WO2023208081A1

WO2023208081A1 - Substituted fluorosulfate and use thereof

Info

Publication number: WO2023208081A1
Application number: PCT/CN2023/091020
Authority: WO
Inventors: Heng Zhang; Peng Chen; Miao PEI; Yu Han; Su Zhao; Xueyin LU
Original assignee: Shenzhen Bay Laboratory
Priority date: 2022-04-28
Filing date: 2023-04-27
Publication date: 2023-11-02
Also published as: CN117279889A

Abstract

Disclosed are a substituted fluorosulfate and use thereof, and fluorines substituted fluorosulfate and non-natural amino acid (covalent amino acid, PrUAA) with enhanced reactivity.

Description

Substituted fluorosulfate and use thereof

BACKGROUND OF THE INVENTION

Covalent drugs have been drawing attentions from both basic research and industry. Covalent drugs have two step combined mechanisms: First, the drug binds to the target via reversible non-covalent interaction; then, the drug would react with the electrophilic amino acid residue on the target through proximity mediated reaction, forming a covalent bond linked conjugate. Additional covalent bonds enable the drug molecules to not dissociate from the target, which extends the duration time of the drug molecule. Thus, covalent drugs would have a stronger and durable efficacy when compared to its non-covalent counterpart.

Due to the structure limitation of small molecular drugs, they often bind to the "pocket" of the protein, in the form of inhibitors. However, protein drugs represented by antibodies can specifically bind to large interaction surfaces, making it possible to disrupt protein-protein interaction. In the clinical practice, the intact IgG antibodies have remarkable pharmacokinetics, but can only bind to the surface of the tissue surface due to their large size. The corresponding fragment antibodies have better tissue permeability, but is limited by worse half-life. It may be that if the fragment antibodies covalently bind to the antigen, it is expected to avoid the limitations while remaining its intrinsic advantages.

With the help of genetic codon expansion technology, non-canonical amino acids carrying a new chemical functional group can be introduced into the target protein, which greatly expands the space of protein engineering. In principle, covalent protein drugs should specifically cross-link with target antigens, while avoiding reacting with the active compounds or other proteins in the living system. This property requires the residue of the introduced proximal reactive unnatural amino acid (covalent amino acid, PrUAA) must have moderate reactivity, which can stably present in a complex biological system and react with target after the antibody binding. Obviously, the warhead of the covalent amino acid is the key factor affecting the cross-linking efficiency of covalent protein drugs. However, there are few suitable functional groups meet the above requirements. Therefore, there is a great need for developing biocompatible and high proximal reactive functional groups and related covalent amino acids, which would greatly facilitate the development of covalent protein drugs.

SUMMARY OF THE INVENTION

The present disclosure provides a substituted fluorosulfate and use thereof. And fluorines substituted fluorosulfate and related PrUAAs with enhanced reactivity are developed in the present application. Covalent protein drugs have been drawing extensive attention from both research and industry. while the site-specific incorporation of proximal-reactive unnatural amino acids (PrUAAs) into proteins through genetic code expansion strategy is one of the important methods to endow proteins with covalent binding capacity, lacking of proper unnatural amino acids greatly limit the development of covalent protein drugs. The fluorosulfate has good biocompatibility but relatively low proximal reactivity. Therefore, we chemoenzymatically generated fluorines substituted fluorosulfate and related PrUAAs with enhanced reactivity. On this basis, we further developed series of covalent single-domain antibodies targeting PD-L1 and confirmed their enhanced efficacy in restoring T cell activity, demonstrating the potentiality of covalent antibody as a new generation of biological drugs.

In one aspect, the present application provides a compound having the structure (I) of:

wherein, said n is more than 0, and X is selected from the electron-withdrawing group.

In another aspect, the present application provides a compound having the structure (II) of:

In another aspect, the present application provides a compound having the structure (I-A) of:

In another aspect, the present application provides a compound having the structure (I-P) of:

In another aspect, the present application provides a compound having the structure (II-A) of:

In another aspect, the present application provides a compound having the structure (II-P) of:

In another aspect, the present application provides a compound having the structure of:

wherein, said X is selected from the electron-withdrawing group.

In another aspect, the present application provides a protein comprising the structure of the compound of any of the present applications.

In another aspect, the present application provides a protein comprising an unnatural amino acid having the structure (I) of:

In another aspect, the present application provides a protein comprising an unnatural amino acid having the structure of:

In another aspect, the present application provides a nucleic acid comprising a sequence encoding the protein of any of the present application.

In another aspect, the present application provides a synthetase comprising a mutant at position 366 and/or position 367 as set forth in sequence of SEQ ID NO: 29.

In another aspect, the present application provides a synthetase comprising an alanine at position 366 and/or position 367 as set forth in sequence of SEQ ID NO: 29.

In another aspect, the present application provides a synthetase comprising a sequence of SEQ ID NO: 31 and/or 34.

In another aspect, the present application provides a nucleic acid comprising a sequence encoding the synthetase of any of the present application.

In another aspect, the present application provides a nucleic acid comprising a sequence of SEQ ID NO: 30 and/or 33.

In another aspect, the present application provides a vector comprising the nucleic acid of any of the present application.

In another aspect, the present application provides a combination comprising the synthetase of any of the present application and the compound of any of the present application.

In another aspect, the present application provides a method of preparing the protein of any of the present application, wherein said method comprises providing the synthetase of any of the present application, the compound of any of the present application and/or the combination of any of the present application.

In another aspect, the present application provides a cell comprising the compound of any of the present application, the protein of any of the present application, the nucleic acid of any of the present application, the synthetase of any of the present application, the nucleic acid of any of the present application, the vector of any of the present application, and/or the combination of any of the present application.

In another aspect, the present application provides a composition comprising the compound of any of the present application, the protein of any of the present application, the nucleic acid of any of the present application, the synthetase of any of the present application, the nucleic acid of any of the present application, the vector of any of the present application, the combination of any of the present application and/or the cell of any of the present application, and optionally a pharmaceutically acceptable adjuvant.

In another aspect, the present application provides a kit comprising the compound of any of the present application, the protein of any of the present application, the nucleic acid of any of the present application, the synthetase of any of the present application, the nucleic acid of any of the present application, the vector of any of the present application, the combination of any of the present application, the cell of any of the present application, and/or the composition of any of the present application.

In another aspect, the present application provides a method for inhibiting binding of a PD-L1 protein to a PD-L1 ligand, wherein said method comprises providing the compound of any of the present application, the protein of any of the present application, the nucleic acid of any of the present application, the synthetase of any of the present application, the nucleic acid of any of the present application, the vector of any of the present application, the combination of any of the present application, the cell of any of the present application, the composition of any of the present application, and/or the kit of any of the present application. In another aspect, the present application provides a method for crosslinking PD-L1 protein, wherein said method comprises providing the compound of any of the present application, the protein of any of the present application, the nucleic acid of any of the present application, the synthetase of any of the present application, the nucleic acid of any of the present application, the vector of any of the present application, the combination of any of the present application, the cell of any of the present application, the composition of any of the present application, and/or the kit of any of the present application.

In another aspect, the present application provides a method for activating immune cell, wherein said method comprises providing the compound of any of the present application, the protein of any of the present application, the nucleic acid of any of the present application, the synthetase of any of the present application, the nucleic acid of any of the present application, the vector of any of the present application, the combination of any of the present application, the cell of any of the present application, the composition of any of the present application, and/or the kit of any of the present application.

In another aspect, the present application provides a method of preparing a compound having the structure (M) of:

wherein, said n is more than 0, and X is selected from the electron-withdrawing group; wherein said method comprises providing a ligase comprising sequence selected from the group consisting of: SEQ ID NO: 15, SEQ ID NO: 21, SEQ ID NO: 25, and SEQ ID NO: 27.

In another aspect, the present application provides a ligase comprising sequence selected from the group consisting of: SEQ ID NO: 15, SEQ ID NO: 21, SEQ ID NO: 25, and SEQ ID NO: 27, for use in preparing a compound having the structure (M) of:

Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

INCORPORATION BY REFERENCES

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWING

The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are employed, and the accompanying drawings (also “figure” and “FIG. ” herein) , of which:

FIG. 1 illustrates a general structure of electron-withdrawing fluorine substituted fluorosulfate with enhanced reactivity.

FIG. 2 illustrates chemical structure of fluorine substituted phenyl sulfurofluoridate.

FIGs. 3A-3D illustrate LC analysis of model reaction to determine the proximal reactivity of fluorine substituted phenyl sulfurofluoridate.

FIG. 4 illustrates result of the proximal reactivity of fluorine substituted phenyl sulfurofluoridate.

FIG. 5 illustrates specific activity of TPLs from different sources for 2-fluoro-L-tyrosine synthesis.

FIG. 6 illustrates Cf TPL and Fn TPL demonstrate efficient synthesis of difluorine substituted L-tyrosine.

FIG. 7 illustrates chemical structure of TPL catalyzed synthesis of fluorine substituted L-tyrosine.

FIG. 8 illustrates chemical structure of fluorine substituted fluorosulfate L-tyrosine.

FIGs. 9A-9D illustrate LC analysis of model reaction to determine the proximal reactivity of fluorosulfate L-tyrosine.

FIG. 10 illustrates result of the proximal reactivity of fluorine substituted fluorosulfate L-tyrosine.

FIGs. 11A-11C illustrate Sequencing result of the tRNA synthetase library. One dominant clone V366A for fluorosulfate-2, 6-difluoro-L-tyrosine was obtained from the randomly mutated library after four rounds of positive selection. (A) Before selection. (B) After three rounds of positive selection. (C) After additional one round of positive selection.

FIG. 12 illustrates Amber suppression efficiency tested by a GFP reporter assay. GFP N194TAG is expressed in the presence of tRNA synthetase and corresponding PrUAAs. The fluorescent intensity in each group is measured by a plate reader and normalised by the strongest one.

FIG. 13 illustrates Western blotting analysis of amber suppression efficiency. GFP N194TAG with a 6xHis tag at the C terminal is expressed in the presence of tRNA synthetase and corresponding PrUAAs. The fluorescent intensity in each group is measured by a plate reader and normalised by the strongest one.

FIGs. 14A-14D illustrates result of successful incorporation of PrUAA into Nb-PD-L1 by ESI-MS. (A) For FSY, the measured MW is 16147 Da (calculated 16148 Da) . (B) For m-F FSY, the measured MW is 16166 Da (calculated 16147 Da) . (C) For o-F FSY, the measured MW is 16166 Da (calculated 16166 Da) . (D) For 2, 5-diF FSY, the measured MW is 16183 Da (calculated 16184 Da) .

FIG. 15 illustrates comparison of the crosslinking efficiency of different GlueBodies with PD-L1. Purified proteins were incubated with PD-L1 at 37 ℃ for 1 h before SDS-PAGE and Coomassie staining.

FIG. 16 illustrates quantification analysis of luciferase activities in T cells after coculturing with PD-L1+ aAPCs treated with the indicated protein drugs for 12 h. The box and error bars represent the mean ± standard error of the mean (n=3) .

FIG. 17 illustrates fluorescence images of 293T cells transfected with pCMV EGFP Y40TAG and tRNA synthetase/tRNA. Cells were incubated with corresponding PrUAA for 24 h after transfection followed by fluorescence imaging. Scale bar= 150 μm.

DETAILED DESCRIPTION

While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.

Where substituent groups are specified by their conventional chemical formulae, written from left to right, they equally encompass the chemically identical substituents that would result from writing the structure from right to left, e.g., -CH₂O-is equivalent to -OCH₂-.

The term “alkyl, ” by itself or as part of another substituent, means, unless otherwise stated, a straight (i.e., unbranched) or branched carbon chain (or carbon) , or combination thereof, which may be fully saturated, mono-or polyunsaturated and can include mono-, di-and multivalent radicals. The alkyl may include a designated number of carbons (e.g., C₁-C₁₀ means one to ten carbons) . Alkyl is an uncyclized chain. Examples of saturated hydrocarbon radicals include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, methyl, homologs and isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. An unsaturated alkyl group is one having one or more double bonds or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2- (butadienyl) , 2, 4-pentadienyl, 3- (1, 4-pentadienyl) , ethynyl, 1-and 3-propynyl, 3-butynyl, and the higher homologs and isomers. An alkoxy is an alkyl attached to the remainder of the molecule via an oxygen linker (-O-) . An alkyl moiety may be an alkenyl moiety. An alkyl moiety may be an alkynyl moiety. An alkyl moiety may be fully saturated. An alkenyl may include more than one double bond and/or one or more triple bonds in addition to the one or more double bonds. An alkynyl may include more than one triple bond and/or one or more double bonds in addition to the one or more triple bonds.

The term “alkylene, ” by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from an alkyl, as exemplified, but not limited by, -CH₂CH₂CH₂CH₂-. Typically, an alkyl (or alkylene) group will have from 1 to 24 carbon atoms, with those groups having 10 or fewer carbon atoms being preferred herein. A “lower alkyl” or “lower alkylene” is a shorter chain alkyl or alkylene group, generally having eight or fewer carbon atoms. The term “alkenylene, ” by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from an alkene.

The term “heteroalkyl, ” by itself or in combination with another term, means, unless otherwise stated, a stable straight or branched chain, or combinations thereof, including at least one carbon atom and at least one heteroatom (e.g., O, N, P, Si, and S) , and wherein the nitrogen and sulfur atoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized. The heteroatom (s) (e.g., N, S, Si, or P) may be placed at any interior position of the heteroalkyl group or at the position at which the alkyl group is attached to the remainder of the molecule. Heteroalkyl is an uncyclized chain. Examples include, but are not limited to: -CH₂-CH₂-O-CH₃, -CH₂-CH₂-NH-CH₃, -CH₂-CH₂-N (CH₃) -CH₃, -CH₂-S-CH₂-CH₃, -CH₂-CH₂, -S (O) -CH₃, -CH₂-CH₂-S (O) ₂-CH₃, -CH═CHO-CH₃, -Si (CH₃) ₃, -CH₂-CH═N-OCH₃, -CH═CH-N (CH₃) -CH₃, -O-CH₃, -O-CH₂-CH₃, and -CN. Up to two or three heteroatoms may be consecutive, such as, for example, -CH₂-NH-OCH₃ and -CH₂-O-Si (CH₃) ₃. A heteroalkyl moiety may include 1, 2, 3, 4 or 5 heteroatom (e.g., O, N, S, Si, or P) . A heteroalkyl moiety may include up to 8 optionally different heteroatoms (e.g., O, N, S, Si, or P) . The term “heteroalkenyl, ” by itself or in combination with another term, means, unless otherwise stated, a heteroalkyl including at least one double bond. A heteroalkenyl may optionally include more than one double bond and/or one or more triple bonds in additional to the one or more double bonds. The term “heteroalkynyl, ” by itself or in combination with another term, means, unless otherwise stated, a heteroalkyl including at least one triple bond. A heteroalkynyl may optionally include more than one triple bond and/or one or more double bonds in additional to the one or more triple bonds.

The term “heteroalkylene, ” by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from heteroalkyl, as exemplified, but not limited by, -CH₂-CH₂-S-CH₂-CH₂-and -CH₂-S-CH₂-CH₂-NH-CH₂-. For heteroalkylene groups, heteroatoms can also occupy either or both of the chain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, and the like) . Still further, for alkylene and heteroalkylene linking groups, no orientation of the linking group is implied by the direction in which the formula of the linking group is written. For example, the formula -C (O) ₂R′-represents both -C (O) ₂R′-and -R′C (O) ₂-. As described above, heteroalkyl groups, as used herein, include those groups that are attached to the remainder of the molecule through a heteroatom, such as -C (O) R′, -C (O) NR′, -NR′R″, -OR′, -SR′, and/or -SO₂R′. Where “heteroalkyl” is recited, followed by recitations of specific heteroalkyl groups, such as -NR′R″or the like, it will be understood that the terms heteroalkyl and -NR′R″are not redundant or mutually exclusive. Rather, the specific heteroalkyl groups are recited to add clarity. Thus, the term “heteroalkyl” should not be interpreted herein as excluding specific heteroalkyl groups, such as -NR′R″or the like.

The terms “cycloalkyl” and “heterocycloalkyl, ” by themselves or in combination with other terms, mean, unless otherwise stated, cyclic versions of “alkyl” and “heteroalkyl, ” respectively. Cycloalkyl and heterocycloalkyl are not aromatic. Additionally, for heterocycloalkyl, a heteroatom can occupy the position at which the heterocycle is attached to the remainder of the molecule. Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples of heterocycloalkyl include, but are not limited to, 1- (1, 2, 5, 6-tetrahydropyridyl) , 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl, 2-piperazinyl, and the like. A “cycloalkylene” and a “heterocycloalkylene, ” alone or as part of another substituent, means a divalent radical derived from a cycloalkyl and heterocycloalkyl, respectively.

In embodiments, the term “cycloalkyl” means a monocyclic, bicyclic, or a multicyclic cycloalkyl ring system. In aspects, monocyclic ring systems are cyclic hydrocarbon groups containing from 3 to 8 carbon atoms, where such groups can be saturated or unsaturated, but not aromatic. In aspects, cycloalkyl groups are fully saturated. Examples of monocyclic cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl. Bicyclic cycloalkyl ring systems are bridged monocyclic rings or fused bicyclic rings. In aspects, bridged monocyclic rings contain a monocyclic cycloalkyl ring where two non-adjacent carbon atoms of the monocyclic ring are linked by an alkylene bridge of between one and three additional carbon atoms (i.e., a bridging group of the form (CH₂) _w, where w is 1, 2, or 3) .

In embodiments, a cycloalkyl is a cycloalkenyl. The term “cycloalkenyl” is used in accordance with its plain ordinary meaning. In aspects, a cycloalkenyl is a monocyclic, bicyclic, or a multicyclic cycloalkenyl ring system. In aspects, monocyclic cycloalkenyl ring systems are cyclic hydrocarbon groups containing from 3 to 8 carbon atoms, where such groups are unsaturated (i.e., containing at least one annular carbon carbon double bond) , but not aromatic.

In embodiments, a heterocycloalkyl is a heterocyclyl. The term “heterocyclyl” as used herein, means a monocyclic, bicyclic, or multicyclic heterocycle. The heterocyclyl monocyclic heterocycle is a 3, 4, 5, 6 or 7 membered ring containing at least one heteroatom independently selected from the group consisting of O, N, and S where the ring is saturated or unsaturated, but not aromatic.

The terms “halo” or “halogen, ” by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom.

The term “acyl” means, unless otherwise stated, -C (O) R where R is a substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

The term “aryl” means, unless otherwise stated, a polyunsaturated, aromatic, hydrocarbon substituent, which can be a single ring or multiple rings (preferably from 1 to 3 rings) that are fused together (i.e., a fused ring aryl) or linked covalently. A fused ring aryl refers to multiple rings fused together wherein at least one of the fused rings is an aryl ring. The term “heteroaryl” refers to aryl groups (or rings) that contain at least one heteroatom such as N, O, or S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom (s) are optionally quaternized.

Each of the above terms (e.g., “alkyl, ” “heteroalkyl, ” “cycloalkyl, ” “heterocycloalkyl, ” “aryl, ” and “heteroaryl” ) includes both substituted and unsubstituted forms of the indicated radical.

Substituents for rings (e.g., cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene) may be depicted as substituents on the ring rather than on a specific atom of a ring (commonly referred to as a floating substituent) .

Two or more substituents may optionally be joined to form aryl, heteroaryl, cycloalkyl, or heterocycloalkyl groups. Such so-called ring-forming substituents are typically, though not necessarily, found attached to a cyclic base structure.

In embodiments, each substituted group described in the compounds herein is substituted with at least one substituent group. More specifically, in aspects, each substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene, and/or substituted heteroarylene described in the compounds herein are substituted with at least one substituent group. In aspects, at least one or all of these groups are substituted with at least one size-limited substituent group. In aspects, at least one or all of these groups are substituted with at least one lower substituent group.

Certain compounds of the present disclosure possess asymmetric carbon atoms (optical or chiral centers) or double bonds; the enantiomers, racemates, diastereomers, tautomers, geometric isomers, stereoisometric forms that may be defined, in terms of absolute stereochemistry, as (R) -or (S) -or, as (D) -or (L) -for amino acids, and individual isomers are encompassed within the scope of the present disclosure. The compounds of the present disclosure do not include those that are known in art to be too unstable to synthesize and/or isolate. The present disclosure is meant to include compounds in racemic and optically pure forms. Optically active (R) -and (S) -, or (D) -and (L) -isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques. When the compounds described herein contain olefinic bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers.

As used herein, the term “isomers” refers to compounds having the same number and kind of atoms, and hence the same molecular weight, but differing in respect to the structural arrangement or configuration of the atoms.

The term “tautomer, ” as used herein, refers to one of two or more structural isomers which exist in equilibrium and which are readily converted from one isomeric form to another.

Unless otherwise stated, structures depicted herein are also meant to include all stereochemical forms of the structure; i.e., the R and S configurations for each asymmetric center. Therefore, single stereochemical isomers as well as enantiomeric and diastereomeric mixtures of the present compounds are within the scope of the disclosure.

Unless otherwise stated, structures depicted herein are also meant to include compounds which differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures except for the replacement of a hydrogen by a deuterium or tritium, or the replacement of a carbon by ¹³C-or ¹⁴C-enriched carbon are within the scope of this disclosure.

The compounds of the present disclosure may also contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds. For example, the compounds may be radiolabeled with radioactive isotopes, such as for example tritium (³H) , iodine-125 (¹²⁵I) , or carbon-14 (¹⁴C) . All isotopic variations of the compounds of the present disclosure, whether radioactive or not, are encompassed within the scope of the present disclosure.

It should be noted that throughout the application that alternatives are written in Markush groups, for example, each amino acid position that contains more than one possible amino acid. It is specifically contemplated that each member of the Markush group should be considered separately, thereby comprising another embodiment, and the Markush group is not to be read as a single unit.

“Analog, ” or “analogue” is used in accordance with its plain ordinary meaning within Chemistry and Biology and refers to a chemical compound that is structurally similar to another compound (i.e., a so-called “reference” compound) but differs in composition, e.g., in the replacement of one atom by an atom of a different element, or in the presence of a particular functional group, or the replacement of one functional group by another functional group, or the absolute stereochemistry of one or more chiral centers of the reference compound. Accordingly, an analog is a compound that is similar or comparable in function and appearance but not in structure or origin to a reference compound.

The terms “a” or “an, ” as used in herein means one or more. In addition, the phrase “substituted with a [n] , ” as used herein, means the specified group may be substituted with one or more of any or all of the named substituents. For example, where a group, such as an alkyl or heteroaryl group, is “substituted with an unsubstituted C₁-C₂₀ alkyl, or unsubstituted 2 to 20 membered heteroalkyl, ” the group may contain one or more unsubstituted C₁-C₂₀ alkyls, and/or one or more unsubstituted 2 to 20 membered heteroalkyls.

Moreover, where a moiety is substituted with an R substituent, the group may be referred to as “R-substituted. ” Where a moiety is R-substituted, the moiety is substituted with at least one R substituent and each R substituent is optionally different. Where a particular R group is present in the description of a chemical genus (such as Formula (I) ) , a Roman alphabetic symbol may be used to distinguish each appearance of that particular R group. For example, where multiple R¹³ substituents are present, each R¹³ substituent may be distinguished as R^13A, R^13B, R^13C, R^13D, etc., wherein each of R^13A, R^13B, R^13C, R^13D, etc. is defined within the scope of the definition of R¹³ and optionally differently.

Descriptions of compounds of the present disclosure are limited by principles of chemical bonding known to those skilled in the art. Accordingly, where a group may be substituted by one or more of a number of substituents, such substitutions are selected so as to comply with principles of chemical bonding and to give compounds which are not inherently unstable and/or would be known to one of ordinary skill in the art as likely to be unstable under ambient conditions, such as aqueous, neutral, and several known physiological conditions. For example, a heterocycloalkyl or heteroaryl is attached to the remainder of the molecule via a ring heteroatom in compliance with principles of chemical bonding known to those skilled in the art thereby avoiding inherently unstable compounds.

A person of ordinary skill in the art will understand when a variable (e.g., moiety or linker) of a compound or of a compound genus (e.g., a genus described herein) is described by a name or formula of a standalone compound with all valencies filled, the unfilled valence (s) of the variable will be dictated by the context in which the variable is used. For example, when a variable of a compound as described herein is connected (e.g., bonded) to the remainder of the compound through a single bond, that variable is understood to represent a monovalent form (i.e., capable of forming a single bond due to an unfilled valence) of a standalone compound (e.g., if the variable is named “methane” in an embodiment but the variable is known to be attached by a single bond to the remainder of the compound, a person of ordinary skill in the art would understand that the variable is actually a monovalent form of methane, i.e., methyl or -CH₃) .

As described herein the complementarity of sequences may be partial, in which only some of the nucleic acids match according to base pairing, or complete, where all the nucleic acids match according to base pairing. Thus, two sequences that are complementary to each other, may have a specified percentage of nucleotides that are the same (i.e., about 60%identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region) .

The term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, -carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an α carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid. The terms “non-naturally occurring amino acid” and “unnatural amino acid” refer to amino acid analogs, synthetic amino acids, and amino acid mimetics which are not found in nature.

The terms “polypeptide, ” “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues, wherein the polymer may in embodiments be conjugated to a moiety that does not consist of amino acids. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.

For example, an electron-withdrawing group (EWG) may draw electron away from a reaction center. When this center is an electron rich carbanion or an alkoxide anion, the presence of the electron-withdrawing substituent has a stabilizing effect. Examples of electron withdrawing groups may be halogens (e.g., F, Cl) .

For example, the compound may be a biomolecule. For example, the compound may be a protein with a structure of the above structure. For example, the compound may be a protein with an unnatural amino acid having a structure of the above structure. For example, the compound may have the above structure as an intermolecular linker. For example, the compound may have the above structure as an intramolecular linker.

For example, wherein said structure (I) and/or structure (II) is linked to a linkage group R₁, said R₁ is optionally substituted substitution. For example, the compound may be a protein with an unnatural amino acid having a structure of the above structure. For example, the R₁ may be R chain of the amino acid. For example, said structure (I) and/or structure (II) may be linked to the R chain of the amino acid.

For example, said R₁ is a bond or is selected from group consisting of: -S (O) ₂-, -NR^1A-, -O-, -S-, -C (O) -, -C (O) NR^1A-, -NR^1AC (O) -, -NR^1AC (O) NR^1B-, -C (O) O-, -OC (O) -, optionally substituted alkylene, optionally substituted heteroalkylene, optionally substituted cycloalkylene, optionally substituted heterocycloalkylene, optionally substituted arylene, and optionally substituted heteroarylene, each said R^1A and R^1B is independently hydrogen, optionally substituted alkyl, optionally substituted heteroalkyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl.

For example, wherein said linkage group R₁ is linked to a linkage group R₂, said R₂ is optionally substituted substitution. For example, the compound may be a protein with an unnatural amino acid having a structure of the above structure. For example, the R₂ may be peptide bond of the amino acid.

For example, said R₂ is a bond or is selected from group consisting of: optionally substituted peptidyl moiety, optionally substituted nucleic acid moiety, and optionally substituted carbohydrate moiety.

For example, wherein said structure (I) and/or structure (II) is linked to said R₂ via optionally substituted tyrosine.

For example, wherein said n is selected from the group consisting of: 1, 2, 3 and 4. For example, wherein said n is 1. For example, wherein said n is 2. For example, wherein said n is 3. For example, wherein said n is 4.

For example, wherein said compound having the structure of:

For example, wherein said compound having the structure of: For example, said structure may have better reactivity.

For example, wherein said compound having the structure of:

In another aspect, the present application provides a compound having the structure of:wherein, said X is selected from the electron-withdrawing group.

In another aspect, the present application provides a compound having the structure of: wherein, said X is selected from the electron-withdrawing group.

For example, wherein each of said X is independently halogen.

For example, wherein each of said X is independently F.

For example, wherein said compound having the structure of:

For example, wherein said compound having the structure of: For example, wherein said compound having the structure of:

For example, wherein said compound having the structure of:

For example, wherein said protein comprises an unnatural amino acid having the structure of the compound of any of the present application.

For example, wherein said protein comprises said unnatural amino acid at position 108 as set forth in sequence of PD-L1 binding protein or the fragment thereof.

For example, wherein said protein comprises said unnatural amino acid at position 108 as set forth in sequence of anti-PD-L1 nanobody or the fragment thereof.

For example, wherein said protein comprises said unnatural amino acid at position 108 as set forth in sequence of SEQ ID NO: 1. For example, wherein said protein may further comprise p62, LC3, nucleotide 8-nitrocyclic guanosine monophosphate (8-nitro-cGMP) , mannose-6-phosphate, N-acetylgalactosamine (GalNAc) , cell-penetrating peptide and/or lysosome-sorting sequence.

In another aspect, the present application provides a synthetase comprising an alanine at position 366 and/or position 367 as set forth in sequence of SEQ ID NO: 29. For example, the amino acid of synthetase of the present application comprise an alanine instead of the original chFSYRS. For example, the amino acid of synthetase of the present application comprise an alanine instead of the original amino acid of SEQ ID NO: 29.

For example, wherein said immune cell comprises T cell.

For example, wherein said activating is tested via luciferase assay.

For example, wherein said method may be an in vitro method.

For example, wherein said method comprises providing a ligase encoded by sequence selected from the group consisting of: SEQ ID NO: 14, SEQ ID NO: 20, SEQ ID NO: 24, and SEQ ID NO: 26.

For example, wherein said n is selected from the group consisting of: 1, 2, 3 and 4.

For example, wherein said compound comprises the structure of:

For example, wherein each of said X is independently halogen.

For example, wherein each of said X is independently F. For example, wherein said compound comprises the structure of:

The following examples are set forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc. ) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations may be used, e.g., bp, base pair (s) ; kb, kilobase (s) ; pl, picoliter (s) ; s or sec, second (s) ; min, minute (s) ; h or hr, hour (s) ; aa, amino acid (s) ; nt, nucleotide (s) ; i.m., intramuscular (ly) ; i.p., intraperitoneal (ly) ; s.c., subcutaneous (ly) ; and the like.

Example 1

Development of fluoro substituted fluorosulfate with enhanced proximal reactivity

5.5 g fluorine substituted phenol was dissolved in 50 mL DCM and 10 mL Et₃N (1.5 eq) . The mixture was stirred at room temperature for 10 min. Then, the atmosphere above the solution was removed with gentle vacuum, and SO₂F₂ gas was introduced by a needle from a balloon filled with the gas. The reaction mixture was vigorously stirred at room temperature for 6-72 hours, monitoring by TLC.

Phenyl sulfurofluoridate

¹H NMR (400 MHz, DMSO) δ 7.63 –7.55 (m, 4H) , 7.55 –7.47 (m, 1H) . ¹³C NMR (101 MHz, DMSO) δ 150.22 (s) , 131.33 (s) , 129.70 (s) , 121.48 (s) . ¹⁹F NMR (377 MHz, DMSO) δ 38.24. MS: 175.0 [C₆H₄FO₃S] ^-.

As shown in Fig. 1, the present application provides an electron-withdrawing fluorine substituted fluorosulfate with enhanced reactivity.

As shown in Fig. 2, the present application provides chemical structure of fluorine substituted phenyl sulfurofluoridate.

2-fluorophenyl sulfurofluoridate

¹H NMR (400 MHz, DMSO) δ 7.70 –7.57 (m, 2H) , 7.43 (d, 2H) . ¹³C NMR (101 MHz, DMSO) δ 163.88, 161.41, 150.13, 132.65, 118.15 –116.96, 116.89, 110.22, 110.02 –108.68.¹⁹F NMR (377 MHz, DMSO) δ 38.79. MS: 193.0 [C₆H₃F₂O₃S] ^-.

3-fluorophenyl sulfurofluoridate

¹H NMR (400 MHz, DMSO) δ 7.79 (t, 1H) , 7.64 –7.52 (m, 2H) , 7.44 –7.35 (m, 1H) . ¹³C NMR (101 MHz, DMSO) δ 154.39, 151.89, 136.83, 131.65, 126.60, 124.15, 118.51.¹⁹F NMR (377 MHz, DMSO) δ 39.41, -129.92. MS: 193.0 [C₆H₃F₂O₃S] ^-.

2,6-difluorophenyl sulfurofluoridate

1H NMR (400 MHz, DMSO) δ 7.64 (dt, 1H) , 7.49 (t, 2H) . ¹³C NMR (101 MHz, DMSO) δ 155.53, 153.00, 131.68, 125.76, 114.19.¹⁹F NMR (377 MHz, DMSO) δ 41.12, -126.70. MS: 211.0 [C₆H₂F₃O₃S] ^-.

2,5-difluorophenyl sulfurofluoridate

¹H NMR (400 MHz, DMSO) δ 7.92 (ddd, 1H) , 7.68 (td, 1H) , 7.47 (ddt, 1H) . ¹³C NMR (101 MHz, DMSO) δ 159.24, 156.79, 151.27, 148.81, 136.47, 119.44, 118.36, 55.24.¹⁹F NMR (377 MHz, DMSO) δ 40.29, -113.99, -134.31. MS: 211.0 [C₆H₂F₃O₃S] ^-.

2,3-difluorophenyl sulfurofluoridate

¹H NMR (400 MHz, DMSO) δ 7.73 –7.59 (m, 2H) , 7.44 (tdd, 1H) . ¹³C NMR (101 MHz, DMSO) δ 152.02, 149.53, 143.76, 141.31, 126.01, 119.67, 119.08, 55.25.¹⁹F NMR (377 MHz, DMSO) δ 39.90, -134.30, -152.73. MS: 211.2 [C₆H₂F₃O₃S] ^-.

3,5-difluorophenyl sulfurofluoridate

¹H NMR (400 MHz, DMSO) δ 7.65 –7.55 (m, 2H) , 7.50 (tt, 1H) . ¹³C NMR (101 MHz, DMSO) δ 164.26, 150.17, 131.27, 107.20, 107.00, 105.97.¹⁹F NMR (377 MHz, DMSO) δ 39.50, -106.03. MS: 211.1 [C₆H₂F₃O₃S] ^-.

2, 3, 6-trifluorophenyl sulfurofluoridate

¹H NMR (400 MHz, DMSO) δ 7.77 (tdd, 1H) , 7.57 (tdd, 1H) . ¹³C NMR (101 MHz, DMSO) δ151.73, 149.24, 148.40, 145.95, 144.63, 142.08, 126.53, 118.81, 113.25.

¹⁹F NMR (377 MHz, DMSO) δ 41.61, -130.78, -138.40, -147.59. MS: 229.1 [C₆H₁F₄O₃S] ^-.

LC analysis of the model reaction to determine the proximal reactivity of phenyl sulfurofluoridate derivatives

To test the proximal reactivity of fluorine substituted phenyl sulfurofluoridate toward nucleophilic group, the model reaction was designed, in which the concentration of imidazole was 100-fold than sulfurofluridate to accelerate the reaction. In detail: 100 μL 10 mM fluorine substituted phenyl sulfurofluoridate solution in CH₃CN was mixed with 900 μL 110 mM imidazole solution (pH 8.5) in a 1.5 mL EP tube. The tube was placed in a thermal shaker and the reaction was conducted at 30 ℃ at 1000 rpm for 4 h. The mixture was analyzed by UPLC and absorption at 260 nm from benzene group was monitored and the conversion rate was calculated with the formula: Area of product / (Area of product + Area of reactant) x 100%.

As summarized in the (Figs. 3A-3D and Fig. 4) , both position and amounts of electro withdrawing fluorine atom have influence on the reactivity of sulfurofluridate. Especially, single fluorine substitution only slightly accelerates the reaction, while difluoro significantly accelerates the reaction.

Example 2

Chemoenzymatic synthesis of fluoro substituted fluorosulfate -L-tyrosine

Construction of plasmids encoding tyrosine phenol lyase (TPL) mutants

The Amino acid sequences encoding TPLs were obtained from Uniprot (www. uniprot. org) database, patents (CN109897845A) and literatures (European Journal of Organic Chemistry 2020, 8, 1050-1054., Journal of Industrial Microbiology and Biotechnology 2019, 12, 1631-1641. ) . The corresponding DNA sequences were synthesized by GENEWIZ (Shanghai, China) with codon optimization against E. coli as host. The synthesized gene with a 6 × His-tag at N terminal was flanked with the endonuclease sites of Nco I and Not I, which was digested by the two corresponding restriction enzymes, respectively and inserted into pre-digested vector pET-28a (+) with the same restriction enzymes termed pET-28a (+) -XX TPL plasmid.

Preparation of E. coli cells overexpressing TPLs

Plasmids pET-28a (+) -XX TPL was transformed into BL21 (DE3) by heat shock method. The transformants were plated on an LB-Kanamycin agar plate and incubated overnight at 37 ℃. A single colony was inoculated into 10 mL of LB medium containing 50 μg/mL kanamycin at 37 ℃. On the following day, 10 mL of overnight cell culture was diluted into 1 L fresh LB medium agitated vigorously at 37 ℃. When OD600 reached 0.4～0.6, IPTG was added to the final concentration of 0.5 mM, then induced at 30 ℃ for 12h. The cells were then harvested by centrifugation at 6,000g for 20 min and stored at -80 ℃ before usage.

General procedure of TPL catalyzed synthesis of fluorine substituted tyrosine by fed-batch method

Biosynthesis of fluorine substituted tyrosine was performed in 100 mL reaction mixture containing 50 mM sodium pyruvate, 50 mM ammonium chloride, 10 mM fluorophenol, 1.0 mM PLP, 0.1g/L TritonX-100, 2.0 g/L EDTA sodium and 15.0 g/L recombinant E. coli cells at pH 8.5 adjusted by ammonia. The reaction was carried out at 30 ℃ with shaking at 170 rpm for 24 h. During the reaction process, sodium pyruvate, fluorophenol and ammonium chloride were fed every 4 hours with a concentration of 5.0 g/L, 2.0 g/L and 3.5 g/L, respectively. The reaction can be monitored with ¹⁹F NMR.

After the synthesis reaction was completed, the pH was adjusted to 1-2 with 6 mol/L HCl solution and stirred vigorously for 1h, followed by filtration and remove the precipitation. The filtrate was then collected and gradually titrated up to pH7.0 with ammonia water (25-28 %) . Removing excess water under reduced pressure until there are about 100 mL residue. Thereafter, the L-tyrosine derivatives that evolved during the ammonia titration were recovered by filtration, washed twice with ice-cold water, and dried at 60 ℃ after being washed once more with ethanol.

As shown in Fig. 5, the present application provides specific activity of TPLs from different sources for 2-fluoro-L-tyrosine synthesis.

As shown in Fig. 6, the present application shows that Cf TPL and Fn TPL demonstrate efficient synthesis of difluorine substituted L-tyrosine.

As shown in Fig. 7, the present application provides chemical structure of TPL catalyzed synthesis of fluorine substituted L-tyrosine.

3-fluoro-L-tyrosine

¹H NMR (400 MHz, D₂O) δ 6.59 (d, 1H) , 6.54 –6.45 (m, 2H) , 3.89 –3.81 (m, 1H) , 2.78 (dd, 5.6 Hz, 1H) , 2.67 (dd, 1H) ; ¹⁹F NMR (377 MHz, D₂O) δ -136.80 (dt, 7.7 Hz) ; MS: 198.3 [C₉H₉FNO₃] ^-.

2-fluoro-L-tyrosine

¹H NMR (400 MHz, D₂O) δ 6.71 (dd, 1H) , 6.25 –6.19 (m, 2H) , 3.87 (t, 1H) , 2.85 (dd, 1H) , 2.71 (dd, 1H) ; ¹³C NMR (101 MHz, D₂O) δ 170.79, 162.65, 160.23, 156.69, 132.15, 112.00 –111.29, 103.10, 102.85, 52.95, 28.58; ¹⁹F NMR (377 MHz, D₂O) δ -115.85 –-116.00; MS: 198.2 [C₉H₉FNO₃] ^-.

3,5-difluoro-L-tyrosine

¹⁹F NMR (377 MHz, D₂O) δ -132.86 –-133.09 (m) ; MS: 216.3 [C₉H₈F₂NO₃] ^-.

2,5-difluoro-L-tyrosine

¹H NMR (400 MHz, D₂O) δ 6.72 (dd, 1H) , 6.43 (dd, 1H) , 3.97 (t, 1H) , 2.93 (dd, 1H) , 2.79 (dd, 1H) ; ¹⁹F NMR (377 MHz, D₂O) δ -121.24 –-121.43, -142.09; MS: 218.1 [C₉H₁₀F₂NO₃] ⁺ .

2,3-difluoro-L-tyrosine

¹H NMR (400 MHz, D₂O) δ 6.46 (td, 1H) , 6.30 (t, 1H) , 3.85 (t, 1H) , 2.86 (dd, 1H) , 2.72 (dd, 1H) ; ¹⁹F NMR (377 MHz, D₂O) δ -141.33, -161.29 –-161.54; MS: 216.3 [C₉H₈F₂NO₃] ^-.

2,6-difluoro-L-tyrosine

¹H NMR (400 MHz, D₂O) δ 6.24 –6.14 (m, 2H) , 3.94 (t, 1H) , 2.93 (dd, 1H) , 2.86 (dd, 1H) ; ¹⁹F NMR (377 MHz, D₂O) δ -114.41 (d, J = 9.4 Hz) ; MS: 216.2 [C₉H₈F₂NO₃] ^-.

General synthesis procedure of fluorine substituted fluorosulfate-L-tyrosine from fluorine substituted L-tyrosine

5 g fluorine substituted L-tyrosine was dissolved in 50 mL 1M NaOH and 50 mL dioxane, followed by the dropwise addition of 10 mL (Boc) ₂O (1 eq) for 2 h under stirring at room temperature. The mixture was further stirred for 1 h. Then, the solvent was removed under reduced pressure and the residue was dissolved in100 mL DCM, washed with 0.1 N HCl (2 × 75 mL) , saturated NaCl solution (2 × 75 mL) step by step. The aqueous layer was collected and solvent removal for following reaction without further purification.

To a 2 L two-neck round-bottom flask containing a magnetic stir bar was added 5 mmol Boc protected fluorine substituted L-tyrosine, 200 mL of CH₂Cl₂ and 800 mL of a saturated Borax solution. The mixture was stirred vigorously for 20 minutes. The reaction system was vacuumed until the biphasic solution started to degas and refilled with SO₂F₂ for three times. The reaction mixture was stirred vigorously at room temperature overnight. CH₂Cl₂ was carefully removed using a rotary evaporator under reduced pressure. Then 1 M aqueous HCl (210 mL) was slowly added to the reaction mixture while stirring and white solid precipitated. The mixture was filtered and the solid was washed with ice cold water (3 × 75 mL) . The white solid was dried under vacuum overnight affording Boc protected fluorine substituted fluorosulfate-L-tyrosine, which was directly used in the next step without further purification. 5 mmol Boc protected fluorine substituted fluorosulfate-L-tyrosine was treated with 4 M HCl in dioxane (11 mL) and the reaction mixture was stirred for 4 h at room temperature, after which 50 mL ether was added to the mixture if there is no white solid precipitated. The solid was filtered and washed by cool ether (2 × 10 mL) , affording the targeted fluorine substituted L-tyrosine HCl salt. The product was analyzed with LC-MS and further purified by reverse phase preparative HPLC.

As shown in Fig. 8, the present application provides chemical structure of fluorine substituted fluorosulfate L-tyrosine.

Fluorosulfate-L-tyrosine

¹H NMR (400 MHz, D₂O) : δ (ppm) 3.23-3.41 (m, 2H) , 4.32-4.34 (m, 1H) , 7.45-7.53 (m, 4H) ; ¹³C NMR (400 MHz, D₂O) : δ (ppm) 38.9, 57.2, 125.0, 135.3, 139.5, 153.5, 173.3; ¹⁹F NMR (400 MHz, D₂O) : δ (ppm) 38.9, 57.2; MS: 264.0 [M+H] ⁺.

Fluorosulfate-L-tyrosine

¹H NMR (400 MHz, D₂O) δ 7.38 (s, 4H) , 4.28 (dd, 1H) , 3.34 –3.24 (m, 1H) , 3.19 (dd, 1H) ; ¹³C NMR (101 MHz, D₂O) δ 171.04, 149.44, 135.18, 131.51, 121.59, 66.53, 53.82, 34.87; ¹⁹F NMR (377 MHz, D₂O) δ 37.49; MS: 262.2 [C₉H₉FNO₅S] ^-.

Fluorosulfate-3-fluoro-L-tyrosine

¹H NMR (500 MHz, D₂O) : δ (ppm) 3.21-3.25 (m, 1H) , 3.32-3.36 (m, 1H) , 4.21-4.24 (t, 1H) , 7.22-7.58 (m, 3H) ; ¹³C NMR (500 MHz, D₂O) : δ (ppm) 35.24, 54.28, 118.79, 123.79, 126.55, 136.30, 137.86, 153.14, 171.66; ¹⁹F NMR (500 MHz, D₂O) : δ (ppm) -128.22, 38.82; MS: 282.0 [C₉H₁₀F₂NO₅S] ⁺, 305.0 [C₉H₉F₂NO₅SNa] ⁺ .

Fluorosulfate-2-fluoro-L-tyrosine

¹H NMR (500 MHz, D₂O) : δ (ppm) 3.19-3.23 (m, 1H) , 3.33-3.37 (m, 1H) , 4.18-4.20 (t, 1H) , 7.26-7.46 (m, 3H) ; ¹³C NMR (500 MHz, D₂O) : δ (ppm) 29.24, 53.34, 109.67, 117.50, 122.79, 133.19, 149.32, 161.54, 171.48; ¹⁹F NMR (500 MHz, D₂O) : δ (ppm) -112.16, 37.85; MS: 282.0 [C₉H₁₀F₂NO₅S] ⁺, 305.0 [C₉H₉F₂NO₅SNa] ⁺ .

Fluorosulfate-2, 3-difluoro-L-tyrosine

¹H NMR (400 MHz, D₂O) δ 7.35 (t, J = 7.7 Hz, 1H) , 7.25 –7.18 (m, 1H) , 4.31 –4.21 (m, 1H) , 3.38 (dd, J = 14.7, 6.4 Hz, 1H) , 3.25 (dd, J = 14.7, 7.1 Hz, 1H) ; ¹³C NMR (101 MHz, D₂O) ) δ 170.81, 151.13, 148.65, 144.03, 141.49, 137.09, 126.26, 124.79, 118.48, 66.53, 52.77, 29.16; ¹⁹F NMR (377 MHz, D₂O) δ 39.15, -136.33, -150.44; MS: 298.1 [C₉H₇F₃NO₅S] ^-, 597.2 [C₁₈H₁₅F₆N₂O₁₀S₂] ^-.

Fluorosulfate-2, 5-difluoro-L-tyrosine

¹H NMR (400 MHz, D₂O) δ 7.02 (dd, J = 8.8, 6.4 Hz, 1H) , 6.94 (dd, J = 10.1, 6.6 Hz, 1H) , 3.89 (t, J = 6.8 Hz, 1H) , 2.92 (dd, J = 14.7, 6.4 Hz, 1H) , 2.80 (dd, J = 14.7, 7.1 Hz, 1H) ; ¹³C NMR (151 MHz, D₂O) δ 170.16, 150.23, 148.58, 135.64, 123.73, 119.80, 111.21, 52.53, 28.77; ¹⁹F NMR (377 MHz, D₂O) δ 39.04, -117.77, -133.37;

MS: 298.2 [C₉H₇F₃NO₅S] ^-, 597.4 [C₁₈H₁₅F₆N₂O₁₀S₂] ^-.

Fluorosulfate-3, 5-difluoro-L-tyrosine

¹H NMR (500 MHz, D₂O) : δ (ppm) 3.17-3.20 (m, 1H) , 3.28-3.32 (m, 1H) , 4.20-4.22 (t, 1H) , 7.14-7.16 (d, 3H) ; ¹³C NMR (500 MHz, D₂O) : δ (ppm) 35.31, 53.87, 114.22, 125.22, 138.00, 153.28, 171.25; ¹⁹F NMR (500 MHz, D₂O) : δ (ppm) -124.96, 40.65; MS: 300.0 [C₉H₉F₃NO₅S] ⁺, 323.0 [C₉H₈F₃NO₅S Na] ⁺ .

Fluorosulfate-2, 6-difluoro-L-tyrosine

¹H NMR (400 MHz, DMSO) δ 7.68 (t, 2H) , 4.03 –3.92 (m, 1H) , 3.27 (dd, 1H) , 3.18 (dd, 1H) . 13C NMR (101 MHz, DMSO) δ 170.05, 162.85, 160.37, 148.77, 113.91, 106.88, 66.81, 56.45, 51.44 , 23.75, 19.00.19F NMR (377 MHz, DMSO) δ (ppm) 40.28, -109.06.

MS: 299.8 [C₉H₉F₃NO₅S] +.

LC analysis of the model reaction to determine the proximal reactivity of Fluorosulfate-L-tyrosine derivatives

Similar reaction was done to test the proximal reactivity of fluorine substituted L-tyrosine toward nucleophilic group: 100 μL 10 mM fluorine substituted L-tyrosine solution was mixed with 900 μL 110 mM imidazole solution (pH 8.5) in a 1.5 mL EP tube. The tube was placed in a thermal shaker and the reaction was conducted at 30 ℃ at 1000 rpm for 4 h. The mixture was analyzed by UPLC and absorption at 260 nm was monitored. The conversion rate was calculated with formula: Area of product/ (Area of product + Area of reactant) x 100%.

The result was summarized in the (Figs. 9A-9D and 10) , both position and amounts of electro withdrawing fluorine atom have influence on the reactivity of sulfurofluridate.

Example 3

Aminoacyl-tRNA Synthetase Selection against fluorosulfate-3, 5-difluoro-L-tyrosine

Construction of the pBK-chFSYRS S4 mutant library plasmids

pBK chFSYRS plasmid was generated by introducing the chFSYRS encoding gene from pSup chFSYRS into pBK vector via ligation independent cloning. Briefly, the chFSYRS gene was amplified with following primers, purified, and ligated into pBK vectors with Exnase II (Vazyme, Cat: C112-01) .

Considering the crystal structure of OMEY-MmPylRS synthetase, four residues are proximal to the backbone of tyrosine derivate, which may have interaction with newly introduced fluoro atoms. Therefore, the pBK-chFSYRS S4 mutant library (S364NNK, V366NNK, G384NNK, G386NNK) of chimera Mm/MbPylRS was constructed using whole plasmid amplification of the iterative saturation mutagenesis (ISM) approach, which introduce mutations during PCR by using synthetic DNA oligonucleotides containing one or more degenerate codons at the target residue. Firstly, primer S364-V366-NNK Forward and primer S364-V366-NNK Reverse containing degenerate codons (NNK) were used to amplify the pBK-chFSYRS plasmid. Then, the resultant library was treated with DpnI to eliminate the parental methylated DNA strands and transformed into DH10B cells. The cells were collected and extracted the plasmids with miniprep Kit to get the pBK-chFSYRS S2 mutant library. The above process was repeated with primer G384-G386-NNK Forward and primer G384-G386-NNK Reverse containing degenerate codons (NNK) using pBK-chFSYRS S2 mutant library as the template, resulting the final The pBK-chFSYRS S4 mutant library with 10⁷ colones.

Selections of active chimeric FSYRS variants for UAAs were carried out with three rounds of positive selection and subsequently negative selection. chPheRS libraries in pBK vectors were firstly electroporated into DH10B competent cells harbouring the negative selection plasmid pPOS-CAT112TAGchPheT-GFP190TAG that contains CAT and GFP dual reporter genes with an amber codon (112TAG for CAT, 190TAG for GFP) , respectively. DH10B cells (100 μL) harboring the pPOS selection reporter was further transformed with 100 ng of pBK-chFSYRS S4 mutant library via electroporation. The electroporated cells were immediately recovered with 1 mL of pre-warmed SOC media and agitated vigorously at 37 ℃ for 1 h, followed by addition of fluorosulfate-3, 5-difluoro-L-tyrosine to the final concentration of 1 mM and agitated vigorously at 37 ℃ for another 1 h. The recovered cells were directly plated on a LB-agar selection plate supplemented with 1 mM fluorosulfate-3, 5-difluoro-L-tyrosine, 12.5 μg mL-1 of 50 μg/ml kanamycin (Kan) , 100 μg/mL of ampicillin (Amp) and 34 μg/mL of chloramphenicol (Cm) . The selection plate was incubated at 37 ℃for 48 h and then stored at room temperature. 57 clones present fluorosulfate-3, 5-difluoro-L-tyrosine -dependent growth were considered as hits and further characterized by Sanger-sequencing. During the positive selection, the pBK-chFSYRS S4 mutant library was parallelly electroporated into DH10B competent cells harboring the negative selection plasmid pNEG-Barnase-Q3TAG-D45TAG-chPheT. The transformed cells were recovered for 1 h at 37 ℃ then plated on LB agar containing 50 μg/mL kan, 100 μg/mL Amp and 34 μg/ml Cm and plated on a LB-agar selection plate. After 24 h of incubation at 37 ℃, there is no clone was found on the plate, which suggest that the synthetases are orthogonal to natural amino acids. Finally, a smaller synthetase library was generated based on the sequencing result and one more round of positive selection was conducted.

A unique amino acid sequence (S364S, V366A, G384G, G386G) was found in the sequencing result. Although the four sites were randomly mutated, three of them were enriched as the original residues, which suggest that they are highly conserved. pSup chFSYRS V366A plasmid was generated by site specifically introducing the Alanine mutation into chFSYRS using the following primers:

pSup chFSYRS V366A (Figs. 11A-11C) was subjected to further characterization to determine the fidelity and processivity of other fluoro substituted unnatural amino acids.

Assessment of amber suppression efficiency in E. coli

The plasmid pBAD GFP 149TAG bearing the GFP-149TAG with a 6xHis tag and the plasmid pSup chFSYRS V366A or pSup chFSYRS V366A carrying the corresponding chimeric synthetase were co-transformed into chemically competent DH10B cells, respectively. The transformed cells were recovered in SOC medium for 1 h with shaking at 37 ℃ and plated on LB agar containing 34 μg/ml chloramphenicol and 100 μg/ml ampicillin for 12 h at 37 ℃. A single colony was picked and grown in 2 ml of LB medium containing required antibiotics at 37 ℃ until OD600 reaching 0.4～0.6 and 1 mM of corresponding unnatural amino acids was added, then incubated at 30 ℃ for 30 min. The cell culture was induced with 0.2%arabinose and incubated at 30 ℃ for 10 h. After induction, 1 ml of cell cultures were collected by centrifuging, and then lyzed by 150 μl BugBuster Protein Extraction Reagent (Millipore) for 10 min at 37 ℃. The supernatant of the lysate (100 μl) was transferred to a 96-well cell culture plate (Costar) . And GFP signals of the supernatant were recorded by TECAN SPARK microplate reader with a background subtraction and normalized by the bacterial density (OD600) that was measured by TECAN SPARK microplate reader as well.

At the same time, 1 ml of cell cultures were collected by centrifuging and resuspended with 100 μL 1xloading buffer, heated at 95 ℃ for 30 min. 10 μL of samples were loaded in 4-20%or 8-16%sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) for electrophoresis and transferred to a 0.22 μm polyvinylidene fluoride (PVDF) membrane (Millipore) . After protein transfer, the membrane was blocked with 5%bovine serum albumin in TBST buffer (20 mM Tris, pH 8.0, 150 mM NaCl, 0.1%Tween-20) for 1 h at room temperature with gentle shaking. The membrane was incubated with primary antibody (1: 1,000; His tag antibody) at room temperature for 1 h. After incubation with primary antibody, the membrane was washed three times (each for 5 min) with TBST buffer. The membrane was then incubated with horseradish peroxidase (HRP) -conjugated rabbit anti-mouse IgG antibodies (1: 5,000 dilution) for 1 h at room temperature. Finally, the membrane was washed three times (each for 5 min) with TBST buffer. The western blot bands were detected by using an electro-chemiluminescence (ECL) western blotting substrate (Millipore, Cat#WBKLS0500) .

Comparing with chFSYRS, chFSYRS V366A variant showed a 9-fold increase in GFP fluorescence incorporated with fluorosulfate-3, 5-difluoro-L-tyrosine, which validate the positive result of the above screening (Figs. 12-13) . Besides, chFSYRS V366A demonstrated enhanced efficiency in incorporating fluorosulfate-2-difluoro-L-tyrosine, fluorosulfate-2, 5-difluoro-L-tyrosine and fluorosulfate-2, 3-difluoro-L-tyrosine, but a decreased efficiency in incorporating fluorosulfate-L-tyrosine and fluorosulfate-3-difluoro-L-tyrosine than chFSYRS. Western blotting analysis of the expression of full length GFP confirm the fluorescence intensity. We proposed that the residue of amino acid in synthetase position 366 is proximal to the meta atom, while fluorine atom is a little larger than hydrogen atom, a minimized residue of alanine will contribute to a lower steric hindrance and methyl group of alanine may have interaction with fluorine, which stabilize the intermediate. Unfortunately, chFSYRS V366A could not mediate the incorporate the fluorosulfate-2, 6-difluoro-L-tyrosine, although this PrUAA is not the one with best proximal reactivity among difluoride added fluorosulfate-L-tyrosine.

Assessment of amber suppression efficiency in mammalian cells

Fluoro substituted fluorosulfate-3, 5-difluoro-L-tyrosine incorporation into proteins in mammalian cells was further tested. The EGFP with TAG mutation at site Y40 was synthesized by (GENWIZ, China) and inserted into pcDNA3.4 vector under the control of CMV, resulting pcDNA3.4-EGFP-Y40TAG as reporter plasmid and suppression of the Y40TAG codon would produce full-length EGFP rendering cells fluorescent. The chimeric synthetases and tRNA genes with codon optimization for human cells were synthesized by (GENWIZ, China) and inserted into pCMV vector by Gibson Assembly under the control of CMV and U6 promoter, resulting, pCMV 8tRNA-chFSYRS and pCMV 8tRNA-chFSYRS V366A, respectively. HEK 293T cells were grown in DMEM medium supplemented with 10%foetal bovine serum and 1%penicillin–streptomycin. Cells were co-transfected with the synthetase plasmids and the reporter plasmid at ratio 1: 1 (μg: μg) . Transfections were performed by PEI according to the manufacturer’s protocol with or without the addition of the corresponding amino acids. Imaging was performed 24 h after transfection. Live HEK 293T cells were imaged XD Inverted Fluorescence Microscope equipped with a 10× objective lens (PlanFluor, SOPTOP) at FITC channel. All images were analysed and processed with ImageJ software (National Institutes of Health) .

Strong EGFP fluorescence was observed from cells transfected with chFSYRS and chFSYRS V366A plasmids and when corresponding UAA was added, which is consistent with the amber suppression result in E. coli. Notably, cell morphology remained normal, which suggest that there is no obvious toxicity of fluoro substituted fluorosulfate-3, 5-difluoro-L-tyrosine to HEK 93T cells, a valuable characteristic of fluoro substituted fluorosulfate-3, 5-difluoro-L-tyrosine possibly due to the extremely low background reactivity of aryl fluorosulfate inside cells. These results demonstrate that fluoro substituted fluorosulfate-3, 5-difluoro-L-tyrosine was incorporated into proteins in mammalian cells using our evolved tRNA synthetase with high efficiency and specificity without causing detrimental effects.

In summary, a series of biorthogonal fluoro substituted fluorosulfate-L-tyrosine with enhanced proximal reactivity and the corresponding tRNA synthetase was developed. Their genetic encoding capacity was validated in both E. coli and mammalian cells. Since these PrUAA could react with residues of His, Lys and Tyr via SuFEx reaction, which are often found at protein surface and interface, our invention would have broad application in the development of covalent protein drugs.

Example 4

Covalently engineered PD-L1 blocking nanobody (GlueBody) with enhanced T cell activity restoration

Expression and purification of PrUAA incorporated Anti PD-L1 VHH (GlueBody)

Plasmids pBAD-Nb-PD-L1-His (TAGs) were separately co-transform with plasmid pSupAR-chFSYRS or pSupAR-chFSYRS V366A into DH10B competent cells. The transformants were plated on an LB-Ampicillin-Chloramphenicol agar plate and incubated overnight at 37 ℃. A single colony was inoculated into 10 mL of LB medium containing 100 μg/mL Ampicillin and 34 μg/mL Chloramphenicol at 37 ℃. On the following day, 10 mL of overnight cell culture was diluted into 1 L fresh Rich nutrition medium (15 g Na₂HPO₄·12H₂O, 6 g KH₂PO₄, 20 g Tryptone, 5 g Yeast extracts, 5 g NaCl, 200 mg CaCl₂, 200 mg MgCl₂, 8 g Glycerol, 0.5 g Glucose) and agitated vigorously at 37 ℃. The bacteria were incubated with 1 mM PrUAA when OD₆₀₀ reached 0.6 and induced with 0.2%arabinose when OD₆₀₀ reached 0.8-1.0. After induction of expression at 30℃ for 12 h, bacteria were harvested by centrifugation at 6000 rpm for 30min. Then, resuspending cells with 100 mL binding buffer, (50 mM Tris-HCl pH 8.0, 500 mM NaCl, 20 mM imidazole, lysozyme 1 mg/mL and protease inhibitors) . The cell suspension was sonicated with Sonic Dismembrator (SCIENTZ, 30%output, 30 min, 1 sec off, 1 sec on) in an ice-water bath, followed by centrifugation (15,000 g, 30 min, 4 ℃) . The soluble fractions were collected and loaded onto a pre-packed Ni-NTA column, eluted with NTA 250 buffer (50 mM Tris-HCl pH 8.0, 500 mM NaCl, 250 mM imidazole) followed by further purification on a superdrex 75 column immediately, and stored at -80℃ for future analysis. Molecular weight of the intact wild type and UAA-incorporated proteins were analyzed by a Waters ACQUITY UPLC I-Class SQD 2 MS spectrometer with electrospray ionization (ESI) . LC separation for sortase-mediated protein conjugation was carried out with a BEH300 C4 Acquity column (1.7 μm, 2.1 × 100 mm) , and positive mode was chosen for ESI-MS to analyze all samples. The total mass of proteins was calculated using MassLynx V4.1 software (Waters) .

ESI-MS analysis showed correct incorporation of four PrUAAs and there is no VHHs with natural amino acids or VHHs with active molecule added detected, which verified the incorporation fidelity and confirmed the biorthogonality of our PrUAAs (Figs. 14A-14D) .

In vitro crosslinking of GlueBodies with PD-L1

The purified GlueBodies was incubated with PD-L1 (Sino Biological, Cat#10084-HNAH) at the molar ratio of 1: 1 in PBS buffer at 37℃ for 5 h. The amount of PD-L1 was 2 μg. 5x reduced loading buffer (CWBio, Cat#CW0027) was added into the tube and heated at 95℃ for 15 min. These samples were then separated by 8-16%SDS-PAGE gel followed by staining with Coomassie brilliant blue. The gray intensity of the band was quantified on ImageJ software.

Since covalent bond cannot be disrupted by denature conditions, SDS-PAGE was used to distinguish the covalent binding and non-covalent binding. The results showed that all the Nb-PD-L1 with PrUAAs at 108 site cross-linked with the target PD-L1 as the new band with molecular weight corresponding to Nb/Ag conjugate was observed on SDS-PAGE. Notably, Nb-PD-L1 with fluorosulfate-2, 5-difluoro-L-tyrosine (28%) and fluorosulfate-2-fluoro-L-tyrosine (26%) showed about 2-fold increasing in the crosslinking ratio in comparison with FSY (15%) , suggesting fluoro addition accelerate the covalent bond formation (Fig. 15) . Notably, although fluorosulfate-3-fluoro-L-tyrosine demonstrate enhanced proximal reactivity than that of fluorosulfate-2-fluoro-L-tyrosine towards electrophilic imidazole, GlueBodies does not behave theoretical covalent binding capacity, which may be resulted from the additional interaction from the introduced fluorine and residues in antigen.

PD-1/PD-L1 blockage assays

To verify that the increased crosslinking will contribute to the enhanced T cell activation restoration, we conducted the following PD-1/PD-L1 blockage assays. PD-1 NFAT-luciferase/Jurkat cells were cultured in RPMI 1640 medium with 10%FBS, 1%Pen-Strep. PD-L1 aAPC/CHO-K1 cells were cultured in Ham’s F-12 with 10%FBS, 1%Penn-Strep (growth medium) . To perform the blockage assay, PD-L1 aAPC/CHO-K1 cells were seeded at a density of 10,000 cells per well into white 96-well microplate in 100 μL of growth medium. After cell attachment, the medium was removed from the CHO-K1 cells, and cells were incubated with the fresh growth medium supplemented with HBSS, 20 nM GlueBodies for 22 h, followed by washing away the antibody and adding 10,000 PD-1 NFAT-luciferase/Jurkat cells in assay medium (RPMI 1640, 10%FBS, 1%Pen-Strep) with same drugs or control. After 5-6 hours of co-culture, cells were lysed and luciferase assay was performed using Luciferase Reporter Gene Assay Kit (Yeasen, Cat#11401ES76) . Luminescence was measured using a TECAN Spark microplate reader.

The addition of antibodies disrupting PD-1/PD-L1 interactions between the engineered Jurkat cells and CHO-K1 cells would relieve the inhibitory signal and restore T-cell receptor (TCR) signaling. Therefore, CHO-K1 cells were separately treated with GlueBodies or controls. When compared to the non-covalent counterpart, covalently engineered both Nb-PD-L1 L108FSY and Nb-PD-L1 L108m-F FSY showed an enhancement of T-cell activation (Fig. 16) and stronger covalent binding would contribute to a stronger activation as Nb-PD-L1 L108m-F FSY behave better than that of Nb-PD-L1 L108FSY. Traditional antibody binds to the target in a dynamic manner, whereas the dissociation of antibody is not what desired. On the contrary, GlueBody binds to the target irreversibly and accumulation of blocking antibody on cancer cells provided a stronger and more sustainable effect.

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

Claims

A compound having the structure (I) of:

wherein, said n is more than 0, and X is selected from the electron-withdrawing group.
A compound having the structure (II) of:

wherein, said n is more than 0, and X is selected from the electron-withdrawing group.
The compound of any of claims 1-2, wherein said structure (I) and/or structure (II) is linked to a linkage group R₁, said R₁ is optionally substituted substitution.
The compound of claim 3, said R₁ is a bond or is selected from group consisting of: -S (O) ₂-, -NR^1A-, -O-, -S-, -C (O) -, -C (O) NR^1A-, -NR^1AC (O) -, -NR^1AC (O) NR^1B-, -C (O) O-, -OC (O) -, optionally substituted alkylene, optionally substituted heteroalkylene, optionally substituted cycloalkylene, optionally substituted heterocycloalkylene, optionally substituted arylene, and optionally substituted heteroarylene, each said R^1A and R^1B is independently hydrogen, optionally substituted alkyl, optionally substituted heteroalkyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl.
The compound of any of claims 3-4, wherein said linkage group R₁ is linked to a linkage group R₂, said R₂ is optionally substituted substitution.
The compound of claim 5, said R₂ is a bond or is selected from group consisting of: optionally substituted peptidyl moiety, optionally substituted nucleic acid moiety, and optionally substituted carbohydrate moiety.
The compound of any of claims 5-6, wherein said structure (I) and/or structure (II) is linked to said R₂ via optionally substituted tyrosine.
A compound having the structure (I-A) of:

wherein, said n is more than 0, and X is selected from the electron-withdrawing group.
A compound having the structure (I-P) of:

wherein, said n is more than 0, and X is selected from the electron-withdrawing group.
A compound having the structure (II-A) of:

wherein, said n is more than 0, and X is selected from the electron-withdrawing group.
A compound having the structure (II-P) of:

wherein, said n is more than 0, and X is selected from the electron-withdrawing group.
The compound of any of claims 1-11, wherein said n is selected from the group consisting of: 1, 2, 3 and 4.
The compound of any of claims 1-12, wherein said compound having the structure of:
The compound of any of claims 1-13, wherein said compound having the structure of:
The compound of any of claims 1-14, wherein said compound having the structure of:
A compound having the structure of: wherein, said X is selected from the electron-withdrawing group.
A compound having the structure of: wherein, said X is selected from the electron-withdrawing group.
The compound of any of claims 1-17, wherein each of said X is independently halogen.
The compound of any of claims 1-18, wherein each of said X is independently F.
A protein comprising the structure of the compound of any of claims 1-19.
A protein comprising an unnatural amino acid having the structure (I) of:

wherein, said n is more than 0, and X is selected from the electron-withdrawing group.
The protein of any of claims 20-21, wherein said protein comprises an unnatural amino acid having the structure of the compound of any of claims 1-19.
The protein of any of claims 20-22, wherein said protein is derived from antigen-binding protein or the fragment thereof.
The protein of any of claims 21-23, wherein said protein comprises said unnatural amino acid at position 108 as set forth in sequence of PD-L1 binding protein or the fragment thereof.
The protein of any of claims 21-24, wherein said protein comprises said unnatural amino acid at position 108 as set forth in sequence of anti-PD-L1 nanobody or the fragment thereof.
The protein of any of claims 21-25, wherein said protein comprises said unnatural amino acid at position 108 as set forth in sequence of SEQ ID NO: 1.
The protein of any of claims 20-26, wherein said protein further comprises proteolysis targeting moiety.
The protein of any of claims 20-27, wherein said protein further comprises mannose-6-phosphate, N-acetylgalactosamine (GalNAc) , cell-penetrating peptide and/or lysosome-sorting sequence.
A nucleic acid comprising a sequence encoding the protein of any of claims 20-28.
A synthetase comprising a mutant at position 366 and/or position 367 as set forth in sequence of SEQ ID NO: 29.
A synthetase comprising an alanine at position 366 and/or position 367 as set forth in sequence of SEQ ID NO: 29.
A synthetase comprising a sequence of SEQ ID NO: 31 and/or 34.
A nucleic acid comprising a sequence encoding the synthetase of any of claims 30-32.
A nucleic acid comprising a sequence of SEQ ID NO: 30 and/or 33.
A vector comprising the nucleic acid of any of claims 29 and 33-34.
A combination comprising the synthetase of any of claims 30-32 and the compound of any of claims 1-19.
A method of preparing the protein of any of claims 20-28, wherein said method comprises providing the synthetase of any of claims 30-32, the compound of any of claims 1-19 and/or the combination of claim 36.
A cell comprising the compound of any of claims 1-19, the protein of any of claims 20-28, the nucleic acid of any of claims 29 and 33-34, the synthetase of any of claims 30-32, the nucleic acid of any of claims 29 and 33-34, the vector of claim 35, and/or the combination of claim 36.
A composition comprising the compound of any of claims 1-19, the protein of any of claims 20-28, the nucleic acid of any of claims 29 and 33-34, the synthetase of any of claims 30-32, the nucleic acid of any of claims 29 and 33-34, the vector of claim 35, the combination of claim 36 and/or the cell of claim 38, and optionally a pharmaceutically acceptable adjuvant.
A kit comprising the compound of any of claims 1-19, the protein of any of claims 20-28, the nucleic acid of any of claims 29 and 33-34, the synthetase of any of claims 30-32, the nucleic acid of any of claims 29 and 33-34, the vector of claim 35, the combination of claim 36, the cell of claim 38, and/or the composition of claim 39.
A method for inhibiting binding of a PD-L1 protein to a PD-L1 ligand, wherein said method comprises providing the compound of any of claims 1-19, the protein of any of claims 20-28, the nucleic acid of any of claims 29 and 33-34, the synthetase of any of claims 30-32, the nucleic acid of any of claims 29 and 33-34, the vector of claim 35, the combination of claim 36, the cell of claim 38, the composition of claim 39, and/or the kit of claim 40.
A method for activating immune cell, wherein said method comprises providing the compound of any of claims 1-19, the protein of any of claims 20-28, the nucleic acid of any of claims 29 and 33-34, the synthetase of any of claims 30-32, the nucleic acid of any of claims 29 and 33-34, the vector of claim 35, the combination of claim 36, the cell of claim 38, the composition of claim 39, and/or the kit of claim 40.
The method of claim 42, wherein said immune cell comprises T cell.
The method of any of claims 42-43, wherein said activating is tested via luciferase assay.
A method of preparing a compound having the structure (M) of:

wherein, said n is more than 0, and X is selected from the electron-withdrawing group; wherein said method comprises providing a ligase comprising sequence selected from the group consisting of: SEQ ID NO: 15, SEQ ID NO: 21, SEQ ID NO: 25, and SEQ ID NO: 27.
The method of claim 45, wherein said method comprises providing a tyrosine phenol lyase encoded by sequence selected from the group consisting of: SEQ ID NO: 14, SEQ ID NO: 20, SEQ ID NO: 24, and SEQ ID NO: 26.
The method of any of claims 45-46, wherein said n is selected from the group consisting of: 1, 2, 3 and 4.
The method of any of claims 45-47, wherein said compound comprises the structure of:
The method of any of claims 45-48, wherein each of said X is independently halogen.
The method of any of claims 45-49, wherein each of said X is independently F.