Functionalized metathesis polymers

Abstract

A compound of formula (I) as a single-addition monomer for chain-end functionalization of metathesis polymers. Also provided is a polymer of formula (II) formed using the compound of formula (I). Further disclosed are two methods for functionalizing a metathesis polymer that utilize a propagating monomer, a Ru catalyst, and a compound of formula (I) to obtain a functionalized metathesis polymer.

Description

[0001] Docket No. BCOT-012-WO1

[0002] BC2025.016.Niu

[0003] FUNCTIONALIZED METATHESIS POLYMERS

[0004] CROSS-REFERENCE TO RELATED APPLICATIONS This Application claims the benefit of and priority to U.S. Application No. 63 / 789,725, filed on April 16, 2025. The entire teachings of the above application is incorporated herein by reference.

[0005] GOVERNMENT SUPPORT

[0006] This invention was made with government support under CHE-2305566 awarded by the National Science Foundation. The government has certain rights in the invention.

[0007] BACKGROUND

[0008] Functional groups enhance the utility of a polymer by introducing new properties to the polymer backbone. They also enable the construction of more sophisticated molecular architectures.

[0009] Ring-opening metathesis polymerization (ROMP) is a living polymerization technique useful for preparing a polymer with functional groups at its chain end. However, ROMP typically produces an unstable Ru-carbene intermediate at the cochain end that requires chain-end capping / quenching.

[0010] There are two classes of reagents for the functionalization of the co-chain end of metathesis polymers.

[0011] Reagents in the first class convert the Ru-carbene into a less-reactive olefin and cleave the Ru species from the polymer backbone. Examples include internal cisol efins, vinylene carbonates, 37 / -furanone, 2-m ethoxy-3, 4-dihydro-27 / -py ran, linear enol ethers, enynes, diazoacetates, and allyl halides. Nevertheless, with the exception of enynes, reagents of this class often suffer from low chain-end functionalization efficiency and can only install a limited scope of functional groups. Although enynes can achieve efficient chain-end capping, they require lengthy synthesis and result in dead chain ends that cannot be further extended.

[0012] Reagents in the second class are single-addition monomers that react with the Ru-carbene propagating species and form a less reactive Ru-carbene species unable toDocket No. BCOT-012-WO1

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[0014] homo-propagate. Reported single-addition monomers are limited to cyclopropane derivatives, oxanorb ornadiene derivatives, and oxanorbomene imides. They either require costly syntheses or offer poor functional group compatibility.

[0015] The need exists for effective single-addition monomers for chain-end functionalization of metathesis polymers.

[0016] SUMMARY

[0017] Provided to meet the need set out above are compounds of formula (I):

[0018]

[0019] in which Ri is H, C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C3-C20 cycloalkyl, 3-to 20-membered heterocycloalkyl, aryl, C7-C20 aralkyl, heteroaryl, or C2-C20 acyl.

[0020] Also provided are polymers of formula (II):

[0021] O"R2

[0022]

[0023] in which PA contains a first polymer backbone having at least 10 first repeat units; R2 is H, C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C3-C20 cycloalkyl, 3- to 20-membered heterocycloalkyl, aryl, C7-C20 aralkyl, heteroaryl, C2-C20 acyl, or PB; X is Ru, alkylidene, or PC; PB contains a second polymer backbone having at least 5 second repeat units; and PC contains a third polymer backbone having at least 5 third repeat units.

[0024] Further disclosed is a first method for functionalizing a metathesis polymer by providing a mixture that contains a first propagating monomer, a Ru catalyst, and a compound of formula (I) and initiating a polymerization reaction to obtain a functionalized metathesis polymer.

[0025] A second method of functionalizing a metathesis polymer is disclosed by providing a metathesis polymer obtained by polymerizing a first propagating monomer in the presence of a Ru catalyst, adding a compound of formula (I) to the

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[0028] metathesis polymer, and causing functionalization of the metathesis polymer to obtain the functionalized metathesis polymer.

[0029] The details of one or more embodiments of the invention are set forth in the drawings and description below. Other features, objects, and advantages of the invention will be apparent from the description and the claims. All references cited herein are hereby incorporated by reference in their entirety.

[0030] DETAILED DESCRIPTION

[0031] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in art to which the subject matter herein belongs. As used herein, the following definitions are supplied in order to facilitate the understanding of the present invention.

[0032] The term “halo” or “halogen” herein refers to a fluoro, chloro, bromo, or iodo radical. Examples include F, Cl, and Br.

[0033] The term “alkyl” refers to a straight or branched hydrocarbon group, preferably containing 1-20 carbon atoms (e.g., Ci-6 and C1-3) and a monovalent radical center derived by the removal of a hydrogen atom from a carbon atom of a parent alkane. Exemplary alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, iso-butyl, tert-butyl, n-pentyl, and n-hexyl.

[0034] The term “haloalkyl” refers to alkyl substituted with one or more halo atoms. Representative examples of haloalkyl include, but are not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, fluoroethyl (e.g., 1-fluoroetyl and 2-fluoroethyl), difluoroethyl (e.g., 1,1-, 1,2-, and 2,2-difluoroethyl), and trifluoroethyl (e.g., 2,2,2-tri fluoroethyl).

[0035] The term “alkylene” refers to a straight or branched hydrocarbon group, preferably containing 1-20 carbon atoms (e.g., C1-6 and C1-3) and two monovalent radical centers or a bivalent radical center derived by the removal of two hydrogen atoms from one or more carbon atoms of a parent alkane. Exemplary alkylene groups include, but not limited to -CH2-, -CH2CH2-, -CH(CH3)-, -C(CH3)2-, -CH2CH2CH2-, and -CH(CH3)CH2-.

[0036] The term “alkenyl”, as employed herein as such or as part of another group, refers to an aliphatic hydrocarbon group having at least 2 carbon atoms and

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[0039] containing one or several double bonds. Representative examples include, but are not limited to, ethenyl, propenyl, and butenyl.

[0040] The term “alkynyl”, as employed herein as such or as part of another group, refers to an aliphatic hydrocarbon group having at least 2 carbon atoms and containing one or several triple bonds. Representative examples include, but are not limited to, ethynyl, propynyl, and butynyl.

[0041] The term “alkoxy” refers to an -O-alkyl group. Representative examples of Ci-7 alkoxy include, but are not limited to methoxy, ethoxy, propoxy, butoxy, isobutoxy, sec-butoxy, and Ze / 7-butoxy. Alkoxy also includes haloalkoxy, namely, alkoxy substituted with one or more halogens, e.g., -O-CH2CI, -O-CF3, and -O-CHCICH2CI.

[0042] The term “cycloalkyl” refers to a nonaromatic, saturated or partially unsaturated monocyclic, bicyclic, tricyclic or tetracyclic hydrocarbon group preferably containing 3 to 12 carbons (e.g., C3-10 and C3-6). Examples are cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl.

[0043] The term “cycloalkylene” refers to a nonaromatic, saturated or partially unsaturated monocyclic, bicyclic, tricyclic or tetracyclic hydrocarbon group preferably containing 3 to 12 carbons (e.g., C3-10 and C3-6) and two monovalent radical centers or a bivalent radical center derived by the removal of two hydrogen atoms from one or two carbon atoms of a parent cycloalkane. Examples include cyclopropylenes

[0044] * «<vX '■■■■•■ / • . , y> (e.g.,

[0045]

[0046] and ) and cyclobutylenes (e.g., , andx z).

[0047] The term “heterocyclyl” refers to a nonaromatic, saturated or partially unsaturated, 3-8 membered monocyclic, 6-12 membered bicyclic, or 11-14 membered tricyclic ring system having one or more heteroatoms (e.g., O, N, P, and S).

[0048] Heterocyclyl may include monovalent, bivalent, and multiple valent radical centers. Examples include oxycyclobutyl, azacyclobutyl, aziridinyl, azetidinyl, pyrrolidinyl, dihydrofuranyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydrothiophenyl, tetrahydro-2-H-thiopyran- 1,1 -di oxidyl, piperazinyl, piperidinyl, morpholinyl,

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[0051] imidazolidinyl, azepanyl, dihydrothiadiazolyl, dioxanyl, isoindolinyl, 2, 5 -dihydro- 1H-pyrrolyl, imidazolidinyl-2-one, phosphinanyl, 1,2,3,4-tetrahydroisoquinolinyl, 2,7-diazaspiro[3.5]nonanyl, 5,6-dihydro-4H-thieno[2,3-c]pyrrolyl, 5,6-dihydro-4H-pyrrolo[3,4-d]thiazolyl, 4,5,6,7-tetrahydro-2H-pyrazolo[4,3-c]pyridinyl, 4, 5,6,7-tetrahydropyrazolo[l,5-a]pyrazinyl, and quinuclidinyl.

[0052] “Cycloalkyl”, “cycloalkylene” and “heterocyclyl” also include fused, bridged, and spiro ring systems. They further include substituted groups such as halocycloalkyl and haloheterocyclyl.

[0053] The term “aryl” refers to a 6-carbon monocyclic, 10-carbon bicyclic, 14-carbon tricyclic aromatic ring system wherein each ring can have one or more (e.g., 1 to 10, 1 to 5, and 1 to 3) substituents. Aryl may include monovalent, bivalent, or multiple valent radical centers. Examples include phenyl, biphenyl, 1- or 2-naphthyl, 1,2-dihydronaphthyl, 1,2, 3, 4- tetrahydronaphthyl, indenyl, and indanyl.

[0054] The term “aralkyl” refers to alkyl substituted with an aryl group.

[0055] The term "heteroaryl" refers to 5 to 14 membered heteroaromatic system comprising 1 to 4 heteroatoms selected from O, S and N. The heteroaryl is preferably 5 to 10 membered heteroaryl; and more preferably, the heteroaryl is an aromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having one or more heteroatoms (e.g., O, N, P, and S). Heteroaryl may include monovalent, bivalent, or multiple valent radical centers. Examples include pyridinyl, pyrimidinyl, quinolinyl, isoquinolinyl, indolyl, benzimidazolyl, benzoxazolyl, benzothiophenyl, benzofuranyl, pyrazolyl, triazolyl, oxazolyl, thiadi azolyl, tetrazolyl, oxazolyl, isoxazolyl, carbazolyl, furyl, imidazolyl, indazolyl, thienyl, thiazolyl, azaindolyl, azabenzimidazolyl, pyrrolo[l,2-a]pyrazinyl, and benzothiazolyl.

[0056] The term “acyl” refers to -C(O)-R’, in which R’ is alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, aralkyl, and heteroaryl.

[0057] Alkyl, alkylene, alkoxy, cycloalkyl, cycloalkylene, heterocyclyl, aryl, aralkyl, heterocyclylalkyl, and heteroaryl mentioned herein include both substituted and unsubstituted moi eties, unless specified otherwise. Examples of a substituent include deuterium (D), hydroxyl (OH), halogen (e.g., F, Cl, and Br), amino (NH2), oxo (=0), cyano (CN), nitro (NO2), alkyl, alkenyl, alkynyl, alkoxy, cycloalkyl, acylamino,

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[0060] alkylamino, aminoalkyl, haloalkyl (e.g., trifluoromethyl), hydroxyalkyl, heterocyclyl, alkoxycarbonyl, amido, carboxy (COOH), alkanesulfonyl, alkylcarbonyl, alkenylcarbonyl, carbamido, carbamyl, carboxyl, thioureido, thiocyanato, sulfonamido, P(O)2, P(=O)(alkyl)2, aryl, arylamino, aralkyl, and heteroaryl. All substitutes can be further substituted.

[0061] The term “azide” refers to radical -N3.

[0062] The term “succinimido” refers t

[0063]

[0064] o

[0065] The term “maleimido” refers t

[0066]

[0067] o O

[0068] Ru-based catalysts include ruthenium trichloride, the first-generation Grubbs catalyst (synthesized from RuC12(PPh3)3, phenyldiazomethane, and tricyclohexylphosphine), the second-generation Grubbs catalyst (based on N-heterocyclic carbene (NHC) such as saturated N-heterocyclic carbene (l,3-bis(2,4,6-trimethylphenyl)dihydroimidazole) and unsaturated N-heterocyclic carbene (1,3-bis(2,4,6-trimethylphenyl)imidazole)), the first-generation Hovey da-Grubbs catalyst, the second-generation Hovey da-Grubbs catalyst, the secondgeneration Hoveyda-Grubbs catalysts (prepared from the 1st generation Hovey da-Grubbs catalyst by the addition of the NHC), the Zhan catalyst, and the third-generation Grubbs catalyst (based on a ruthenium center and pyridine ligands such as pyridine and 3-bromopyridine).

[0069] As set forth above, a compound of formula (I)

[0070]

[0071] is provided in which Ri is H, C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C3-C20 cycloalkyl, 3- to 20-membered heterocycloalkyl, aryl, C7-C20 aralkyl, heteroaryl, or C2-C20 acyl.

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[0074] In certain embodiments, each of alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, aralkyl, heteroaryl, and acyl, independently, is unsubstituted or substituted with one or more groups selected from D, halo, hydroxyl, amino, CN, azide, succinimido, carboxyl, Ci-Ce alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C2-C6 carboxylate, Ci-Ce alkylamino, C2-C10 dialkylamino, C2-C6 amido, Ci-Ce alkoxy, aryl, heteroaryl, aryloxy, C5-C6 cycloalkyl, or 5- to 6-membered heterocycloalkyl.

[0075] In particular compounds, Ri is acyl represented by formula -C(O)-Ra-LKa-Rb; Ra is Ci-Ce alkylene, phenylene, or benzylene; LKa is absent, -C(O)-, -C(O)O-, or -C(O)NRc, Rc being H or Ci-Ce alkyl; and Rb is H, halo, azide, Ci-Ce alkyl, C2-C6 alkenyl, C2-C6 alkynyl, or succinimido, each of alkylene, phenylene, benzylene, alkyl, alkenyl, alkynyl, independently, being unsubstituted or substituted with a leaving group selected from halo, cyano, azide, succinimido, maleimido, and dibenzocyclooctyne-amino.

[0076]

[0077] Also provided are polymers of formula (II):

[0078] O"R2

[0079]

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[0082] in which PA contains a first polymer backbone having at least 10 first repeat units; R2 is H, C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C3-C20 cycloalkyl, 3- to 20-membered heterocycloalkyl, aryl, C7-C20 aralkyl, heteroaryl, C2-C20 acyl, or PB; X is Ru, alkylidene, or PC; PB contains a second polymer backbone having at least 5 second repeat units; and PC contains a third polymer backbone having at least 5 third repeat units.

[0083] Each of the first, second, and third polymer backbones, independently, can be a norbornene-derived backbone, a norbornene exo-dicarboximide-derived backbone, a PEGylated norbomene exo-dicarboximide-derived backbone, a norbomene N-isopropyl-exo-dicarboximide-derived backbone, a cis-cyclooctene-derived backbone, a 2,3 -dihydrofuran-derived backbone, a 3,4-dihydropyran-derived backbone, an enyne-derived backbone, a diyne-derived backbone, a polystyrene backbone, and a biopolymer backbone.

[0084] Further examples can be found in following reference. Feist and Xia, J. Am. Chem. Soc. 2020, 142, 1186-1189; Feist, Lee, and Xia, Nat. Chem. 2022, 14, 53-58; Tashiro et al., J. Am. Chem. Soc. 2023, 145, 2941-2950; An, Ryu, and Choi, Angew. Chem., Int. Ed. 2023, 62, e202309632; Sui et al., J. Am. Chem. Soc. 2020, 142, 12942-12947; Sui and Gutekunst, ACS Macro Lett. 2022, 11, 630-635; and Pal, Mandal, and Kilbinger, ACS Macro Lett. 2022, 11, 847-853.

[0085] In an embodiment of the polymer, X is alkylidene or PC. In other embodiments, X is CH2 or PC, and each of PA, PB, and PC, independently, has 10 -100,000 repeat units. In a preferred embodiment, each of PA, PB, and PC, independently, has 20 - 50,000 repeat units. In a more preferable embodiments, each of PA, PB, and PC, independently, has 25 - 10,000 repeat units. Further, in an even more preferable embodiment, each of PA, PB, and PC, independently, has 30 - 5,000 repeat units.

[0086] In certain embodiments of the polymer, R2 is PB containing a second polymer backbone that connects to the oxygen atom on the tetrahydrofuran ring through a second linker, and the second linker is C1-C20 alkyl, C3-C20 cycloalkyl, 3- to 20-membered heterocycloalkyl, aryl, C7-C20 aralkyl, heteroaryl, or C2-C20 acyl.

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[0089] As summarized above, within the scope of the invention is a method of functionalizing a metathesis polymer in which the method includes the steps of (la) providing a mixture that contains a first propagating monomer, a Ru catalyst, and a compound of formula (I) and (2a) initiating a polymerization reaction to obtain a functionalized metathesis polymer.

[0090] In the above method, the polymerization can be achieved, e.g., via a ringopening metathesis polymerization.

[0091] Examples of the first propagating monomer are norbornene exo-dicarboximide, PEGylated norbornene exo-dicarboximide, norbomene N-isopropyl-exo-dicarboximide, cis-cyclooctene, and cyclopentene.

[0092] Suitable Ru catalysts include the first-generation Grubbs catalystfl], the second-generation Grubbs catalyst[2], the first-generation Hovey da-Grubbs catalyst[3], the second-generation Hovey da-Grubbs catalyst[4], and the third-generation Grubbs catalyst[5],

[0093] [1] Grubbs, R. H. et al. Angew. Chem. Int. Ed. 1995, 34, 2039-2041.

[0094] [2] Grubbs, R. H. et al. Org. Lett. 1999, 1, 953-956.

[0095] [3] Hoveyda, A. H. et al. J. Am. Chem. Soc. 1999, 121, 791-799.

[0096] [4] Hoveyda, A. H. et al. J. Am. Chem. Soc. 2000, 122, 8168-8179.

[0097] [5] Grubbs, R. H. et al. Angew. Chem. Int. Ed. 2002, 41, 4035-4037.

[0098] Also summarized above is another method of functionalizing a metathesis polymer that includes the steps of (lb) providing a metathesis polymer obtained by polymerizing a first propagating monomer in the presence of a Ru catalyst,

[0099] (2b) adding a compound of formula (I) to the metathesis polymer, and (3b) causing functionalization of the metathesis polymer to obtain a functionalized metathesis polymer.

[0100] Particular embodiments of both of the above methods further include the step of (4) connecting a second or third polymer backbone to the functionalized metathesis polymer.

[0101] The second or third polymer backbone can be connected via a linker reaction. In particular embodiments, the second or third polymer backbone is connected via a chain extension reaction using a second or third propagating monomer.

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[0104] Finally, the second or third polymer backbone can be derived from a biopolymer.

[0105] The polymers of this invention can be prepared by synthetic methods well known in the art. See, e.g., R. Larock, Comprehensive Organic Transformations (3rd Ed., John Wiley and Sons 2018); P. G. M. Wuts and T. W. Greene, Greene’s Protective Groups in Organic Synthesis (4th Ed., John Wiley and Sons 2007); L. Fieser and M. Fieser, Fieser and Fieser’s Reagents for Organic Synthesis (John Wiley and Sons 1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis (2nd ed., John Wiley and Sons 2009) and subsequent editions thereof.

[0106] One aspect of this invention relates to 3,6-anhydrogalactal as a single-addition monomer for chain-end functionalization in metathesis polymers introducing novel aspects and improvements over existing technologies. See Scheme I below.

[0107]

[0108] Disclosed herein is novel 3,6-anhydrogalactal as a bicyclic enol ether monomer in ring-opening metathesis polymerization (ROMP). Unlike other singleaddition monomers such as cyclopropenes or oxanorbomadienes, 3,6-anhydrogalactal features a unique bicyclic structure that provides increased ring strain energy (5.2 kcal / mol compared to 0.5 kcal / mol for simple galactals). Not to be bound by any theory, this structural characteristic promotes efficient ring-opening and leads to the

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[0111] formation of a stabilized Fischer-type ruthenium carbene, inherently preventing homo-propagation and making the process highly selective and efficient.

[0112] Synthesis of 3,6-anhydrogalactal can be achieved by a simple, two-step process starting from inexpensive, biomass-derived D-galactal.

[0113] Another advantage is the ability to introduce a 3,6-anhydrogalactal monomer simultaneously with propagating monomers, such as norbomene derivatives, during ROMP. The simultaneous addition does not interfere with polymerization kinetics or chain growth.

[0114] As a result, the need for sequential monomer addition is eliminated, simplifying the process and making it more amenable to scale-up for industrial applications.

[0115] The 3,6-anhydrogalactal monomers also exhibit broad functional group compatibility, supporting a diverse array of functionalities including acyl, azide, alkyne, and NHS esters. This versatility enables a wide range of applications such as block copolymer synthesis, polymer-polymer coupling, and bioconjugation.

[0116] The monomers provide enhanced reactivity and efficiency, enable rapid and complete chain-end functionalization, and can achieve quantitative end-capping with a small amount (e.g., 1.5 equivalents) of the capping reagent. Not to be bound by any theory, the high efficiency is attributed to the favorable steric and electronic properties of the monomers.

[0117] The monomers and polymerization methods are suitable to construct block copolymers, facilitate polymer coupling via click chemistry, and enable the bioconjugation of proteins such as superfolder green fluorescent protein (sfGFP).

[0118] In addition to cost-effectiveness, sustainability is achieved by using renewable, biomass-derived galactals. The monomers are compatible with standard industrial ROMP processes due to its functional group tolerance and ability to be added simultaneously with other monomers. The monomers enable precise and well-defined chain-end functionality, as confirmed by analytical methods such as MALDITOF mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy.

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[0121] The monomers of this invention are suitable for preparing drug delivery systems utilizing precise bioconjugation of polymers to targeting ligands or antibodies, creating "smart" carriers with stimuli -responsive release mechanisms.

[0122] The monomers of this invention are useful in preparing polymers functionalized with reactive chain ends (e.g., azides) for self-healing materials that repair cracks via click chemistry. Sustainable packaging materials can be prepared from the monomers of this invention, creating biodegradable block copolymers with tunable hydrophobic / hydrophilic segments.

[0123] Without further elaboration, it is believed that one skilled in the art can, based on the disclosure herein, utilize the present invention to its fullest extent. The following specific examples are, therefore, to be construed as merely descriptive, and not limitative of the remainder of the disclosure in any way whatsoever.

[0124] EXAMPLES

[0125] Example 1: Materials and methods

[0126] All manipulations of air- and moisture-sensitive materials were carried out under nitrogen in a glovebox. Compounds I1, 21, Ml2, M23, M33, and SI4were prepared according to literature reports. Grubbs 1stCatalyst, Grubbs 3rdCatalyst and Hovey da-Grubbs Catalyst M721 (HG2-Me) were purchased from Sigma. Deuterated chloroform purchased from Cambridge Isotope Laboratory and stored in a nitrogen gas filled glovebox. Solvents for air sensitive reactions, such as dichloromethane (DCM), tetrahydrofuran (THF), and dimethylformamide (DMF), were purchased from Fisher Scientific (HPLC grade) and used after purification by a dry solvent system (Pure Process Technology). Otherwise, solvents ( / / -hexane, EtOAc, acetone, and MeOH) were used as received. All other chemicals were purchased from Alfa Aesar, Sigma-Aldrich, Acros, Fisher Scientific, or TCI chemical companies and used as received without further purification. DBCO-PEG8-DBCO was purchased from Broadpharm.

[0127] o o H .o. .o. .o. N. N ■o- N ■o- ■o' ■o H - o o

[0128]

[0129] DBCO-PEG8-DBCO

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[0132] 1H,13C, NMR — Spectra were recorded on a Bruker® AVANCE NEO (500 MHz) NMR spectrometer. Chemical shifts (6) for1H and13C NMR spectra were referenced to protons of the residual solvent (for1H) or deuterated solvent itself (for13C).

[0133] High-resolution mass spectrometry (HRMS) — Analyses were performed on JEOL® AccuTOF DART Micromass LCT ESI-MS.

[0134] Matrix-Assisted Laser Desorption / Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) — Analyses were performed on a Bruker® Auto Flex Max instrument (positive mode). a-Cyano-4-hydroxy cinnamic acid was used as matrix, with Nal or AgTFA serving as the cation source. Three stock solutions were prepared in THF: matrix (20 g / L), cation additive (5 g / L), and polymer (10 g / L). In an Eppendorf tube, matrix (15 pL), polymer (10 pL), and cation solution (1 pL) were combined. A small aliquot (0.5 pL) was then applied to the plate for analysis.

[0135] Size -exclusion chromatography (SEC) — Analyses were carried out using a Tosoh’s high performance SEC system HLC-8320GPC with a refractive index detector and a Wyatt® miniDAWN multiangle light scattering (MALS) detector. The Tosoh SEC system was equipped with three TSKgel AlphaM® columns at 50°C and a flow rate of 0.6 mL / min. and calibrated using polystyrene standards (ReadyCal® Kit, Sigma- Aldrich #81434). A procedure developed by Tosoh Bioscience was used to measure the dn / dc value. First, a polystyrene standard solution in DMF (cinj = 1 mg / mL) was run through SEC system with the injection volume Vinj of 100 pL. The integrated area RIarea was detected. The dn / dc value of polystyrene in DMF at 25°C is 0.158 mL / g. Thus, the RI constant KRI for the system was calculated based on the following equation. KRI = RIarea / (cinj • Vinj • dn / dc).

[0136] To measure the dn / dc value of polymer, the analyte solution was run through the SEC system with injection volume Vinj from 25 pL to 100 pL with the interval of 25 pL. SEC elution profiles with different injection volumes were obtained. A plot of RIarea / (KRI • cinj) to Vinj was made and the slope is the dn / dc value. Using this method, the dn / dc values of polyMl, P4, P7, and P12 were measured to be 0.1256, 0.1145, 0.1558, and 0.0755 mL / g, respectively.

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[0139] Liquid chromatography-mass spectrometry (LC-MS) analysis — LC-MS was done on an 1260 Infinity® ESI-TOF, Agilent Technologies, Phenomenex, Aeris™ 3.6 pm WIDEPORE® XB-C8 column, LC Column 100 x 4.6 mm. Separation was done by a flow rate of 0.2 mL / min, and with mobile phase of solvent A (water-MeCN-0.1% trifluoroacetic acid in 95:5:0.1 ratio) and solvent B (MeCN-water-0.1% trifluoroacetic acid in 95: 5: 0.1 ratio); 0-1 min 5% B, 1-8 min 99% B, 8-9 min 99% B to 1% B, 9-10 min 1% B to 5% B, 10-14 min 5% B. Mass deconvolution was performed on Magtran software.

[0140] Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE): — Approximately 2.5 pg of sfGFP were loaded per lane. The samples were heated in SDS-loading buffer for 5 min, then analyzed with 12% SDS-PAGE gel. Proteins were stained with SYPRO™ Orange Protein Stain (Thermo Fisher Scientific) and imaged on the ChemiDoc® MP (BioRad) under Dylight 550.

[0141] Example 2: (2R,3R,4R)-2-((tosyloxy)methyl)-3,4-dihydro-2H-pyran-3,4-diyl diacetate (3)

[0142] / K .CL / K HO || 1. TsCI, pyridine, DCM, 0 - 22 °C, 1.5 h TsO jT |]

[0143] 2. AC2O, 0 - 22 °C, 6 h AcCT y OH OAc

[0144]

[0145] D-Galactal 3

[0146] To a solution of D-galactal (10.0 g, 68.4 mmol) in pyridine (88 mL, 1.09 mol), a solution of 4-methylbenzenesulfonyl chloride (19.6 g, 102.6 mmol) in DCM (100 mL) was added dropwise at 0 °C. The mixture was allowed to stir for 1.5 hours at 22°C, followed by the addition of acetic anhydride (32.2 mL, 342 mmol) at 0°C. The mixture was allowed to stir for 6 hours at 22°C. The mixture was concentrated by blowing air through the flask. The crude product was purified by flash chromatography ( / r-hexane / DCM / acetone = 6:3:2) to afford product 3 as pale-yellow oil. (13.90 g, 36.3 mmol, 53% yield). 'HNMR (500 MHz, CDC13) 87.79 (d, 2H), 7.36 (d, 2H), 6.35 (d, J= 6.3 Hz, 1H), 5.72 - 5.43 (m, 1H), 5.43 - 5.27 (m, 1H), 4.71 (dd, J = 6.4, 3.1, 1H), 4.13 (dd, J= 10.4, 4.3 Hz, 1H), 2.45 (s, 3H), 2.04 (s, 3H), 2.01 (s, 3H).13C NMR (126 MHz, CDCI3) 6 170.07, 169.77, 145.15, 145.14, 132.51, 129.90,

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[0149] 128.02, 98.85, 72.40, 66.67, 63.75, 63.42, 21.65, 20.74, 20.52. HRMS (DART-MS) m / z calculated, for CI7H20O8S [M+NH4]+: 402.1217, found: 402.12136.

[0150] Example 3: (lR,5R,8R)-2,6-dioxabicyclo[3.2.1]oct-3-en-8-ol (4)

[0151] Ambersep 900 ion exchange resin

[0152]

[0153] Methanol, 22 °C, 1 h

[0154]

[0155]

[0156]

[0157] To a solution of 3 (0.390 g, 1.01 mmol) in methanol (10 mL), Ambersep 900 ion exchange resin (0.8 g) was added. The mixture was allowed to stir at 22°C for 1 hour. The mixture was concentrated under vacuum. The crude product was purified by flash chromatography ( / r-hexane / DCM / acetone = 4:4:2) to afford product 4 as pale-yellow oil. (0.108 g, 0.84 mmol, 83% yield). 'H NMR (600 MHz, CDC13) 86.25 (d, J= 5.6 Hz, 1H), 4.95 (d, J= 5.5 Hz, 1H), 4.52 (d, J= 8.0 Hz, 1H), 4.45 - 4.39 (m, 1H), 4.26 (d, J = 10.7 Hz, 1H), 4.18 (dd, J = 10.9, 4.1 Hz, 1H), 4.11 (d, J = 6.2 Hz, 1H), 1.89 (dd, J= 8.0, 1.1 Hz, 1H).13C NMR (126 MHz, CDCI3) 6 145.23, 101.35, 79.27, 73.63, 73.27, 71.52. HRMS (DART-MS) m / z calculated, for C6H8O3[M+H-H2O]+: 111.0440, found: 111.04409.

[0158] Example 4: (lR,5R,8R)-8-methoxy-2,6-dioxabicyclo[3.2.1]oct-3-ene (5)

[0159] 1. NaH, THF, 0 °C, 30 min

[0160] 2. Mel, 22 °C, 1 h

[0161]

[0162] 5

[0163] 4

[0164] To a solution of 4 (0.63 g, 4.43 mmol) in anhydrous THF (21 mL), sodium hydride (132 mg, 5.54 mmol) was added at 0°C under N2 atmosphere. The mixture was allowed to stir at 0°C for 20 minutes, followed by the addition of methyl iodide (0.723 g, 5.1 mmol). The mixture was allowed to stir at 22°C for 3 hours, and subsequently concentrated under vacuum. The crude product was purified by flash chromatography ( / 7-hexane / ethyl acetate = 3:1) to afford product 5 as pale-yellow oil. (0.480 g, 3.07 mmol, 70% yield). 'H NMR (500 MHz, CDCI3) 66.27 (d, J= 5.4 Hz, 1H), 5.00 (dd, J= 6.3, 5.4 Hz, 1H), 4.49 (d, J= 4.1 Hz, 1H), 4.33 (d, J= 6.3 Hz, 1H),

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[0167] 4.27 (d, J= 10.6 Hz, 1H), 4.21 (d, J= 1.5 Hz, 1H), 4.17 (dd, J= 10.6, 4.1 Hz, 1H), 3.46 (s, 3H).13C NMR (126 MHz, CDCI3) 6 145.18, 101.98, 79.84, 77.52, 74.11, 70.47, 56.29. HRMS (DART-MS) m / z calculated, for C7H10O3 [M+H]+: 143.0703, found: 143.07075.

[0168] Example 5: (lR,5R,8R)-2,6-dioxabicyclo[3.2.1]oct-3-en-8-yl (2,5-dioxopyrrolidin-l-yl) succinate (6)

[0169] 1. Succinic anhydride, THF, 22 °C, 12 h d o-

[0170]

[0171] 2. N-hydroxysuccinimide, DCC, THF, 22 °C, 12 h

[0172]

[0173] O u

[0174]

[0175] 6 To a solution of 4 (0.2 g, 1.56 mmol) and triethylamine (157 mg, 1.56 mmol) in anhydrous THF (1.34 mL), succinic anhydride (195 mg, 1.95 mmol) was added at 22 °C under N2 atmosphere. The mixture was allowed to stir at 22°C for 12 hours. The mixture was concentrated under vacuum to afford white solid which was dissolved in anhydrous THF (3.59 mL). TV, TV' -Dicyclohexylcarbodiimide (326 mg, 1.58 mmol) and TV-hydroxy succinimide (182 mg, 1.58 mmol) were added at 22°C and the mixture was allowed to stir at 22°C for 12 hours. The reaction was diluted with toluene (5 mL) and filtered by passing through celite pad. The filtrate was concentrated under vacuum and purified by flash chromatography ( / r-hexane / acetone = 2:1) to afford 6 as off-white solid. (0.310 g, 0.95 mmol, 61% yield). 'H NMR (500 MHz, CDCI3) 86.28 (d, J= 5.4 Hz, 1H), 5.54 - 5.43 (m, 2H), 5.02 (dd, J= 6.4, 5.5 Hz, 1H), 4.57 (d, J= 4.0 Hz, 4H), 4.34 (d, J= 6.3 Hz, 1H), 4.30 (d, J= 10.8 Hz, 1H), 4.18 (dd, J= 10.8, 4.1 Hz, 1H), 2.97 (d, J= 6.8 Hz, 2H), 2.88 - 2.75 (m, 5H).13C NMR (126 MHz, CDCI3) 6 170.23, 168.87, 167.56, 145.18, 101.90, 77.16, 74.70, 74.22, 71.79, 28.76, 26.25, 25.58. HRMS (DART-MS) m / z calculated, for CuHisNOs [M+H]+: 326.0870, found: 326.08735.

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[0178] Example 6: (lR,5R,8R)-2,6-dioxabicyclo[3.2.1]oct-3-en-8-yl 4-bromobutanoate (7)

[0179] 4-Bromobutyryl chloride

[0180] Pyridine

[0181]

[0182] DCM, 0 -22 °C, 1 h

[0183]

[0184]

[0185] To a solution of 4 (0.28 g, 2.19 mmol) and pyridine (276 mg, 3.5 mmol) in anhydrous DCM (5 mL), 4-bromobutyryl chloride (445 mg, 2.4 mmol) was added dropwise at 0°C under N2 atmosphere. The mixture was allowed to stir at 22°C for 1 hour and was subsequently concentrated under vacuum. The crude product was purified by flash chromatography ( / 7-hexane / acetone = 4:1) to afford product 7 as pale-yellow oil. (0.491 g, 1.77 mmol, 82% yield). 'H NMR (500 MHz, CDCI3) 86.28 (d, J= 5.5 Hz, 1H), 5.52 - 5.41 (m, 1H), 5.11 -4.99 (m, 1H), 4.53 (d, J= 4.0 Hz, 1H), 4.40 - 4.28 (m, 2H), 4.17 (dd, J = 10.7, 4.1 Hz, 1H), 3.47 (d, J= 6.4 Hz, 2H), 2.57 (d, J= 7.3 Hz, 2H), 2.18 (t, J= 7.3, 6.4 Hz, 2H).13C NMR (126 MHz, CDCI3) 6 171.75, 145.13, 101.97, 74.18, 74.15, 71.84, 32.42, 32.40, 27.52. HRMS (DART-MS) m / z calculated, for CioHi3Br04[M+H]+: 277.0070, found: 277.00747.

[0186] Example 7: (lR,5R,8R)-2,6-dioxabicyclo[3.2.1]oct-3-en-8-yl 4-(((2-bromo-2-methylpropanoyl)oxy)methyl) benzoate (8)

[0187]

[0188] To a solution of 4 (0.15 g, 1.17 mmol), 4-(((tert-butyldimethylsilyl)oxy) methyl)benzoic acid (SI, 343 mg, 1.29 mmol), and 7V-(3-dimethylaminopropyl)-7V'-ethylcarbodiimide hydrochloride (EDC HC1, 269 mg, 1.4 mmol) in anhydrous DCM (5.5 mL), 4-(dimethylamino)pyridine (14.3 mg, 0.12 mmol) was added dropwise at 22°C under N2 atmosphere. The mixture was allowed to stir at 22°C for 12 hours and

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[0191] was subsequently concentrated under vacuum. The crude product was purified by flash chromatography (w-hexane / acetone = 4:1) to afford product S2 as white solid. (0.337 g, 0.90 mmol, 76% yield). 'H NMR (500 MHz, CDC13) 88.00 (d, J= 8.3 Hz, 2H), 7.40 (d, J= 8.4 Hz, 2H), 6.33 (d, J= 5.4 Hz, 1H), 5.69 (s, 1H), 5.16 - 5.02 (m, 1H), 4.79 (s, 2H), 4.67 (d, J= 4.1 Hz, 1H), 4.45 (d, J= 6.3 Hz, 1H), 4.38 (d, J= 10.7 Hz, 1H), 4.28 (dd, J= 10.7, 4.1 Hz, 1H), 0.95 (s, 9H), 0.10 (s, 6H).13C NMR (126 MHz, CDCI3) 6 165.63, 147.46, 145.17, 129.81, 128.00, 125.71, 102.18, 77.51, 74.36, 74.28, 71.98, 64.45, 25.88, 18.37, -5.31. HRMS (DART-MS) m / z calculated, for C2oH2805Si [M+H]+: 377.1784, found: 377.17727.

[0192] To a solution of S2 (0.3 g, 0.8 mmol) in anhydrous THF (3 mL), tetrabutyl ammonium fluoride (218 mg, 0.84 mmol) was added at 22°C. The mixture was allowed to stir at 22°C for 1 hour. The mixture was concentrated under vacuum. The crude was purified by flash chromatography ( / r-hexane / acetone = 2: 1) to afford product S3 as white solid. (0.2 g, 0.76 mmol, 95% yield). To a solution of S3 (0.165 g, 0.63 mmol) and pyridine (65 mg, 0.82 mmol) in anhydrous THF (5 mL), a-bromoisobutyryl bromide (159 mg, 0.69 mmol) was added dropwise at 0 °C under N2 atmosphere. The mixture was allowed to stir at 22 °C for 1 hour and was subsequently concentrated under vacuum. The crude product was purified by flash chromatography (w-hexane / acetone = 4:1) to afford product 7 as pale-yellow oil. (0.122 g, 0.3 mmol, 48% yield).XH NMR (500 MHz, CDCI3) 68.04 (d, J= 8.4 Hz, 2H), 7.46 (d, J= 8.5 Hz, 2H), 6.33 (d, J= 5.5 Hz, 1H), 5.72 - 5.66 (m, 1H), 5.10 (dd, J= 6.2, 5.5 Hz, 1H), 4.67 (d, J= 4.1 Hz, 1H), 4.45 (d, J= 6.3 Hz, 1H), 4.38 (d, J= 10.8 Hz, 1H), 4.27 (dd, J= 10.8, 4.1 Hz, 1H), 1.96 (s, 6H).13C NMR (126 MHz, CDCI3) 8 171.31, 165.25, 145.20, 141.02, 130.13, 129.34, 127.49, 102.12, 77.44, 74.50, 74.33, 71.95, 66.64, 55.34, 30.72. HRMS (DART-MS) m / z calculated, for Ci8Hi9BrO6[M+H]+: 411.0438, found: 411.04473.

[0193] Example 8: Representative procedure for sequential addition method

[0194] To a solution of Ml (10 mg, 56.4 pmol) in CDCI3 (0.25 mL), a solution of G3 (6.24 mg, 7.05 pmol) in CDCI3 (312 pL) was added at 22°C in a glovebox filled with nitrogen. The mixture was allowed to stir at 22°C for 5 minutes, followed by the addition of 5 (1.5 mg, 10.58 pmol). The mixture was allowed to stir in a preheated oil

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[0197] bath at 45°C for 2 hours. An aliquot (15 pL) of the mixture was diluted with CDCh under nitrogen atmosphere and subjected to NMR analysis. The reaction was quenched by the addition of ethyl vinyl ether (50 pL) to the rest of the mixture and the mixture was allowed to stir for 10 minutes. The mixture was added to methanol (10 mL) and off-white solid was precipitated through centrifuging at 5000 rpm for 10 minutes. The solid was dissolved in CHCI3, precipitated again in methanol and dried under vacuum, affording off-white solid (8.0 mg, yield 80%).

[0198] Example 9: Representative procedure for one-pot method

[0199] To a solution of Ml (10 mg, 56.4 pmol) and 5 (1.5 mg, 10.58 pmol) in CDCI3 (0.25 mL), a solution of G3 (6.24 mg, 7.05 pmol) in CDCI3 (312 pL) was added at 22°C in a glovebox filled with nitrogen. The vial was immediately put into a preheated oil bath at 45°C and the mixture was allowed to stir at this temperature for 2 hours. An aliquot (15 pL) of the mixture was diluted with CDCI3 under nitrogen atmosphere and subjected to NMR analysis. The reaction was quenched by the addition of ethyl vinyl ether (50 pL) to the rest of the mixture and the mixture was allowed to stir for 10 minutes. The mixture was added to methanol (10 mL) and off-white solid was precipitated through centrifuging at 5000 rpm for 10 minutes. The solid was dissolved in CHCI3, precipitated again in methanol and dried under vacuum, affording off-white solid (8.1 mg, yield 81%). P5, P6, and P7 were prepared under identical conditions, with the only variation being the use of different monomers or bicyclic enol ethers.

[0200] Example 10: Representative Initiation tests with different Grubbs-type catalysts To a solution of 2 (10 mg, 70.4 pmol) in CDCI3 (100 pL), a solution of G3 (12.4 mg, 14.1 pmol) in CDCI3 (420 pL) was added at 22°C in a glovebox filled with nitrogen. The mixture was transferred into an air-tight NMR tube and was subjected to NMR analysis at varying times.

[0201] Example 11: Chain extension ofP2 by M3 (PSI)

[0202] To a solution of Ml (5 mg, 28.3 pmol) and 5 (0.7 mg, 5.3 pmol) in CDCI3 (0.125 mL), a solution of G3 (3.1 mg, 3.5 pmol) in CDCI3 (156 pL) was added at 22°C in a glovebox filled with nitrogen. The vial was immediately put into a preheated oil bath at 45°C and the mixture was allowed to stir at this temperature for 2

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[0205] hours. A solution of M3 (23.2 mg, 112.9 pmol) in anhydrous THF (0.45 mL) was added to this mixture at 22°C in a glovebox filled with nitrogen. The mixture was allowed to stir at 22°C for 4 hours. An aliquot (15 pL) of the mixture was diluted with CDCh under nitrogen atmosphere and subjected to NMR analysis. The reaction was quenched by the addition of ethyl vinyl ether (50 pL) to the rest of the mixture and the mixture was allowed to stir for 10 minutes. A small portion of the mixture was analyzed by NMR, revealing full conversion of M3. The mixture was added to methanol (10 mL) and off-white solid was precipitated through centrifuging at 5000 rpm for 10 minutes. The solid was dissolved in CHCI3, precipitated again in methanol and dried under vacuum, affording off-white solid (20 mg, yield 79%).

[0206] Example 12: Chain extension ofP2 by DHF and M3 (P4)

[0207] To a solution of Ml (10 mg, 56.4 pmol) and 5 (1.5 mg, 10.58 pmol) in CDCI3 (0.25 mL), a solution of G3 (6.24 mg, 7.05 pmol) in CDCI3 (312 pL) was added at 22°C in a glovebox filled with nitrogen. The vial was immediately put into a preheated oil bath at 45°C and the mixture was allowed to stir at this temperature for 2 hours. A solution of DHF (31.6 mg, 451.4 pmol) and M3 (46.3 mg, 225.7 pmol) in anhydrous THF (0.9 mL) was added to this mixture at 22°C in a glovebox filled with nitrogen. The mixture was allowed to stir at 22°C for 4 hours. An aliquot (15 pL) of the mixture was diluted with CDCI3 under nitrogen atmosphere and subjected to NMR analysis. The reaction was quenched by the addition of ethyl vinyl ether (50 pL) to the rest of the mixture and the mixture was allowed to stir for 10 minutes. A small portion of the mixture was analyzed by NMR, revealing an 81% conversion of M3. The mixture was added to methanol (10 mL) and off-white solid was precipitated through centrifuging at 5000 rpm for 10 minutes. The solid was dissolved in CHCI3, precipitated again in methanol and dried under vacuum, affording off-white solid (23 mg, yield 65%).

[0208] Example 13: Degradation ofP4 under acidic condition (P4-degraded)

[0209] To a solution of P4 (13 mg, 1.81 pmol) in THF (200 pL), HC1 aqueous solution (I M, 11 pL) was added at 22°C. The mixture was allowed to stir at 22°C for 12 hours. The mixture was added to methanol (10 mL) and off-white solid was precipitated through centrifuging at 5000 rpm for 10 minutes. The solid was dissolved

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[0212] in CHCh, precipitated again in methanol and dried under vacuum, affording off-white solid (3 mg, yield 71%).

[0213] Example 14: Chain extension through ATRP of styrene (P7)

[0214] A solution of P7 (10 mg, 4.4 pmol), styrene (91.1 mg, 874.4 pmol) and PMDETA (1.52 mg, 8.74 pmol) was degassed by freeze-pump-thaw and backfilled with nitrogen. To the frozen mixture, CuBr (0.63 mg, 4.4 pmol) was added under nitrogen flow to the vial. The mixture was evacuated-backfilled with nitrogen twice. The mixture was thawed and placed into a preheated oil bath at 110°C. The reaction was allowed to stir at 110°C for 4 hours and was subsequently allowed to cool to 22°C. A small portion of the mixture was analyzed by NMR, revealing 31% conversion of styrene. The mixture was added to / / -hexane (10 mL) and off-white solid was precipitated through centrifuging at 5000 rpm for 10 minutes. The solid was dissolved in CHCh, precipitated again in / / -hexane and dried under vacuum, affording off-white solid (32.2 mg, yield 83%).

[0215] Example 15: Post-polymerization transformation (P8 andP9)

[0216] Synthesis of P8: To a solution of P5 (16.8 mg, 7.2 pmol) in DMF (140 pL), sodium azide (9.4 mg, 144.2 pmol) was added at 22°C. The mixture was allowed to stir at 22°C for 12 hours. The mixture was filtered with syringe filter (PTFE, 0.22 pm) and added to methanol (10 mL) and off-white solid was precipitated through centrifuging at 5000 rpm for 10 minutes. The solid was dissolved in CHCI3, precipitated again in methanol and dried under vacuum, affording off-white solid (12.0 mg, yield 72%).

[0217] Synthesis of P9: To a solution of P6 (12.5 mg, 4.3 pmol) in DMF (100 pL), propargylamine (473 pg, 0.55 pL, 8.6 pmol) was added at 22°C. The mixture was allowed to stir at 22°C for 12 hours and was subsequently added to methanol (10 mL) and off-white solid was precipitated through centrifuging at 5000 rpm for 10 minutes. The solid was dissolved in CHCI3, precipitated again in methanol and dried under vacuum, affording off-white solid (11.2 mg, yield 91%).

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[0220] Example 16: Polymer -polymer conjugation through CuAAC (P 11)

[0221] To a solution of P8 (2.27 mg, 0.76 pmol) and P9 (2.16 mg, 0.76 pmol) in anhydrous THF (100 pL), a solution of CuBr (0.22 mg, 1.5 pmol), sodium ascorbate (0.75 mg, 3.8 pmol), and TV, N, N N", 7V''-pentamethyldiethylenetriamine (PMDETA) (0.26 mg, 1.5 pmol) in anhydrous THF (67 pL) was added at 22°C under nitrogen atmosphere. The mixture was allowed to stir at 22°C for 12 hours and was subsequently added to methanol (10 mL) and off-white solid was precipitated through centrifuging at 5000 rpm for 10 minutes. The solid was dissolved in CHCh, precipitated again in methanol and dried under vacuum, affording off-white solid (3.6 mg, yield 81%).

[0222] Example 17: Preparation of water-soluble Pl 3

[0223] Synthesis of P12: To a solution of M2 (44.2 mg, 0.1 mmol) and 7 (5.2 mg, 18.8 pmol) in CDCI3 (225 pL), a solution of G3 (11.1 mg, 12.5 pmol) in CDCI3 (275 pL) was added at 22°C in a glovebox filled with nitrogen. The vial was immediately put into a preheated oil bath at 45°C and the mixture was allowed to stir at this temperature for 2 hours. The reaction was quenched by the addition of ethyl vinyl ether (50 pL) to the rest of the mixture and the mixture was allowed to stir for 10 minutes. The mixture was diluted with methanol (0.5 mL), precipitated in n-hexane / ethyl ether (10 mL, v / v 4: 1) and brown solid was precipitated through centrifuging at 5000 rpm for 10 minutes. The solid was dissolved in CHCI3, precipitated again in w-hexane / ethyl ether (10 mL, v / v 4: 1) and dried under vacuum, affording brown solid (37.8 mg, yield 77%).

[0224] Synthesis of P13: To a solution of P12 (23 mg, 4.80 pmol) in DMF (400 pL), sodium azide (6.3 mg, 95.9 pmol) was added at 22°C. The mixture was allowed to stir at 22 °C for 12 hours and was subsequently filtered with syringe filter (PTFE, 0.22 pm) and diluted with methanol (0.5 mL). The mixture was precipitated in n-hexane / ethyl ether (10 mL, v / v 4: 1) and brown solid was precipitated through centrifuging at 5000 rpm for 10 minutes. The solid was dissolved in CHCI3, precipitated again in w-hexane / ethyl ether (10 mL, v / v 4: 1) and dried under vacuum, affording brown solid (20.3 mg, yield 70%).

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[0227] Example 18: Protein expression for sfGFP-151-AzK:

[0228] sfGFP-151-AzK expression was done using previously described methods. In brief, plasmid containing sfGFP-151-TAG gene and plasmid containing an Methanosarcina mazei pyrrolysyl-tRNA synthetase / tRNA pair were co-transformed into E. coli BL21 cells. Protein expression was induced with 0.5 mM IPTG in LB supplemented with 1 mM A6-((2-azidoethoxy)carbonyl)-L-lysine (AzK) at OD600 ~ 0.6 at 22°C for 16 hours. Cells were lysed with B-PER™ Bacterial Protein Extraction Reagent (Thermo Scientific) and proteins were purified by Ni-NTA (Thermo Scientific) affinity chromatography.

[0229] Example 19: Protein-polymer bioconjugation:

[0230] 200 pM sfGFP-151AzK and 10 mM DBCO-PEG8-DBCO (Broadpharm) were diluted in DMSO / DPBS (1 :3) (pH = 7.4) to a final concentration of 5 pM and 250 pM, respectively. Conjugations were done in 250 pL scale in 0.6 mL tubes by nutating the mixtures at 22°C for overnight. The DBCO-labeled sfGFP, sfGFP-151-DBCO, was buffer exchanged in DPBS using an Amicon 10 kDa molecular weight cut-off centrifugal filter device to remove excess DBCO probes. 5 mM N3 -Polymer in water was diluted in 25 pM DBCO-prelabeled sfGFP to the final concentration of 250 pM. Conjugations were done in 50 pL scale in 0.6 mL tubes by nutating the mixtures at 22°C for overnight.

[0231] Results

[0232] First, a 3,6-anhydro-D-glucal (2) was prepared from 3,4-di-O-acetyl-6-O-toluene- / ?-sulphonyl-D-glucal (1) in the presence of basic OH resin. Intermediate 1 could be readily synthesized starting from inexpensive D-glucal through tandem deacetylation and cyclization (See Scheme II below). Following the same route, 3,6-anhydro-D-galactal (4), a geometric isomer of 2, was also prepared in high yield (83%) (Scheme II).

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[0235] (i) TsCI, Pyridine AMBERJET 4400

[0236] (ii) AC2O, Pyridine OH resin 50% 75% (i) TsCI, Pyridine AMBERJET 4400 (ii) AC2O, Pyridine OH resin 53% 83%

[0237]

[0238] Scheme II

[0239] The metathesis reactivity of 2 and 4 was found to differ significantly. Nearly no initiation was observed when 2 was treated with Grubbs first-generation catalyst (Gl) at room temperature (R.T.), or Hovey da-Grubbs second-generation catalyst (HG2), at 50°C more than 24 hours. A slow initiation was observed when Grubbs third-generation catalyst (G3) was employed (43% conversion, 24 hours, R.T.) (Scheme III). The 'H NMR signal of the product at 13.51 ppm was assigned to the alkylidene proton of Fischer-type Ru-carbene. The low reactivity of 2 was attributed to steric hindrance and the Ru coordination by the -OH group on C4 endo to the enol moiety, impeding monomer coordination to the Ru center. Consistent with this rationale, 4 with a -OH group on C4 exo to the enol moiety demonstrated a significantly faster initiation with G3, reaching 100% conversion into the Fischer-type Ru-carbene in an hour (Scheme III). Only one equivalent (relative to the catalyst) of 4 was consumed after 24 hours, suggesting that 4 did not undergo homo-propagation and was a single-addition monomer in ROMP.

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[0242] Scheme III

[0243]

[0244] Scheme IV

[0245] ROMP of a model system consisting of 4-methyl-3,6-anhydro-D-galactal 5 as the single- addition monomer and norbornene exo-dicarboximide Ml as the propagating monomer was studied (Scheme IV). The polymerization of Ml was rapid, consuming all monomers in 5 minutes and generating polymer Pl with an Ru-alkylidene peak at 18.54 ppm inXH NMR. After the addition of 2 equiv. of 5, the Ru-alkylidene signal (18.54 ppm) was converted into a Fischer-type Ru-carbene signal at 13.76 ppm over four hours at 22°C (polymer P2). Complete conversion could also be accomplished using 1.5 equiv. of 5 under 45°C in 2 hours. A polymer with a stable cochain end, P3, was formed by quenching the Fischer-type Ru-carbene in P2 with ethyl vinyl ether. MALDI-TOF mass spectrum of P3 showed only one series of peaks

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[0248] corresponding to the expected mass. InXH NMR, the ratio of the integrals of the w-chain end to the a-chain end was consistent with the stoichiometry expected from the molecular formula corresponding to the number of repeat units such as n is 9, 10, 11, 12, 13, 14, 15, 16, or 17. The mass for the molecular formula where n is 13 is calculated as 2655.0571 [M+Ag]+and found 2654.8043 [M+Ag]+. Taken together, all evidence suggested that quantitative co-chain end functionalization of the polymer.

[0249] Given the reactivity difference between 5 and Ml, it was tested whether they can be added into the reaction simultaneously without affecting the reaction outcome. (Scheme V).

[0250] Simultaneous addition Sequential addition G3 (1.0 equiv.) CDCI3, N2, RT, 5 min M1 * 5 (1.5 equiv.) (8 ecluiv-) CDCI3, N2, 45 °C, 2 h

[0251]

[0252] equ ■iv. x) mlieenicvcthen EVEScheme V

[0253] It was found that, at a degree of polymerization (DP) of 8, when 5 and Ml were added simultaneously, a polymer was generated with identical

[0254]

[0255] NMR spectrum and molecular weight compared to the one generated by the sequential addition of these two monomers (Scheme V). Chain-end incorporation of 5 that was added simultaneously with Ml remains highly efficient at a higher DP of 90.

[0256] Similarly, successful co-chain end functionalization was also achieved for the simultaneous addition of 5 and a PEGylated norbornene exo-dicarboximide M2, or cv.s-cyclooctene (COE). It is believed that 3,6-anhydrogalactal represents the first single-addition monomer in ROMP that can be added simultaneously with the propagating monomer without compromising the latter’s reactivity.

[0257] Density functional theory (DFT) calculations provided mechanistic insights into the reactivity of 5 in the presence of propagating monomers in ROMP (Scheme vi).

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[0260] DG in kcal / mol

[0261] M06(D3) / def2-TVZPP / PCM(CHClj) / / BP86(D3) / def2-SVP

[0262] G10

[0263]

[0264] G9 Precat 1 G5

[0265] Scheme VI

[0266] Compared to Ml (Precat 1 to tSG8_G9 DG' = 19.9 kcal / mol), the initiation of 5 (Precat 1 to tSG3_G4 DG' = 22.7 kcal / mol) required to overcome a higher energy barrier, with a DDG of 2.8 kcal / mol, suggesting that the initiation of Ml was faster than that of 5. Moreover, double addition of 5 was also met with a high energy barrier (G5 to tSGi2 _G13 DG' = 16.5 kcal / mol), and was significantly endergonic (DG = 10.8 kcal / mol) from the single-addition product G5, making the double addition of 5 energetically disfavored. In addition, calculations also suggest that the bicyclic enol ether structure of 5 raised the ring strain energy (RSE = 5.2 kcal / mol) compared to that of a simple galactal (RSE = 0.5 kcal / mol). The higher RSE was necessary for 5 to

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[0269] react efficiently with the propagating Ru-carbene. When tri-O-acetyl-D-galactal was used to react with the Fischer-type Ru-carbene at the chain end of P2, more than one Ru-carbene peaks (19.10, 18.54, 13.22 and 13.17 ppm) were observed after the treatment of 45 °C for 2 hours, indicating that monocyclic galactals could not achieve efficient chain-end functionalization.

[0270] Despite the reduced reactivity of the Fischer-type Ru-carbenes, reports have shown that cyclic enol ether such as DHF could ready react with this species in ROMP. Indeed, chain extension of P2 by a mixture of DHF and norbornene N-isopropyl-exo-dicarboximide (M3) successfully generated a block copolymer P4, with a number-average molecular weight ( / n) of 7.6 kg / mol and dispersity (£)) of 1.08 (Scheme VII).

[0271]

[0272] p2DHF M3 P4

[0273] Scheme VII

[0274] Diffusion-ordered spectroscopy (DOSY) NMR analysis showed only one diffusion signal corresponding to the1H NMR signals from all repeating units, confirming the block copolymer structure. Consistent with previous reports on the ROMP of DHF, it was also found that the vinyl ether motif from the incorporated DHF conferred degradability for this block copolymer under acidic conditions.

[0275] Leveraging their single-addition reactivity, a series of derivatives of 3,6-anhydro-D-galactal was prepared for the chain-end functionalization. It was found that 3,6-anhydro-D-galactals 6, 7, and 8, consisting of an NHS ester, a bromide, and a 2 -bromoisobutyryl group, respectively, each achieved successful chain end functionalization when they were added with Ml simultaneously. The1H NMR and MALDLTOF analyses confirmed the successful chain-end functionalization of the resulting polymers, P5, P6, and P7. The NHS ester group and bromide could readily react with various nucleophiles, enabling diverse functional groups to be introduced at the w-chain ends of metathesis polymers. For example, the NHS group of P5 could react with propargyl amine to generate P8 with an alkyne chain end, while the

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[0278] bromide group of P6 can react with sodium azide to generate P9 with an azido chain end. (Scheme VIII). Facile coupling between P8 and P9 could be achieved via copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction to give P10 with a molecular weight consistent with the sum of those of P8 and P9. Next, P7 with a a-bromoisobutyryl chain-end group was prepared using a 3,6-anhydro-D-galactal 8 bearing this group. Subsequent atom-transfer radical polymerization (ATRP) of styrene using P7 as the macroinitiator generated block copolymer Pl 1 Mn= 10.4 kg / mol, D = 1.07). DOSYNMR analysis confirmed a single polymer species consisting of both repeating units.

[0279]

[0280] Finally, the utility of the chain-end functionalization strategy for polymer bioconjugation was investigated. A water-soluble polymer P13 was prepared using M2 and 7 followed by chain-end azidation. A green fluorescent protein with an azido lysine incorporated at the position 151 (sfGFP-151-AzK) by non-canonical amino acid (ncAA) mutagenesis was functionalized with a dicyclooctyne DBCO-PEG8-DBCO, before coupling with P13. Both SDS-PAGE and MALDI-TOF analyses indicated that P13 was successfully conjugated to sfGFP-151-AzK in 87% yield.

[0281] Compounds 3,6-anhydrogalactals as demonstrated above are useful singleaddition monomers for efficient and versatile chain-end functionalization of metathesis polymers. Readily synthesized from biomass-derived galactal, these 3,6-anhydrogalactals exhibit excellent single-addition reactivity even when introduced simultaneously with the propagating monomer. Compatible with diverse functional groups, 3,6-anhydrogalactals facilitated chain-end functionalization of metathesis

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[0284] polymers for a variety of applications, such as block copolymer synthesis, polymerpolymer coupling, and bioconjugation. DFT calculations elucidated the unique reactivities of 3,6-anhydrogalactals underlying their single-addition behavior in the presence of a propagating monomer. These findings underscore the potential of 3,6-anhydrogalactals as cost-effective and sustainable reagents for advanced polymers in material science and bioconjugation.

[0285] OTHER EMBODIMENTS

[0286] All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.

[0287] From the above description, one skilled in the art can easily ascertain the essential characteristics of the present invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, other embodiments are also within the scope of the following claims.

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Claims

DocketNo. BCOT-012-WO1BC2025.016.Niu -31-CLAIMS1. A compound of formula (I):(I),in which Ri is H, C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C3-C20 cycloalkyl, 3-to 20-membered heterocycloalkyl, aryl, C7-C20 aralkyl, heteroaryl, or C2-C20 acyl.

2. The compound of claim 1, wherein each of alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, aralkyl, heteroaryl, and acyl, independently, is unsubstituted or substituted with one or more groups selected from D, halo, hydroxyl, amino, CN, azide, succinimido, carboxyl, Ci-Ce alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C2-C6 carboxylate, Ci-Ce alkylamino, C2-C10 dialkylamino, C2-C6 amido, Ci-Ce alkoxy, aryl, heteroaryl, aryloxy, C5-C6 cycloalkyl, or 5- to 6-membered heterocycloalkyl.

3. The compound of claim 1 or 2, wherein Ri is acyl that is represented by formula -C(O)-Ra-LKa-Rb; Ra is Ci-Ce alkylene, phenylene, or benzylene; LKa is absent, -C(O)-, -C(O)O-, or -C(O)NRc, Rc being H or Ci-Ce alkyl; and Rb is H, halo, azide, Ci-Ce alkyl, C2-C6 alkenyl, C2-C6 alkynyl, or succinimido, each of alkylene, phenylene, benzylene, alkyl, alkenyl, alkynyl, independently, being unsubstituted or substituted with a leaving group selected from halo, cyano, azide, succinimido, maleimido, and dibenzocyclooctyne-amino.4920-7647-9649, v. 1DocketNo. BCOT-012-WO1BC2025.016.Niu -32-4. The compound of any one of claims 1 to 3, wherein Ri is H, methyl,5. A polymer of formula (II):crR2(II),in whichPA contains a first polymer backbone having at least 10 first repeat units; R2 is H, C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C3-C20 cycloalkyl, 3- to 20-membered heterocycloalkyl, aryl, C7-C20 aralkyl, heteroaryl, C2-C20 acyl, or PB;X is Ru, alkylidene, or PC;PB contains a second polymer backbone having at least 5 second repeat units; andPC contains a third polymer backbone having at least 5 third repeat units.

6. The polymer of claim 5, wherein X is alkylidene or PC.

7. The polymer of claims 5 or 6, wherein X is CH2 or PC, each of PA, PB, and PC, independently, has 10 - 100,000 repeat units, preferably 20 - 50,000 repeat units, more preferably 25 - 10,000 repeat units, and even more preferably 30 - 5,000 repeat units.4920-7647-9649, v. 1Docket No. BCOT-012-WO1BC2025.016.Niu -33-8. The polymer of any one of claims 5-7, wherein R2 is PB containing a second polymer backbone that connects to the oxygen atom on the tetrahydrofuran ring through a second linker, and the second linker is C1-C20 alkyl, C3-C20 cycloalkyl, 3- to 20-membered heterocycloalkyl, aryl, C7-C20 aralkyl, heteroaryl, or C2-C20 acyl.

9. A method of functionalizing a metathesis polymer, the method comprising the steps of:(la) providing a mixture that contains a first propagating monomer, a Ru catalyst, and a compound of any one of claims 1-4, and(2a) initiating a polymerization reaction to obtain a functionalized metathesis polymer.

10. A method of functionalizing a metathesis polymer, the method comprising the steps of:(lb) providing a metathesis polymer obtained by polymerizing a first propagating monomer in the presence of a Ru catalyst,(2b) adding a compound of any one of claims 1-4 to the metathesis polymer, and(3b) causing functionalization of the metathesis polymer thereby obtaining a functionalized metathesis polymer.

11. The method of claim 9, wherein the polymerization is achieved via a ringopening metathesis polymerization.

12. The method of any one of claims 9-11, further comprising the step of (4) connecting a second or third polymer backbone to the functionalized metathesis polymer.

13. The method of claim 12, wherein the second or third polymer backbone is connected via a linker reaction.4920-7647-9649, v. 1DocketNo. BCOT-012-WO1BC2025.016.Niu -34-14. The method of claim 13, wherein the second or third polymer backbone is derived from a biopolymer.

15. The method of claim 12, wherein the second or third polymer backbone is connected via a chain extension reaction using a second or third propagating monomer.4920-7647-9649, v. 1

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