J Am. Chem, Soc., 2019, 141:1446) of hydrogels has offered new ways to incorporate degradable hydrogels into biomedical applications including but not limited to injectable hydrogels (C. Wang ei al.. Biomaterials. 2016. 104:129; V. Pertici et al., Biomacromolecules, 2019, 20:149), mechano-responsive hydrogels for stem cell differentiation (Y. Ma etal., Adv. Mater., 2018, 30:el705911; W. J. Hadden et al.. Proc. Natl. Acad. Sci., 2017, 114:5647), and synchronized drug-releasing hydrogels (M. K. Gupta et al.. Adv. Funct. Mater., 2017, 27:04107; G W. Ashley et l.. Proc. Natl. Acad. Sci.. 2013, 110:2318).1AFSDOCS:304108277.1Attorney Docket No. 046094-798001 WO Date of Deposit: December 18, 2025

[0004] Despite the potential of hydrogels to possess similar water content to native biological tissues, most hydrogels exhibit poor material toughness, which limits their use in load bearing and mechanically deforming biomedical settings. Over the past two decades, new approaches have been developed to enhance the toughness of hydrogels, including double-network (DN) hydrogels (J. P. Gong et al., Adv. Mater., 2003, 15:1155; J.-Y. Sun et al., Nature 2012. 489:133; Q. Chen el al.. J. Mater. Chem. B, 2015, 3:3654; D. T. Wu el al., J. Dent. Res., 2023, 102:497). nanocomposite hydrogels (S. Merino et al.. ACS Nano, 2015, 9:4686; A. K. Gaharwar el al.. Biomacromolecules. 2011, 12:1641), and slide-ring hydrogels (Y. Okumura et al., Adv. Mater., 2001, 13:485). The enhanced toughness of hydrogels has afforded application in cartilage repair (X. Li et al.. Adv. Healthc. Mater., 2024:e2400431; L. Lei et al., Adv, Healthc. Mater., 2024, 13:e2302551), soft robotics (M, Baumgartner et al., Nat. Mater., 2020, 19:1102; M. Cianchetti et al.. Nat. Rev. Mater., 2018, 3:143; R. Nasseri et al., Nat. Common., 2023, 14:6108), contact lenses (X. Deng et al., ACS Appl. Mater. Interfaces, 2016, 8:22064; D. Kang et al., ACS Nano, 2022, 16:15827), and wound dressings (S, O, Blacklow et al., Sci. Adv., 2019, 5:eaaw3963; M. Kuddushi et al,, ACS Appl. Bio Mater.. 2023. 6:3810; P. Rao etal., Adv. Mater., 2018, 30:el801884).

[0005] One approach to achieving high mechanical toughness in hydrogels involves creating interpenetrating networks that combine a physically crosslinked polymer chain with a covalently crosslinked polymer network. DN hydrogels of polyacrylamides (PAAm) covalently crosslinked with bisacrylamides and alginates (Alg) physically crosslinked with divalent cations have been reported to be capable of stretching >20 times their initial length with fracture energies >6 kJ m2(J.-Y. Sun et al., Nature, 2012, 489:133). DN hydrogels exhibit high toughness through energy dissipation from physical crosslink dissociation and stress distribution via the elastic covalent network. Tough and robust hydrogels have been employed in a wide range of in vivo applications, including implantable hydrogel devices (Y. Zhao et al., Adv. Healthc. Mater., 2019, 8:el900709; F. Wang et al., Adv. Funct. Mater., 2024, 34:02314471), biosensors (A. Herrmann et al., Adv. Healthc. Mater., 2021, 10:e2100062), vascular grafts (S. Dimitrievska et al.. Adv, Funct. Mater,. 2020, 30:201908963; C. Zhang et al.. Adv. Healthc. Mater., 2021, 10:e2100839), and tissue spacers (M. Uhl et al., Radiat. Oncol., 2014, 9:96). In recent advancements of tough hydrogels, degradability has been a key focus, as it enables these materials to fulfill their functions and then be disintegrated safely, minimizing potential long-term adverse effects and the need for additional surgical interventions for removal.AFSDOCS:304108277.1Attorney Docket No. 046094-798001 WO Date of Deposit: December 18, 2025

[0006] Heat, pH, and magnetism can trigger material degradation, and these stimuli have been used in combination with bioorthogonal dissociation chemistries to develop a variety of degradable soft materials. Notably, hydrogels derived by crosslinking thiol based multi -arm PEG and oxanorbomadi ene dicarboxylate reagents undergo degradation via retro-Diels-Alder reaction with mild heating (C. J. Higginson el al., J. Am. Chem. Soc., 2015, 137:4984). Additionally, tetraz.ine modified alginate hydrogels coupled to trans-cyclooctene prodrugs have been used for targeted drug delivery7(J. M. M. Oneto et al., ACS Cent. Sci., 2016, 2:476). Other examples include thermo-magnetically responsive grippers used in tissue biopsies (S. R. Goudu et al.. Adv. Funct. Mater., 2020, 30:202004975) and pH-triggered gastric-resident devices (J. Liu et al., Nat. Commun., 2017, 8:124).

[0007] Although these stimulus-responsive hydrogels are effective for certain applications, they present drawbacks that limit their use in some in vivo settings. Heat-based stimuli may cause unintended damage to adjacent tissues, while magnetic stimuli could disrupt standard medical imaging equipment, pH-based stimuli have limited spatial effectiveness and have the potential to harm pH sensitive tissues. Photo-responsive hydrogels require a light source to induce degradation, which can be challenging when hydrogels are implanted deep within tissues. Biodegradable hydrogels generally exhibit relatively slow and uncontrolled degradation. And although useful for applications such as therapeutics delivery, they are not ideal for applications that require repetitive removal. Moreover, these hydrogels gradually hydrolyze over time without induction at a specific timepomt, negatively impacting the mechanical integrity of the hydrogels prior to the end of their useful lifetime. In cases where preserving the integrity of the hydrogel is desired until the moment of degradation, chemically responsive hydrogels are favored. Such chemically induced degradation processes must be compatible with the hydrogel environment, and the chemical stimuli should be bioorthogonal. Furthermore, previously reported degradable hydrogels degrade over several hours to day s Processes for improving the kinetics of the degradation of hydrogels are needed.SUMMARY OF DISCLOSURE

[0008] The current disclosure solves the problems mentioned above by producing chemically responsive PAAm / Alg hydrogels that can degrade upon exposure to chemical stimulus with fast kinetics and high biocompatibility.3AFSDOCS:304108277.1Attorney Docket No. 046094-798001 WO Date of Deposit: December 18, 2025

[0009] A first aspect of the present disclosure is directed to a compound having a structure represented by formula 1:R2 P" LIx>xR2-O R., (I), or a pharmaceutically acceptable salt or stereoisomer thereof as a substantially pure regioisomer,wherein:o is 0, 1, or 2;p is 0, 1, or 2;q is 0, 1, or 2;Ri is (Ci-Cs) alkyl, (C3-C10) carbocyclyl, or 4- to 10-membered heterocyclyl comprising 1 to 3 heteroatoms selected from O, N, and S, wherein said alky l, carbocyclyl or heterocyclyl is further optionally substituted;each R2 is independently a polymerizable moiety;Li is absent or a linker;L2 is absent or a linker; andX is a leaving group.

[0010] In some embodiments, the compound having a structure represented by formula I:X W,R;(I), or a pharmaceutically acceptable salt or stereoisomer thereof, as a substantially pure regioisomer, may be prepared by a method, comprising:i) reacting a compound of formula II,(II). and a compound offormula (III), form a compound of formula IV,ii) subjecting the reaction product of i) to a lithium halogen exchange followed by protonation,iii) removing the nitrogen protecting groups, andiv) acylating the terminal nitrogen atoms.wherein:AFSDOCS:304108277.1Attorney Docket No. 046094-798001 WO Date of Deposit: December 18, 2025o is 0, 1, or 2;p is 0, 1, or 2;q is 0, 1, or 2;R i is (Ci-Cs) alkyl, (C3-C10) carbocyclyl, or 4- to 10-membered heterocyclyl comprising 1 to 3 heteroatoms selected from O, N, and S, wherein said alkyd, carbocyclyl or heterocyclyl is further optionally substituted;each R?is independently a polymerizable moiety;each R3 is independently a nitrogen protecting group;R4 is chloro, bromo, or iodo;Li is absent or a linker;I. / 2 is absent or a linker; andX is a leaving group. Accordingly, the compound of formula (I) prepared via the method may be considered as a related aspect of the disclosure.

[0011] A second aspect of the present disclosure is directed to a compound of formula IV,R4H0“1 P LW NXZX" P'3H / \Rl(IV). The compound of formula (IV) may be used as a reactant, e g., a starting material, in the preparation of a compound of formula (I).

[0012] A third aspect of the present disclosure is directed to a hydrogel which is the reaction product of: / \i) a compound of formula I:(I), as a substantially pure regioisomer,ii) a polymerizable reagent, andiii) an initiator.

[0013] Further aspects of the present disclosure are directed to methods of degrading hydrogels, that entail contacting the hydrogel with a diboron reagent.

[0014] The bioorthogonally degradable tough hydrogels can function in both dry and wet environments, and in vitro and in vivo. Employing enamine N-oxide bisacrylamide as a covalent crosslinker for PAArn / Alg or PNiPAAm / Alg hydrogels enables the hydrogels to be degraded by treatment with a diboron reagent (e.g., B2(OH)4). The degradation is inducible, and the degradation kinetics are rapid. More specifically and as disclosed in working examples, hydrogels crosslinking agents of formula (I) are more susceptible to degradation 5AFSDOCS:304108277.1Attorney Docket No. 046094-798001 WO Date of Deposit: December 18, 2025with a diboron reagent, both in terms of extent and rate of degradation, compared to known hydrogels, including Applicant's prior hydrogels. High biocompatibility’ of both the hydrogels and B2(OH)4 provides utility in wound dressings and allows for repeated atachment and detachment to the adhered tissue without causing secondary skin damage.BRIEF DESCRIPTION OF THE DRAWINGS

[0015] FIG. 1A-FIG. IK shows the fabrication and mechanical properties of degradable double network PAAm / Alg hydrogels using crosslinkers ENBAA-1 -3. (FIG. 1 A) Illustration highlighting monomers, crosslinkers, and polymerization reagents used to fabricate degradable PAAm / Alg hydrogels. The effect of crosslinker length on the (FIG. IB) maximum stresses, (FIG. 1C) maximum strains, (FIG. ID) fracture energies, and (FIG. IE) Young’s moduli of PAAm / Alg hydrogels fabricated with varying covalent crosshnker concentrations (MBAA, ENBAA-1-3) measured under tensile loading. Mean values are shown, and error bars represent ± SD (n=3). (FIG. IF) Stress-strain curves comparing PAAm / Alg hydrogels cast with MBAA and varying concentrations of ENBAA-1 crosslinker relative to that of MBAA. The maximum (FIG. 1G) stress and (FIG. Ill) strain of PAAm / A]g hydrogels cast with ENBAA-1 crosslinker and stored at 37°C for 15 d are graphed Mean values are shown, and error bars represent ± SD (n=3). (FIG. II) Viscoelastic responses of PAAm / Alg hydrogels cast with MBAA and ENBAA-1 crosslinkers measured by dynamic mechanical analysis (DMA). Mean values are shown, and error bars represent ± SD, (n:::3). (FIG. 1J) Compressive stresses at 80% strain of PAAm / Alg hydrogels cast with MBAA and ENBAA-1 crosslinkers measured under compressive loading. Mean values are shown, and error bars represent ± SD (n=3). (FIG. IK) Illustration of PAAm / Alg hydrogels crosslinked with ENBAA-1 crosslinker.

[0016] FIG. 2A-FIG. 2F shows the degradation profiles of degradable 1.5 mm thick PAAm / Alg hydrogels. (FIG. 2A) Photos of PAAm / Alg hydrogels before and after exposure to aqueous B2(OH)4 (500 pM) for 20 mm. Degradation of PAAm / Alg hydrogels cast with ENBAA-1 crosslinkers evaluated by tensile tests. (FIG. 2B) Stress-strain curves, (FIG. 2C) maximum stresses, (FIG. 2D) fracture energies, and (FIG. 2E) Young’s moduli of PAAm / Alg hydrogels exposed to various concentrations of aqueous B2(OH)4 for 10 min. (FIG. 2F) Photos of PAAm / Alg hydrogels exposed to aqueous B2(OH)4 (500 pM) for 10 min under tensile loading. Before tensile pull (left) and at 3.4 mm / mm strain (right). Mean values are shown, and error bars represents ± SD (n = 3). Statistical analysis by a one-way AN OVA 6AFSDOCS:304108277.1Attorney Docket No. 046094-798001 WO Date of Deposit: December 18, 2025with post hoc t-tests with Bonferroni correction, ****p < 0.0001, ** * < 0.01, *P < 0.05, ns = no significance between groups.

[0017] FIG. 3A-FIG. 3G shows the degradation profiles of 1 0 mm thick and 1.5 mm thick PAAm / Alg hydrogels evaluated with tensile and theology tests. (FIG. 3A) Stress-strain curves, (FIG. 3B) maximum stresses, (FIG. 3C) fracture energies, and (FIG. 3D) Young's moduli of PAAm / Alg hydrogels of vaiying thickness exposed to various concentrations of aqueous 62(011)4 for 10 min. (FIG. 3E) Shear storage moduli of 1.0 mm thick, and (FIG. 3F) 1.5 mm thick PAAm / Alg hydrogels cast with ENBAA-1 exposed to aqueous B2(OH)4. (FIG.3G) Fraction storage modulus of 1.0 mm and 1.5 mm thick PAAm / Alg hydrogels cast with ENBAA-1 exposed to aqueous 62(011)4. Mean values are shown, and error bars represents ± SD (n = 3). Statistical analysis by a one-way ANOVA with post hoc Mests with Bonferroni correction, **** » < 0.0001, **P < 0.01, *P < 0.05, ns = no significance between groups.

[0018] FIG. 4A-FIG. 4D shows the biocompatibility7of a degradable PAAm / Alg hydrogel and 62(01-1) reagent. (FIG. 4A) Illustration of the biocompatibility assay of PAAm / Alg hydrogels. (FIG. 4B) Fluorescence microscope images of cells stained with Calcein AM (left, green), ethidium homodimer- 1 (middle, red), and overlay of two channels (right). (FIG. 4C) Cell viability’ of L929 cells incubated with PAAm / Alg hydrogels and exposed to aqueous 62(01-1)4 (500 pM) for 30 min. Cell viability was calculated using a custom pipeline on CeliProfiler, (FIG. 4D) L929 fibroblasts exposed to Bp / OHfi were evaluated for cell viability¬ using the CellTiter-Glo 2.0 assay.

[0019] FIG. 5A-FIG. 5C shows the analysis of the strength of adhesion of degradable hydrogels to tissues. (FIG. 5 A) Illustration of T-peel test on porcine skin. (FIG. 5B) Adhesion energies of degradable hydrogels on porcine skin. Energies were measured using the T-peel test. Loctite, Periacryl. chitosan + EDC / sulfo-NHS were used as adhesives. Mean values are shown, and error bars represent ± SD, (n==3). (FIG 5C) H& E images of mice skin after adhesion and removal of PAAm / Alghydrogels cast with ENBAA-1 crosslinker. Aqueous B2(OH)4 (500 pM, 30 min) was used to degrade the hydrogels.

[0020] FIG. 6A-FIG. 6H shows PNiPAAm / Alg hydrogels employed as intraoral hydrogel wound dressings. (FIG. 6A) Illustration of the use of PNiPAAm / Alg hydrogels as antiswelling and degradable intraoral hydrogel wound dressings together with aqueous tetrahydroxydiboron as the chemical inducer applied as a mouth wash. (FIG. 6B) Swelling ratios of PAAm / Alg and PNiPAAm / Alg hydrogels swollen in PBS, pH 7.4 at 23°C or 37°C for 3 d. Mean values are shown, and error bars represent ± SD (n::::3). (FIG. 6C) Photos of 7AFSDOCS:304108277.1Attorney Docket No. 046094-798001 WO Date of Deposit: December 18, 2025PAAm / Alg and PNiPAAm / Alg hydrogels before and after swelling in PBS, pH 7.4 at 37°C for 3 d. (FIG. 6D) Maximum stress. (FIG. 6E) maximum strain, and (FIG. 6F) Young's modulus of swollen PAAm / Alg and PNiPAAm / Alg hydrogels in PBS, pH 7.4 at 37 °C for 3 d. Mean values are shown, and error bars represent ± SD (n=3). (FIG. 6G) Viscoelastic responses of swollen PAAm / Alg and PNiPAAm / Alg hydrogels in PBS, pH 7.4 at 37 °C for 3 d. Mean values are shown, and error bars represent ± SD (n=3). (FIG. 6H) Lap shear stress of swollen PAAm / Alg and PNiPAAm / Alg hydrogels in PBS, pH 7.4 at 37 °C for 3 d on porcine skin pre-treated with pooled human saliva. Adhesives used include Loctite. Periacryl, chitosan + EDC / sulfo-NHS and Ora- Aid. Mean values are shown, and error bars represent ± SD (n=3), as analyzed by a one-way ANOVA with post hoc / -tests with Bonferroni correction, ****p < 0.0001.

[0021] FIG. 7A-FIG. 7E shows the degradation profiles of swollen PNiPAAm / Alg hydrogels. (FIG. 7 A) Stress-strain curves of swollen PNiPAAm / Alg hydrogels exposed to various concentrations of Bz( OII)4 for 10 min. (FIG. 7B) Maximum stress, (FIG. 7C) fracture energy, and (FIG. 7D) Young's modulus of swollen PNiPAAm / Alg hydrogels exposed to various concentrations of B2(OH)4 for 10 min. Mean values are shown and error bars represent ± SD (n=3). (FIG. 7E) Ex vivo application of PNiPAAm / Alg hydrogels adhered to and removed from pork tongue. Hydrogels were adhered using chitosan + EDC / sulfo-NHS. Solution of B2(OH)4 (500 pM) was applied in 3 different ways for 10 min: 1) submerged, 2) sprayed or 3) applied by wet gauze.

[0022] FIG. 8A-FIG. 8C shows a maximum tolerated dose (MTD) study for administration of 62(01-1)4 by oral gavage in mice. (FIG 8A) Illustration of oral gavage and timeline of the MTD study. (FIG. 8B) Survival rate and (FIG. 8C) weight change (%) of mice administered with B2(OH)4 every 24 h for 5 consecutive days.

[0023] FIG. 9A-FIG 9E shows the mechanical properties of PAAm / Alg hydrogels cast with ENBAA-1 covalent crosslinker. (FIG. 9A) Stress-strain curves, (FIG. 9B) max stresses, (FIG. 9C) max strains, (FIG. 9D) fracture energies, and (FIG. 9E) elastic moduli of PAAm / Alg hydrogels with varying ENBAA-1 stoichiometries. Mean values are shown, and error bars represent ± SD («:::3 samples per group).

[0024] FIG. 10A-FIG. 10E shows the mechanical properties of PAAm / Alg hydrogels cast with ENBAA-2 covalent crosslinker. (FIG 10A) Stress-strain curves, (FIG. 10B) max stresses, (FIG. 10C) max strains, (FIG. 10D) fracture energies, and (FIG. 10E) elastic moduli8AFSDOCS:304108277.1Attorney Docket No. 046094-798001 WO Date of Deposit: December 18, 2025of PAAm / Alg hydrogels with varying ENBAA-2 stoichiometries. Mean values are shown, and error bars represent ± SD (n = 3 samples per group).

[0025] FIG. 11 A-FIG. 1 IE shows the mechanical properties of PAAm / Alg hydrogels cast with ENBAA-3 covalent crosslinker. (FIG 11 A) stress-strain curves, (FIG 11B) max stresses, (FIG. 11C) max strains, (FIG. 11D) fracture energies, and (FIG. HE) elastic moduli of PAAm / Alg hydrogels with vaiying ENBAA-3 stoichiometries. Mean values are shown, and error bars represent ± SD n ------ 3 samples per group).

[0026] FIG. 12 A-FIG I2E shows the mechanical properties of PAAm / Alg hydrogels cast with MBAA covalent crosslinker. (FIG. 12A) stress-strain curves, (FIG. 12B) max stresses, (FIG. 12C) max strains, (FIG. 12D) fracture energies, and (FIG. 12E) elastic moduli of PAAm / Alg hydrogels with varying covalent crosslinker equivalents. Mean values are shown, and error bars represent ± SD n = 3 samples per group).

[0027] FIG. 13A-FIG. 131 shows further optimization of ENBAA-1 crosslinker stoichiometries relative to MBAA. Mechanical properties of PAAm / Alg hydrogels cast with ENBAA-1 covalent crosslinker. (FIG. 13 A) Stress-strain curves, (FIG. 13B) max stresses, (FIG. 13C) max strains, (FIG. 13D) fracture energies, and (FIG. 13E) elastic moduli of PAAm / Alg hydrogels with vaiying ENBAA-1 stoichiometries. Direct comparison of (FIG.13F) max stresses, (FIG. 13G) max strains, (FIG. 13H) fracture energies, and (FIG. 131) elastic moduli between PAAm / Alg hydrogels cast with lx MBAA or 1.2x ENBAA-1, Mean values are shown, and error bars represent ± SD (n = 3 samples per group). P-values were obtained with unpaired / -tests.

[0028] FIG. 14A-FIG. 14C shows the stability of PAAm / Alg hydrogels cast with the ENBAA-1 crosslinker was assessed by tensile tests. (FIG. 14A) Max stresses, (FIG. 14B) max strains, and (FIG. 14C) elastic moduli of PAAm / Alg hydrogels cast with l MBAA or 1.2x ENBAA-1. Mean values are shown, and error bars represent i SD (n = 6 samples per group) as analyzed by a one-way ANOVA with post hoc / -tests with Bonferroni correction. Not significant (ns) if no pairwise comparison is shown.

[0029] FIG. 15 is a representative stress-strain curve of PAAm / Alg hydrogels cast with l. Ox MBAA or 1.2x ENBAA-1 covalent crosslinker.

[0030] FIG. 16 is a series of photographs of inversion tests of PAAm hydrogels cast with either MBAA or ENBAA-1 crosslinker and exposed to aqueous B2(OH)4 (10 mM) for 10 min.9AFSDOCS:304108277.1Attorney Docket No. 046094-798001 WO Date of Deposit: December 18, 2025

[0031] FIG. 17 is a series of photographs of PAAm / Alg hydrogels submerged in various concentrations of B2(OH)4 at various time points.

[0032] FIG. 18A-FIG. 18E shows the degradation of PAAm / Alg hydrogels with varying concentrations of B2.(OH)4 for 3 min was evaluated through tensile tests, (FIG. 18A) Representative stress-strain curves of PAAm / Alg hydrogels exposed to B2(OH)4for 3 min. (FIG. 18B) Inset of stress-strain curve in panel FIG. 18A. (FIG. 18C) Maximum stresses, (FIG. 18D) fracture energies, and (FIG. 18E) elastic moduli of PAAm / Alg hydrogels exposed to B2.(OH)4 for 3 min. Mean values are shown, and error bars represents ± SD (n = 3 samples per group) as analyzed by a one-way ANOVA wrth post hoc / -tests with Bonferroni correction, ****P < 0.0001, ***P < 0.001 between groups.

[0033] FIG. 19A-FIG. 19D shows the degradation of PAAm / Alg hydrogels using varying concentrations of B (OH)4 for 20 min, was evaluated through tensile tests. (FIG. 19A) Representative stress-strain cruxes of PAAm / Alg hydrogels exposed to B2(OH)4 for 20 min. (FIG. 19B) Maximum stresses, (FIG. 19C) fracture energies, and (FIG. 19D) elastic moduli of PAAm / Alg hydrogels exposed to B2(OH)4 for 20 min. Mean values are shown and error bars represent ± SD (n = 3 samples per group).

[0034] FIG. 20A-FIG 20C show's the degradation of 1.5 mm and 1.0 mm thick PAAm / Alg hydrogels using varying concentrations of B 2(011)4 for 10 min was evaluated through tensile tests. (FIG. 20 A) Representative stress-strain curves of PAAm / Alg hydrogels exposed to 1 mM B2(OH)4 for 10 mm. (FIG. 20B) Representative stress-strain curves of PAAm / Alg hydrogels exposed to 500 pM B2(OH)4 for 10 min. (FIG. 20C) Representative stress-strain curves of PAAm / Alg hydrogels exposed to 100 pM B2(OH)4 for 10 min. Mean values are shown, and error bars represent ± SD (n = 3 samples per group).

[0035] FIG. 21A-FIG. 21H shows discrete absolute storage and loss moduli of 1.5 mm and 1.0 mm thick PAAm / Alg hydrogels exposed to various aqueous [B2(OH)4|. Storage and loss moduli of 1.5 mm and 1.0 mm thick PAAm / Alg hydrogels cast with ENBAA-l exposed to (FIG. 21 A) [B2(OH)4] 0 mM, (FIG. 2 IB) 100 pM, (FIG. 21 C) 500 tiM. (FIG. 2 ID) 1 mM, (FIG. 21E) 3 mM, (FIG. 21F) 10 mM. Storage and loss moduli of 1.5 mm and 1.00 mm thick PAAm / Alg hydrogels cast with MBAA exposed to (FIG. 21G) [62(011)4 0 mM, and (FIG.21H) 10 mM. All measurements were performed at 20°C with 1 Hz frequency and 5% shear strain amplitude. Mean values are shown and error bars represent ± SD (n = 3 samples per group).10AFSDOCS:304108277.1Attorney Docket No. 046094-798001 WO Date of Deposit: December 18, 2025

[0036] FIG. 22A-FIG. 22B shows discrete fraction storage and loss moduli of 1.5 mm and 1.0 mm thick PAAm / Alg hydrogels exposed to various concentrations of aqueous B2OH4 solution. Storage moduli of (FIG. 22A) 1.0 mm and (FIG. 22B)1.5 mm thick PAAm / Alg hydrogels cast with ENBAA-1 or MBAA crosslinker then exposed to B2(OH)4 concentrations of 0 mM, 100 pM, 500 pM, 1 mM. 3 mM and 10 mM. Mean values are shown and error bars represent ± SD (« = 3 samples per group).

[0037] FIG. 23 is a bar graph showing adhesion energies of PAAm / Alg hydrogels on porcine skin Loctite, Periacryl, and chitosan with EDC and sulfo-NHS adhesives were applied to porcine skin followed by PAAm / Alg hydrogel. Mean values are shown and error bars represents ± SD (n = 3 samples per group).

[0038] FIG. 24A-FIG. 24D are a series of H& E images of mice skin after adhesion and removal of PAAm / Alg hydrogels cast with MBAA. (FIG. 24A) Loctite, (FIG. 24B) Periacryl and (FIG. 24C) chitosan with EDC and (FIG. 24D) sulfo-NHS adhesives wnre applied to mice skin followed by removal after treatment with B2(OH)4 (500 pM in H2O, 20 mL).

[0039] FIG, 25A-FIG. 25D shows the swelling ratios of PAAm / Alg and PNiPAAm / Alg hydrogels covalently crosslinked with lx MBAA. Hydrogels were swollen in (FIG. 25A) H2O, (FIG. 25B) PBS, pH 7.4, (FIG. 25C) MEM + 10% human serum, and (FIG. 25D) MEM + 10% huma saliva, and incubated at 23°C or 37°C. Mean values are shown, and error bars represent ± SD (n = 3 samples per group).

[0040] FIG. 26A-FIG. 26D shows the swelling ratios of PAAm / Alg and PNiPAAm / Alg hydrogels covalently crosslinked with 1.2x ENBAA-1 covalent crosslinker. Hydrogels were swollen in (FIG. 26A) H2O, (FIG. 26B) PBS, pH 7.4, (FIG. 26C) MEM + 10% human serum, and (FIG. 26D) MEM + 10% huma saliva, and incubated at 23°C or 37°C. Mean values are shown, and error bars represent ± SD (n = 3 samples per group).

[0041] FIG. 27A-FIG. 27C shows unswollen PNiPAAm / Alg hydrogels' mechanical properties. (FIG. 27A) Maximum stress, (FIG. 27B) maximum strain, and (FIG. 27C) elastic modulus of PNiPAAm / Alg hydrogels cast with either lx MBAA or 1.2x ENBAA-1 covalent crosslinker. Mean values are shown, and error bars represent ± SD (« = 3 samples per group).

[0042] FIG. 28A-FIG. 28D show^s viscoelastic responses of swollen PAAm / Alg and NiPAAm / Alg hydrogels cast with lx MBAA covalent crosslinker. Hydrogels were swollen in (FIG. 28A) H2O. (FIG. 28B) PBS, pH 7.4. (FIG. 28C) MEM + 10% human serum, or (FIG. 28D) MEM + 10% human saliva at 37°C for 3 d before measurement. Mean values are shown, and error bars represent ± SD (n = 3 samples per group).11AFSDOCS:304108277.1Attorney Docket No. 046094-798001 WO Date of Deposit: December 18, 2025

[0043] FIG. 29A-FIG. 29D shows viscoelastic responses of swollen PAAm / 'Alg and NiPAAm / Alg hydrogels cast with 1.2x ENBAA-1 covalent crosslinker. Hydrogels were swollen in (FIG. 29 A) H2O, (FIG. 29B) PBS, pH 7.4, (FIG. 29C) MEM + 10% human serum, or (FIG. 29D) MEM + 10% human saliva at 37°C for 3 d before measurement. Mean values are shown, and error bars represent ± SD (n = 3 samples per group).

[0044] FIG. 30A-FIG. 30D shows the degradation profiles of swollen PNiPAAm / Alg hydrogels. (FIG. 30 A) Representative stress-strain curves of swollen PNiPAAm / Alg hydrogels exposed to various concentrations of an aqueous solution of B2(OH)4 for 3 min. (FIG. 30B) Maximum stresses, (FIG. 30C) fracture energies, and (FIG. 30D) elastic moduli of swollen PNiPAAm / Alg hydrogels exposed to various concentrations of an aqueous solution of B2(OH)4for 3 min. Mean values are shown, and error bars represent ± SD (n = 3 samples per group).

[0045] FIG. 31A-FIG. 31B shows biocompatibility of a degradable PAAm / Alg hydrogel and B2(OH)4. (FIG. 31 A) Fluorescence microscopy images of cells stained with Calcein AM (left, green), ethidium homodimer- 1 (middle, red), and overlay of the two channels (right). (FIG.3 IB) Cell viability of L929 cells incubated with PAAm / Alg hydrogels and exposed to B2(OH)4for 30 min. Cell viability was calculated using a custom pipeline on Cell Profiler.

[0046] FIG. 32 shows adhesion and removal of PNiPAAm / Alg hydrogels cast with MBAA on pork tongue. Hydrogels were adhered with Loctite. Hydrogels were cast with blue food coloring for visualization. Hydrogel-tissue adducts were then treated with either water or aqueous B2(OH)4 (500 pM) for removal.

[0047] FIG. 33 shows adhesion and removal of PNiP Am / Alg hydrogels cast with MBAA on pork tongue. Hydrogels were adhered with Periaciyl. Hydrogels were cast with blue food coloring for visualization. Hydrogel-tissue adducts were then treated with either water or aqueous B2(OH)4(500 pM) for removal.

[0048] FIG. 34 show’s adhesion and removal of PNiPAAm / Alg hydrogels cast with MBAA on pork tongue. Hydrogels were adhered with chitosan + EDC / sulfo-NHS. Hydrogels were cast with blue food coloring for visualization. Hydrogel -tissue adducts were then treated with either water or aqueous B2(OH)4(500 pM) for removal

[0049] FIG 35 shows adhesion and removal of PNiPAAm / Alg hydrogels cast with ENBAA-1 on pork tongue. Hydrogels were adhered with Loctite. Hydrogel-tissue adducts were then treated with either ater or aqueous B2(OH)4(500 pM) for removal.12AFSDOCS:304108277.1Attorney Docket No. 046094-798001 WO Date of Deposit: December 18, 2025

[0050] FIG. 36 shows adhesion and removal of PNiPAAm / Alg hydrogels cast with ENBAA-1 on pork tongue. Hydrogels were adhered with Periacryl. Hydrogel-tissue adducts were then treated with either water or aqueous B2(OH)4(500 pM) for removal.

[0051] FIG. 37 shows adhesion and removal of PNiPAAm / Alg hydrogels cast with ENBAA-1 on pork tongue. Hydrogels were adhered with chitosan + EDC / sulfo-NHS. Hydrogels were cast with blue food coloring for visualization. Hydrogel-tissue adducts were then treated with either water or aqueous B2(OH)4(500 pM) for removal.

[0052] FIG. 38 shows fit NMR of the regioselective, 2ndgeneration version of ENBAA-1 as compared to that of the 1stgeneration version of ENBAA-1.DETAILED DESCRIPTION

[0053] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the subject matter herein belongs. As used in the specification and the appended claims, unless specified to the contrary, the following terms have the meaning indicated in order to facilitate the understanding of the present disclosure.

[0054] As used in the description and the appended claims, the singular forms ‘‘a”, “‘an”, and “the” include plural referents unless the context clearly dictates otherwise. Therefore, for example, reference to “a composition” includes mixtures of two or more such compositions, reference to “an inhibitor'’ includes mixtures of two or more such inhibitors, and the like.

[0055] Unless stated otherwise, the term "about” means within 10% (e.g, within 5%. 2%, or 1%) of the particular value modified by the term “about.”

[0056] The transitional term “comprising,” which is synonymous with “including,” “containing,” or “characterized by,” is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. When used in the context of the number of heteroatoms in a heterocyclic structure, it means that the heterocyclic group that that minimum number of heteroatoms. By contrast, the transitional phrase “consisting of’ excludes any element, step, or ingredient not specified in the claim. The transitional phrase “consisting essentially of” limits the scope of a claim to the specified materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the disclosure.

[0057] With respect to compounds of the present disclosure, and to the extent the following terms are used herein to further describe them, the following definitions apply.13AFSDOCS:304108277.1Attorney Docket No. 046094-798001 WO Date of Deposit: December 18, 2025

[0058] The phrase “as a substantially pure regioisomer,” as used herein in the specific context of a compound having a structure represented by formula I:R2 P" LIx>xR2-O R., (1), means that the compound of formula (I) is the single regioisomer detectable by proton nuclear magnetic resonance (1H NMR) as conducted in accordance with the disclosure in the section entitled '''chemical instrumentation” that appears in the working examples, and is free of its regioisomer, i.e., the compound having a structure represented by formula V:_0 Ri (V), which is not detectable via the1H NMR.

[0059] As used herein, the term “alkyl’’ refers to a saturated linear or branch ed-chain monovalent hydrocarbon radical. In some embodiments, the alkyl radical is a Ci-Ce group. In some embodiments, and to the extent not disclosed otherwise for any one or more groups of the compounds of formula (I-V) or the hy drogels disclosed herein, the alkyl radical is a Co-Ce. C0-C5, C0-C3, Ci-Ce, C1-C5, C1-C4 or C1-C3 group (wherein Co alkyl refers to a bond). Examples of alkyl groups include methyl, ethyl, 1 -propyl, 2-propyl, i-propyl, 1 -butyl, 2-methyl-1 -propyl, 2-butyl, 2-methyl-2-propyl, 1 -pentyl, n-pentyl, 2-pentyl, 3-pentyl, 2-methyl- 2-butyl, 3-methyl-2-butyl, 3-methyl-l-butyl, 2-methyl-l -butyl, 1 -hexyl, 2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 3-methyl-3-pentyl, 2-methyl-3-pentyl, 2.3-dimethyl-2-butyl, and 3,3-dimethyl-2-butyl. In some embodiments, an alkyl group is a C1-C3 alkyl group. In some embodiments, an alkyl group is a C1-C2 alkyl group. In some embodiments, an alkyl group is a methyl group.

[0060] As used herein, the term “alkylene"’ refers to a straight or branched divalent hydrocarbon chain linking the rest of the molecule to a radical group, consisting solely of carbon and hydrogen, containing no unsaturation and having from one to six carbon atoms, for example, methylene, ethylene, propylene, n-butylene, and the like. The alkylene chain may be attached to the rest of the molecule through a single bond and to the radical group through a single bond. In some embodiments, and to the extent not disclosed otherwise for any one or more groups of the compounds of formula (I-V) or the hydrogels disclosed herein, an alkylene group contains one to four carbon atoms (C1-C4 alkylene). In other embodiments, an alkylene contains one to three carbon atoms (C1-C3 alkylene). In other embodiments, anAFSDOCS:304108277.1Attorney Docket No. 046094-798001 WO Date of Deposit: December 18, 2025alkylene group contains one to two carbon atoms (C 1-C2 alkylene). In other embodiments, an alkylene group contains one carbon atom (Ci alkylene).

[0061] As used herein, the term "alkenyl" refers to a linear or branched-cham monovalent hydrocarbon radical with at least one carbon-carbon double bond. An alkenyl includes radicals having "cis" and "trans" orientations, or alternatively, " E" and " Z" orientations. In some embodiments, the alkenyl radical is a C2-C15 group. In some embodiments, and to the extent not disclosed otherwise for any one or more groups of the compounds of formula (I-V) or the hydrogels disclosed herein, the alkenyl radical is a C2-C12, C2-C10, C2-C8, C2-C6 or C2-C4 group. Examples include ethenyl or vinyl, prop-l-enyl, prop-2-enyl, 2-methylprop-l-enyl, but-l-enyl, but-2-enyl, but-3-enyl. buta-1, 3-dienyl, 2-methylbuta-1,3-diene, hex-l-enyl, hex- 2-enyl, hex-3-enyl, hex-4-enyl and hexa-1, 3-dienyl.

[0062] As used herein, the term "alkynyl" refers to a linear or branched monovalent hydrocarbon radical with at least one carbon-carbon triple bond. In some embodiments, the alkynyl radical is a C2-C15 group. In some embodiments, and to the extent not disclosed otherwise for any one or more groups of the compounds of formula (I-V) or the hydrogels disclosed herein, the alkynyl radical is C2-C12, C2-C10. C2-C8. C2-C6 or C2-C3. Examples include ethynyl prop-l-ynyl, prop-2-ynyl, but-l-ynyl, but-2-ynyl and but-3-ynyl.

[0063] The terms “alkoxy 1” or “alkoxy” as used herein refer to an alkyl group, as defined above, having an oxygen radical attached thereto, and which is the point of attachment. In some embodiments, the alkoxyl group is methoxy, ethoxy, propyloxy, or tert-butoxy. An “ether” is two hydrocarbyl groups covalently linked by an oxygen. Accordingly, the substituent of an alkyl that renders that alkyl an ether is or resembles an alkoxyl, such as can be represented by one of -O-alkyl, -O-alkenyl. and -O-alkynyl.

[0064] As used herein, the term “halogen” (or “halo” or “halide”) refers to fluorine, chlorine, bromine, or iodine.

[0065] As used herein, the term “carbocyclic” (also "carbocyclyl") refers to a group that used alone or as part of a larger moiety, contains a saturated, partially unsaturated, or aromatic ring system having 3 to 12 carbon atoms. The term carbocyclyl includes mono-, bi-, tri-, fused, bridged, and spiro-ring systems, and combinations thereof. In one embodiment, carbocyclyl includes 3 to 10 carbon atoms (C3-C10). In one embodiment, carbocyclyl includes 3 to 6 carbon atoms (C3-C6). In one embodiment, carbocyclyl includes 5 to 6 carbon atoms (C5-C6). In some embodiments, carbocyclyl, as a bicycle, includes Ce-Cio. In another embodiment, carbocyclyl, as a spiro system, includes C5-C11. Representative examples of 15AFSDOCS:304108277.1Attorney Docket No. 046094-798001 WO Date of Deposit: December 18, 2025monocyclic carbocyclyls include cyclopropyl, cyclobutyl, cyclopentyl, 1 -cyclopent- 1-enyl, 1- cyclopent-2-enyl, l-cyclopent-3-enyl, cyclohexyl, 1 -cyclohex- 1-enyl, l-cyclohex-2-enyl. 1- cyclohex-3-eny 1, cyclohexadienyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, cycloundecyl, and phenyl; bicyclic carbocyclyls having 7 to 11 ring atoms include [4,3], [4,4], [4,5], [5,5], [5,6] or [6,6] ring systems, such as for example bicyclo[2.2 1]heptane, bicyclo[2.2.2]octane, naphthalene, and bicyclo[3.2.2]nonane. Representative examples of spiro carbocyclyls include spiro[2.2]pentane, spiro[2.3]hexane, spiro[2.4]heptane, spiro[2.5]octane and spiro

[0045] decane. The term carbocyclyl includes and ring systems as defined herein. The term carbocyclyl also includes cycloalkyl rings (e.g., saturated or partially unsaturated mono-, bi-, or spiro-carbocycles). The term carbocyclic group also includes a carbocyclic ring fused to one or more (e g., 1. 2 or 3) different cyclic groups (e.g., aryl or heterocyclic rings), where the radical or point of attachment is on the carbocyclic ring.

[0066] Therefore, the term carbocyclic also embraces carbocyclylalkyl groups which as used herein refer to a group of the formula — Rc-carbocyclyl where R° is an alkylene chain. The term carbocyclic also embraces carbocyclylalkoxy groups which as used herein refer to a group bonded through an oxygen atom of the formula -O-Rc-carbocyclyl where Rcis an alky lene chain.

[0067] As used herein, the term "aryl" refers to a group that includes monocyclic, bicyclic or tricyclic, carbon ring system, that includes fused rings, wherein at least one ring in the system is aromatic. In some embodiments, the aralkoxy group is a benzoxy group. The term "aryl" may be used interchangeably' with the term "aryl ring". In one embodiment, aryl includes groups having 6-12 carbon atoms. In another embodiment, aryl includes groups having 6-10 carbon atoms. Examples of ary l groups include phenyl, naphthyl, biphenyl, 1,2,3,4-tetrahydronaphthalenyl, and the like, w’hich may be substituted or independently substituted by one or more substituents described herein. A particular ary l is phenyl. In some embodiments, an aryl group includes an aryl ring fused to one or more (e.g, 1, 2 or 3) different cy clic groups (e.g., carbocyclic rings or heterocyclic rings), where the radical or point of attachment is on the aryl ring.

[0068] Aralkyl groups (e.g., benzyl) refer to a group of the formula -Rc-aryl where Rcis an alkylene chain such as methylene or ethylene. In some embodiments, the aralkyl group is an optionally substituted benzy l group. Aralkoxy groups refer to a group bonded through an oxygen atom of the formula — 0 - Rc— ary l where Rcis an alky lene chain such as methylene or ethylene.16AFSDOCS:304108277.1Attorney Docket No. 046094-798001 WO Date of Deposit: December 18, 2025

[0069] As used herein, the term "heterocyclyl” refers to a "carbocyclyl" that used alone or as part of a larger moiety, contains a saturated, partially unsaturated or aromatic ring system, wherein one or more (e.g, 1, 2, 3, 4, or 5) carbon atoms have been replaced with a heteroatom or heteroatom-containing group (e.g.. O, N, N(O), S, S(O), or S(O)₂.). The term heterocyclyl includes mono-, bi-, tri-, fused, bridged, and spiro-ring systems, and combinations thereof. In some embodiments, a heterocyclyl refers to a 3- to 12-membered heterocyclyl ring system. In some embodiments, a heterocyclyl refers to a saturated ring system, such as a 3- to 12-membered saturated heterocyclyl ring system In some embodiments, a heterocyclyl refers to a heteroaryl ring system, such as a 5- to 12-membered heteroaryl ring system. The term heterocyclyl also includes C2-C8 heterocycloalkyl, which is a saturated or partially unsaturated mono-, bi-, or spiro-ring system containing 2-8 carbons and one or more (e.g, 1, 2, or 3) heteroatoms.

[0070] In some embodiments, a heterocyclyl group includes 3-12 ring atoms and includes monocycles, bicycles, tricycles and spiro ring systems, wherein the ring atoms are carbon, and one to 5 ring atoms is a heteroatom such as nitrogen, sulfur or oxygen. In some embodiments, heterocyclyl includes 3- to 7-membered monocycles having one or more heteroatoms selected from O, N, and S. In some embodiments, heterocyclyl includes 4- to 6-membered monocy cles having one or more heteroatoms selected from O, N, and S. In some embodiments, heterocyclyl includes 3-membered monocycles. In some embodiments, heterocyclyl includes 4-membered monocycles. In some embodiments, heterocyclyl includes 5- to 6-membered monocycles. In some embodiments, the heterocyclyl group includes 0 to 3 double bonds. In any of the foregoing embodiments, heterocyclyl includes I, 2, 3 or 4 heteroatoms. Any nitrogen or sulfur heteroatom may optionally be oxidized (e.g., NO, SO, SO2). and any nitrogen heteroatom may optionally be substituted (e.g, methyl, isopropyl) and / or quatemized (e.g., [NR+J’Cr, [NR+f OH') Representative examples of heterocyclyls include oxiranyl, aziridinyl, thiiranyl, azetidinyl, oxetanyl, thietanyl, 1,2-dithietanyl, 1,3-dithietanyl, pyrrolidinyl, dihydro- IH-pyrrolyl, dihydrofuranyl, tetrahydropyranyl, dihydrothienyl. tetrahydrothienyl, imidazolidinyl, piperidinyl, piperazinyl. morpholinyi, thiomorpholinyl. 1, 1-dioxo-thiomorpholinyl, dihydropyranyl, tetrahydropyranyl, hexahydrothiopyranyl, hexahydropyrimidinyl, oxazinanyl, thiazinanyl, thioxanyl, homopiperazinyl, homopiperidinyl, azepanyl, oxepanyl, thiepanyl, oxazepinyl, oxazepanyl, diazepanyl, 1,4-diazepanyl, diazepinyl, thiazepinyl, thiazepanyl, tetrahydrothiopyranyl, oxazolidinyl. thiazolidinyl, isothiazolidinyl. 1,1-dioxoisothiazolidinonyl, oxazolidinonyl,17AFSDOCS:304108277.1Attorney Docket No. 046094-798001 WO Date of Deposit: December 18, 2025imidazolidinonyl, 4,5,6,7-tetrahydro[2H]indazolyl, tetrahydrobenzoimidazolyl, 4, 5,6,7- tetrahydrobenzo|d]imidazolyl, 1,6-dihydroimidazol[4,5-d]pyrrolo[2,3-b]pyridinyl, thiazinyl, thiophenyl, oxazinyl, thiadiazinyl, oxadiazinyl, dithiazinyl, dioxazinyl, oxathiazinyl, thiatriazinyl, oxatriazinyl, dithiadiazinyl, imidazolinyl, dihydropyrimidyl, tetrahydropyrimidyl, 1-pyrrolinyl, 2-pyrrolinyl, 3-pyrrolinyl, indolinyl, thiapyranyl, 2H-pyranyl, 4H-pyranyl, dioxanyl, 1,3-dioxolanyl, pyrazolinyl, pyrazolidinyl, dithianyl, dithiolanyl, pyrimidinonyl, pyrimidindionyl, pyrimidin-2,4-dionyl, piperazinonyl, piperazindionyl, pyrazolidinylimidazolinyl, 3-azabicyclo[3.1.0]hexanyl, 3,6-diazabicyclo[3.1. l]heptanyl, 6-azabicyclo[3.1. l]heptanyl, 3-azabicyclo[3.1.1]heptanyl, 3-azabicyclo[4.1.0]heptanyl, azabicyclo[2.2.2]hexanyl, 2-azabicyclo[2.2.1]octanyl, 8-azabicyclo[3.2. IJoctanyl, 2-azabicyclo[2.2.2]octanyl, 8-azabicyclo[2.2.2]octanyl, 7-oxabi cyclo [2.2.1] heptane, azaspiro[3.5]nonanyl, azaspiro[2.5]octanyl, azaspiro[4.5]decanyl, l-azaspiro[4.5]decan-2-only, azaspiro[5.5]undecanyl, tetrahydroindolyl, octahydroindolyl, tetrahydroisoindolyl, tetrahydroindazolyl, 1,1-dioxohexahydrothiopyranyl. Examples of 5- membered heterocyclyls containing a sulfur or oxygen atom and one to three nitrogen atoms are thiazolyl (e.g, thiazol-2-yl), thiadiazolyl (e g., l,3,4-thiadiazol-5-yl and 1,2,4-thiadiazol-5-yl), oxazolyl (e.g., oxazol-2-yl), and oxadiazolyl (e g.. l,3,4-oxadiazol-5-yl and 1,2,4-oxadiazol-5-yl). Example of 5-membered heterocyciyls containing 2 to 4 nitrogen atoms include imidazolyl (e.g., imidazol-2-yl), triazolyl (e.g., 1,3,4-triazol-5-yl, l,2,3-triazol-5-yl, and l,2,4-triazol-5-yl), and tetrazolyl (e.g., lH-tetrazol-5-yl). Representative examples of benzo-fused 5 -membered heterocyclyls include benzoxazol-2-yl, benzthiazol-2-yl and benzimidazol-2-yl. Example of 6-membered heterocyclyls containing one to three nitrogen atoms and optionally a sulfur or oxygen atom are pyridyl (e.g., pyrid-2-yl, pyrid-3-yl, and pyrid-4-yl), pyrimidyl (e.g, pyrimi d-2-yl and pyrimid-4-yl), triazinyl (e g., l,3,4-triazin-2-yl and 1.3,5-triazin-4-yl). pyridazinyl (e.g., pyridazin-3-yl), and pyrazinyl. In some embodiments, a heterocyclic group includes a heterocyclic ring fused to one or more (eg., 1 or 2) different cyclic groups (e.g, carbocyclic rings or heterocyclic rings), where the radical or point of attachment is on the heterocyclic ring, and in some embodiments wherein the point of attachment is a heteroatom contained in the heterocyclic ring.

[0071] Therefore, the term heterocyclic embraces N-heterocyclyl groups which as used herein refer to a heterocyclyl group containing at least one nitrogen atom and where the point of attachment of the heterocyclyl group to the rest of the molecule is through a nitrogen atom in the heterocyclyl group. Representative examples of N-heterocyclyl groups include 1- 18AFSDOCS:304108277.1Attorney Docket No. 046094-798001 WO Date of Deposit: December 18, 2025morpholinyl, 1-piperidinyl, 1-piperazinyl, 1-pyrrolidinyl, l-pyrazolidinyl, 1 -imidazolinyl and 1-imidazolidinyl. The term heterocyclic also embraces C-heterocyclyl groups which as used herein refer to a heterocyclyl group containing at least one heteroatom and where the point of atachment of the heterocyclyl group to the rest of the molecule is through a carbon atom in the heterocyclyl group. Representative examples of C-heterocyclyl radicals include 2- or 3-morpholinyl, 2- or 3- or 4-piperidinyl, 2-piperazmyl. and 2- or 3-pyrrolidinyl. The term heterocyclic also embraces heterocyclylalkyl groups which as disclosed above refer to a group of the formula -Rc-heterocyclyl where Rcis an alkylene chain. The term heterocyclic also embraces heterocyclylalkoxy groups which as used herein refer to a radical bonded through an oxygen atom of the formula — O— Rc-heterocyclyl where Rcis an alky lene chain.

[0072] As used herein, the term "heteroaryl" refers to a monocyclic, bicyclic or tricyclic ring system having 5 to 12 ring atoms, wherein at least one ring is aromatic and contains at least one heteroatom (e.g., O, N, or S). In one embodiment, heteroaryl includes 5- to 6-membered monocyclic aromatic groups where one or more ring atoms is O, N, or S. Representative examples of heteroaryl groups include thienyl, furyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, triazolyl. thiadiazolyl. oxadiazolyl, tetrazolyl, thiatriazolyl, oxatriazolyl, pyridyl, pyrimidyl, imidazopyridyl, pyrazinyl, pyridazinyl, triazinyl, tetrazinyl, tetrazolo[l,5-b]pyridazinyl, purinyl, deazapurinyl, benzoxazolyl, benzofuryl, benzothiazolyl, benzothiadiazolyl, benzotriazolyl, benzoimidazolyl, indolyl, 1,3-thiazol-2-yl, 1,3,4-triazol-5-yl, 1,3-oxazol-2-yl, 1,3,4-oxadiazol-5-yl, 1,2,4-oxadiazol-5-yl, 1,3,4-thiadiazol-5-yl, 1H-tetrazol-5-yl, and 1,2,3-triazol-5-yl. The term "heteroaryl" also includes groups in which a heteroaryl is fused to one or more cyclic (e.g., carbocyclyl, or heterocyclyl) rings, where the radical or point of attachment is on the heteroaryl ring. Nonlimiting examples include indolyl. indolizinyl, isoindolyl, benzothienyl, benzothiophenyl, methylenedioxyphenyl, benzofuranyl, di benzofuranyl, indazolyl. benzimidazolyl, benzodi oxazolyl, benzthiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinohzinyl, carbazolyl, acridmyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl and pyrido[2,3-b]-1,4-oxazin-3(4H)-one. A heteroaryl group may be mono-, bi- or tri-cyclic. In some embodiments, a heteroaryl group includes a heteroaryl ring fused to one or more (e.g, 1 or 2) different cyclic groups (e.g, carbocyclic rings or heterocyclic rings), where the radical or point of attachment is on the heteroaryl ring, and in some embodiments wherein the point of attachment is a heteroatom contained in the heterocyclic ring.19AFSDOCS:304108277.1Attorney Docket No. 046094-798001 WO Date of Deposit: December 18, 2025

[0073] Therefore, the term heteroaryl embraces N-heteroaryl groups which as used herein refer to a heteroaryl group as defined above containing at least one nitrogen and where the point of attachment of the heteroaryl group to the rest of the molecule is through a nitrogen atom in the heteroaryl group. The term heteroaryl also embraces C-heteroaryl groups which as used herein refer to a heteroaryl group as defined above and where the point of attachment of the heteroaryl group to the rest of the molecule is through a carbon atom in the heteroaryl group.

[0074] As used herein, the term "nitrogen protecting group" is a reversibly formed derivative of an existing nitrogen-containing functional group in a compound. Representative examples of nitrogen protecting groups include formamide, acetamide, chloroacetamide, trichloroacetamide, trifluoroacetamide, phenylacetamide, 3-phenylpropanamide, picolinamide. 3-pyridylcarboxamide, an N-benzoylphenylalanyl derivative, benzamide, p- phenylbenzarmde, o-nitrophenyl acetamide, o-nitrophenoxyacetamide, acetoacetamide, (N’-dithiobenz loxyacylamino)acetamide, 3-(p-hydroxyphenyl)propanamide, 3-(o- nitrophenyl)propanamide, 2-methyl-2-(o-nitrophenoxy)propanamide, 2-methyl-2-(o-phenylazophenoxy)propanamide, 4-chlorobutanamide, 3-methyl-3-nitrobutanamide, o-nitrocinnamide, an N-acetylmethionine derivative, o-nitrobenzamide, o-(benzoyloxymethyl)benzamide, methyl carbamate, ethyl carbamate, 9-fluorenylmethyl carbamate (Fmoc), 9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluorenylmethyl carbamate, 2,7-di-t-buty l-[9-( 10, 10-dioxo- 10.10,10, 10-tetrahydrothioxanthy l)]methyl carbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc), 2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate (Teoc), 2-phenylethyl carbamate (hZ), 1- (1 -adamant}’ 1)-1 -methylethyl carbamate (Adpoc), l,l-dimethyl-2-haloethyl carbamate, 1,1-dimethyl-2,2-di bromoethyl carbamate (DB-t-BOC), 1, 1-dimethy 1-2,2, 2 -tri chloroethyl carbamate (TCBOC), 1-methyl-1-(4-biphenylyl)ethyl carbamate (Bpoc), 1-(3,5-di-t-butylphenyl)-1-methylethyl carbamate (t-Bumeoc), 2-(2’- and 4’-pyridyl)ethyl carbamate (Pyoc), 2-(N, N-dicyclohexylcarboxamido)ethyl carbamate, t-butyl carbamate (BOC or Boc), 1-adamantyl carbamate (Adoc), vinyl carbamate (Voc), allyl carbamate (Alloc), 1-isopropylallyl carbamate (Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate (Noc), 8-quinolyl carbamate. N-hydroxypiperidinyl carbamate, alkyldi thio carbamate, benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz), p-nitrobenzyl carbamate, p-bromobenzyl carbamate, p-chlorobenzyl carbamate, 2,4-dichlorobenzyl carbamate, 4- methylsulfinylbenzyl carbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl 20AFSDOCS:304108277.1Attorney Docket No. 046094-798001 WO Date of Deposit: December 18, 2025carbamate, 2-methylthioethyl carbamate, 2-methylsulfonylethyl carbamate, 2-(p-toluenesulfonyl)ethyl carbamate, [2-(l.3 -dithianyl)] methyl carbamate (Dmoc). 4-methylthiophenyl carbamate (Mtpc), 2,4-dimethylthiophenyl carbamate (Bmpc), 2-phosphonioethyl carbamate (Peoc), 2-triphenylphosphonioisopropyl carbamate (Ppoc), 1,1-dimethyl-2-cyanoethyl carbamate, m-chloro-p-acyloxybenzyl carbamate, p-(dihydroxyboryl)benzyl carbamate, 5-benzisoxazolylmethyl carbamate, 2-(trifluoromethyl)-6-chromonylmethyl carbamate (Tcroc), m-nitrophenyl carbamate, 3,5-dimethoxybenzvl carbamate, o-nitrobenzyl carbamate, 3,4- dimethoxy-6-nitrobenzyl carbamate, phenyl (o-nitrophenyl)methyl carbamate, t-amyl carbamate, S-benzyl thiocarbamate, p-cyanobenzyl carbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentyl carbamate, cyclopropylmethyl carbamate, p-decyloxybenzyl carbamate, 2,2-dimethoxyacylvinyl carbamate, o-(N, N-dimethylcarboxamido)benzyl carbamate, l,l-dimethyl-3-(N, N-dimethylcarboxamido)propyl carbamate, 1,1-dimethylpropynyl carbamate, di(2-pyridyl)methyl carbamate, 2-furanylmethyl carbamate, 2-iodoethyl carbamate, isoborynl carbamate, isobutyl carbamate, isonicotinyl carbamate. p-(p'-methoxyphenylazo)benzyl carbamate. 1 -methylcyclobutyl carbamate. 1 -methylcyclohexyl carbamate, 1 -methyl- 1-cyclopropylmethyl carbamate, l-methyl-l-(3,5-dimethoxyphenyl)ethyl carbamate, 1-methyl-l-(p-phenyiazophenyl)ethyl carbamate, 1-methyl-l-phenylethyI carbamate, 1 -methyl- 1 -(4- pyridyljethyl carbamate, phenyl carbamate, p-(phenylazo)benzyl carbamate, 2,4,6-tri-t-butylphenyl carbamate, 4-(trimethylammonium)benzyl carbamate, and 2,4,6-trimethylbenzyl carbamate.

[0075] To the extent not disclosed otherwise for any particular group(s), representative examples of substituents may include alkyl (e.g., Ci-Ce, Ci-Cs, C1-C4, C1-C3, Cj-Cz, Ci), substituted alkyl (e.g., substituted Ci-Cg, C1-C5, C1-C4, C1-C3, C1-C2, Ci), alkoxy (e.g.. Ci-Cs, C1-C5, C1-C4, Ci-C3, C1-C2, Ci), substituted alkoxy (e.g.. substituted Ci-C6, C1-C5, C1-C4, Ci- C3, C1-C2. Ci), haloalkyl (e g., CF3), alkenyl e.g, C2-C6, C2-C5, C2-C4, C2-C3, C2), substituted alkenyl (e.g., substituted Ch-Ce, C2-C5, C2-C4, C2-C3, C2), alkynyl (e.g, C -Cg, C2-C5, C2-C4, C2-C3, C2), substituted alkynyl (e.g, substituted C2-C-6, C2-C5, C2-C4, C2-C3, C2), cyclic (e.g., C3-C12, Cs-Ce). substituted cyclic (e.g., substituted C3-CJ2, CS-CA). carbocyclic (e.g., C3-C12. C5-C6), substituted carbocyclic (e.g, substituted Ca-Ci?., C3-C6), heterocyclic (e.g., 3- to 12-membered, 5-to 6-membered), substituted heterocyclic (e., substituted 3- to 12-membered, 5-to 6-membered), aryl (e.g., benzyl and phenyl), substituted aryl (e.g, substituted benzyl or substituted phenyl), heteroaryl (e.g., pyridyl or pyrimidyl), substituted 21AFSDOCS:304108277.1Attorney Docket No. 046094-798001 WO Date of Deposit: December 18, 2025heteroaryl (e.g., substituted pyridyl or substituted pyrimidyl), aralkyl (e.g, benzyl), substituted aralkyl (e.g., substituted benzyl), halo, hydroxyl, aryloxy (e.g, C6-C12. Ce), substituted aryloxy (e.g, substituted C6-C12, Cr,), alkylthio (e.g, Ci-Cg), substituted alkylthio (e.g., substituted C1-C0), atylthio (e.g., Cg-Ci?., Ce), substituted arylthio (e.g, substituted Cg-C12, Co), cyano, carbonyl, substituted carbonyl, carboxyl, substituted carboxyl, amino, substituted amino, amido, substituted amido, thio, substituted thio, sulfinyl, substituted sulfinyl, sulfonyl, substituted sulfonyl, sulfinamide. substituted sulfinamide, sulfonamide, substituted sulfonamide, urea, substituted urea, carbamate, substituted carbamate, amino acid, and peptide groups. Terminal substituents, unless otherwise stated, may include C i-Ce alkyl, Ci-Cs alkoxy, halo, hydroxyl, cyano or amino.

[0076] In one aspect, compounds of the disclosure are represented by formula I: / \R' (I), or a pharmaceutically acceptable salt or stereoisomer thereof, as a substantially pure regioisomer,wherein:0 is 0, 1, or 2;p is 0, 1. or 2;q is 0, 1, or 2;Ri is (Ci-Cs) alkyd, (C3-C10) carbocyclyl, or 4- to 10-membered heterocyclyl comprising 1 to 3 heteroatoms selected from O, N, and S, wherein said alkyl, carbocyclyl or heterocyclyl is further optionally substituted;each R2is independently a polymerizable moiety:Li is absent or a linker;L2 is absent or a linker; andX is a leaving group.

[0077] Compounds of the disclosure represented by formula I:Pl 44 + L-S 'o-'R2P L'f q N X" ”0_0(I), or a pharmaceutically acceptable salt or stereoisomer thereof, as a substantially pure regioisomer, may be prepared by a method, comprising:AFSDOCS:304108277.1Attorney Docket No. 046094-798001 WO Date of Deposit: December 18, 2025Hi) reacting a compound of formula II,Rxi(II), and a compound ofR-N L^N'RIH Lformula (III), Cl'1, to form a compound of formula IV,R4H3'N' Nx^ X''R3H / \~°R1 (IV),ii) subjecting the reaction product of i) to a lithium halogen exchange and subsequent protonation,hi) removing the nitrogen protecting groups, andiv) acylating the terminal nitrogen atoms,wherein:o is 0, 1, or 2;p is 0, 1, or 2;q is 0, 1, or 2;Ri is (Ci-Cs) alkyl, (C3-C10) carbocyclyl, or 4- to 10-membered heterocyclyl comprising 1 to 3 heteroatoms selected from O, N, and S, wherein said alkyd, carbocyclyl or heterocyclyl is further optionally substituted;each R?is independently a polymerizable moiety;each R? is independently a nitrogen protecting group;R-i is chloro, bromo, or iodo;Li is absent or a linker;L?. is absent or a linker; andX is a leaving group.

[0078] The compounds of formula (I) are regioselective, i.e., they are substantially' pure regioisomers as defined herein. Unlike the compounds disclosed in Applicant’s co-pending International Application No. PCT / US2024 / 035794, they are free of the regioisomerR 2^ TlX' R2represented by formula V:(V), As explained and demonstrated in working Example 2 herein, the compounds of the co-pending application, which bear the identical structure, have now been found to be a physically inseparable mixture of two positional isomers (also referred to herein as regioisomers). The present compounds of 23AFSDOCS:304108277.1Attorney Docket No. 046094-798001 WO Date of Deposit: December 18, 2025formula (I) are substantially pure regioisomers, as determined byNMR (as defined herein).

[0079] In some embodiments, Li is absent.

[0080] In some embodiments, L2is absent

[0081] In some embodiments, Li is a linker.

[0082] In some embodiments, L2is a linker.

[0083] The linker provides a covalent attachment between the two atoms to which the linker is bound, and which is nonreactive with the other groups in the compound.

[0084] In some embodiments, the linker is an alkydene chain, which may be interrupted by, and / or terminate (at either or both termini) in at least one of -O -, -S -, -N(R’) -, -C==C-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-. -C(NOR’)-. -C(O)N(R’)-, -C(O)N(R’)C(O)~, - R'C(O)N(R')R’~. -C(O)N(R’)C(O)N(R’)-, ~N(R’)C(O)~, ~N(R’)C(O)N(R’)~, - N(R’)C(O)O -, -OC(O)N(R’) -, -C(NR')--, -N(R’)C(NR’)~, -C(NR’)N(R’)-, -• N(R’)C(NR’)N(R’)~, -OB(Me)O~, -S(O)2-, -OS(O)-, -S(O)O-, -S(O)-, -OS(O)2- - S(O)2O- -N(R‘)S(O)2-. -S(O)2N(R')-, -N(R')S(O)-, -S(O)N(R')-, -N(R’)S(O)2N(R')-, -N(R’)S(O)N(R>. -OP(O)O(R’)O~, ~-N(R’)P(O)N(R’R’)N(R’)~, C3-C12 carbocyclyl, 3- to 12-membered heterocyclyl, 5- to 12-membered heteroaryl or any combination thereof, wherein each R’ is independently H or optionally substituted C1-C24 alkyl, wherein the interrupting and the one or both terminating groups may be the same or different.

[0085] In some embodiments, the alkylene chain is a C0-C50 alkylene chain. In some embodiments, the alkylene chain is a C0-C10 alkylene chain. In some embodiments, the alkylene chain is a C11-C50 alkylene chain. In some embodiments, the alkylene chain is a Ci-C2.4 alky lene chain. In some embodiments, the alkylene chain is a Ci-Cis alkylene chain. In some embodiments, the alkylene chain is a C1-C12 alkylene chain. In some embodiments, the alkylene chain is a C1-C10 alkylene chain. In some embodiments, the alkylene chain is a Ci-Cs alkylene chain. In some embodiments, the alkylene chain is a Ci-C« alkylene chain In some embodiments, the alkyd ene chain is a C1-C4 alkyd ene chain. In some embodiments, the alky lene chain is a C1-C2 alky lene chain. In some embodiments, the alkylene chain is interrupted by, and / or terminates (at either or both termini) in at least one of-N(R’)-, -C(O}~, -C(O)O-. -OC(O)- -C(O)N(R’)- -N(R’)C(O)- -N(R’)C(O)O- -OC(O)N(R’)-, -S(O)2-, -N(R')S(O)2“, ~S(O)2N(R')~, 4- to 6-membered heterocyclyl, or a combination thereof. In some embodiments, the alkylene chain is interrupted by, and / or terminates (at either or both termini) with - (R' j-. In some embodiments, the alkylene chain is interrupted by, and / or 24AFSDOCS:304108277.1Attorney Docket No. 046094-798001 WO Date of Deposit: December 18, 2025terminates (at either or both termini) with -C(O)~. In some embodiments, the alkylene chain is interrupted by, and / or terminates (at either or both termini) with C(O)O In some embodiments, the alkylene chain is interrupted by, and / or terminates (at either or both termini) with -C(O)N(R')- In some embodiments, the alkylene chain is interrupted by, and / or terminates (at either or both termini) with -N(R’ S(O)2“ In some embodiments, the alkylene chain is interrupted by, and / or terminates (at either or both termini) with a 4- to 6-membered heterocyclyl.

[0086] Tn some embodiments, the linker is a polyethylene glycol chain, which may be interrupted by, and / or terminate (at either or both termini) in at least one of O, -S - N(R’)-, -C=C-, -C(O)-> C(O)O-, OC(O) OC(O)O C(NOR )-, -C(O)N(R‘)-, - C(O)N(R')C(O)-, -R’C(O)N(R’)R -C(O)N(R')C(O)N(R’)- -N(R')C(O)-. - N(R’)C(O)N(R’)-, -N(R’)C(O)O- -OC(O)N(R’)-. -C(NR’)-. -N(R')C(NR’)-. - C(NR’)N(R’). -N(R’)C(NR’)N(R’)-, 0B(Me)0, -S(O>2--, OS(O) S(O)O -S(O)-, - OS(O)2- -S(O)2O-, -N(R’)S(O)2- -S(O)2N(R’)-, -N(R’)S(O)~, -S(O)N(R’)-, - N(R')S(O)2N(R')- -N(R’)S(O)N(R')- -OP(O)O(R')O- -N(R')P(O)N(R'R')N(R )- C3-C12 carbocyclyl. 3- to 12-membered heterocyclyl, 5- to 12-membered heteroaryl or any combination thereof, wherein each R’ is independently H or optionally substituted C1-C24 alkyl, wherein the interrupting and the one or both terminating groups may be the same or different.

[0087] In some embodiments, the polyethylene glycol chain has 0 to 50 ~-(CH2CH2-O)-units. In some embodiments, the polyethylene glycol chain has 0 to 10 (CH2CH2-O)- units. In some embodiments, the polyethylene glycol chain has 11 to 50 -(CH2CH2-O)- units. In some embodiments, the polyethylene glycol chain has 1 to 20 -(CH2CH2-O)- units. In some embodiments, the polyethylene glycol chain has 1 to 15 --(CH2CH2-O)- units. In some embodiments, the polyethylene glycol chain has I to 10 -~(CH2CII2-O)- units. In some embodiments, the polyethylene glycol chain has 1 to 6 -(CH2CH2-O)- units. In some embodiments, the polyethylene glycol chain has 1 to 2 (CH2CH2-O)- emits. In some embodiments, the polyethylene glycol is interrupted by, and / or terminates (at either or both termini) in at least one of -N(R’)-, ~C(O)~, -C(O)O- -OC(O)-~. -C(O)N(R’)~. - N(R’)C(O)- -N(R’)C(O)O- -OC(O)N(R’)-, -S(O)2- -N(R’)S(O)2- -S(O)2N(R’)- 4- to 6-membered heterocyclyl, or a combination thereof. In some embodiments, the polyethylene glycol chain is interrupted by, and / or terminates (at either or both termini) with N(R’) - In some embodiments, the polyethylene glycol chain is interrupted by. and / or terminates (at 25AFSDOCS:304108277.1Attorney Docket No. 046094-798001 WO Date of Deposit: December 18, 2025either or both termini) with -C(O)~ In some embodiments, the polyethylene glycol chain is interrupted by, and / or terminates (at either or both termini) with C(O)O In some embodiments, the polyethylene glycol chain is interrupted by, and / or terminates (at either or both termini) with -C(O)N(R’)~. In some embodiments, the polyethylene glycol chain is interrupted by, and / or terminates (at either or both termini) with ~N(R’)S(O)2~. In some embodiments, the polyethylene glycol chain is interrupted by, and / or terminates (at either or both termini) with 4- to 6-membered heterocyclyl.

[0088] In some embodiments, Ri is (Ci-Cs) alkyl. In some embodiments, Ri is methyl.

[0089] In some embodiments, R4 is chloro. In some embodiments, R4 is bromo.

[0090] In some embodiments, X is a leaving group, which as known in the art refers to an atom or group of atoms which breaks away from the rest of the molecule, taking with it the electron pair winch used to be the bond between the leaving group and the rest of the molecule. Representative examples of leaving groups include esters, carbonates, carbamates, sulfoxides, sulfonates, sulfates, sulfones, thioesters, and thionoesters. In some embodiments, the leaving group is OR9, SR$>, -OC(O)R9. -OC(O)OR9, -OC(O)NR9R9. -OC(S)R9, --OC(S)OR9, -OC(S)NR9R9, -OS(O)2R9, -OS(O)2OR9. -OP(O)OR9OR9. -OP(O)R9R9, - SC(O)R9, -SC(O)SR9, or -SC(S)SR9, wherein each R9is independently hydrogen, (Ci-Cs) alkyl, (C3-C10) carbocyclyl, or 4- to 7-membered heterocyclyl, wherein said alkyl, carbocyclyl, or heterocyclyl is optionally substituted.

[0091] As known in the art, polymenzable moieties refer to a molecule that reacts with another molecule, which may the same or different, to form a polymer and a co-polymer, respectively. Representative examples of polymerizable moieties / reagents that may be suitable for use in preparing the disclosed polymers / hydrogels include:Cl vinyl clqethylene oxide vinyl pyrrolidone acrylamide N-acryloylmorpholine N, N-dimethyI acrylamide AFSDOCS:304108277.1Attorney Docket No. 046094-798001 WO Date of Deposit: December 18, 2025.\'(2-hydro.xyethyi)-2-methyle:iebutanamide 2-hydroxyethyl methacrylate 3-hydroxypropyl methacrylate OHO Oglycerol methacrylate acrylic acid N, N’-Melhyienebistictyiamide carboxymethyl cellulose, hydroxypropyl methyl cellulose, arid hydroxy ethyl cellulose.

[0092] In some embodiments, each R? is acrylamide.

[0093] In some embodiments, the optional substituent for a compound of formula 1 is independently alkyd, alkenyl, alkynyl, halo, haloalkyl, cycloalkyl, heterocycloalkyl, hydroxy, alkoxy, cycloalkoxy, heterocycloalkoxy, haloalkoxy, aryloxy, heteroaryl oxy, aralkyloxy. alkyenyloxy, alkynyloxy, ammo, alkylamino, cycloalkylamino, heterocycloalkydamino, aiylamino, heteroaiyiamino, aralkylammo, N-alkyl-N-arylamino, N-alkyl-N-heteroaryl amino, N-alkyl-N-aralkylamino, hydroxyalkyl, aminoalkyl, alkylthio, haloalkylthio, alkylsulfonyl, haloalkylsulfonyl, cycloalk lsulfonyl, heterocycloalkyl sulfonyl, arylsulfonyl, heteroarylsulfonyl, aminosulfonyl, alkylaminosulfonyl, cycloalkylaminosulfonyl, heterocycloalk laminosulfonyl, ai laminosulfonyl, heteroarylaminosulfonyl, N-alkyl-N-aryl aminosulfonyl, N-alkyl-N-heteroar laminosulfonyl, formyl, alkylcarbonyl, haloalkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, carboxy, alkoxycarbonyl, alkydcarbonyloxy, ammo, alkylsulfonylammo, haloalkylsulfonylamino, cycloalkylsulfonylamino, heterocycloalkydsulfonylamino, arylsulfonylamino, heteroarydsulfonylamino, aralkylsulfonylamino, alkylcarbonylamino,27AFSDOCS:304108277.1Attorney Docket No. 046094-798001 WO Date of Deposit: December 18, 2025haloalkylcarbonylamino, cycloalkylcarbonylamino, heterocycloalkylcarbonylamino, arylcarbonylamino, heteroarylcarbonylamino, aralkylsulfonylamino, aminocarbonyl, alkylaminocarbonyl, cycloalkylaminocarbonyl, heterocycloalkylaminocarbonyl, aiylaminocarbonyl, heteroarylaminocarbonyl, N-alkyl-N-arylaminocarbonyl, N-alkyl-N- heteroarylaminocarbonyl, cyano, nitro, and azido.

[0094] Hie disclosed compounds also embrace combinations of the specific Ri, R2, o, p, q, Li, and L2 groups disclosed above

[0095] Tn some embodiments, the compound of formula I is of formula la or lb:or(lb), or a stereoisomer thereof, wherein n is an integer from 1-6.

[0096] In some embodiments, the compound of formula I is:U -U Me O

[0097] Another aspect of the present disclosure is directed to a hydrogel which is the reaction product of:Z \i) a compound of formula I:Ri (I), as a substantially pure regioisomer,ii) a polymerizable reagent, andrii) an initiator,wherein:o is 0, 1, or 2;28AFSDOCS:304108277.1Attorney Docket No. 046094-798001 WO Date of Deposit: December 18, 2025p is 0, 1, or 2;q is 0, 1, or 2;Ri is (Ci-Cs) alkyl, (C3-C10) carbocyclyl, or 4- to 10-inembered heterocyclyl comprising 1 to 3 heteroatoms selected from O, N, and S, wherein said alkyl, carbocyclyl or heterocyclyl is further optionally substituted;each R2is independently a polymerizable moiety,Li is absent or a linker;L?. is absent or a linker; andX is a leaving group. Hydrogels crosslinking agents of formula (I) are more susceptible to degradation with a di boron reagent, both in terms of extent and rate of degradation, compared to known hydrogels.

[0098] In some embodiments, Li is absent.

[0099] In some embodiments, L2is absent.

[0100] In some embodiments, Li is a linker.

[0101] In some embodiments, L? is a linker.

[0102] In some embodiments, the linker is an alkylene chain, which may be interrupted by, and / or terminate (at either or both termini) in at least one of -O -, -S-, -N(R’)-, -C=C~ C(O) C(O)O OC(O) OC(O)O, -C(NOR’)-, -C(O)N(R’)-, -C(O)N(R’)C(O)-, R’C(O)N(R’)R’- -C(O)N(R')C(O)N(R')-, -N(R’)C(O)-, -N(R')C(O)N(R’)-, - N(R’)C(O)O- -OC(O)N(R')-_ -C(NR )-, -N(R')C(NR')-, -C(NR’)N(R’)-, - N(R’)C(NR’)N(R’)-, OB(Me)O -, -S(O)2-. OS(O)-, -S(O)O-, -S(O)--, -OS(O)2-, - S(O)2O-, -N(R’)S(O)2-. -S(O)2N(R’)-, -N(R’)S(O)-, -S(O)N(R’)-, -N(R’)S(O)2N(R')-, - N(R’)S(O)N(R’)-, -OP(O)O(R )O~, -N(R’)P(O)N(R’R )N(R )- C3-C12 carbocyclyl, 3- to 12-membered heterocyclyl, 5- to 12-membered heteroaiyl or any combination thereof, wherein each R’ is independently H or optionally substituted C1-C24 alkyl, wherein the interrupting and the one or both terminating groups may be the same or different.

[0103] In some embodiments, the alky lene chain is a C1-C24 alkylene chain. In some embodiments, the alkylene chain is a Ci-Cis alkylene chain. In some embodiments, the alkylene chain is a C1-C12 alkylene chain In some embodiments, the alkylene chain is a Ci-C10 alkylene chain In some embodiments, the alkylene chain is a Ci-Cs alkylene chain In some embodiments, the alkylene chain is a Ci-Cs alkylene chain, hi some embodiments, the alkylene chain is a C1-C4 alkylene chain. In some embodiments, the alky lene chain is a C1-C2 alkylene chain. In some embodiments, the alkylene chain is interrupted by, and / or terminates 29AFSDOCS:304108277.1Attorney Docket No. 046094-798001 WO Date of Deposit: December 18, 2025(at either or both termini) in at least one of -N(R’)--, ~C(O)~, ~-C(O)O-, -OC(O)--, - C(O)N(R')~, -N(R')C(O}-. -N(R’)C(O)O OC(O)N(R ) -, -S(O)2-, -N(R")S(O)2--, -S(O)2N(R’)--, 4- to 6-membered heterocyclyi, or a combination thereof. In some embodiments, the alkylene chain is interrupted by, and / or terminates (at either or both termini) with -N(R’)-. In some embodiments, the alkylene chain is interrupted by, and / or terminates (at either or both termini) with -C(O) In some embodiments, the alkylene chain is interrupted by, and / or terminates (at either or both termini) with ~C(O)O~. In some embodiments, the alkylene chain is interrupted by, and / or terminates (at either or both termini) with ~C(O)N(R’)~. In some embodiments, the alkylene chain is interrupted by, and / or terminates (at either or both termini) with -N(R’)S(O)2---. In some embodiments, the alkylene chain is interrupted by. and / or terminates (at either or both termini) with a 4- to 6-membered heterocyclyi.

[0104] In some embodiments, the linker is a polyethylene glycol chain, which may be interrupted by, and / or terminate (at either or both termini) in at least one of -O-, -S-, - N(R')-, -C=C-, -C(O)-, -C(O)O- -OC(O)-, -OC(O)O-, -C(NOR')-, -C(O)N(R')-, -C(O)N(R’)C(O)- ~R’C(O)N(R’)R -C(O)N(R')C(O)N(R )- ~N(R’)C(O)~. - N(R’)C(O)N(R’)-, -N(R’)C(O)O~ -OC(O)N(R’)--, -C(NR’)-, -N(R’)C(NR’)--, - C(NR’)N(R’)~, -N(R’)C(NR’)N(R’)-, -OB(Me)O~, -S(O)2~, OS(O>, S(O)O -, -S(O)-, - OS(O)2-, -S(O)2O-, -N(R')S(O)2-, -S(O)2N(R")-, -N(R’)S(O)-. -S(O)N(R’)-, -N(R’)S(O)2N(R’)-, -N(R')S(O)N(R’)~, -OP(O)O(R’)O~. -N(R’)P(O)N(R'R’)N(R’)-, C3-Ci2 carbocyclyl, 3- to 12-membered heterocyclyi. 5- to 12-membered heteroaryl or any combination thereof, wherein each R’ is independently II or optionally substituted C1-C24 alky l, wherein the interrupting and the one or both terminating groups may be the same or different.[00105| In some embodiments, the polyethylene glycol chain has 1 to 20 --(CH2CH2-O)-units. In some embodiments, the polyethylene glycol chain has 1 to 15 -(CH2CH2-O)- units. In some embodiments, the polyethylene glycol chain has 1 to 10 (CH2CH2-O)- units. In some embodiments, the polyethylene glycol chain has 1 to 6 (CH2CH2-O)- units. In some embodiments, the polyethylene glycol chain has 1 to 2 -(CH2CH2-O)- units. In some embodiments, the polyethylene glycol is interrupted by, and / or terminates (at either or both termini) in at least one of ~N(R’)-, -C(O)~, ~C(O)O~, -OC(O)~, -C(O)N(R')- - N(R’)C(O) -, -N(R’)C(O)O -, -OC(O)N(R’) -, -S(O)2--, -N(R’)S(O)2-, -S(O)2N(R’)--, 4- to 6-membered heterocyclyi, or a combination thereof. In some embodiments, the polyethylene 30AFSDOCS:304108277.1Attorney Docket No. 046094-798001 WO Date of Deposit: December 18, 2025glycol chain is interrupted by, and / or terminates (at either or both termini) with ~N(R’)“. In some embodiments, the polyethylene glycol chain is interrupted by. and / or terminates (at either or both termini) with -C(O)-. In some embodiments, the polyethylene glycol chain is interrupted by, and / or terminates (at either or both termini) with -C(O)O- In some embodiments, the polyethylene glycol chain is interrupted by, and / or temiinates (at either or both termini) with -C(O)N(R’) In some embodiments, the polyethylene glycol chain is interrupted by. and / or terminates (at either or both termini) with -N(R')S(O)2-. hi some embodiments, the polyethylene glycol chain is interrupted by, and / or terminates (at either or both termini) with 4- to 6-membered heterocyclyl.[001061 In some embodiments, Ri is (Ci-Cs) alkyl. In some embodiments, Ri is methy l.

[0107] In some embodiments, each R2 is acrylamide.

[0108] In some embodiments. X is a leaving group, which as known in the art refers to an atom or group of atoms which breaks away from the rest of the molecule, taking with it the electron pair which used to be the bond between the leaving group and the rest of the molecule. Representative examples of leaving groups include esters, carbonates, carbamates, sulfoxides, sulfonates, sulfates, sulfones, thioesters, and thionoesters. In some embodiments, the leaving group is OR9, SR9, -OC(O)R9, -OC(O)OR9, -OC(O)NR9R9, -OC(S)R9, --OC(S)OR9, -OC(S)NR9R9, -OS(O)2R9, -OS(O)2OR9, -OP(O)OR9OR9, -OP(O)R9R9, - SC(O)R9, -SC(O)SR9, or -SC(S)SR9, wherein each R9is independently hydrogen, (Ci-Co) alkyl. (C3-C10) carbocyclyl, or 4- to 7-membered heterocyclyl, wherein said alkyl, carbocyclyl, or heterocyclyl is optionally substituted.

[0109] In some embodiments, the polymerizable reagent is the same as R2. In such cases, the hydrogel is a homopolymer.

[0110] In some embodiments, the polymerizable reagent is different than R2. and the hydrogel is a co-polymer, e.g., a random or block co-polymer. In some embodiments, the polymerizable reagent comprises two or more different polymerizable moieties, e.g, vinyl chloride and vinyl alcohol, styrene and acrylamide, or vinyl chloride, styrene, and acrylamide.

[0111] In some embodiments, the polymerizable reagent is AfX’-methylenebisaciy lamide (MBAA) or an enamine / V-oxide bisacrylamide (ENBAA).

[0112] As known in the art, initiators are chemical species that react w ith a polymerizable monomer to form an intermediate compound that is capable of linking successively with a large number of other polymerizable monomers. Representative examples of initiators 31AFSDOCS:304108277.1Attorney Docket No. 046094-798001 WO Date of Deposit: December 18, 2025include peroxides (e.g., benzoyl peroxide, di-tert-butyl peroxide, methyl ethyl ketone peroxide, acetone peroxide, and peroxydisulfate) and aliphatic azo compounds (e.g., azobisisobutyronitrile (AIBN), azobis(cyclohexanecarbonitrile (ACHN), and diethyldiazene).[001131 The disclosed hydrogels also embrace combinations of the specific polymerizable reagents and Ri, R2, 0, p, q, Li, and L2 groups disclosed above.

[0114] Other aspects of the present disclosure are directed to a compound of formula IV:(IV),wherein:o is 0, 1, or 2;p is 0, 1, or 2:q is 0, 1, or 2:Ri is (Ci-C's) alkyl, (C3-C10) carbocyclyl. or 4- to 10-membered heterocyclyl comprising 1 to 3 heteroatoms selected from O, N, and S, wherein said alkyl, carbocyclyl or heterocyclyl is further optionally substituted;each R? is independently a nitrogen protecting group:R4 is chloro, bromo, or iodo;Lj is absent or a linker;L is absent or a linker: andX is a leaving group.

[0115] In some embodiments, the compound of formula IV is of formula Iva or Ivb:N N(Iva) or (Ivb), wherein n is an integer from 1-6.[00116| In some embodiments, the compound of formula IV is:N NV NH+ H N N Nused as reactants, e.g, starting materials, in the synthesis of compounds of formula (I).AFSDOCS:304108277.1Attorney Docket No. 046094-798001 WO Date of Deposit: December 18, 2025

[0117] Compounds of the present disclosure may be in the form of a free acid or free base, or a pharmaceutically acceptable salt. A pharmaceutically acceptable salt of the compounds of this disclosure can be formed, for example, by reaction of an appropriate free base of a compound of the disclosure and an appropriate pharmaceutically acceptable acid in a suitable solvent under standard conditions well known in the art. See, for example, Gould, P. L., “Salt selection for basic drugs,’" International Journal of Pharmaceutics, 5:201-217 (1986); Bastin, R. J., et al., “Salt Selection and Optimization Procedures for Pharmaceutical New Chemical Entities,” Organic Process Research and Development, 4-A21-4 5 (2000); and Berge, S. M., et al., “Pharmaceutical Salts,” Journal of Pharmaceutical Sciences, 66:1-19 (1977).

[0118] Compounds of the present disclosure may have at least one chiral center and thus may be in the form of a stereoisomer, which as used herein, embraces all isomers of individual compounds that differ only in the orientation of their atoms in space. The term stereoisomer includes mirror image isomers (enantiomers which include the (R-) or (S-) configurations of the compounds), mixtures of mirror image isomers (physical mixtures of the enantiomers, and racemates or racemic mixtures) of compounds, geometric (cis / trans or E / Z, R / S) isomers of compounds and isomers of compounds with more than one chiral center that are not mirror images of one another (diastereoisomers). The chiral centers of the compounds may undergo epimerization in vivo,' thus, for these compounds, administration of the compound in its (R-) form is considered equivalent to administration of the compound in its (S-) form. Accordingly, the compounds of the present disclosure may be made and used in the form of individual isomers and substantially free of other isomers, or in the form of a mixture of various isomers, e.g., racemic mixtures of stereoisomers.

[0119] In some embodiments, the compound of formula I or the hydrogel is an isotopic derivative m that it has at least one desired isotopic substitution of an atom, at an amount above the natural abundance of the isotope, i.e., enriched. In one embodiment, the compound includes deuterium or multiple deuterium atoms. As used herein, the term “compound” embraces isotopic derivatives.Methods of Synthesis

[0120] In another aspect, the present disclosure is directed to a method for making a compound of formula I as a substantially pure regioisomer or IV and a hydrogel which is the reaction product of a compound of formula I as a substantially pure regioisomer, a polymerizable reagent, and an initiator. Broadly, the compounds and their pharmaceutically 33AFSDOCS:304108277.1Attorney Docket No. 046094-798001 WO Date of Deposit: December 18, 2025acceptable salts and stereoisomers may be prepared by any process known to be applicable to the preparation of chemically related compounds. The compounds of the present disclosure will be better understood in connection with the synthetic schemes that are described in various working examples and which illustrate non-limiting methods by which the compounds may be prepared, e.g. compounds of formula I as a substantially pure regioisomer or IV and hydrogels which are the reaction product of a compound of formula I as a substantially pure regioisomer, a polymerizable reagent, and an initiator.

[0121] In another aspect, the present disclosure is directed to methods for preparing hydrogels which are the reaction product of:i) a compound of formula I:(I), as a substantially pure regioisomer,li) a polymerizable reagent, andiii) an initiator.

[0122] In some embodiments, the reacting is carried out in the presence of a solvent.[001231 In some embodiments, the solvent is non-aqueous solvent. In some embodiments, the non-aqueous solvent is DCM, CHCh, CCh, DCE. toluene, MeCN, or THF.

[0124] In some embodiments, the solvent is an aqueous solvent In some embodiments, the aqueous solvent is a sodium alginate solution. In some embodiments, the aqueous solvent is a buffered solvent. In some embodiments, tire buffered solvent is phosphate-buffered saline. In some embodiments, the pH of the phosphate-buffered saline is about 7.4.

[0125] In some embodiments, the solvent is a protic solvent In some embodiments, the protic solvent is water, MeOH, EtOH, iPrOH, nBuOH, TFE, or HFIP.

[0126] In some embodiments, the reacting is carried in the solvent with a concentration of is 1-25 wt%. In some embodiments, the concentration is I, 5, 10, or 15 wt%,

[0127] In some embodiments, the reacting of formula I with the polymer is in a ratio of 1-20 wt% In some embodiments, the ratio of formula I to polymer is 3, 6, or 12 wt%

[0128] In some embodiments, the reaction is carried out over a week. In some embodiments, the reaction is carried out over five days. In some embodiments, the reaction is carried out over three days. In some embodiments, the reaction is carried out over a period of 24 hours. In some embodiments, the reaction is carried out over a period of 18 hours. In some embodiments, the reaction is carried out over a period of 12 hours. In some embodiments, the reaction is carried out over a period of 6 hours. In some embodiments, the reaction is carried 34AFSDOCS:304108277.1Attorney Docket No. 046094-798001 WO Date of Deposit: December 18, 2025out over a period of 3 hours. In some embodiments, the reaction is carried out over a period of 2 hours. In some embodiments, the reaction is carried out over a period of 1 hour. In some embodiments, the reaction is carried out over a period of 45 minutes In some embodiments, the reaction is carried out over a period of 30 minutes. In some embodiments, the reaction is earned out over a period of 15 minutes. In some embodiments, the reaction is carried out over a period of 5 minutes. In some embodiments, the reaction is carried out over a period of 1 minute.

[0129] In some embodiments, the methods of preparing the hydrogels comprises the following operations or steps: 1) dissolving a polymerizable reagent (e.g., acrylamide) in water at a desired weight per volume and stirring for a period of time; 2) the solution obtained in step 1) is added to a mixture containing a compound of formula I, a catalyst (e.g., tetramethylethylenediamine (TMEDA)), an initiator (e.g.. ammonium persulfate (APS)), and calcium sulfate (CaSCri) and stirred for a period of time; 3) the mixture obtained in step 3 is stored at room temperature for a period of time, forming the hydrogel. Room temperature is defined as 22.5 ± 2.5°C.

[0130] Methods of synthesizing hydrogels are known in the art. Varying the properties of the hydrogels, e.g, molecular weight and viscosit, to suit the clinical need at hand, is also within the level of skill in the art. See, Freedman el al.. Adv. Mater., 2021, 33(7^:e2008553 and Ahmed, J, Adv. Res., 2015, 6(2)'.105-121. For example, a hydrogel of desired viscosity' is formed by adding appropriate amounts of water. To obtain a gel that changes viscosity m vivo, calcium citrate tetrahydrate can be added to the water component or added after gel formation to obtain gels with higher viscosity. See, PCT Publication No. WO 2013 / 112381. Other parameters that can be varied to create hydrogels with the desired specific properties include: %w / v crosslinker, %w / v monomer, solvent, mol% initiator, reaction time, temperature, and pH.Methods of Use

[0131] As known in the art, hydrogels may be used as a suitable drug delivery' system for drugs due to their tunable properties, controllable degradation, and ability to protect labile drugs. See, Vigata et al.. Pharmaceutics, 2020. 12(12 1188. Accordingly, in some embodiments, effective amounts of a therapeutically active agent may be added at some point in the preparation of the hydrogels. As known in the art, modifiable non-hydrogel polymers may be used as films and plastics. In some aspects, the present disclosure is directed to35AFSDOCS:304108277.1Attorney Docket No. 046094-798001 WO Date of Deposit: December 18, 2025methods of degrading hydrogels. In some embodiments, the present method comprises contacting the hydrogel with a diboron reagent.

[0132] In some embodiments, the diboron reagent is a symmetrica] diboron reagent. In some embodiments, the diboron reagent is an unsymmetrical diboron reagent In somecf' / embodiments, the diboron reagent is EhlOH / , Ehpiit,^6,Other representative examples of diboron reagents include bi s(catecholato)diboron, bis(h exylene gly colato)diboron. bis [ (-)pinanediolato] di boron, bis(diisopropyl-l-tartrate g]ycolato)diboron, bis(N, N, N’, N’-tetramethyl-d-tartaramide glycolato)diboron, and 2,2'-bi-l,3,2-dioxaborinane. Yet other diboron reagents which may be suitable for use in the present disclosure are disclosed in Ali et al.. Studies in Inorganic Chemistry, “Chapter 1 - Chemistry of the diboron compounds7’ 22:1-57 (2005); Neeve et al., Chem, Rev. i ' 1 ’6( 16 / 9091-9161 (2016): Ding et al.. Molecules 2-7(7 / 1325 (2019).

[0133] In some embodiments, the diboron reagent is used at a concentration of about 1 pM to about 1 M. In some embodiments, the diboron reagent is used at a concentration of about 1 pM to about 100 mM. In some embodiments, the diboron reagent is used at a concentration of about 1 pM to about 10 mM. In some embodiments, the diboron reagent is used at a concentration of about 1 pM to about 1 mM. In some embodiments, the diboron reagent is used at a concentration of about 1 pM to about 100 pM. In some embodiments, the diboron reagent is used at a concentration of about 1 pM to about 10 pM. In some embodiments, the diboron reagent is used at a concentration of about 1 pM to about 1 pM, In some embodiments, the diboron reagent is used at a concentration of about I pM to about 100 nM. In some embodiments, the diboron reagent is used at a concentration of about 1 pM to about 10 nM. In some embodiments, the diboron reagent is used at a concentration of about 1 pM to about I nM. In some embodiments, the diboron reagent is used at a concentration of about 1 pM to about 100 pM. In some embodiments, the diboron reagent is formulated in DMSO.

[0134] In some embodiments, the biboron solution contains a calcium chelator. In some embodiments, the calcium chelator is ethylenediaminetetraacetic acid (EDTA) or citric acid.AFSDOCS:304108277.1Attorney Docket No. 046094-798001 WO Date of Deposit: December 18, 2025

[0135] In some embodiments, the diboron reagent is formulated, e.g., as a solution, in water or saline. In some embodiments, the solution further comprises a solubilizing additive, e g., DMSO. In some embodiments, the diboron agent is formulated as a solid.

[0136] These and other aspects of the present disclosure will be further appreciated upon consideration of the following Examples, which are intended to illustrate certain particular embodiments of the disclosure but are not intended to limit its scope, as defined by the claims.EXAMPLES

[0137] Example 1: Summary'

[0138] The enamine A’-oxide motif described herein are used to engineer biocompatible materials whose polymeric network are decrosslinked by a diboron chemical trigger. The non-degradable tough hydrogel was converted to a degradable tough hydrogel by replacing the non-degradable acrylate functionalized crosslinker (A, AT’-methylenebis(acrylamide), MBAA) with the mducibly degradable analog enamine A-oxide bis(acrylamide) (ENBAA).

[0139] Example 2: Synthesis of ENBAA Crosslinkers

[0140] Enamine Ar-oxides can be synthesized by intermolecular retro-Cope elimination reaction between hydroxylammes and alkynes (D. Kang, J. Kim, J. Am. Chem. Soc., 2021, 143:5616); however, we have now discovered that the regioselectivities achieved through this reaction are non-ideal for making degradable hydrogels that require complete or thorough degradation. In instances where complete polymer decrosslinking is desired for thorough material degradation, exclusive formation of enamine A-oxides with the anti-Markovnikov regiochemistry is paramount. Unlike its counterpart, the regioisomeric Markovmkov addition product does not undergo rapid fragmentation upon treatment with aqueous diboron reagents under equivalent conditions. While the regioselectivity of the hydroamination reaction is strongly governed by propargylic substituent inductive effects on the alkyne component and judicious choice of such substituents is known to improve regioselectrvity in some applications (D Kang el al., Angew. Chem Int. Ed., 2021, 60:16947), here, the composition of the alkyne is constrained by the need for a propargylic carbamate at the propargylic position where it senes as both a convenient linkage and a superb leaving group.37AFSDOCS:304108277.1Attorney Docket No. 046094-798001 WO Date of Deposit: December 18, 2025Hydroamination of propargylic carbamates do not produce enamine A-oxides with regioisomeric purity.[00141 Schemes 1A and IB (below) describe the first-generation route to bisacrylamide enamine N-oxide crosslinkers and the presently disclosed (second generation) bisacrylamide enamine N-oxide crosslinkers of formula (I), respectively.Schemes 1A and B. Synthesis of enamine A-oxide-based bioorlhogonally degradable bisacrylamide crosslinkers.OH, N, NHBoc BocHN Me 1 OBocHN NN H BOCEocHlW ' N 16 NUHDBoc " O MeH 8 3: 1"d Me o 3, major 4, minor BocHN NHBoc N'MeH2N BocHN H NHBoc 5 6Reagents and conditions: (a) Hydroxylamine 1 (2 equiv), alkyne 2 (1 equiv), 20% TFE / CHCfo (v / v), 60°C, 56%, (b) B2(OH)4, HJO, (C) MBS, AgNO3, acetone, 0°C, 91%, (d) 1 (2.00 equiv), 20% TFE / CHCh (v / v), 50°C, 92%, (e) nBuLi, THF. -78°C; H, O, 23°C. 76%, (f) 20% TFA / CH2C12(vAty 23°C; A-acryloxysuccinimide, NaHCO3, H2O. 23°C, 80% (2-step).[00142| Scheme 1A describes the first-generation route to bisacrylamide enamine7-oxide crosslinkers. Hydroamination of alkyne 2 featuring a propargylic carbamate with hydroxylamine 1 resulted in formation of an 8.3:1 ratio of regioisomers favoring the desiredAFSDOCS:304108277.1Attorney Docket No. 046094-798001 WO Date of Deposit: December 18, 2025anti-Markovnikov product 3 over the undesired Markovmkov product 4. Notably, the regioisomers were inseparable by both standard flash column chromatography and reverse phase high performance liquid chromatography (HPLC). Reduction of each with an aqueous solution of tetrahydroxy diboron (Bz(OH)4) reagent resulted in reductive fragmentation of the former to amines 5 and 6 while affording the still enamine 7 from the latter, poignantly highlighting the challenges of advancing a regioisomeric mixture. These first-generation crosslinkers, such as ENBAA-1, are also described in Applicant’s pending on International Application No. PCT / US2024 / 035794. The “first-gen” crosslinkers, e.g., ENBAA-1, are single diastereomers, which refers to the E / Z olefin isomerism but as also explained above, are not pure from a regioisomeric standpoint (which refers to positional isomerism). That is, the first-gen crosslinkers such as ENBAA-1 have now been found to include a physically inseparable mixture of two regioisomers, namely the compound of formula (I) and the compound of formula (V). As explained below’, the crosslinkers of the present disclosure (compounds of formula (I), e.g., ENBAA-1, are pure regioisomers as defined herein (FIG.38).

[0143] Alternatively, prior studies demonstrated that terminally halogenated alkynes undergo rapid and regioselective hydroamination with, A-dialkylhydroxylamines to produce regioisomerically pure enamine A-oxides with the anti-Markovnikov constitution (D. Kang et al., Angew. Chem. Int. Ed., 2021, 60:16947; S. Siriwongsup et al.. Chem, 2024, 10:1306), While it was determined that a-haloenamine A-oxides featuring a primary carbamate at the allylic position cannot be directly employed for polymer degradation as currently designed since their reductive activation does not lead to complete and rapid dissociation, dehalogenation afforded the enamine A-oxide crosslinker precursor 3 that as targeted,

[0144] Scheme IB describes an embodiment of the efficient regioselective synthesis of enamine iV-oxide bisaciy larmde (ENBAA) crosslinkers of the present disclosure, that avoids production of compound 4 as an intermediate. Retro-Cope elimination reaction between hydroxylamine 1 and bromoalkyne 8 yielded bromoenamine A-oxide 9 in 92% yield as a single regioisomer (in contrast to the production of the two regioisomers (compounds 3 and 4) in scheme I A). Subsequent lithium-bromine exchange using n-BuLi follow ed by protonation of the vinylhthium intermediate with water afforded enamine A-oxide 3. Then, Boc-deprotection followed by acryloylation using A-acryloxysuccimmide in basic aqueous solution afforded enamine A-oxide bisacrylamide ENBAA-1 in 80% yield over two steps as a single regio- and diastereoisomer. The presently disclosed method produces compounds of 39AFSDOCS:304108277.1Attorney Docket No. 046094-798001 WO Date of Deposit: December 18, 2025formula (I) as single, substantially pure regioisomers and diastereomers that avoids production of the intermediate compound 4. By avoiding compound 4, the regioisomer which is the undesired compound of formula (V) is not produced (as determined by ’ll NMR as defined herein).

[0145] To synthesize crosslinkers of varying lengths, hydroxylamines S8 and S9 were employed in the hydroamination reaction with bromoalkyne 8. These ENBAA crosslinkers were then used to fabricate PAAm / Alg hydrogels.

[0146] ENBAA Linker Length Study and Fabrication of Degradable Tough Hydrogel

[0147] To fabricate PAAm / Alg hydrogels, high and low molecular weight sodium alginates were physically crosslinked using calcium sulfate. Acrylamides were covalently crosslinked using either nondegradable MBAA or degradable enamine A-oxide bis acrylamides (ENBAA) 1-3 (FIG 1 A).

[0148] To evaluate the effect of the covalent crosslinker length on the mechanical properties of PAAm / Alg hydrogels, crosslinkers ENBAA 1-3 were used to fabricate the hydrogels (Table 1). Stress-strain curves were obtained under tensile loading, and the maximum stress and strain, fracture energies, and Young’s moduli were compared (FIG. 1B-FIG. IE and FIG. 9-FIG. 12). Fracture energies were determined using a method introduced by Rivlin (R. S Rivlin, A. G. Thomas, J. Polym. Sci., 1953, 10:291), and the Young's modulus was determined by calculating the initial slope of the stress-strain curve. Nondegradable hydrogels fabricated with MBAA crosslmkers have been reported to give a maximum stress of 156 kPa, maximum strain of >20 mm / mm, and a fracture energy of >6 kJ m2(J.-Y. Sun, et al., Nature, 2012, 489:133). Linker length studies revealed that as the covalent crosslinker length increases, greater stoichiometries of the crosslinkers are necessary to obtain a similar stress, strain, fracture energy, and Young’s modulus. When comparing the fracture energies of hydrogels made with different crosslinkers, hydrogels made with ENBAA- 1 at lx mol equivalent relative to MBAA had fracture energies that were twice as large as those of hydrogels made with ENBAA-3 at lx mol equivalent relative to MBAA. Moreover, hydrogels cast with ENBAA-3 required more than 2x the amount of crosslinkers to obtain similar mechanical properties compared to those cast with MBAA. As the covalent crosslinker lengths increases, more stoichiometries of the crosslinker is necessary to achieve comparable mechanical properties (B. R. Freedman et al.. Adv. Mater., 2021, 33:e2008553; E. Mariner et al.. Biointerphases, 2019, 14:031002; M.-J. Xie et al., Polymer. 2024, 290:126550).40AFSDOCS:304108277.1Attorney Docket No. 046094-798001 WO Date of Deposit: December 18, 2025

[0149] Because the intrinsic physicochemical properties of the enamine A7-oxide crosslinkers are different from those of MBAA, it was essential to optimize the relative amount of the crosslinker to obtain hydrogels with mechanical properties comparable to those of the non-degradable tough hydrogels. Hydrogels cast with 1.2x ENBAA-1 crosslinkers behaved most like those cast with MBAA (FIG. IF and FIG. 13). Moreover, degradable tough hydrogels maintained their mechanical properties even after incubation at 37°C for 15 d (FIG. 1G-FIG 1H and FIG. 14). highlighting the stability7of enamine A-oxide crosslinkers under physiologically relevant conditions for extended periods of time. Viscoelastic responses measured by dynamic mechanical analysis (DMA) and compressive stresses of hydrogels further confirmed the similar mechanical properties between the non-degradable and degradable tough hydrogels (FIG. 11-F1G. I J and FIG. 15).

[0150] Example 3: Degradation of PAAm / Alg Tough Hydrogels

[0151] The degradable PAAm hydrogels were exposed to 10 mM aq. B?.(0H)4, and complete dissolution of the hydrogel was observed within 20 mm by vial inversion tests (FIG. 16)

[0152] PAAm / Alg hydrogels cast with blue food coloring were exposed to 500 pM aq. B.?(0H)4, and these gels were degraded considerably w ithin 20 mm (FIG. 2A and FIG. 17). The mechanical properties of hydrogels exposed to B2(OH)4 were evaluated further by obtaining their stress-strain curves (FIG. 2B-FIG 2E). Significant degradation of the hydrogels were observed e\en when they were exposed to 500 pM aq. B2(OH)4 for 10 min with a decrease in maximum stress from 90 to 18 kPa, and fracture energy from 6.8 to 0.3 kJ nr2Hydrogels exposed to B2(OH)4 for 3 and 20 min were also evaluated (FIG. 18 and FIG. 19).

[0153] FIG. 2F shows images of PAAm / Alg hydrogels exposed to 500 pM aq. B2(OH)4 for 10 min then placed under tension. Diboron induced degradation of the PAAm / Alg hydrogels was apparent. The gels clearly lost most of their mechanical properties as evidenced by formation of holes in the middle of the gel even at low strain.

[0154] The mechanical properties remained largely intact for hydrogels exposed to 100 pM aq. B2(OH)4. Although a greater than 2-fold decrease in fracture energy was observed, the maximum stress was maintained at 80 kPa at >35 mm / mm strain. The increase in strain and a decrease in Young's modulus are both reflected in the stress-strain curves, which suggests41AFSDOCS:304108277.1Attorney Docket No. 046094-798001 WO Date of Deposit: December 18, 2025that only partial degradation has occurred despite the excess amount of B2(OH)4 used relative to the crosslinker.

[0155] To determine whether the marginal degradation was due to poor diffusion of the diboron solution into the hydrogel, the degradation of 1.5 ram and 1.0 mm thick PAAm / Alg hydrogels were evaluated at varying concentrations of B2(OH)4 (FIG. 3A-FIG. 3D and FIG.20). 1.0 mm thick hydrogels reacted with B2(OH)4inore effectively across all concentrations. The greatest difference was observed at 100 pM B2(OH)4 where the maximum stress decreased below' 20 kPa and the fracture energy decreased to less than 1 kJ irr2for 1,0 mm thick gels.

[0156] To better understand the effect of gel thickness on the rate of degradation, the hydrogels’ (1.0 mm or 1.5 mm thick, 5 mm diameter) shear moduli 'ere measured in the presence of aqueous B2(OH)4 (FIG. 3E-FIG. 3F and FIG. 21). Discrete rheology' measurements of hydrogels exposed to B 2(011)4 with concentrations ranging from 100 pM to 10 mM showed that 1.0 mm thick gels responded to B2(OH)4 faster across all concentrations. At 500 pM [B2(OH)4], a 10-fold decrease in G’ was achieved by 45 min. To better visualize the relative trends between samples, the fraction storage modulus 'as calculated and plotted by normalizing the modulus with the initial storage modulus (FIG. 3G and FIG. 22). 1.0 mm thick gels responded to B2.(OH)4 across all concentrations. Even for 100 pM and 500 pM concentrations of aq. B2(OH)4 tested, the storage moduli of both 1.0 mm and 1.5 mm thick gels all decreased by more than half in less than 10 mm. Tensile testing data coupled with viscoelastic responses of hydrogels with these dimensions and geometry' strongly suggested that PAAm / Alg hydrogels fabricated with ENBAA-1 as the chemical crosslinker underwent induced degradation using B2(OH)4 within 10 min. Moreover, the data indicated that the rate of degradation is limited by the rate of diffusion of the diboron solution into the hydrogel for gels with these dimensions and geometry-.

[0157] Example 4: Biocompatibility and Removability of Degradable Tough Hydrogels

[0158] To assess the biocompatibility of the PAAm / Alg hydrogels, the gels were placed on top of a monolayer of L929 fibroblast cells in tissue culture. After incubation for 72 h, the gels W'ere degraded 'ith 500 M aq. B2(OH)4 then stained with Calcein AM and ethidium homodimer 1 (FIG. 4A). Tire cells were imaged with a fluorescence microscope, showing live and dead cells (FIG. 4B). The cell viability was calculated for cells treated with or without the degradable hydrogel, and the results showed no statistical differences between the 42AFSDOCS:304108277.1Attorney Docket No. 046094-798001 WO Date of Deposit: December 18, 2025two groups (FIG. 4C). Similarly, the cytotoxicity of B2(OH)4 was also evaluated on L929 cells. L929 cells were treated with JtyCOHty at concentrations up to 50 mM, incubated for 0.5 or 3 h, then assayed by the CellTiter-Glo cell viability assay. Cells treated with diboron for 0.5 h showed an IC50 value of >20 mM, and cells treated with diboron for 3 h showed an IC50 value of >9 mM, both well above the concentrations and exposure times required to effectively degrade the PAAm / Alg hydrogels (FIG. 4D).

[0159] The ability of ENBAA-crosslinked degradable tough hydrogels to adhere to porcine skin was evaluated by T-peel test using three adhesives: 1) chitosan with EDC and sulfo-NHS, 2) commercially available gel-type cyanoacrylate adhesive Loctite 4541, and 3) commercially available high viscosity liquid-type cyanoacrylate adhesive Periacryl 90HV (FIG. 5 A). While many types of chitosan adhesives have been reported in the literature, including covalent adhesives based on chitosan solution supplemented with EDC and sulfo- NHS (J. Li et al., Science, 2017, 357:378), pH-responsive topological adhesives based on chitosan adsorbed on a dissipative matrix (J. A. Cintron-Cruz et al.. Adv. Mater., 2022, 34:e2205567), or chitosan-based films (B. R. Freedman el al.. Proc. Natl Acad. Sci., 2024, 121:e2304643121), chitosan adhesives supplemented with EDC and sulfo-NHS were used.

[0160] Adhesion energies were determined by taking twice the plateau value of the ratio of the force to width (R. S. Rivlin, A. G. Thomas, J. Polym. Sci., 1953, 10:291). Of the three adhesives tested, chitosan + EDC / sulfo-NHS proved to be the most effective adhesive with an adhesion energy of 710 J m-2, followed by Periacryl with 550 fm2. then Loctite with 290 J nr2. Moreover, when comparing the adhesion energies of nondegradable and degradable hydrogels on porcine skin, no significant differences were observed (FIG. 5B).

[0161] Using the degradable gels described herein, chitosan was confirmed to act as a strong adhesive even on wet surfaces and that the adhesion energies are not compromised in the presence of blood as previously reported (J. Li et al.. Science, 2017, 357:378). In contrast, the cyanoacrylate-based adhesives Loctite and Periacryl exhibited poor adhesion energies when blood was applied to the surface of tissues prior to adhesion (FIG. 23). This is consistent with the known incompatibility of cyanoacrylate adhesives with wet surfaces. Both adhesives solidify immediately upon exposure to water and form plastics that cannot accommodate dynamic movements of the tissues.

[0162] Strong adhesion energies are important for holding a material in place; however, they can be a problem when detachment is required. For example, the removal of strong adhesives from various tissues risks fibrosis and other forms of secondary tissue damage. An 43AFSDOCS:304108277.1Attorney Docket No. 046094-798001 WO Date of Deposit: December 18, 2025effective approach to this challenge is to degrade the underlying biomaterial to which the adhesive has been applied. When a material’s mechanical properties are compromised, its ability to adhere also decreases as the toughness of a material is linked to the strength of its adhesion to biological tissues, and the two cannot be decoupled (B. R Freedman et al., Adv. Mater., 2021, 33:e2008553; H. Yang etal., ACS Appl. Bio Mater., 2019, 2:1781).

[0163] To evaluate the effect of hydrogel degradation on the adhesion energies, hydrogels were adhered to mice skin then removed with or without degradation. The tissues were then stained and assessed by histology7(FIG. 5C and S16). Removal of both MBAA and ENBAA-1 crosslinked PAAm / Alg hydrogels from mice skin without degradation resulted in the detachment of the outer layer of epithelium when adhesives such as Periacryl and chitosan were used; however, the epithelia were observed to be intact when the hydrogels were peeled after submerging the tissue in an aqueous solution of 500 pM B2(OH)4 for 30 min.

[0164] Example 5: PNiPAAtn / Alg Intraoral Hy drogels

[0165] Although PAAm / Alg hydrogels are tough and powerful in dry7environments, once introduced to a wet environment where the hydrogel can soak up solvent, the material's toughness decreases due to swelling (X Ni et al., ACS Macro Lett. 2021, 10:180). To minimize the effect that swelling has on the intrinsic properties of the material, degradable thermoresponsive poly(7V-isopropylactylamide) (NiPAAm) / Alg hydrogels have also been fabricated using the same ENBAA-1 crosslinker. NiPAAms undergo phase transition above its low critical solution temperature (LCST) which allows for de-swellmg, maintaining the material’s original mechanical properties (R. P. Dumitriu et al., J. Appl. Polym. Sci., 2014, 131:40733; R. P. Dumitriu et al, Solid State Phenom., 2009, 154:17). And because NiPAAm’s LCST is below 37°C or a normal body temperature, NiPAAm / Alg hydrogels are suitable for applications in vivo under wet environments (H. Zhang et al.. Colloid Polym. Set, 2016, 294:1959; H. K. Ju et al., J. Appl. Polym. Sci., 2002, 83:1128; W. J. Zheng et al., ACS Appl. Mater. Interfaces, 2015, 7:1758).

[0166] Tough degradable hy drogel wound dressings are especially needed in applications where the surrounding environment undergoes consistent deformations and where repeated attachment and detachment are favorable. The intraoral cavity is one organ where hydrogel wound dressings can have an impact (FIG. 6A). Attachment of tough hydrogels to oral lesions such as mouth ulcers, sores, or post-surgical sutures can be beneficial to protect the site of injury from food or beverage as well as from secondary infections (J. Wu et al.. Adv.44AFSDOCS:304108277.1Attorney Docket No. 046094-798001 WO Date of Deposit: December 18, 2025Mater,. 2022, 34:e2200115; Y. Ding et al., Adv. Healthc. Mater., 2024, e2400912); however, because the intraoral cavity is constantly wet from saliva, hydrogels that undergo extreme swelling are not ideal materials for oral wound dressings. For example, PAAm / Alg hydrogels undergo significant swelling when immersed in water (M. Yu. Gorshkova et al., Mendeleev Commun., 2022, 32:189).

[0167] To evaluate the swelling of PAAm / Alg and NiPAAm / Alg hydrogels in various aqueous media, the swelling ratios for these hydrogels were measured (FIG. 6B). Hydrogels were immersed in PBS, pH 7.4 at either 23°C or 37C'C for 72 h, and the swelling ratios were recorded. At room temperature, both PAAm / Alg and PNiPAAm / Alg hydrogels showed swelling ratios up to 40-fold. At 37°C, although PAAm / Alg hydrogels showed similar final swelling ratios. PNiPAAm / Alg hydrogels showed de-swelling from the originally fabricated state, with a swelling ratio of 0.6-fold. This data is consistent with other PNiPAAm-based hydrogels that are swelled above their LCST. The A-isopropyl groups interfere with hydrogen bonding and causes deswelling. Photos of hydrogels swollen at 37°C m PBS show that PNiPAAm / Alg hydrogels maintain their size while PAAm / Alg hydrogels swell to almost 3 times their original size (FIG. 6C). Swelling behaviors w ere similar in other aqueous media including cell culture media + 10% human serum or media + 10% pooled human saliva, suggesting that the swelling behavior of NiPAAm / Alg hydrogels will be similar in an intraoral environment (FIG. 25 and FIG. 26).

[0168] Sw elling or desw^elling of hydrogels influences their mechanical properties. When comparing the maximum stress, strain. Young’s modulus, and viscoelastic properties of swollen hydrogels, swollen PNiPAAm / Alg hydrogels dramatically outperform swollen PAAm / Alg hydrogels. Moreover, swollen PNiPAAM / Alg hydrogels outperformed unswollen PNiPAAm / Alg hydrogels (FIG. 27), reaching maximum stress and strain of 48 kPa and 11 mm / mm. respectively (FIG. 6D-FIG. 6F). Viscoelastic responses of swollen PAAm / Alg and PNiPAAm / Alg hydrogels in various media were also evaluated (FIG. 6G, FIG. 28, and FIG.29). The storage modulus G' of PNiPAAm / Alg hydrogels was nearly 10-fold greater compared to that of PAAm / Alg hydrogels. Additionally, hydrogels that were swollen in media + human serum or media + human saliva showed comparable G' and G" to hydrogels swollen in PBS, pH 7.4, suggesting that the enamine Ar-oxides are stable in biological media.

[0169] To evaluate the adhesive energies most applicable for intraoral applications, lap shear stress of swollen hydrogels was measured (FIG. 6H). Lap shear stress of PNiPAAm / Alg hydrogels on porcine skin were an order of magnitude higher than those of 45AFSDOCS:304108277.1Attorney Docket No. 046094-798001 WO Date of Deposit: December 18, 2025PAAm / Alg hydrogels as well as those of the commercially available intraoral hydrogel adhesive Ora-Aid. The tissues were pre-incubated with pooled human saliva before adhesion for 2 min. The saliva had minimal effect on the adhesion energies of all three adhesives.

[0170] Degradation of PNiPAAm / Alg hydrogels was also evaluated in a similar manner to that of PAAm / Alg hydrogels. Because ENBAA-1 was used to fabricate the PNiPAAm / Alg hydrogels, the degradation trends were similar to those of PAAm / Alg hydrogels (FIG. 7 A). Stress-strain curves of hydrogels exposed to aqueous B2(OH)4 for 10 min showed a concentration dependent degradation profile The maximum stresses, fracture energies, and Young's moduli of PNiPAAm / Alg hydrogels were indicative of the significant degradation of mechanical properties as the concentration of the B2(OH)4 was increased (FIG. 7B-FIG.7D). It was difficult to measure the fracture energies of hydrogels exposed to B2(OH)4 with concentrations greater than 100 pM because rapid degradation led to formation of an amorphous gel. Hydrogels exposed to 100 pM aq. B2(OH)4 showed no loss in Young’s modulus, which suggested incomplete degradation; however, the stress-strain curves showed a sharp drop in the stress as the strain starts to increase after the inflection point. A certain force is required to observe loss in mechanical properties of hydrogels m tensile tests (H. Yang ef al.. ACS Appl. Bio Mater, 2019, 2:1781; C. J. Higginson et al.. J Am. Chem Soc., 2015, 137:4984), but this does not mean that the dissociation of the ENBAA-1 crosslinker is absent. Degradation of PNiPAAm / Alg hydrogels exposed to various concentrations of aqueous B2(OH)4 for 3 min was also explored (FIG. 30).

[0171] Cell viability assays were also performed with PNiPAAm / Alg hydrogels to confirm their biocompatibility. L929 cells were exposed to PNiPAAM / Alg hydrogels that had been pre-incubated in PBS, pH 7.4 for 3 d (FIG. 31). The cells were incubated with the hydrogels for 72 h. then treated with B2(OH)4 to induce hydrogel degradation. After 0.5 h, the cells were treated with Calcein AM and ethidium homodimer 1 to visualize live and dead cells. Cells that were treated with both the hydrogel and aqueous B2(OH)4 showed cell viabilities >95%.

[0172] To evaluate the feasibility of PNiPAAm / Alg hydrogels from tissue, the diboron-mediated hydrogel degradation approach was evaluated on pork tongue in an ex vivo setting (FIG. 7E and FIG. 32-FIG.37). Hydrogel disks were first adhered to pork tongue using Loctite, Periacryl, or chitosan + EDC / sulfo-NHS. Then, aqueous solutions of B2(OH)4 (500 pM) were applied to the tissue in three different ways: 1) the tissue was submerged in a diboron solution to simulate the delivery of the reagent as a mouth wash; 2) the diboron 46AFSDOCS:304108277.1Attorney Docket No. 046094-798001 WO Date of Deposit: December 18, 2025solution was sprayed directly onto the hydrogel with a spray bottle, completely covering the surface of the tissue; and 3) a wet gauze saturated with diboron solution was placed over the hydrogel. Photos showed that when the hydrogel-tissue adduct was submerged in water without diboron treatment, the hydrogel remained adhered. Application of diboron solution by any of the three methods resulted in quick and easy removal of the hydrogel. Submerging the hydrogel in the aqueous 62(06)4 solution proved to be most effective at degrading the hydrogel, but applying solutions of diboron via either spray bottle or wet gauze was enough to degrade the hydrogel for easy removal. Chemically inducible and degradable hydrogels with its fast degradation times, and efficient removability coupled with the biocompatibility of the diboron reagent are effective in circumstances where repeated attachment and detachment of hydrogels is necessary, like in the oral cavity.

[0173] PNiPAAm / Alg hydrogels were designed to function as a degradable intraoral wound dressing m the presence of diboron reagents, so it was necessary to assess the safety of 62(011)4 when swallowed. In vivo maximum tolerated dose studies of 62(06)4 were conducted in mice to prove the safety of the B2.(OH)4 solutions (FIG. 8A). Nude mice were administered 0 (H2O. vehicle), 25, 75 and 150 mg / kg doses of aqueous 62(06)4 solution via oral gavage. Five consecutive daily doses were administered, and the survival rate, weight, and food consumption of the mice were recorded over 14 d (FIG. 8-FIG. 8C). All of the mice in the study survived and showed no signs of weight loss or restriction in their diet Tire byproduct of 62(06)4 when reacted with A-oxides is boric acid, which is widely used as a buffering component m pharmaceutical formulations. These MTD studies show that 62(06)4 is safe for ingestion by mice even at 200 mM concentrations and volumes up to 200 pL repeatedly over multiple days.[00174| Example 6: Experimental Methods

[0175] Synthesis ofbis-Boc-bromoenamine N-oxide (9)

[0176] 2,2,2-Trifluoroethanol (TFE) in chloroform (20% v / v, 20 mL) was added to a 25 mL round bottom flask charged with hydroxylamine 1 (3.47 g, 18.2 mmol, 2.00 equiv) and bromoalkyne 8 (2.94 g, 9.14 mmol, 1 equiv). The vial was flushed with nitrogen, sealed with a septum cap and Parafilm, then heated to 50°C in an oil bath. After 3 h, the reaction was cooled to room temperature and concentrated under reduced pressure. The crude oil was purified by flash column chromatography on silica gel (eluent: 10^40% CMA in chloroform) to give the title compound as a white foam (4.30 g, 92%). Tl NMR (500 MHz,47AFSDOCS:304108277.1Attorney Docket No. 046094-798001 WO Date of Deposit: December 18, 2025CD3OD) 87.48 (t, J = 5.6 Hz, 1H), 4.72 (qd, J = 14.5, 5.7 Hz, 2H), 4.07-3.90 (m, IH), 3.52- 3.35 (m, 6H), 3.17 (d, J = 4.9 Hz, 2H). 3.14 (d, J = 4.8 Hz, 2H). 1.43 (s, 9H), 1.43 (s. 9H).!3C NMR (126 MHz, CD3OD) 8 158.7, 158.5, 158.3, 131.5, 128.7, 80.5, 80.3, 69.0, 63.9, 60.9, 42.1, 41.3, 36.2, 28.9, 28.9. FTIR (thin film) cm’1: 3332 (br), 2984 (w), 1692 (s), 1524 (m), 1454 (w), 1252 (m), 1170 (s), 1030 (w). HRMS (ESI) (m / zy'. cak’d for CwHssBrNrCh [M+H]’: 511.1762, found: 511.1786. TLC (30% CMA in chloroform), Rf 0.14 (KMnO4).

[0177] Synthesis of his-Boc-enamine N-oxide (3)

[0178] A round bottom flask was charged with bis-Boc-bromoenamine A-oxide 9 (3.27 g, 6.36 mmol, 1 equiv) and purged with nitrogen. Tetrahydrofuran (200 mL) was then added via syringe, the reaction mixture was cooled to -78°C using an acetone-dry ice bath, and n-butyllithium (1.9 M in hexanes. 3.68 mL, 7.00 mmol. 1.10 equiv) was added dropwise via syringe. After 20 mm, the reaction was quenched with deionized water (1 mL) and warmed to room temperature by removing the acetone-dry ice bath. The resulting crude mixture was concentrated under reduced pressure and purified by flash column chromatography on silica gel (eluent: 30— *60% CMA in chloroform) to give the title compound as a white foam (2.09 g, 76%).[H NMR (500 MHz, CDjOD) 5 6.56 (dt. J = 13.3. 5.0 Hz. IH), 6.49 (d. J = 13.4 Hz, 1H), 4.68 (d, J = 4.9 Hz, 2H), 3.61-3.38 (m, 4H), 3.25 (s, 3H), 3.22-2.97 (m, 4H), 1 43 (s, 9H), 1.43 (s, 9H).13C NMR (126 MHz, CD3OD) 8 157.2, 156.9, 156.7, 140.7, 123.0, 79.1, 78.7, 68.9, 60.6, 58.1, 40.5, 39.8, 34.9, 27.4, 27.3. FTIR (thin film) cm’1: 3343 (br), 2974 (w), 1700 (s), 1524 (m), 1415 (m), 1364 (m). 1252 (m), 1167 (s), 1039 (w), 779 (w ). HRMS (ESI) (m / z): cak’d for C19H36 4O7 ]M+H]+: 433.2657, found: 433.2655. TLC (60% CMA in chloroform), R / : 0.29 (KMnCL).

[0179] Synthesis ofbisacrylamide-enamine N-oxide (ENBAA-1)

[0180] Trifluoroacetic acid in dichloromethane (20% v / v, 3 mL) was added to a round bottom flask charged with bis-Boc-enamme A-oxide 3 (521 mg, 1.21 mmol, 1 equiv) at room temperature. After 3 h, the reaction mixture was concentrated under reduced pressure. To the crude oil was added deionized water (8 mL) followed by sodium bicarbonate (709 mg, 8.44 mmol, 7.00 equiv) portion-wise at room temperature. The flask was covered in foil, and N-acryloxysuccinimide (428 mg. 2.53 mmol, 2.10 equiv) was added to the solution. After 1 h, the reaction mixture was purified by automated Cis reverse phase column chromatography (30 g Cis silica gel, 25 pm spherical particles, eluent: H2O + 0.2% EtsN (2 CV), gradient 0- *100% MeCN / H2O + 0.2% EtsN (10 CV) to give the title compound as a white solid (329 mg, 80%, 2 steps). fll NMR (500 MHz, CD3OD) 06.60 (dt, J - 13.0, 5.0 Hz, IH), 6.56-6.5148AFSDOCS:304108277.1Attorney Docket No. 046094-798001 WO Date of Deposit: December 18, 2025(m, 1H), 6.29-6.17 (m, 4H), 5.76-5.63 (m, 2H). 4.70 (dd, J = 5.1, 1.6 Hz, 2H), 3.76-3.57 (m, 3H), 3.57-3.49 (m, 1H). 3.38 (td. J = 6.1, 3.6 Hz, 2H), 3.30-3.24 (m, 5H).13C NMR (126 MHz, CD?OD) 6 167.0, 166.8, 157.0, 140.6, 130.7, 130.4. 125.7. 125.4, 123.3, 68.6, 60.7, 58.4, 40.1, 38.9, 33.9. FTIR (thin film) cm4: 3340 (br), 2974 (w) 1696 (s), 1528 (m), 1453 (m), 1364 (m), 1252 (m), 1170 (m), 998 (w), 779 (w). HRMS (ESI) (m'zy'. calc’d for C15H24N4O5 [M+H]2 341.1819, found: 341.1823.

[0181] Preparation of PAAm / Alg Hydrogels

[0182] 2.2% sodium alginate solution was prepared by dissolving high-molecular-weight sodium alginate (110 mg, MVG, NovaMatrix) and low -molecular- weight sodium alginate (110 mg, VLVG, NovaMatrix) in Hank's Balanced Salt Solution (HBSS, 10 mL, pH 7.4). acrylamide (1.35 g). AA’, A', A’'-tetramethyIethylenediamine (TEMED. 8 pL) and methylene bisacrylamide (MBAA, 123 mM in water, 36 pL) or enamine A-oxide bisacrylamide (ENBAA-1, 156 mM in water, 36 pL) covalent crosslinker was added to the fully dissolved sodium alginate solution and drawn into a syringe. A second syringe containing ammonium persulfate (APS, 226 pL of 6.6% w7v in water) and calcium sulfate dihydrate (CaSCH ^HzO, 0.75 M in water, 191 pL) was connected to the syringe containing the alginates, acrylamide, TEMED, and covalent crosslinker with a female-female luer connector and syringe mixed. For ENBAA-2 and ENBAA-3, refer to Table S 1. The gel was cast into glass molds (75 mm x 80 mm x 1.5 mm, w*1xd), placed in a sealed plastic bag away from light, and left to crosslink for 24 h. After 24 h. the gel was removed from the mold, placed m a sealed plastic bag, and stored at 4CC.

[0183] Hydrogel Mechanical Properties

[0184] Tensile tests were performed m tension (Instron 3400-SC1, 100 N load cell) and stress-strain curves were obtained to evaluate the maximum stretch, stress, and elastic energy of the hydrogels. The hydrogel was glued between two glass plates at each end, resulting in rectangular specimens measuring 70 mm x 5 mm x 1.5 mm (wxjxd). All tensile tests were performed in air at room temperature using a tensile testing machine with a 100 N load cell. The rate of tension was fixed at 50 mm min1and the hydrogel was pulled until rupture. From the stress-strain curve, the maximum stretch, maximum strain, and the elastic energy were calculated. The initial slopes of the stress-strain curves were used to derive the elastic energy.

[0185] The fracture energy of a gel was assessed following the method outlined by Rivlin and Ihomas (R. S. Rivlin, A. G. Thomas. J. Polym. Sei., 1953, 10:291). Two samples of identical gel, one unnotched and one notched, were evaluated. Rectangular hydrogel 49AFSDOCS:304108277.1Attorney Docket No. 046094-798001 WO Date of Deposit: December 18, 2025specimens with dimensions: width (w) = 70 mm, thickness (t) = 1,5 mm, and length ( / ') = 5 mm underwent the tensile testing protocol outlined above. The unnotched gel was placed under tension until rupture, and the resultant force-length curve was analyzed to determine the work done (U(L)). For the notched sample, a 40 mm-long notch was created using a razor blade, and the critical clamp distance (Lc) at which the notch transitions into a running crack was measured. The fracture energy was then calculated using equation 1:Fracture energy (kJ / m2) = 17(LC) / (w)( / ) Equation 1

[0186] Viscoelastic Responses of PAAm / Alg Hydrogels determined by DMA

[0187] The viscoelastic responses of PAAm / Alg hydrogels were determined using a Mettler Toledo DMA 1 instrument. Compression frequency sweep tests at 0.1% strain were carried out over the frequency range of 0.1-30 Hz. PAAm / Alg gels were cast following the general procedure. Hydrogels were punched into disks (8.0 mm diameter. 1.5 mm thickness) for each measurement. The storage modulus E' and loss modulus E" were recorded.

[0188] Degradation of PAAmAlg Hydrogels with aq. B2( H)

[0189] PAAm / Alg hydrogels were cast following the general procedures. Tensile tests were performed following the general procedures to determine the maximum stress and elastic modulus Fracture energy w7as determined following the general procedures. Prior to tensile test, only the sample region undergoing stress was submerged in aq. [BjfOHh] for the specified time.

[0190] Discrete Viscoelastic Responses of PAAm / Alg Hydrogels Exposed to aq. B2(OH)4

[0191] PAAm / Alg hydrogels were cast following the general procedures. Rheology tests were performed on TA Instruments Discovery HR 2 to evaluate the trends of hydrogels exposed to aqueous [B2.(OH)4] ranging from 10 mM to 100 pM. A rubber spacer (5 mm diameter. 5 mm thick) was attached to the bottom plate. A 150Cw sandpaper was fixed on the bottom plate and 800Cw sandpaper w7as fixed to the top spindle to provide sufficient grip on the samples during measurement. Viscoelastic responses of nondegradable and degradable hydrogels aqueous B2(OH)4 were measured, and the shear moduli were recorded. Hydrogels disks (1.0 mm or 1.5 mm thick, 5 mm diameter) were prepared for evaluation. Six [B2(OH)4| were chosen: 10 mM, 3 mM, 1 mM. 500 pM, 100 pM and 0 pM. Initial oscillation tests were performed to obtain initial storage and loss moduli The top spindle was raised to 5 mm height, and aqueous B?(OH)4 (5 mL) solution was added to the bottom plate. Subsequently, when the spindle reached the geometry gap, the gel's moduli were recorded. The geometry7gap was set at 800 pm for the 1.0 mm thick samples and at 1200 pm for the 1.5 mm thick 50AFSDOCS:304108277.1Attorney Docket No. 046094-798001 WO Date of Deposit: December 18, 2025samples. After each measurement, the top spindle was sufficiently raised to increase the contact surface area of aqueous B2(OH)4to the gel disks. It was essential to allow the diboron solution to diffuse into the gel from the bottom and the side surface of the hydrogel disk. This procedure was repeated for 36 cycles. The holding time was 10 seconds for the first 6 cycles and 1 minute for the remaining cycles. For each cycle, the oscillation test was performed at 20°C with 1 Hz frequency and 5% shear strain amplitude.

[0192] Cell Viability Assay

[0193] For cell culture. L929 fibroblasts were cultured in modified MEM 1 * with 1-glutamine containing 10% horse serum (Fisher™), 100 umts / mL penicillin and 0.1 mg / mL streptomycin (Sigma™) in a humidified chamber at 37°C under an ambient atmosphere with 5% CO2 unless otherwise stated. All cell lines were acquired from ATCC. Ceils were passaged and dissociated with 0.25% trypsin, 0.1% EDTA in HBSS (Coming). All cells tested negative for mycobacteria with the MycoAlert® PLUS Mycoplasma Detection Kit (Lonza) following manufacturer’s protocol. To prepare 96-well plate, an aqueous solution of human fibronectin (10 pg mL1, 100 uL, Coming) was added to each well. The plate was gently shaken to distribute the solution evenly, incubated at room temperature for 0.5 h, washed using autoclaved deionized water (100 pL) then autoclaved PBS, pH 7.4 (100 pL), and air-dried for 0.5 h at room temperature.

[0194] L929 fibroblasts were seeded in an opaque 96-well plate at a density of 10,000 cells per well in media (100 pL, MEM containing 10% horse serum, 100 units mL-1penicillin, 0.1 mg mL1streptomycin). PBS (pH 7.4, 100 pL) was added to the edge wells. The cells were incubated in a humidified chamber at 37CC with 5% ( () ■. After 24 h, the media was aspirated and replaced with media (100 pL, MEM supplemented with 10% FBS, 100 units mL1penicillin, 0.1 mg mL1streptomycin) containing tetrahydroxydiboron. Treatment concentrations started at 50 niM and was serially diluted 2.5-fold across nine wells. Vehicle controls corresponding to the treatment consisted of complete growth media (100 pL). The treated plates were incubated at 37°C with 5% CO2 for 0.5 h or 3 h after which the media w as removed, the cells were washed twice with PBS pH 7.4, and the solution -was replaced with complete growth media (100 pL). After incubating at 37°C w ith 5% CO2 for 24 h, the plates w ere removed from the incubator and allowed to equilibrate at room temperature for 10 min. CeUTiter-Glo® 2,0 reagent (50 pl,, Promega) was added directly to each well and mixed gently. The plates were incubated at room temperature for 10 min to stabilize the51AFSDOCS:304108277.1Attorney Docket No. 046094-798001 WO Date of Deposit: December 18, 2025luminescence signal and then analyzed by a microplate reader (Clariostar Plus, BMG Labtech).

[0195] LIVE / DEAD Cell Viability Assay

[0196] To prepare a 24-well plate, an aqueous solution of human fibronectin (10 pg mL-1, 500 pL, Coming) was added to each well. The plate was gently shaken to distribute the solution evenly, incubated at room temperature for 0.5 h. washed using autoclaved deionized water (500 pL) then autoclaved PBS, pH 7.4 (500 pL), and air-dried for 0.5 h at room temperature. L929 fibroblasts were seeded in a 24-well plate at a density7of 5,000 cells per well in media (500 pL, MEM containing 10% horse serum, 100 units mL1penicillin, 0.1 mg mL1streptomycin). The cells were incubated in a humidified chamber at 37°C with 5% CO2, After 24 h, the media 'as aspirated and replaced with media (100 pL, MEM supplemented with 10% FBS, 100 units / mL penicillin, 0.1 mg mL-1streptomycin) and hydrogel disks were introduced to each well. Vehicle controls corresponding to the treatment consisted of complete growth media (500 pl..). The treated plates were incubated at 37°C with 5% CO?„ After 72 h. the media was aspirated and replaced with media (1 mL, MEM supplemented with 10% FBS, 100 units mL ' penicillin, 0.1 mg mLJstreptomycin, 500 pM tetrahydroxy diboron), and incubated at 37°C with 5% CO?. After 0.5 h, the plates were removed from the incubator and allowed to equilibrate at room temperature for 10 min. The media was aspirated and replaced with an aqueous solution of Calcein AM and ethidium homodimer-1 (EthD-1) (500 pL, 2 pM Calcein AM. 4 pM EthD-1, Invitrogen) then mixed gently. The plates were incubated at room temperature for 20 min and then imaged by fluorescence microscopy.

[0197] Fluorescence Microscopy and Cell Counting

[0198] Wells were imaged at the Confocal and Light Microscopy Core at Dana-Farber Cancer Institute using a Zeiss 980 confocal microscope. Images were acquired with a 10* objective at 0.16 micron pixel1using a Hamamatsu camera. Cells stained with Calcein AM were imaged with a 482 / 35 filter (excitation) and a 536 / 75 filter (emission) and false-colored green and cells stained with Ethd-1 were imaged with a 560 / 40 filter (excitation) and a 630 / 75 filter (emission) and false-colored red. Five images per well, (w;:::3) were analyzed using CellProfiler software with a custom pipeline. Cells stained with Calcein AM, CA and cells stained with Ethd-L CD were counted. Cell viability was calculated from Equation 2:Cell viability' (%) = CA / A + CD) >' 100 Equation 2

[0199] Adhesive Application and Adhesion Energy Measurement52AFSDOCS:304108277.1Attorney Docket No. 046094-798001 WO Date of Deposit: December 18, 2025

[0200] Loctite (4541, Henkel, gel -type cyanoacrylate), Periaciyl (high viscosity cyanoacrylate), and chitosan (2% w / v in 0.5 M MES buffer, pH 4.5) with l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, 12 mg ml"1) and A-hydroxy sulfosuccinimide sodium salt (sulfo-NHS, 12 mg mL1) were used as adhesives. For Loctite and Periaciyl, 21 mg crnr1was applied to the surface of the tissue, and PAAm / Alg hydrogel was applied and compressed for 20 min. For chitosan with EDC and sulfo-NHS, 25 pL cm2was applied to the surface of the tissue, and PAAm / Alg hydrogel was applied and compressed for 20 min.

[0201] Adhesion energy was measured by T-peel test using an Instron 3400 tensile testing instrument with a 100 N load cell under a uniaxial tension with a rate of 50 mm min1in air at room temperature. A stnp of hydrogel (70 1 x 1.5 mm3) was adhered to pork skin with one end open, forming a bilayer with an edge crack. The back of the hydrogel was glued to a rigid polyethylene terephthalate film (3M) with Krazy Glue to ensure that all work done by the machine would be equal to the energy dissipated at the crack tip. The free ends were attached in between two glass plates to which the machine grips were attached. Force¬ displacement curves were obtained, and adhesion energy was calculated using Equation 3:Adhesion energy (J nr2):::2 * (plateau value of F) / width Equation 3[00202| Ex Vivo Evaluation of Application and Removal of PAAm / Alg Hydrogel

[0203] Ex vivo application and removal of PAAm / Alg hydrogels were evaluated on the skin of euthanized mice. Nude mice were donated by Dana-Farber Cancer Institute, Animal Resources Facility. Mice were euthanized with CO2 followed by cervical dislocation. Mice were skinned and the skin was used without any further treatment. Loctite (4541. Henkel, gel-type cyanoacrylate-based adhesive), Periacryl (high viscosity cyanoacry late-based adhesive) and chitosan (2% w / v in 0,5 M MES pH 4.5) with I -ethyl-3-(3-dimethylammopropyl)carbodiimide (EDC, 12 mg mL3) and A-hydroxysulfosuccinimide sodium salt (sulfo-NHS, 12 mg mL-1) were used as adhesives. A strip of hydrogel (40 * 10 x 1.5 mm3) was adhered to the surface of mice skin. For Loctite and Periacryl, 21 mg cm1was applied to the surface of the tissue. Then PAAm / Alg hydrogel was applied and compressed for 20 mm. For chitosan with EDC and sulfo-NHS, 25 pL cm2was applied to the surface of the tissue. Then P AAm / Alg hydrogel was applied and compressed for 20 min.

[0204] Hydrogels were either removed with or without degradation. For removal of hydrogels without degradation, the back of the hydrogel was glued to a rigid polyethylene terephthalate film (3M) with KrazyGlue to ensure that all work done when removing the hydrogel would be equal to the energy dissipated at the crack tip. For removal of hydrogels 53AFSDOCS:304108277.1Attorney Docket No. 046094-798001 WO Date of Deposit: December 18, 2025after degradation, the tissue with adhered hydrogel was submerged in an aqueous solution of B2(OH)4 (500 pM in H2O, 20 mL) for 30 min in air at room temperature. The tissue was then removed from the B2(OH)4 solution, and the degraded hydrogel was wiped away with a tissue (KimTech). The tissues were collected and submerged in 10% formalin in PBS pH 7.4 (5 mL) at 4°C for 24 h. Tissues were then washed with PBS pH 7.4 (2 * 10 mL), then stored in 70% ethanol (5 mL) at 4°C for hematoxylin and eosin (H& E) staining.

[0205] Tissue samples were embedded in paraffin, sectioned, mounted, and stained with hematoxylin and eosin (H& E) by the Rodent Histopathology Core at Harvard Medical School. H& E slides were imaged at the Confocal and Light Microscopy Core at Dana-Farber Cancer Institute using a Nikon Ti Eclipse. Images were acquired with a 10x objective at 5.68-micron pixel-1using a DMC6200 camera from Leica.[002061 Preparation of PNiPAAm Alg Hydrogels

[0207] 2.2% Sodium alginate solution was prepared by dissolving high-molecular- weight sodium alginate (110 mg. MVG, NovaMatrix) and lo -molecular-weight sodium alginate (110 mg, VLVG, NovaMatrix) m HBSS (10 mL, pH 7.4). To a fully dissolved sodium alginate solution were added AMsopropyl acrylamide (1.35 g), N. N. N', N'~ tetrametihyletliylenediamine (TEMED, 8 pL) and methylene bisacrylamide (MBAA, 123 mM in water, 36 pL) or enamine A-oxide bisacrylamide (ENBAA-1, 156 mM in water, 36 pL) covalent crosslinker and drawn into a syringe. A second syringe containing ammonium persulfate (APS, 226 pL of 6.6% w / v m water) and calcium sulfate dihydrate (CaSCU 2H2O, 075 M in water, 191 uL) was connected to the syringe containing the alginates, aciylamide, TEMED and covalent crosslinker with a female-female luer connector and syringe mixed. The gel was cast into glass molds (75 x 1.5 x so mm’), placed in a sealed plastic bag away from light, and left to crosslink for 24 h. After 24 h, the gel was removed from the mold and placed in a container filled with PBS pH 7.4. The gel was stored in PBS pH 7.4 at 37°C for 72 h prior to use.[002081 Swelling Ratios of Degradable Hydrogels

[0209] The swelling ratios of the degradable tough hydrogels were evaluated. Cylindrical specimens (8 mm diameter, 1.5 mm height) w'ere immersed in PBS pH 7.4 (TO mL) and incubated at 23°C or 37°C. The mass of swollen hydrogels w-as weighed over the course of 72 h. The swelling ratio w7as calculated using Equation 4:Q (%) = ( v. — wi) / wi x 100 Equation 454AFSDOCS:304108277.1Attorney Docket No. 046094-798001 WO Date of Deposit: December 18, 2025where Q (%) is swelling ratio. W2 is the weight of the hydrogel after swelling, and w i is the weight of the hydrogel before swelling.

[0210] Single Lap Shear Stress Measurement

[0211] Lap shear tests were conducted to evaluate the adhesion strength between the hydrogels and experimental biological surfaces. The lap shear stresses examine the resistance of hydrogels to shear forces when adhered to biological tissues. Loctite (4541, Henkel, geltype cyanoacrylate), Periacryl (high viscosity cyanoacrylate), and chitosan (2% w7v in 0.5 M MBS buffer, pH 4.5) with l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, 12 mg mL ’) and Ar-hydroxysulfosuccinimide sodium salt (sulfo-NHS, 12 mg mL ’) were used as adhesives. For Loctite and Periacryl, 20 mg cm-2was applied to the surface of either swollen PAAm / Alg or NiPAAm / Alg hydrogels in PBS, pH 7.4 at 37°C over 3 d and was applied to the porcine skin and compressed for 20 min. For chitosan with EDC and sulfo-NHS, 25 uL cm-2was applied to the surface of either swollen PAAm / Alg hydrogel or NiPAAm / Alg hydrogels in PBS pH 7.4 at 37°C over 3 d and was applied to the porcine skin and compressed for 20 min. Prior to hydrogel application, the tissues were treated with pooled human saliva (200 pL). For Ora- Aid, no adhesives were applied.

[0212] Lap shear tests were conducted with an Instron 3400 tensile testing instrument w ith a 100 N load cell under a uniaxial tension with a rate of 50 mm min1in air at room temperature. A strip of hydrogel (70 mm x 15 mm * 1.5 mm, 1 x w h) was adhered to porcine skin, creating an overlap between the hydrogel and the tissue with an area of 50 mm x 1.5 mm. The back of the hydrogel was glued to a rigid polyethylene terephthalate film (3M) with Krazy Glue to ensure that all work done by the machine would be equal to the energy dissipated at the crack tip. The free ends were attached in between tw o glass plates to which the machine grips were attached. Force-displacement curves were obtained, and lap shear stresses were calculated using Equation 5:Lap shear stress (kPa) = maximum force / bond area Equation 5

[0213] Ex Vivo Evaluation of Adhesion and Removal of PNiPAAm / Alg Hydrogel

[0214] To evaluate the degradable and removable PNiPAAm / Alg hydrogels in an ex vivo context, PNiPAAm / Alg hydrogels were adhered to pork tongue and an aqueous solution of B2(OH)4 was applied. NiPAAm / Alg were cast following the general procedures for hydrogel preparation and swollen in PBS pH 7.4 at 37°C for 3 d. Blue food coloring was added during hydrogel preparation for visualization. Hydrogels were punched into disks (10 mm diameter, 1.0 mm thickness). Three adhesives were employed: 1) Loctite, 2) Periacryl and 3) chitosan +55AFSDOCS:304108277.1Attorney Docket No. 046094-798001 WO Date of Deposit: December 18, 2025EDC and sulfo-NHS. For Loctite and Periacryl, 20 mg cm ' n as applied to the surface of the hydrogel. For chitosan with EDC and sulfo-NHS, 25 pL cur1was applied to the surface of the hydrogels. Pooled human saliva (70 pL) was applied to the pork tongue followed by the hydrogel. The hydrogel was compressed for 20 min. Hydrogel-tissue adducts were then treated with a 500 pM solution of B2.(OH)4 in water in three different ways: 1) submerging the hydrogel / tissue m a solution of fhiOHLto mimic swishing; 2) spraying FhfOHh solution using a spray bottle; or 3) applying a gauze saturated with B2(OH)4 solution.

[0215] In Vivo Maximum Tolerable Dose (MTD) Study ofB2-(OH)4

[0216] All animal experiments were performed according to procedures and protocols approved by the Dana-Farber Cancer Institute Institutional Animal Care and Use Committee (Protocol #20-019). 8-week-old, female, homozygous NU / J mice (Jackson Laboratory) were dosed with aqueous solution of B2(OH)4 via oral gavage using a reusable stainless feeding needle, 20G-1.5" curved. All B?(OH)4 solutions were prepared with autoclaved deionized water and were syringe filtered (UltraCruz™. P VDF 0.22 pm. 13 mm diameter) before oral gavage. Four groups were dosed with different B2(OH)4 concentrations: 1) vehicle (0 mgs kg-1, 0 rnM), 2) low (25 mgs kg-1, 34.3 mM), 3) medium (75 mgs kg-1, 96.2 mM), and 4) high (150 mgs kg-1, 205.8 mM). The highest dosage was near the solubility limit of B2(OH)4 in water. B2(OH)4 was dosed via oral gavage every 24 h for 5 consecutive days. Mice survival, body weight, and food intake were recorded every day for the first 5 d, then every 2-3 d for 9 d. Mice were euthanized at day 14 with CO2 followed by cervical dislocation.

[0217] The swelling property of PAAm / Alg hydrogels we circumvented by switching the covalently linked polymer network to PNiPAAm polymers. This allowed the PNiPAAm / Alg gels to be used in constantly wet and damp biological environments PNiPAAm / Alg hydrogels also demonstrated inducible degradation with fast kinetics. PNiPAAm / Alg hydrogels exhibited use in intraoral hydrogel wound dressings. In vivo MTD studies involving the administration of diboron solution through oral gavage demonstrated the low toxicity of tetrahydroxydiboron.

[0218] Example 7: General Information

[0219] Synthesis Information

[0220] All reactions were conducted in flame-dried round bottom flasks under a positive pressure of nitrogen unless otherwise stated. Gas-tight syringes with stainless steel needles or cannulae 'ere used to transfer air- and moisture-sensitive liquids. Flash column 56AFSDOCS:304108277.1Attorney Docket No. 046094-798001 WO Date of Deposit: December 18, 2025chromatography was performed using granular silica gel (60-A pore size, 40-63 pm, Silicycle). Analytical thin layer chromatography (TLC) was performed using glass plates pre¬ coated with 0.25 mm silica gel impregnated with a fluorescent indicator (254 nm, Silicycle). TLC plates were visualized by exposure to short wave ultraviolet light (254 nm) and / or an aqueous solution of potassium permanganate (KMnO-i). Organic solutions were concentrated at 20 °C on rotary evaporators capable of achieving a minimum pressure of ~2 torr unless otherwise stated. Reaction heating was performed using a UCON fluid heating bath.

[0221] Chemical Materials

[0222] All solvents were purchased from Fisher Scientific or Sigma- Aldrich. Unless otherwise stated chemical reagents were purchased from Fisher Scientific, Sigma-Aldrich, Alfa Aesar, Oakwood Chemical, Acres Organics, Combi-Blocks, TCI America, or Chem- Impex. Low molecular weight (PRONOVA UP VLVG) and high molecular weight (PRONOVA UP MVG) sodium alginates were purchased from Nov aMatrix. CMA refers to a solution of 80: 18:2 v / v / v chloroform: methanol: ammonium hydroxide (28-30% ammonia solution). Chloroform used in CMA solutions and as co-eluents in silica gel column chromatography were stabilized with 0.75% v / v ethanol. Chloroform used in all hydroamination reactions were stabilized with pentene.

[0223] Chemical Instrumentation

[0224] Proton nuclear magnetic resonance ('H NMR) spectra, recorded with a 500 MHz Avance III Spectrometer with multi-nuclear Smart probe, are reported in parts per million on the 5 scale, and are referenced from the residual protium in the NMR solvent (CDCf: 5 7.24, CD3OD: 8 3.31) Data are reported as follows: chemical shift [multiplicity (s = singlet, d = doublet, t::::triplet, q::::quartet, p::::quintet, dd::::doublet of doublets, dt::::doublet of triplets, dq = doublet of quartets, ddd = doublet of doublets of doublets, ft = triplet of triplets, td = triplet of doublets, m = multiplet), coupling constant(s) in Hertz, integration, assignment]. Carbon-13 nuclear magnetic resonance (13C NMR) spectra are referenced from the carbon resonances of the solvent (CDCL: 8 77.23, CD3OD: 6 49.15). Infrared data (IR) were obtained with a Can.7630 Fourier transform infrared spectrometer equipped with a diamond ATR objective and are reported as follows: frequency of absorption (cnr1), intensity of absorption (s = strong, m = medium, w = weak, br = broad). High resolution mass spectra (HRMS) were recorded on a Q Exactive™ Plus Hybrid Quadrupole-Orbitrap™ Mass Spectrometer using an electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), or electron ionization (El) source. Automated C18 reverse phase 57AFSDOCS:304108277.1Attorney Docket No. 046094-798001 WO Date of Deposit: December 18, 2025chromatography was performed using an Isolera One (Biotage) purification system. High performance liquid chromatography (HPLC) purification was performed using an Agilent 1260 Infinity system

[0225] Mechanical Instrumentation

[0226] Tensile, compression, adhesion, and lap-shear forces were measured using Instron Single Column 3400 with a load cell of 100 N. Shear moduli were measured using TA Instruments Discovery HR 20 or 2 rheometer. Dynamic mechanical analysis was performed using Metler Toledo DMA 1

[0227] Example 8: Synthetic Procedures and Compound Characterization

[0228] tert-Butyl (2-(hydroxy(melhyl)amino)ethyl)carbarmte (I)MeNHOH-HC!Et3N, DMSO OHBocHN go °C BGCHN MeS174%1

[0229] A round bottom flask was charged with / e -butyl (2-bromoethyl)carbamate (M. J. Nutt et al., J. Org. Chem., 2023, 88:11968) (SI, 275 g, 123 mmol. 1 equiv) and. V-methyl hydroxylamme hydrochloride (16.3 g, 196 mmol, 1.60 equiv) then purged with nitrogen.. Anhydrous dimethyl sulfoxide (150 mL) and triethylamine (54.8 mL, 392 mmol, 3.20 equiv) were then sequentially added via syringe, and the reaction mixture was heated to 60°C. After 3 h, the mixture was cooled to 0°C using an ice-water bath then diluted with deionized water (150 mL) and saturated aqueous sodium bicarbonate solution (150 mL). The aqueous layer was extracted with ethyl acetate (3 200 mL). The combined organic layers were washed with deionized water (4 x 300 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel (eluent: 20--»40% acetone in hexanes) to give the title compound as a white solid (15.8 g, 74%).NMR (500 MHz, CDClj) 8 5.44 (s, 1H), 3.27 (app s, 2H), 2.64 (I, J= 5.4 Hz, 2H), 2.57 (s, 3H). 1.37 (s, 9H).nC NMR (126 MHz, CDCh) 8 156.7, 79.5, 61.6, 48.8, 38.4, 28.6. FTIR (thin film) cm1: 3362 (br), 2979 (w), 1684 (s), 1513 (m). 1394 (m). 1248 (m), 1170 (s), 1025 (m), 861 (w), 730 (w). HRMS (ESI) ( z): calc’d for CsHis ^Ch [M+H]: 191.1390, found: 191.1394. TLC (20% acetone in hexanes), R 0.23 (KMnO4).

[0230] tert-Butyl-l-prop-2-yn-l-yl ethane-L2-diyldicarbamate (2)58AFSDOCS:304108277.1Attorney Docket No. 046094-798001 WO Date of Deposit: December 18, 2025propargyl chloroformateEtgN, CH2CI2 p;U —!i 93%06 2

[0231] Triethylamine (31.1 mL, 223 mmol, 1.20 equiv) was added via syringe to a solution of te / 7-butyl A-(2-aminoeth l) carbamate (6, 31.2 g, 195 mmol, 1.05 equiv) in dichloromethane (400 mL) The solution w'as cooled to 0°C using an ice-water bath. Propargyl chloroformate (18.1 mL. 186 mmol, 1 equiv) was then added dropwise via syringe. After 15 min, the reaction mixture was warmed to room temperature by removing the ice¬ water bath. After 5 h, die reaction mixture was diluted with saturated aqueous sodium bicarbonate solution (400 mL), and the aqueous layer was extracted with di chloromethane (3 x 300 mL), The combined organic layers were washed with brine (500 mL), dried over sodium sulfate, filtered, and concentrated under reduced pressure. The solid crude mixture was purified by flash column chromatography on silica gel (eluent: 20% ethyl acetate in hexanes) to give the title compound as a white solid (41.8 g, 93%).1H NMR (500 MHz, CDCh) 6 5.55 (t, J= 5.7 Hz, 1H). 5,06 (s, 1H). 4.61 (d, J= 2.5 Hz, 2H). 3.24 (t, J= 5.6 Hz, 2H), 3.19 (d, J = 8.0 Hz, 2H), 2.42 (t, J = 2.5 Hz, 1H), 1.37 (s, 9H).15C NMR (126 MHz, CDCh) 8 156.6, 156.1, 79.7, 78.4, 74.8, 52.6, 41.7, 40.6, 28.5. FTIR (thin film) cm: 3309 (br), 1685 (m), 1525 (m), 1367 (w), 1264 (m), 1144 (m), 1025 (w), 865 (w), 779 (w). HRMS (ESI) (m zy. cak'd for CuHisNzC [M+H]+: 243.1339, found: 243.1342. T EC (20% ethyl acetate in hexanes). R / i 0.21 (KMnOr).

[0232] 3-Bromoprop-2-yn-l-yl tert-butyl ethane- 1,2-diyldicarbamate (8)NBS, AgNO3H acetone H zxJL - *-, Ck BocHN KrUaV? %° 91%02 8

[0233] Acetone (85 mL) was added to a round bottom flask charged with alkyne 2 (4.01 g, 16.6 mmol, 1 equiv) and silver nitrate (281 mg, 1.66 mmol, 0.10 equiv). The solution was then cooled to 0°C using an ice-water bath A-bromosuccinimide (NBS, 3.24 g, 18.2 mmol, 1.10 equiv) was then added. After 1 h, the resulting mixture was concentrated at 4 °C and diluted with saturated aqueous sodium thiosulfate solution (150 mL) and saturated aqueous sodium bicarbonate solution (50 mL). The aqueous layer was extracted with dichloromethane (3 x 200 mL). The combined organic layers were washed with brine (300 mL), dried over 59AFSDOCS:304108277.1Attorney Docket No. 046094-798001 WO Date of Deposit: December 18, 2025sodium sulfate, filtered, and concentrated under reduced pressure. Tire resulting crude mixture was purified by flash column chromatography on silica gel (eluent: 20% ethyl acetate in hexanes) to give the title compound as a white solid bromoalkyne § (4.84 g, 91%).NMR (500 MHz, CDCL,) 5 5.46 (t, J = 5.8 Hz, 1H), 499 (s, 1H), 4.64 (s, 2H), 3.25 (t, J = 5.6 Hz, 2H), 3.22 (app s, 2H), 1.39 (s, 9H).13C NMR (126 MHz, CDCI3) 0 156.7, 156.0, 79.8, 74.9, 53.5, 46.9, 41.8, 40.6, 28.5. FTIR (thm film) cm4: 3314 (br), 2974 (w), 1681 (s), 1547 (m), 1454 (w). 1326 (w), 1263 (m), 1148 (m), 992 (w). HRMS (ESI) (m / z) cak’d for CuHnBrNjC [M+H]: 321.0444, found: 321.0446. TLC (20% ethyl acetate in hexanes), R ’ 0.21 (KMnO4).[00234| Bis-Boc-enamine N-oxide ( 3)”O MeH~Q H BocHN % NHBoc BocHN T NHBoc Br O H? O, 23’C 6976%3[002351 A round bottom flask was charged with bis-Boc-bromoenamine A-oxide 9 (3.27 g, 6.36 mmol, 1 equiv) and purged with nitrogen. Tetrahydrofuran (200 mL) was then added via syringe, the reaction mixture was cooled to -78°C using an acetone-dry ice bath, and n- butyllithium (1.9 M in hexanes. 3.68 mL. 7.00 mmol. 1.10 equiv) was added drop wise via syringe. After 20 min, the reaction was quenched with deionized water (1 mL) and warmed to room temperature by removing the acetone-dry ice bath. The resulting crude mixture was concentrated under reduced pressure and purified by flash column chromatography on silica gel (eluent: 30~*60% CMA m chloroform) to give the title compound as a white foam (2.09 g. 76%). ^I NMR (500 MHz, CD3OD) 8 6.56 (dt, J === 13.3, 5.0 Hz, 1H), 6.49 (d, J === 13 4 Hz, IH), 4.68 (d, J = 4.9 Hz, 2H), 3.61-3 38 (m, 4H), 3.25 (s, 3H), 3 22-2.97 (m, 4H), 1.43 (s, 9H), 1.43 (s, 9H).i3C NMR (126 MHz, CD3OD) 5 157.2, 156.9, 156.7, 140.7, 123.0, 79.1, 78.7, 68.9, 60.6, 58.1, 40.5, 39.8, 34.9, 27.4, 27.3. FTIR (thm film) cm4: 3343 (br), 2974 (w), 1700 (s). 1524 (m). 1415 (m), 1364 (m). 1252 (m), 1167 (s), 1039 (w). 779 (w). HRMS (ESI) H): calc’d for 9H36N4O7 [M+H]+: 433 2657, found: 433.2655. TLC (60% CMA in chloroform), R 0.29 (KMnO4).[002361 Bisacrylamide-enamine N-oxide (ENBAA-1)1. 20% TFA / CH2CI2, 23 C Q 2 W-Acryloxysucdnimide Q 80% (2 steps)3 ENBAA-160AFSDOCS:304108277.1Attorney Docket No. 046094-798001 WO Date of Deposit: December 18, 2025

[0237] Trifluoroacetic acid in dichloromethane (20% v / v, 3 mL) was added to a round bottom flask charged with bis-Boc-enamine rV-oxide 3 (521 mg, 1.21 mmol, 1 equiv) at room temperature. After 3 h, the reaction mixture was concentrated under reduced pressure. To the crude oil was added deionized water (8 mL) followed by sodium bicarbonate (709 mg, 8.44 mmol, 7.00 equiv) portion-wise at room temperature. The flask was covered in foil, and N-acryloxy succinimide (428 mg, 2.53 mmol, 2.10 equiv) was added to the solution. After 1 h, the reaction mixture was purified by automated Cis reverse phase column chromatography (30 g Cis silica gel, 25 pm spherical particles, eluent: H2O + 0.2% Et? N (2 CV), gradient 0— >100% MeCN / EkO + 0.2% EtsN (10 CV) to give the title compound as a white solid (329 mg, 80%, 2 steps). ’H NMR (500 MHz, CD3OD) 86.60 (dt, J = 13.0, 5.0 Hz, 114), 6.56-6.51 (m. 1H), 6.29-6.17 (m, 4H), 5.76-5.63 (m, 2H), 4.70 (dd. J = 5.1, 1.6 Hz, 2H). 3.76-357 (m, 3H), 3.57-3.49 (m. 1H), 3.38 (td, J = 6.1, 3.6 Hz, 2H), 3.30-3.24 (m. 5H).13C NMR (126 MHz, CD3OD) δ 167.0, 166.8, 157.0, 140.6, 130.7, 130.4, 125.7, 125.4, 123.3, 68.6, 60.7, 58.4, 40.1, 38.9, 33.9. FTIR (thin film) cm: 3340 (br), 2974 (w) 1696 (s), 1528 (m), 1453 (m), 1364 (m), 1252 (m), 1170 (m), 998 (w), 779 (w). HRMS (ESI) (m / z): calc'd for C15H24N4O5 [M+H]+: 341.1819, found: 341.1823.

[0238] Bis-Boc-enamine N-oxide (3) and regioisomer (4)O X Me BocHN''3. major OHBocHN Me _. NHBoc BocHN'' NH4. minor

[0239] 2,2,2-Trifluoroethanol (TFE) in chloroform (20% v / v, 20 mL) was added to a round bottom flask charged with hydroxylamine 1 (3.33 g, 17.5 mmol, 2.00 equiv) and alkyne 2 (2.12 g, 8.75 mmol, 1 equiv). The flask was flushed with nitrogen, sealed with a septum cap and Parafilm, and heated to 60°C in an oil bath. After 48 h, the reaction was cooled to room temperature by removing the oil bath and concentrated under reduced pressure. The crude oil was purified by flash column chromatography on silica gel (eluent: 30→60% CMA in chloroform) to give the title compounds as an inseparable mixture of off- white solid with a regioisomeric ratio of 8.3:1 (2.12 g, 56%). Major regioisomer:1H NMR (500 MHz, CD3OD) δ 6.56 (dt, J = 13.1, 5.0 Hz, 1H), 6.50 (d, J = 13.3 Hz, 1H), 4.68 (d, J = 4.9 Hz. 2H), 3.57-3.41 (m, 4H), 3.25 (s, 3H), 3.18-3.13 (m. 4H), 1.43 (s, 18H).13C NMR (126 MHz, CD3OD) δ 157.1, 156.9, 156.7, 140.8, 123.0, 79.0, 78.7, 68.9, 60.6, 58.1, 40.6,61AFSDOCS:304108277.1Attorney Docket No. 046094-798001 WO Date of Deposit: December 18, 202539.8, 34.9, 27.4 (2C). FTIR (thin film) cm’1: 3284 (br), 3094 (br) 2404 (w), 1707 (m), 1655 (s), 1625 (m), 1551 (m). 1453 (s), 1360 (w), 1249 (m), 1159 (w), 988 (w), 776 (w). HRMS (ESI) (m / z): calc’d for CwH Ch [M+H]H: 433.2657. found: 433.2658. TLC (60% CMA in chloroform), Rf: 0.29 (KMnO₄).

[0240] Bisacrylamide-enamine N-oxide (ENBAA-1) and regioisomer (S3) BocHN / s. NHBoc NH1. 20% TFA / CHaCI?, 23 ‘C 3, major 2 W-Acryloxysuccinimsde ENBAA-1 Nai-lCO3, H2O, 23 ’C BocHNx / ~s IIH,UD76% (2 steps)N If NHBoc " <3 Me o4, minor S3

[0241] Trifluoroacetic acid in dichloromethane (20% v / v, 2 mL) was added to a round bottom flask charged with enamine / V-oxides 3 and 4 (212 mg. 0.49 mmol, 1 equiv) at room temperature. After 3 h. the reaction mixture was concentrated under reduced pressure. To the crude oil was added deionized water (3 mL) and sodium bicarbonate (288 mg, 3.43 mmol, 7.00 equiv) portion-wise at room temperature. The flask was covered in foil, and A7-acryloxysuccinimide (171 mg, 1.01 mmol, 2.10 equiv) was added to the solution. After 1 h, the reaction mixture was purified by automated Cis reverse phase column chromatography (30 g Cis silica gel, 25 pm spherical particles, eluent: H2O + 0.2% EtsN (2 CV), gradient 0— > 100% MeCN / TbO + 0.2% EtsN (10 CV) to give the tide compounds as an inseparable mixture with a regioisomeric ratio of 9.1: 1 (127 mg, 76%, 2 steps)NMR (500 MHz, CD3OD) 8 6.60 (dt, J = 13.3, 5.0 Hz, 1H), 6.56-6.50 (m, 1H), 6.31-6.15 (m, 4H), 5.71-5.66 (m, 2H), 4.70 (dd, J - 5.0. 1.5 Hz, 2H), 3.77-3.57 (m, 3H), 3.52 (ddd, J - 11.2. 7.0, 4.6 Hz, 1H), 3.38 (td, J = 5.8, 3.3 Hz, 2H), 3.31-3.22 (m, 5H).!3C NMR (126 MHz, CD3OD) δ 167.0, 166.8, 157.0, 140.6, 130.7, 130.4, 125.7, 125.3, 123.3, 68.6, 60.7, 58.4, 40.1, 38.9, 33.9. FTIR (thin film) cnT: 3273 (br), 3060 (br), 1714 (m), 1662 (s), 1625 (m), 1547 (s), 1409 (m), 1245 (m), 988 (w). FIRMS (ESI) (m / z): calc’d for C15H24N4O5 [M+H]1: 341.1819, found: 341.1824.

[0242] Boc-PEG3-tosylate (S5)1. Boc₂O, Et3NLoi.H2N OTsS4 DMAP, CH2Cl2S523 °C, 80%AFSDOCS:304108277.1Attorney Docket No. 046094-798001 WO Date of Deposit: December 18, 2025

[0243] Triethylamine (6.51 mL, 46.6 mmol, 3.00 equiv) was added via syringe to a solution of amino-PEG4-OH S4 (3.00 g. 15.5 mmol, 1 equiv) in dichloromethane (150 mL) at room temperature. Di-terf-butyl dicarbonate (4.07 g, 18.6 mmol, 1.20 equiv) was then added in one portion and the reaction mixture was flushed with nitrogen and capped with a rubber septum. After 6 h, the mixture was diluted with saturated aqueous sodium bicarbonate solution (250 mL). The aqueous layer was extracted with dichloromethane (3 x 200 mL). The combined organic layers were washed with aqueous hydrochloric acid (1 N, 100 mL) and brine (300 mL). dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude was used in the next step without further purification.

[0244] The crude oil was dissolved in dichloromethane (150 ml). Then, triethylamine (6.51 mL, 46.6 mmol, 3.00 equiv), 4-dimethylaminopyridine (189 mg. 1.55 mmol, 0.10 equiv), and 4-toluenesulfonyl chloride (3.55 g, 18.6 mmol, 1.30 equiv) at room temperature. The reaction flask was flushed with nitrogen and capped with a rubber septum. After 1 h, the mixture w?as diluted with saturated aqueous sodium bicarbonate (250 mL). The aqueous layer was extracted with dichloromethane (3 x 200 mL). The combined organic layers were washed with brine (300 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel (eluent: 10—>40% acetone in hexanes) to give the title compound as a yellow oil (5.56 g, 80%). ¹H NMR (500 MHz, CDCl₃) δ 7.70 (d, J = 8.4 Hz, 2H), 7.26 (d, J = 8.2 Hz, 2H), 4.07 (t, J = 4.9, 2H), 3.60 (t, J = 4.81 Hz, 2H). 3.50 (m, 8H), 3.43 (t, J = 5.2 Hz, 2H), 3.20 (d, J = 5.4 Hz, 2H), 2.35 (s, 3H), 1.34 (s, 9H).13C NMR (126 MHz, CDCh) 5 156.0. 144.8, 133.0, 129.9, 127.9, 79.1, 70.7, 70.5, 70.5, 70.2, 70.1, 69.3, 68.7, 40.3, 28.4, 21.6. FTIR (thin film) cm’1; 2870 (w), 1707 (m), 1509 (w), 1356 (m), 1248 (m), 1174 (s), 1096 (s), 1013 (m), 916 (s), 816 (m). HRMS (ESI) (m / z): calc'd for C20H33NO8S [M+H]+: 448.2000, found: 448.1997. TLC (50% acetone in hexanes), Rf: 0.63 (UV, KMnO₄)

[0245] Boc-PEG5-tosylate (S7)1. Boc₂O, Et3Nt01 CH2CI2, 23 °C r 1 H2N 2. TsCl, Et3N → BocHN OTsS6 DMAP, CH2Cl2S723 °C, 77%

[0246] Triethylamine (4.47 mL, 32.0 mmol. 3 equiv) was added via syringe to a solution of amino-PEG6-OH S6 (3.00 g, 10.7 mmol, 1 equiv) in di chloromethane (100 mL) at room temperature. Di-r<?rLbutyl dicarbonate (3.02 g, 13.9 mmol, 1.30 equiv) was then added in one63AFSDOCS:304108277.1Attorney Docket No. 046094-798001 WO Date of Deposit: December 18, 2025portion and the reaction mixture was flushed with nitrogen and capped with a rubber septum. After 6 h, the mixture was diluted with saturated aqueous sodium bicarbonate solution (200 mL). The aqueous layer was extracted with dichloromethane (3 × 150 mL). The combined organic layers were washed with aqueous hydrochloric acid (1 N, 100 mL) and brine (300 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude was used in the next step without further purification.

[0247] The crude oil was dissolved in dichloromethane (100 ml). Then, triethylamine (4.47 mL. 32.0 mmol, 3 equiv), 4-dimethylaminopyridine (125 mg, 1.06 mmol, 0.10 equiv), and 4-toluenesulfonyl chloride (2.65 g, 13.9 mmol, 1.30 equiv) were added at room temperature. The reaction flask was flushed with nitrogen and capped with a rubber septum. After 1 h, the mixture was diluted with saturated aqueous sodium bicarbonate (200 mL). The aqueous layer -was extracted with di chloromethane (3 x 150 mL). The combined organic layers were washed with brine (300 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel (eluent: 10—»50% acetone in hexanes) to give the title compound as a yellow oil (4.41 g, 77%). ¹H NMR (500 MHz, CDCl₃) δ 7.75 (d, J = 8.3 Hz, 2H), 7.30 (d, J= 8 1 Hz, 2H), 4.12-4 10 (m, 2H), 3.65-3.63 (m, 2H), 3.60 (s, 4H), 3.58 (qd, J = 4.1, 3.5, 1.8 Hz, 10H), 3.54 (s, 4H), 3.49 (t, J = 5.1 Hz, 2H), 3.26 (d, J = 4.9 Hz, 2H), 2.40 (s, 3H), 1.39 (s, 9H).13C NMR (126 MHz, CDCl₃) δ 156.1, 144.9, 133.2, 130.0, 128.1, 70.9, 70.8, 70.7, 70.7. 70.7. 70.4. 70.3. 69.4. 68.8. 40.5, 28.6, 21.8. FTIR (thin film) cm’1: 1707 (in). 1513 (w), 1453 (w), 1358 (m), 1248 (w). 1174 (s), 1096 (s), 1017 (m). 917 (s), 884 (w), 816 (w). HRMS (ESI) (mA): calc’d for C24H4iNO8S [M+H]’: 536.2524, found: 536.2524. TLC (50% acetone in hexanes), Rf: 0.36 (UV, KMnO₄).

[0248] PEG3-hydroxylamine (S8)MeNHOH’HCI Et3N, DMSOBocHN

[0249] A 50 mL round bottom flask was charged with Boc-PEG3-tosylate S5 (2.20 g, 4.91 mmol, 1 equiv) and A-methylhydroxylamine hydrochloride (820 mg, 9.82 mmol, 2.00 equiv) then purged with nitrogen. Anhydrous dimethyl sulfoxide (6 mL) and triethylamine (2.75 mL, 19.6 mmol, 4.00 equiv) were then sequentially added via syringe, and the reaction mixture was heated to 60°C. After 3 h, the mixture was cooled to 0°C using an ice-water bath then diluted with deionized water (3 mL). The crude mixture was purified by automated CisAFSDOCS:304108277.1Attorney Docket No. 046094-798001 WO Date of Deposit: December 18, 2025reverse phase column chromatography (60 g Cis silica gel, 25 pm spherical particles, eluent: H2O + 0.1% TFA (2 CV), gradient 0→100% MeCN / H2O + 0.1% TFA (10 CV). Product containing fractions were concentrated under reduced pressure and purified by flash column chromatography on silica gel (eluent: 10— >30% CMA in chloroform). Product containing fractions were collected, concentrated under reduced pressure, flash frozen with liquid nitrogen, and lyophilized to give the title compound as a white solid (1.27 g, 80%). ¹H NMR (500 MHz, CDCl₃) δ 3.68 (s, 2H), 3.63-3.62 (m, 4H). 3.61-3.59 (m. 2H), 3.58-3.56 (m, 2H), 3.50 (t, J = 5.1 Hz, 2H), 3.28 (q, J = 5.3 Hz, 2H), 2.80 (t, J = 5.3 Hz, 2H), 2.62 (s, 3H), 1.40 (s, 9H).13C NMR (126 MHz, CDCI3) 8 156.3, 79.2, 70.8, 70.6, 70.6, 70.4, 70.3, 69.0, 61.4, 48.9, 40.6, 28.6. FTIR (thin film) cm-1: 3283 (br), 1692 (m), 1521 (w), 1454 (w), 1274 (m), 1249 (m), 1096 (s). 943 (w), 854 (w). HRMS (ESI) (m / z): calc'd for C14H30N2O6[M+H]+: 323.2177, found: 323.2172. TLC (40% acetone in hexanes), Rf: 0.16 (KMnO₄)

[0250] PEG5-hydroxylamine (S9)MeNHOH-HCILo1 Et3N. DMSO BocHN'l ^' OTs - ’S7 60 °C, 83%

[0251] A 50 mL round bottom flask was charged with Boc-PEG5-hydroxylamine S7 (2.07 g, 3.86 mmol, 1 equiv) and N-methylhydroxylamine hydrochloride (646 mg, 7.74 mmol. 2.00 equiv) then purged with nitrogen. Anhydrous dimethyl sulfoxide (5 mL) and triethylamine (2.16 mL, 15.5 mmol, 4.00 equiv) were then sequentially added via syringe, and the reaction mixture was heated to 60°C. After 3 h, the mixture was cooled to 0°C using an ice-water bath then diluted with deionized water (3 mL). The crude mixture was purified by automated Cis reverse phase column chromatography (60 g Ci8 silica gel. 25 pm spherical particles, eluent: H2O + 0.1% TFA (2 CV), gradient 0→100% MeCN / H2O + 0.1% TFA (10 CV). Product containing fractions were concentrated under reduced pressure and purified by flash column chromatography on silica gel (eluent: 10— >30% CMA in chloroform). Product containing fractions were collected, concentrated under reduced pressure, flash frozen with liquid nitrogen, and lyophilized to give the title compound as an off-white solid (1.57 g, 83%). ¹H NMR (500 MHz, CDCl₃) δ 3.69 (t,.7= 5.2 Hz, 2H), 3.63 (d, 7 = 1.5 Hz, 12H), 3.62-3.60 (m, 2H), 3.60-3.58 (m, 2H), 3.51 (t, J = 5.1 Hz, 2H), 3.29 (q, 7 = 5.4 Hz, 2H), 2.83 (t, 7 = 5.3 Hz, 2H), 2.65 (s, 3H), 1.42 (s, 9H).13C NMR (126 MHz, CDCI3) 8 156.3, 70.8, 70.8, 70.8, 70.8, 70.7, 70.5, 70.5, 70.5, 69.0, 61.5, 49.0, 40.6. 28.7. FTIR (thin film) cm: 3241 (br), 1707 (m),AFSDOCS:304108277.1Attorney Docket No. 046094-798001 WO Date of Deposit: December 18, 20251521 (w), 1274 (w), 1249 (m), 1100 (s), 1044 (w). HRMS (ESI) (m'z): cak’d for CisfhsNzOs M-i-Hf: 411.2701, found: 411.2968. TLC (50% acetone in hexanes), Rf: 0.17 (KMnO4).

[0252] Bis-Boc-PEG3-bromoenamine N-oxide (S10)

[0253] 2,2,2-Trifluoroethanol (TFE) in chloroform (20% v / v, 4 mL) was added to an 8 mL vial with hydroxylamine S3 (810 mg, 2.41 mmol, 1 equiv) and bromoalkyne 8 (807 mg, 2.51 mmol, 1 equiv). The vial was flushed with nitrogen, sealed with a septum cap and Parafilm, then heated to 30°C in an oil bath. After 24 h, the reaction was cooled to room temperature and concentrated under reduced pressure. The crude oil was purified by flash column chromatography on silica gel (eluent: 10—40% CMA in chloroform) to give the title compound as an off-white foam (1,41 g, 87%),!H NMR (500 MHz, CD3OD) 5 7.43 (t, J = 5.6 Hz. 1H), 4.75 (d, J = 5.7 Hz, 2H), 3.99-3.94 (m, 1H), 3.86-3.80 (m, 2H). 3.78-3.73 (m, 1H), 3.66-3.59 (m, 8H). 3.52 (t, ■ / 5.6 Hz, 2H), 3.48 (s, 3H), 3.24 (t, J = 5.5 Hz, 2H), 3.17 (dd, J = 13.0, 4.9 Hz, 4H), 1.46 (s, 18H).13C NMR (126 MHz, CD3OD) 5 157.1, 157.0, 156.9, 131.9, 125.4, 78.7, 78.7, 70.2, 70.1, 70.0, 69.9, 69.7, 68.9, 64.2, 62.4, 59.0, 40.5, 39.9, 39.8, 27.4, 27.4. FTIR (thin film) cm'1: 2974 (w), 1700 (s), 1528 (w), 1424 (m). 1364 (m), 1242 (tn), 1163 (s), 1040 iyx ). HRMS (ESI) (m / z) cak'd for C^IUsBrN^jo [M+H]’: 6442627, found: 644.2605 TLC (60% CMA in chloroform), R / : 0.30 (KMnO4).

[0254] Bis-Boc-PEG5-bromoenamine N-oxide (SI I)Br OHS3u8 S11

[0255] 2,2,2-Trifluoroethanol (TFE) in chloroform (20% v / v, 2 mL) was added to a 4 mL vial charged with hydroxylamine S9 (340 mg, 0.82 mmol, 1 equiv) and bromoalkyne 8 (266 mg, 0.82 mmol, I equiv). The vial was flushed with nitrogen, sealed with a septum cap and Parafilm, then heated to 30°C in an oil bath. After 24 h, the reaction was cooled to room temperature and concentrated under reduced pressure. The crude oil was purified by flash column chromatography on silica gel (eluent: 10—50% CMA in chloroform) to give the title compound as a yellow oil (481 mg, 80%). 'HNMR (500 MHz. CD3OD) 6 7.42 (t,.7 = 5,7 Hz, 1H), 4.75 (d..7 = 5.6 Hz. 2H), 3.95 (ddd,.7= 11.7. 8 6, 4.0 Hz. 1H). 3.85-3.80 (m, 2H), 3.78- 3.73 (m, 1H), 3.66 (d, J = 3.9 Hz, 10H), 3.65-3.62 (m. 6H), 3.61-3.57 (m, 2H), 3.53 (t, J = 5.6 Hz. 2H), 3.48 (s, 3H), 3.24 (t, J = 5.6 Hz, 2H), 3.20-3.18 (m, 2H), 3.17-3.14 (m, 2H),66AFSDOCS:304108277.1Attorney Docket No. 046094-798001 WO Date of Deposit: December 18, 20251.46 (s, 18H). ':C NMR (126 MHz, CD3OD) 8 158.7, 158.6, 158.4, 133.5, 127.0, 80.3, 80.2, 71.7, 71.7, 71.7. 71.7. 71.6. 71.4, 71.2, 70.5, 65.7, 64.0, 60.6, 42.1, 41.5, 41.3, 28.9 (2C). FTIR (thin film) cm’1: 2930 (w), 1703 (s), 1532 (w), 1424 (m), 1364 (m), 1163 (s), 1103 (m). HRMS (ESI) ( z): cak’d for CssHseBrTWu [M+H]+: 7323151, found: 732.3122. TLC (60% CMA in chloroform), Rf: 0.29 (KMnO₄).

[0256] Bis-Boc-PEG3-enamine N-oxide (SI 2)THFBr O -78 X; 0,,, NHBOC - * „,,klBocHNqvh N O N H2O 23 °C BocHNq N (j N "’4-O MeH60% ° -O MeHS10 S12

[0257] A round bottom flask was charged with bis-Boc-PEG3-bromoenamine Afoxide S10 (972 mg. 1.51 mmol. I equiv) and purged with nitrogen. Tetrahydrofuran (20 mL) was then added via syringe, the reaction mixture was cooled to -78°C using an acetone-dry ice bath, and n- butyllithium (2.5 M in hexanes, 725 pL, 1.81 mmol, 1.20 equiv) was added dropwise via syringe. After 20 min, the reaction was quenched with deionized water (200 pL) and warmed to room temperature by removing the acetone-dry ice bath. The resulting crude mixture was concentrated under reduced pressure and purified by flash column chromatography on silica gel (eluent: 30-^60% CMA in chloroform) to give the title compound as an off-white oil (531 mg, 60%). ’ll NMR (500 MHz, CD3OD) 8 6.61-6.55 (m, 1H), 6.55-6.45 (m, 1H), 4.69 (d, J = 4.9 Hz, 2H), 3.98 (ddd, J = 12.1, 6.2, 3.2 Hz, 1H). 3.82 (ddd. J = 12.1. 6.5, 3.1 Hz, 1H). 3.63 (dd. J = 9.6, 4.9 Hz, 8H), 3.52 (t, J = 5.6 Hz, 2H). 3.31 (s, 3H), 3.24 (t, J = 5.6 Hz, 2H), 3.18 (m, 6H), 1.46 (s, 18H).13C NMR (126 MHz, CD3OD) 8 157.1, 157.0, 156.9, 146.0, 141.9, 140.5, 122.0, 121.2, 108.4, 70.6, 70.1, 70.0, 69.9, 69.7, 64.4, 60.7, 57.6, 40.5, 39.8, 27.4 (2C). FTIR (thin film) cm'1: 3329 (br), 2974 (w), 1700 (s), 1532 (m), 1433 (w), 1252 (m), 1170 (m). HRMS (ESI) (m / z)'. calc’d for C25H48N4O10 [M+H]1: 565.3443, found: 565.3449. TLC (60% CMA in chloroform), Rf: 0.25 (KMnO₄).

[0258] Bis-Boc-PEG3-enamine N-oxide (SI 3)Br O nBuLi, THF -78 °C; ■O'P" N';^OAN^NHBOCH2O. 23 " C ■’ -o' MeH’ -O MeH83%S11 S13

[0259] A round bottom flask was charged with bis-Boc-PEG5-bromoenamine A-oxide Sil (382 mg, 0.52 mmol, 1 equiv) and purged with nitrogen. Tetrahydrofuran (10 ml) was then added via syringe, the reaction mixture was cooled to -78°C using an acetone-dry ice bath, and w-butyllithium (2.5 M in hexanes, 251 pL, 0.62 mmol, 1.20 equiv) was added dropwise via syringe. After 20 min, the reaction was quenched with deionized water (200 pL) and 67AFSDOCS:304108277.1Attorney Docket No. 046094-798001 WO Date of Deposit: December 18, 2025warmed to room temperature by removing the acetone-dry ice bath. The resulting crude mixture was concentrated under reduced pressure and purified by flash column chromatography on silica gel (eluent: 30— ►60% CMA in chloroform) to give the title compound as a yellow oil (283 mg, 83%). ’H NMR (500 MHz, CD3OD) 3 6.58 (d, J= 12.99 Hz, 1H), 6.55-6.49 (m, 1H), 4.79-4.65 (m, 2H), 3.98 (ddd, J = 12.1, 6.1, 3.2 Hz, 1H), 3.82 (ddd, J = 12.0, 6.5, 3.2 Hz, 1H). 3.66 (q, J - 5.6, 4.4 Hz, 14H), 3.64-3.61 (m, 4H), 3.53 (t, J = 5.6 Hz, 3H), 3.31 (s, 3H), 3.24 (t, J = 5.6 Hz, 3H), 3.17 (tt, J = 13.5, 6.5 Hz, 5H), 1.46 (s, 18H)i3C NMR (126 MHz, CD3OD) 8 157.1, 157.0, 156.9. 141.9, 121 2, 70.7, 70.2, 70.2, 70.1, 70.0, 69.9, 69.9, 69.7, 64.5, 60.7, 57.6, 40.5, 39.9, 39.8, 27.4 (2C). FTIR (thin film) cm’b3317 (br), 1707 (s), 1524 (m), 1453 (w), 1252 (m), 1170 (m), 1111 (m). HRMS (ESI) (ZM'Z): cak’d for C29H56N4O12 [M+H]+: 653.3967, found: 653.3973. TLC (60% CMA in chloroform), Rf: 0.21 (KMnO₄).

[0260] PEG3-bisacrylamide-enamine N-oxide (ENBAA-2)1. 20% TFA / CH2Cl2, 23 °C 2. N-Acryloxysuccinimide NaHCO₃, H₂O, 23 °C NHBoc’O N 69% (2 steps) N'0 Me HENBAA-2

[0261] Trifluoroacetic acid in dichloromethane (20% v / v, 2 mL) was added to a round bottom flask charged with bis-Boc-PEG3-enamine N-oxide S12 (531 mg, 0 82 mmol, 1 equiv) at room temperature. After 2 h, the reaction mixture was concentrated under reduced pressure. To the crude oil was added deionized water (2 mL) followed by sodium bicarbonate (555 mg, 6.61 mmol, 8.00 equiv) portion-wise at room temperature. The flask was covered in foil, and ZV-acryloxysuccinimide (294 mg, 1.73 mmol, 2,10 equiv) was added to the solution. After 1 h, the reaction mixture was purified by automated Cis reverse phase column chromatography (30 g Cis silica gel, 25 pm spherical particles, eluent: H2O H- 0.2% EtsN (2 CV), gradient 0— >100% MeCN / EhO + 0.2% EtsN (10 CV). Product containing fractions were concentrated under reduced pressure and purified by flash column chromatography on silica gel (eluent: 10^30% methanol in dichloromethane, 0.2% EtsN) to give the title compound as an off-white oil (267 mg, 69%, 2 steps). ¹H NMR (500 MHz, CD₃OD) δ 6.58 (dt, J = 13.2, 1.6 Hz, 1H), 6.50 (dt, J = 13.3, 5.3 Hz, 1H), 6.33-6.18 (m, 4H), 5.82-5.35 (m. 2H), 4.69 (dd, J = 5.2, 1.6 Hz. 2H), 3.98 (ddd,.7 = 11.9, 5 9, 2.9 Hz, 1H), 3.82 (ddd, J = 12 1, 6.6, 3.1 Hz, 1H), 3.65 (qd, J= 3.8, 2.3 Hz, 7H), 3.63-3.61 (m, 2H), 3.59 (t, J = 5.5 Hz, 3H), 3.46 (t, J = 5.5 Hz, 2H), 3.38 (t, J = 6.0 Hz, 2H), 3.30 (s, 3H), 3.27 (t, J = 6.2 Hz, 2H).13C NMR (126 MHz, CD₃OD) δ 167.0, 166.8, 156.9, 142.0, 130.6, 125.4, 121.2. 70.6. 70.1, 70.0, 69.9, 69.9,AFSDOCS:304108277.1Attorney Docket No. 046094-798001 WO Date of Deposit: December 18, 202569.1, 64.4, 60.7, 57.6, 46.2, 40.0, 39.0, 38.9, 24.8. FTIR (thin film) cm-1: 3280 (br), 2937 (w), 1714 (m), 1622 (s), 1625 (m), 1550 (m), 1454 (w), 1249 (m), 1118 (m), 992 (m), 961 (w). HRMS (ESI) (ffl'z): calc’d for C2iI-fc6N4O8 [M+H]': 473.2606, found: 473.2607. TLC (30% methanol in dichloromethane, 0.1% Et₃N), Rf: 0.29 (UV, KMnO₄).

[0262] PEG5-bisacrylamide-enamine N-oxide (ENBAA-3)1. 20% TFA / CH2Cl2, 23 °C 2. N-Acryloxysuccinimide NaHCO₃, H₂O, 23 °C BocHNl N O N O Me 71% (2 steps-)S13 ENBAA-3

[0263] Trifluoroacetic acid in dichloromethane (20% v / v, 2 ml.) was added to a round bottom flask charged with bis-Boc-PEG5-enamine / V-oxide S13 (183 mg, 0.28 mmol, 1 equiv) at room temperature.. After 2 h, the reaction mixture was concentrated under reduced pressure. To the crude oil was added deionized water (2 mL) followed by sodium bicarbonate (188 mg, 2.23 mmol, 8.00 equiv) portion-wise at room temperature. The flask was covered in foil, and N-acryloxysuccinimide (99.4 mg, 0.59 mmol, 2.10 equiv) was added to the solution. After 1 h, the reaction mixture was purified by automated Cis reverse phase column chromatography (30 g Cis silica gel, 25 pm spherical particles, eluent: H2O + 0.2% EtsN (2 CV), gradient 0— >100% MeCN / H2O + 0.2% EtjN (10 CV). Product containing fractions were concentrated under reduced pressure and purified by flash column chromatography on silica gel (eluent: 10— >30% methanol in dichloromethane. 0.2% Et? N) to give the title compound as an off-white oil (111 mg, 71 %, 2 steps) ¹H NMR (500 MHz, CD3OD) δ 6.58 (d, J = 13.4 Hz, 1H), 6.50 (dt, J = 13.2, 5.3 Hz, 1H), 6.34-6.17 (m, 4H), 5.78-5.59 (m, 2H), 4.69 (d,.7= 5.3 Hz, 2H), 3.98 (ddd, J = 12.1, 6.2, 3.2 Hz, 1H), 3.82 (ddd, 7 = 12.0, 6.6, 3.1 Hz, 1H), 3.66 - 3.62 (m, 16H), 3.60 (t, J = 5.4 Hz, 4H), 3.46 (t, J = 5.5 Hz, 2H), 3.39 (t, J = 6.2 Hz, 1H). 3.31 (s. 3H), 3.27 (t, 7= 6.2 Hz. 2H).13C NMR (126 MHz, CD3OD) δ 167.0, 166.8, 156.9. 142.0, 130.7, 125.4, 125.3, 121.2, 70.6, 70.2, 70.1, 70.0, 69.9, 69.1, 64.5. 60.7, 57.5, 40.0, 39.1, 38.9. FTIR (thin film) cm-1: 3319 (br), 2870 (w), 1718 (m), 1662 (s), 1625 (m), 1547 (s), 1454 (m), 1349 (w), 1250 (m), 1111 (s), 992 (w), 954 (w). HRMS (ESI) (m / z): calc'd for C25H44N4O10 [M+H]+: 561.3130, found: 561.3134. TLC (30% methanol in dichloromethane, 0.1% EtsN), R / : 0.15 (KMnO4).

[0264] Example 9: Covalent Crosslinker Length Studies

[0265] The mechanical properties of PAAm / Alg hydrogels were investigated to determine the influence of covalent crosslinker length. Hydrogels cast with different crosslinker lengths69AFSDOCS:304108277.1Attorney Docket No. 046094-798001 WO Date of Deposit: December 18, 2025and concentrations were prepared according to the protocol in the general procedures. Table 1 details the concentrations of covalent crosslinkers and the amount used per 10 mL of pre-gel solution.Table 1. Table outlines the molar equivalent, mol, concentration, and volume of covalent crosslinker used to prepare hydrogels with varying crosslinker stoichiometries.H H 0. ENBAA-13, ENBAA-2 I " O n = 5, ENBAA-3MBAA ENBAA Molar equivalent of covalent crosslinker relative to hydrogel cast with MBAA covalent crosslinker Molar equivalent of covalent 0.5x 0.8x l. Ox 1.2x 1.5x 2. Ox 3, Ox crosslinker relative to reportedMBAA hydrogel in 10 mL ofmonomer solutionMol of crosslinker used in 10 2.3 3.7 4.6 5.5 6.9 9.2 13.8 mL of monomer solution(pmol)Concentration of covalent 65 104 130 156 195 260 390 crosslinker (mM)Volume of covalent crosslinker 36 36 36 36 36 36 36 used in 10 mL of monomersolution (pL)

[0266] All patent publications and non-patent publications are indicative of the level of skill of those skilled in the art to which this disclosure pertains. All these publications are herein incorporated by reference to the same extent as if each individual publication were specifically and individually indicated as being incorporated by reference.

[0267] Although the disclosure herein has been described with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of the principles and applications of the present disclosure. It is therefore to be understood that numerous modifications may be made to the illustrative embodiments and that other arrangements may be devised without departing from the spirit and scope of the present disclosure as defined by the appended claims.70AFSDOCS:304108277.1

Claims

Attorney Docket No. 046094-798001 WO Date of Deposit: December 18, 2025What is claimed is;1. A compound having a structure represented by formula I:u Avy, - Rp P Li P N x ‘'oR' (I), or a pharmaceutically acceptable salt or stereoisomer thereof, as a substantially pure regioisomer,wherein:o is 0, 1, or 2;p is 0, 1, or 2;q is 0, 1, or 2;Ri is (Ci-Cs) alkyl, (C3-C10) carbocyclyl, or 4- to 10-membered heterocyclyl comprising 1 to 3 heteroatoms selected from O, N. and S, wherein said alky l, carbocyclyl or heterocyclyl is further optionally substituted;each R2 is independently a polymerizable moiety;Li is absent or a linker;L2 is absent or a linker; andX is a leaving group.

2. A compound having a structure represented by formula I:R2P q q N X" ” / x0-0 R, (I), or a pharmaceutically acceptable salt or stereoisomer thereof, as a substantially pure regioisomer, which is the product of a reaction, comprising:i) reacting a compound of formula II,(II), and a compound ofHformula (III), O1H, to form a compound of formula IV,H / \-° R1 (TV), ii) subjecting the reaction product of i) to a lithium halogen exchange and subsequent protonation,iii) removing the nitrogen protecting groups, and71AFSDOCS:304108277.1Attorney Docket No. 046094-798001 WO Date of Deposit: December 18, 2025iv) acylating the terminal nitrogen atoms,wherein:o is 0, 1, or 2;p is 0, 1, or 2;q is 0, 1, or 2;Ri is (Ci-Cg) alkyl, (C3-C10) carbocyclyl, or 4- to 10-membered heterocyclyl comprising 1 to 3 heteroatoms selected from O, N, and S. wherein said alkyl, carbocyclyl or heterocyclyl is further optionally substituted;each R2 is independently a polymerizable moiety;each R3 is independently a nitrogen protecting group;R4 is chloro, bromo, or iodo;Li is absent or a linker;L2is absent or a linker; andX is a leaving group.

3. The compound of claim 2, wherein X is an ester, a carbonate, a carbamate, a sulfoxide, a sulfonate, a sulfate, a sulfone, a thioester, or a tliionoester.4 The compound of claim 2 or 3, wherein Li is an alkylene chain, which may be interrupted by. and / or terminate (at either or both termini) in at least one of -O-, -S--, - N(R') -, -C-C-, -C(O)--, C(O)O-, -OC(O)--, -OC(O)O--. -C(NOR)--. -C(O)N(R')-, - C(O)N(R')C(O)-, -R'C(O)N(R')R'~, -C(O)N(R')C(O)N(R')-, -N(R')C(O)-, N(R')C(O)N(R)-, -N(R’)C(O)O- -OC(O)N(R')-, -C(NR')~, -N(R')C(NR')-, - C(NR')N(R')-, -N(R')C(NR')N(R')-, -OB(Me)O-, -S(O)2-, -OS(O)-, -S(O)O-, -S(O)-, - OS(O)2-, -S(O)2O-, -N(R')S(O)2~, -S(O)2N(R')-, -N(R')S(O)-, -S(O)N(R’)-, - N(R')S(O)2N(R')-, -N(R')S(O)N(R')-, -OP(O)O(R')O-, -N(R’)P(O)N(R'R )N(R’)-, C3-C12 carbocyclyl, 3- to 12-membered heterocyclyl, 5- to 12-membered heteroaryl or any combination thereof, wherein each R' is independently H or optionally substituted Ci-Ce alkyl, wherein the interrupting and the one or both terminating groups may be the same or different, ora polyethylene glycol chain, which may be interrupted by, and / or terminate (at either or both termini) in at least one of O, --S-, --N(R')-, -C=C-, -C(O) -, -C(O)O-~, --OC(O)-, - OC(O)O-, -C(NOR')-, -C(O)N(R')~. -C(O)N(R')CXO)-, -R'C(O)N(R')R'-. - 72AFSDOCS:304108277.1Attorney Docket No. 046094-798001 WO Date of Deposit: December 18, 2025C(O)N(R')C(O)N(R')-, -N(R')C(0)-, -N(R’)C(0)N(R')-, -N(R')C(O)O- -OC(O)N(R')~, - C(NR')--, -N(R')C(NR')-, -C(NR')N(R')-, N(R')C(NR')N(R') -OB(Me)O-. -S(O)2-, - OS(O)-, -S(O)O- -S(O)-, -OS(O)2- -S(O)2O-, -N(R')S(O)2- -S(O)2N(R')-, -N(R')S(O>-, -S(O)N(R')-, ~N(R,)S(O)2N(R')-, -N(R')S(O)N(R')-, -OP(O)O(R')O- N(R’)P(O)N(R'R’)N(R’)-, C3-C12 carbocyclyl, 3- to 12-membered heterocyclyl, 5- to 12- membered heteroaryl or any combination thereof, wherein each R' is independently H or optionally substituted Ct-Cc, alkyl, wherein the interrupting and the one or both terminating groups may be the same or different.

5. The compound of any one of claims 2-4, wherein L2is an alkylene chain, which may be interrupted by. and / or terminate (at either or both termini) in at least one of -O-, -S-, -N(R’)-, ( (. -C(O)- -C(O)O- -OC(O)-, -OC(O)O-. -C(NOR')-. -C(O)N(R')-, - C(O)N(R')C(O) -, -R'C(O)N(R')R'--, -C(O)N(R')C(O)N(R')-, --N(R')C(O)--, N(R')C(0)N(R!)-, ~N(R')C(O)O-, -OC(O)N(R')-, -C(NR')-, -N(R')C(NR')-. - C(NR')N(R')-, -N(R')C(NR')N(R')-, -OB(Me)O-, -S(O)2- -OS(O)-, -S(O)O-, -S(O)-, -OS(O)2-. -S(O)2O-. -N(R')S(O)2~. -S(O)2N(R')-. -N(R')S(O)-, -S(O)N(R’)-, - N(R')S(O)2N(R')-, -N(R')S(O)N(R’)-. -OP(O)O(R')O -N(R’)P(O)N(R'R’)N(R’)-, C3-C12 carbocyclyl, 3- to 12-membered heterocyclyl, 5- to 12-membered heteroaryl or any combination thereof, wherein each R' is independently H or optionally substituted Ci-Cg alkyl, wherein the interrupting and the one or both terminating groups may be the same or different, ora polyethylene glycol chain, which may be interrupted by, and / or terminate (at either or both termini) in at least one of -O-, -S-, -N(R')--, ~C=C~, -C(O)--, -C(O)O--, ~-OC(O)~, - OC(O)O-, ~C(NOR')“, -C(O)N(R’)-. ~C(O)N(R')C(O)~, -R'C(O)N(R’)R'-. - C(O)N(R')C(O)N(R')-, -N(R')C(O)-, -N(R')C(O)N(R')-, -N(R’)C(O)O- -OC(O)N(R')-. - C(NR')-, -N(R')C(NR')-, -C(NR')N(R')-, -N(R')C(NR')N(R')- -OB(Me)O-, -S(O)2-, -OS(O)~, ~S(O)O~, ~S(O) -, OS(O)22 S(O)2O -, -N(R')S(O)2-, -S(O)2N(R') -, N(R')S(O), -S(O)N(R')--, -N(R!)S(O)2N(R')--, -N(R')S(O)N(R')--, -OP(O)O(R')O-, N(R’)P(O)N(R'R’)N(R’)-. C3-C12 carbocyclyl. 3- to 12-membered heterocyclyl. 5- to 12- membered beteroaryl or any combination thereof, wherein each R' is independently H or optionally substituted Ci-Ce alkyl, wherein the interrupting and the one or both terminating groups may be the same or different.AFSDOCS:304108277.1Attorney Docket No. 046094-798001 WO Date of Deposit: December 18, 20256. The compound of any one of claims 2-5, wherein Ri is (Ci-Cs) alkyl.

7. The compound of claim 2, wherein each R2 is acrylamide.

8. The compound of claim 7, which is of formula lai or Ia2:

10. A hydrogel which is the reaction product of:X" ' ”0 “ / \i) a compound of formula I:(1), as a substantially pure regioisomer,ii) a polymerizable reagent, andiii) an initiator,wherein:o is 0, 1, or 2;p is 0, 1, or 2;q is 0, 1, or 2;74AFSDOCS:304108277.1Attorney Docket No. 046094-798001 WO Date of Deposit: December 18, 2025Ri is (Ci-Cs) alkyl, (C3-C10) carbocyclyl, or 4- to 10-membered heterocyclyl comprising 1 to 3 heteroatoms selected from O, N, and S, wherein said alky l, carbocyclyl or heterocyclyl is further optionally substituted;each R2is independently a polymerizable moiety;Li is absent or a linker;L2 is absent or a linker; andX is a leaving group.

11. The hydrogel of claim 10, wherein X is an ester, a carbonate, a carbamate, a sulfoxide, a sulfonate, a sulfate, a sulfone, a thioester, or a thionoester.

12. The hydrogel of claim 10 or 11. wherein Li is an alkylene chain, which may be interrupted by, and / or terminate (at either or both termini) in at least one of O, -S-, - N(R')~, -G=C- -C(O)-, -C(O)O- -OC(O)-, -OC(O)O- -C(NOR')- -C(O)N(R')-, - C(O)N(R')C(O)-, -R’C(O)N(R')R'-. -C(O)N(R')C(O)N(R')-. -N(R’)C(O)-, N(R’)C(O)N(R')-. -N(R')C(O)O- -OC(O)N(R)-, -C(NR')-, -N(R')C(NR')- - C(NR')N(R')-, -N(R')C(NR')N(R')-. -OB(Me)O-, -S(O)2-, -OS(O)-. -S(O)O-, -S(O)--, - OS(O)2-> -S(O)2O~, -N(R')S(O)2-, -S(O)2N(R')~, -N(R')S(O)-, -S(O)N(R')-, - N(R')S(O)2N(R')-, -N(R')S(O)N(R’)-, -OP(O)O(R’)O-, -N(R’)P(O)N(R'R’)N(R’)-, C3-C12 carbocyclyl, 3- to 12-membered heterocyclyl. 5- to 12-membered heteroaryl or any combination thereof, wherein each R' is independently H or optionally substituted Ci-Ce alkyl, wherein the interrupting and the one or both terminating groups may be the same or different, ora polyethylene glycol chain, which may be interrupted by, and / or terminate (at either or both termini) in at least one of -O-, -S-, -N(R')-, ( C. ~C(O)~, -C(O)O-, -~OC(O)-~, - OC(O)O-, -C(NOR')-, -C(O)N(R')-, -C(O)N(R')C(O)-, -R'C(O)N(R')R'-, - C(O)N(R')C(O)N(R!) -, ~N(R')C(O)~, -N(R')C(O)N(R')~, -N(R')C(O)O~, -OC(O)N(R')~, - C(NR')-, -N(R')C(NR')-, -C(NR')N(R>-, -N(R')C(NR')N(R')-, -OB(Me)O- -. -S(O)2-, - OS(O)-, -S(O)O-. -S(O)-, -OS(O)2~, -S(O)2O-. -N(R')S(O)2-. ~S(O)2N(R')“, -N(R')S(O)~ -S(O)N(R')-. -N(R')S(O)2N(R')-, -N(R,)S(O)N(R’)-, -OP(O)O(R')O- N(R’)P(O)N(R'R')N(R")-, C3-C12 carbocyclyl, 3- to 12-membered heterocyclyl, 5- to 12-membered heteroaryl or any combination thereof, wherein each R' is independently H or75AFSDOCS:304108277.1Attorney Docket No. 046094-798001 WO Date of Deposit: December 18, 2025optionally substituted Ci-Ce alkyl, wherein the interrupting and the one or both terminating groups may be the same or different.

13. The hydrogel of any one of claims 10-12, wherein La is an alkylene chain, which may be interrupted by, and / or terminate (at either or both termini) in at least one of ~O~, ~S~, ~ N(R')-. C=C -C(O)-, C(O)O OC(O)--, -OC(O)O---, -C(NOR') -, -C(O)N(R') - C(O)N(R')C(O)-, -R'C(O)N(R')R'-, -C(O)N(R')C(O)N(R')-, -N(R’)C(O)-, N(R')C(O)N(R')- -N(R')C(O)O-, -OC(O)N(R')-, -C(NR')-, -N(R')C(NR’)- - C(NR')N(R')-, -N(R')C(NR')N(R')--, OB(Me)O -S(O)2--, --OS(O)-, -S(O)O-, --S(O)-, - OS(O)2~, -S(O)2O -, -N(R')S(O)2~, -S(O)2N(R')--, -N(R')S(O)-, -S(O)N(R')-, - N(R')S(O)2N(R')- -N(R')S(O)N(R')-, -OP(O)O(R')O- -N(R )P(O)N(R'R )N(R )-, C3-C12 carbocyclyl. 3- to 12-membered heterocyclyl, 5- to 12-membered heteroaryl or any combination thereof, wherein each R' is independently H or optionally substituted Ci-Ce alkyl, wherein the interrupting and the one or both terminating groups may be the same or different, ora polyethylene glycol chain, which may be interrupted by, and / or terminate (at either or both termini) in at least one of -O-, S. -N(R')--, -C=C-, -C(O)-, -C(O)O~, -OC(O)~, - OC(O)O, C(NOR') -C(O)N(R')-, -C(O)N(R')C(O)~, -R'C(O)N(R')R'-, C(O)N(R')C(O)N(R')- -N(R’)C(O)-, -N(R')C(O)N(R’)-, -N(R')C(O)O-, -OC(O)N(R')-, -C’(NR')-. -N(R')C(NR')- -C(NR')N(R')-, ~N(R')C(NR')N(R')--, ~OB(Me)O~. ~S(O)2~ - OS(O)-, -S(O)O--, --S(O)--. -OS(O)2--, -SCOW-, -N(R')S(O)2--, --S(O)2N(R'}-, -N(R')S(O)~, -S(O)N(R')- -N(R')S(O)2N(R')- -N(R')S(O)N(R')-, -OP(O)O(R')O-, N(R’)P(O)N(R'R,)N(R")--, C3-C12 carbocyclyl, 3- to 12-membered heterocyclyl, 5- to 12- membered heteroaryl or any combination thereof, wherein each R' is independently H or optionally substituted Cj-Cg alkyl, wherein the interrupting and the one or both terminating groups may be the same or different.

14. The hydrogel of any one of claims 10-13, wherein Ri is (Ci-Cs) alkyl.

15. The hydrogel of any one of claims 10-14, wherein each R?. is acrylamide.

16. The hydrogel of any one of claims 10-15, wherein the polymerizable reagent is N,N'-methylenebisacrylamide (MBAA) or an enamine N-oxide bisacrylamide (ENBAA).76AFSDOCS:304108277.1Attorney Docket No. 046094-798001 WO Date of Deposit: December 18, 202517. A process of preparing the hydrogel of any one of claims 10-16. comprising reacting the Q L7^R2R, I \compound of formula 1:-0(I), as a substantially pure regioisomer, a polymerizable reagent, and an initiator,wherein the reacting is carried out in the presence of a solvent.

18. The process of claim 17, wherein the solvent is non-aqueous solvent.

19. The process of claim 17, wherein the solvent is an aqueous sol vent.

20. The process of claim 19, wherein the aqueous solvent is a sodium alginate solution.

21. The process of claim 19, wherein the aqueous solvent is a buffered solvent.

22. The process of claim 21, wherein the buffered solvent is phosphate-buffered saline.

23. The process of claim 22, wherein the pH of the phosphate-buffered saline is about 7.4.

24. A method of degrading the hydrogel of any one of claims 10-16, comprising contacting the hydrogel with a diboron reagent.

25. The method of claim 24. wherein the diboron reagent is a symmetrical diboron reagent.

26. The method of claim 24, wherein the diboron reagent is an unsymmetrical diboron reagent.

27. The method of any one of claims 24-26, wherein the diboron reagent is a solution.

28. A compound of formula IV: (IV).77AFSDOCS:304108277.1Attorney Docket No. 046094-798001 WO Date of Deposit: December 18, 202529. The compound of claim 28, which is:(IVa) or (IVb), wherein n is an integer from 1-6.

30. The compound of claim 29, which is:AFSDOCS:304108277.1

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