CN114409563A - Linker for protein labeling and application thereof in biological medicine - Google Patents

Linker for protein labeling and application thereof in biological medicine Download PDF

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CN114409563A
CN114409563A CN202011173629.8A CN202011173629A CN114409563A CN 114409563 A CN114409563 A CN 114409563A CN 202011173629 A CN202011173629 A CN 202011173629A CN 114409563 A CN114409563 A CN 114409563A
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陈小华
郭安娣
周宾山
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Shanghai Institute of Materia Medica of CAS
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Abstract

The present invention providesA kind of linker is used for protein labeling and the application thereof in biological medicine, in particular to a photoinduced o-nitrobenzyl alcohol linker shown in a general formula (I) and the application. The photoinduced o-nitrobenzyl alcohol linker developed by the invention can specifically mark protein side chain lysine, and then specifically couple affinity markers, fluorescent substances, active drugs and the like to biological macromolecules such as proteins and antibodies, so that a direct way is provided for the development of novel protein drugs and antibody drugs ADC drugs, biological diagnostic reagents, imaging probes and the like.

Description

Linker for protein labeling and application thereof in biological medicine
Technical Field
The invention belongs to the technical field of biological pharmacy and biology, and mainly relates to a novel photoinduced o-nitrobenzyl alcohol compound which is used for selectively marking lysine free amino in polypeptides and proteins and is applied to the purposes of specifically coupling affinity markers, fluorescent substances, active drugs and the like to the polypeptides and the proteins so as to develop targeted tracing diagnostic reagents, tumor targeted therapeutic drugs and the like.
Background
Proteins are an important class of biological macromolecules and are the material basis of life. The specific spatial structure of a protein determines the function of the protein. On the basis of maintaining the integrity and the function of the protein, the protein is subjected to chemical selective marker modification through chemical reaction of functional groups so as to obtain a novel bioconjugate, which is particularly important in the fields of chemical biology and biomedical research. In more and more research reports, not only methods for modifying translated proteins by using a series of enzymes, but also labeling methods by using conjugation of small molecule substances, and selective labeling using natural amino acid residues in proteins is a very cost-effective strategy. Chemical modification processes directly convert the chemical structure of a particular residue in a protein, and because of the presence of various reactive groups in the protein, conjugation can occur at that particular site, especially where the natural amino acids cysteine and lysine both contain nucleophilic functional groups. The free amino group of lysine is also a group with high nucleophilic reactivity in protein molecules, and the chemical modification of the amino group is of great importance in protein sequence analysis and is receiving wide attention. Lysine free amino group is used as a binding site, activated carboxyl functional groups are generally adopted, and N-hydroxysuccinimide ester is particularly adopted, but the methods have the problems of insufficient specificity in selectivity, low coupling efficiency and the like, and a new method for selectively marking and modifying protein lysine residues is still required to be developed in biological application.
Antibody-drug conjugates (ADCs) are an emerging biologic therapy that utilizes a variety of tissue-specific antibodies in combination with a range of linker designs to enable the transport and selective release of cytotoxic drugs in the vicinity of tumors. ADC consists of three parts, an antibody (antibody), a linker (linker) and a cytotoxin (toxin), and is a chemically complex conjugate. ADC drug design is a complex fusion of antibody selection, binding strategies, linker design and payload potency. The choice of antibody is governed by the target antigen, and the linker is chosen according to the type of binding employed and the desired mechanism of action of the payload. Currently, 4 ADC drugs are approved by FDA for marketing, which are: adcetris (2011), Kadcyla (2013), Mylotarg (2017) and bespossa (2017), with over 60 ADC drugs in clinical studies. The antibody modification sites commonly used for ADC drugs in clinical studies are mainly composed of two types, lysine residues and cysteine residues. When cysteine residues are selected as coupling sites, although the antibody has only 4 pairs of interchain disulfide bonds, and DAR values of ADC drugs formed by the cysteine residues generated by reducing the interchain disulfide bonds are theoretically uniform, the uniformity of final products is poor due to poor selectivity of the existing reducing agents (DTT and TCEP) on the interchain disulfide bonds, and the disulfide bonds of the antibody are damaged, so that the stability of the antibody is influenced. When lysine residues are selected as the coupling sites, because an antibody contains more than 40 lysine residues, the chemical selectivity is very poor, and the drug combination sites and the combination number are complex and simultaneously very wide drug loading distribution is obtained. Nevertheless, three of the four ADCs on the market today have successfully adopted a lysine coupling strategy.
Therefore, the development of a novel method for selectively marking and modifying the lysine residues of the protein not only has a very positive effect on the research of the protein per se, but also has unique advantages when the method is used for the site-specific synthesis of ADC drugs. So far, no chemical reaction functional group with photoinduced activity is introduced into the selective labeling modification of protein lysine residues. The light-induced chemically reactive functional groups are inert reactive functional groups in the absence of light, and upon irradiation with light of a specific wavelength, such reactive functional groups produce highly reactive intermediates that undergo a chemical reaction in which they form irreversible covalent bonds with the site of action on the protein to which they are targeted. The photo-crosslinking reaction has the advantages of high speed, simple conditions, suitability for in-situ reaction and the like. Therefore, it is necessary to develop a new method for selectively labeling protein lysine residues with high efficiency, reliability and good selectivity.
Disclosure of Invention
The inventor designs a reaction functional group with photoinduction activity, which has a simple structure and is easy to synthesize, wherein the photoinduction activity functional group mainly contains the structure of o-nitrobenzyl alcohol. The reaction functional group mainly reacts with amino group under the light induction condition, and mainly reacts with side chain amino group of lysine in protein in the protein compound to form Indazolone (Indazolone), so that covalent connection with stable structure is formed. The reaction with the polypeptide or protein is as follows:
Figure BDA0002748074610000021
the o-nitrobenzyl alcohol linker can be reacted with lysine on protein under very mild conditions, and has very high reaction speed and high reaction efficiency (chem.,2019,5,2955-2968.RSC adv.,2019,9, 13249-13253). Therefore, the photoinduced o-nitrobenzyl alcohol linker developed by the invention can specifically mark lysine of corresponding protein, and then specifically couple affinity markers, fluorescent substances, active drugs and the like to polypeptide and protein, thereby providing a direct path for the design and development of novel ADC drugs, biological diagnostic reagents, imaging probes and the like.
An object of the present invention is to provide a compound represented by the general formula (I), a tautomer, an enantiomer, a diastereomer, a racemate, an isotopic compound thereof, and various forms of salts or hydrates thereof.
Another object of the present invention is to provide methods for selectively labeling lysine in polypeptides and proteins using the compounds.
The invention also aims to provide application of the compounds in preparing antibody-fluorescent tracer substance conjugates and antibody-drug conjugates.
Another object of the present invention is to provide a novel method for developing antibody-drug conjugates from such compounds.
The invention provides a compound shown as the following general formula (I), or a tautomer, enantiomer, diastereomer, racemate, isotopic compound, various forms of salts or hydrates thereof.
Figure BDA0002748074610000031
Wherein Y is selected from: -CO-, -NH-CH2-、-O-CO-CH2-、-NH-COO-CH2-、-NH-CO-NH-CH2-、-COOCH2-、-CO-NH-、-O-CH2-、-CH2-、-COO-、-OCO-、-O-、-S-、-SO2-、-C≡C-、-C＝C-、-SO2NH-、-NHCONH-、-NHCSNH-、-NH-、-CONH-CH2-or absent, wherein one end of Y may be attached to the 3,4, 5 or 6 position of the phenyl ring in the ortho-nitrobenzyl alcohol structure;
wherein R is1Is one or more than one substitution of any position except Y substitution position on 3,4, 5 or 6 position in the structure of o-nitrobenzyl alcohol, and when R is polysubstituted1May be the same or different, R1Each independently selected from hydrogen, deuterium, amino, halogen, nitro, cyano, C1-6Alkyl radical, C3-10Cycloalkyl radical, C1-5Alkoxy radical, C1-6Alkylamino or aminoalkyl radical, C1-C6Alkylcarbonyl group, C2-C6Alkoxycarbonyl group, C2-C6Alkylamino carbonyl, C5-8Heterocyclic group, C6-10Aryl radical, C5-6A heteroaryl group,
Figure BDA0002748074610000032
Wherein n is0And n1Is 1, 2, 3,4 or 5, wherein said alkyl, cycloalkyl, alkoxy, alkylamino or aminoalkyl, alkylcarbonyl, alkoxycarbonyl, alkylaminocarbonyl, heterocyclyl, aryl, heteroarylOptionally further substituted by one or more groups selected from halogen, hydroxy, amino, C1-C6Alkoxy, cyano, nitro; in particular, R1Each independently is hydrogen;
r is selected from hydrogen, deuterium, halogen, nitro, cyano, hydroxyl, alkyl hydroxyl, aryl hydroxyl, alkyl amino, aryl amino, sulfydryl, alkyl sulfydryl, aryl sulfydryl, carboxylic acid, alkyl carboxylic acid, aryl carboxylic acid, alkynyl, alkyl alkynyl, aryl alkynyl, azide, alkyl azide, aryl azide, carbonyl, alkyl carbonyl, aryl carbonyl, aldehyde group, alkyl aldehyde group, aryl aldehyde group, alkyl, cycloalkyl, alkoxy, heterocyclic group, aryl, heteroaryl or any combination thereof, wherein the above groups are optionally further substituted by one or more groups selected from halogen, hydroxyl, amino, C1-C6Alkoxy, cyano, nitro.
Preferably, in the compound represented by the general formula (I),
y is selected from: -CO-, -NH-CH2-、-O-CO-CH2-、-NH-COO-CH2-、-NH-CO-NH-CH2-、-COOCH2-、-CO-NH-、-O-CH2-、-CH2-、-COO-、-OCO-、-O-、-S-、-SO2-、-C≡C-、-C＝C-、-SO2NH-、-NHCONH-、-NHCSNH-、-NH-、-CONH-CH2-or absent, wherein one end of Y may be attached to the 3,4, 5 or 6 position of the phenyl ring in the ortho-nitrobenzyl alcohol structure;
wherein R is1Is one or more than one substitution of any position except Y substitution position on 3,4, 5 or 6 position in the structure of o-nitrobenzyl alcohol, and when R is polysubstituted1May be the same or different, R1Each independently selected from hydrogen, deuterium, amino, halogen, nitro, cyano, C1-6Alkyl radical, C3-10Cycloalkyl radical, C1-5Alkoxy radical, C1-6Alkylamino or aminoalkyl radical, C1-C6Alkylcarbonyl group, C2-C6Alkoxycarbonyl group, C2-C6Alkylamino carbonyl, C5-8Heterocyclic group, C6-10Aryl radical, C5-6A heteroaryl group,
Figure BDA0002748074610000041
Wherein n is0And n1Is 1, 2, 3,4 or 5, wherein said alkyl, cycloalkyl, alkoxy, alkylamino or aminoalkyl, alkylcarbonyl, alkoxycarbonyl, alkylaminocarbonyl, heterocyclyl, aryl, heteroaryl is optionally further substituted with one or more substituents selected from the group consisting of halogen, hydroxy, amino, C1-C6Alkoxy, cyano, nitro; in particular, R1Each independently is hydrogen;
r is selected from hydrogen, deuterium, halogen, nitro, cyano, hydroxyl, alkyl hydroxyl, aryl hydroxyl, alkyl amino, aryl amino, sulfydryl, alkyl sulfydryl, aryl sulfydryl, carboxylic acid, alkyl carboxylic acid, aryl carboxylic acid, alkynyl, alkyl alkynyl, aryl alkynyl, azide, alkyl azide, aryl azide, carbonyl, alkyl carbonyl, aryl carbonyl, aldehyde group, alkyl aldehyde group, aryl aldehyde group, alkyl, cycloalkyl, alkoxy, heterocyclic group, aryl, heteroaryl or any combination thereof, wherein the above groups are optionally further substituted by one or more groups selected from halogen, hydroxyl, amino, C1-C6Alkoxy, cyano, nitro.
More preferably, among the compounds represented by the general formula (I)
Wherein Y is selected from: -CO-, -NH-CH2-、-O-CO-CH2-、-NH-COO-CH2-、-NH-CO-NH-CH2-、-COOCH2-、-CO-NH-、-O-CH2-、-CH2-、-COO-、-OCO-、-O-、-S-、-SO2-、-C≡C-、-C＝C-、-SO2NH-、-NHCONH-、-NHCSNH-、-NH-、-CONH-CH2-or absent, wherein one end of Y may be attached to the 3,4, 5 or 6 position of the phenyl ring in the ortho-nitrobenzyl alcohol structure;
wherein R is selected from hydrogen, deuterium, halogen, nitro, cyano, hydroxyl, alkyl hydroxyl, aryl hydroxyl, alkyl amino, aryl amino, mercapto, alkyl mercapto, aryl mercapto, carboxylic acid, alkyl carboxylic acid, aryl carboxylic acid, alkynyl, alkyl alkynyl, aryl alkynyl, azide and alkyl azideNitrogen, aromatic azide, carbonyl, alkylcarbonyl, aromatic carbonyl, aldehyde group, alkylaldehyde group, arylaldehyde group, alkyl, cycloalkyl, alkoxy, heterocyclic group, aryl, heteroaryl or any combination thereof, wherein the above groups are optionally further substituted by one or more groups selected from halogen, hydroxy, amino, C1-C6Alkoxy, cyano, nitro;
wherein R is1Is one or more than one substitution of any position except Y substitution position on 3,4, 5 or 6 position in the structure of o-nitrobenzyl alcohol, and when R is polysubstituted1May be the same or different, R1Each independently selected from hydrogen, deuterium, amino, halogen, C1-3Alkoxy, nitro,
Figure BDA0002748074610000042
Wherein n is0And n1Is 1, 2, 3,4, 5, in particular R1Each independently hydrogen.
Further preferably, the compound of formula (i) is selected from the following formulae:
Figure BDA0002748074610000051
wherein R and R1The definitions of (a) are the same as those described above.
The term "halogen" refers to fluorine, chlorine, bromine or iodine.
The term "hydrocarbyl" refers to a substituent containing only carbon and hydrogen atoms, including, without limitation, methyl, ethyl, isopropyl, propyl, cyclohexyl, phenyl, and the like.
The term "C1-C6" alkyl refers to a straight or branched chain saturated alkyl group having 1 to 6 carbon atoms in the chain, including without limitation methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, and the like.
The term "cycloalkyl" refers to a saturated cyclic alkyl group consisting of carbon atoms, including without limitation cyclobutyl, cyclopentyl, cyclohexyl, and the like.
The term "C3-C10 cycloalkyl" refers to a saturated mono-or poly-cycloalkyl group containing 3 to 10 carbon atoms, including, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl.
The term "C6-C10 aryl" refers to aromatic ring groups containing 6-10 ring atoms, but no heteroatoms in the ring atoms, such as phenyl, naphthyl.
The term "5-8 membered heterocyclyl" means a ring containing one or more saturated and/or partially saturated rings, including 5 to 8 ring atoms, wherein one or more ring atoms are selected from heteroatoms of nitrogen, oxygen or sulfur, the remaining ring atoms being carbon; for example, propylene oxide, tetrahydrofuranyl, pyrrolidinyl, tetrahydropyranyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl.
The term "5-6 membered heteroaryl" refers to a monovalent aromatic ring group containing 5-6 ring atoms and 1-4 heteroatoms in the ring atoms as ring members. The heteroatoms may be selected from nitrogen, oxygen or sulfur.
The term "tautomer" refers to structural isomers that readily interconvert by a chemical reaction with each other being an isomer, which reaction generally results in formal movement of hydrogen atoms or protons with concomitant transformation of single bonds and adjacent double bonds.
The term "enantiomer" refers to stereoisomers that are mirror images of each other and do not overlap.
"diastereomer" refers to a stereoisomer that has two or more chiral neutrals and is not a mirror image.
"racemic" refers to two stereoisomers that are mirror images of each other, but have opposite optical rotations and cancel each other out.
The salt is a salt formed by a molecule and a corresponding organic acid, inorganic acid or organic base and inorganic base, for example, hydrochloric acid, formic acid, trifluoroacetic acid, succinic acid, methanesulfonic acid salt and the like of the compound.
"hydrate" refers to a compound containing water.
The invention further provides the use of the compound shown in the general formula (I) according to the invention, the tautomer, the enantiomer, the diastereomer, the racemate, the isotopic compound, and various forms of salts or hydrates thereof for preparing the marker for selectively marking the free amino group of the lysine on the side chain of the polypeptide or protein. The o-nitrobenzyl alcohol compound shown in the general formula (I) can selectively mark the free amino of the lysine side chain in polypeptide and protein mainly under the light induction condition.
The compound shown in the general formula (I) or tautomer, enantiomer, diastereomer, racemate, precursor compound, isotope compound, salt or hydrate thereof in various forms can form a connecting structure through amino of lysine side chains of polypeptide and protein, and can be used for preparing antibody-drug conjugates, antibody-affinity labels and antibody-fluorescent substances.
Accordingly, in a further aspect, the present invention provides the use of the compounds represented by the general formula (i) of the present invention, tautomers, enantiomers, diastereomers, racemates, isotopic compounds, and salts in various forms or hydrates thereof for the preparation of antibody-drug conjugates, antibody-affinity labels and antibody-fluorescent substances.
The conjugate has a characteristic structural formula (II):
Figure BDA0002748074610000061
wherein, Y, R1The same as defined in the above general formula (I),
a is a polypeptide or protein;
z is L-X, X comprises one or no of affinity marker, tracer fluorescent substance and active drug or their derivatives; in particular, affinity labels such as biotin and folate, and tracer fluorescent substances including, but not limited to, rhodamine, fluorescein, pigment, coumarin. Wherein the active agents include, but are not limited to, Maytansinoids (Maytansinoids), Auristatins (Auristatins), Calicheamicins (Calicheamicins), adriamycins (Doxorubicins), pyrrolobenzodiazepine dimers (PBDs), Triptolide (Triptolide), colchicines (Colchicine), combretastatins (Combretastatin), homoharringtonines (homoharringtonines), camptothecins (camptothecins), taxanes (Paclitaxel), and all agents useful for antibody drug conjugates;
l can be: C1-C9 alkyl, C2-C9 alkenyl, C2-C9 alkynyl, aryl, heteroaryl, C3-C9 cycloalkyl, C3-C9 heterocyclyl, -NR1-, -O-, -S-, -CO-, -OCO-, -COO-, -NHCO-, -CONR1-, -C ═ NR1-, -C ═ S-O-, -C ═ S-NR1-, -CS2-, -NR1CO-, -NR1CSNR2-, -OCONR1-, -OSO-, Val-Val-PAB, Val-Cit-PAB, Val-Ala-PAB, Val-Lys (Ac) -PAB, Phe-Lys (Ac) -PAB, D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn-PAB, Ala-PAB, PAB and any combination or null thereof, wherein R1 and R2 are independently selected from H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, aryl, heteroaryl, C3-C9 cycloalkyl and C3-C9 heterocyclic group, and n3 is 0-23; or L is absent.
Preferably, in the antibody-drug conjugate represented by the general formula (ii), L is selected from the following structures and any combination thereof:
Figure BDA0002748074610000071
wherein n4 is an integer of 0 to 23.
In particular, a above refers to a unit that can bind, reactively associate, or complex a receptor or antigen, including but not limited to a chimeric antibody, a humanized antibody, a human antibody, or an antibody fragment.
In particular, the antibody-drug conjugate is selected from the following structures;
Figure BDA0002748074610000072
Figure BDA0002748074610000081
wherein, Y, L, R1The definition of (a) is the same as the above definition,
Figure BDA0002748074610000082
refers to an Antibody (Antibody) which refers to a unit capable of binding, reactively associating or complexing a receptor or antigen, such as a chimeric Antibody, a humanized Antibody, a human Antibody or an Antibody fragment.
The compound represented by the general formula (I) according to the present invention or the above-mentioned antibody-drug conjugate, antibody-affinity label and antibody-fluorescent substance can be prepared by or by referring to the methods of the following examples or methods similar thereto.
Drawings
FIG. 1 shows the selective reaction of Compound 1G for-NH 2 on the polypeptide.
Figure 2 shows the molecular weight profile of compound 1G-labeled polypeptide product.
FIG. 3 shows the selective reaction of Compound 1F to-NH 2 on the polypeptide.
Figure 4 shows the molecular weight profile of compound 1F-labeled polypeptide product.
FIG. 5 shows ESI-TOF spectra of unlabeled and 1F-labeled polypeptide aptamers.
Fig. 6 shows ESI-TOF spectra of unlabeled nanobody-anti-human epidermal growth factor antibody and 1F-labeled nanobody-anti-human epidermal growth factor antibody.
FIG. 7 shows ESI-TOF spectra of unlabeled ubiquitin and 1F-labeled modified ubiquitin.
FIG. 8a is an ESI-TOF spectrum of unlabeled chymotrypsinogen and chymotrypsinogen labeled with 1F.
FIG. 8 b is an ESI-MS/MS spectrum of the digested peptide fragment after chymotrypsinogen was labeled with Compound 1F.
FIG. 8 c is the marker protein and TAMRA-N3And (4) carrying out fluorescence imaging on the SDS-PAGE gel after coupling.
FIG. 9 a is an ESI-TOF spectrum of unlabeled lysozyme and lysozyme labeled with Compound 1F.
FIG. 9 b is the ESI-MS/MS spectrum of the cleaved peptide fragment after lysozyme was labeled with Compound 1F.
FIG. 9 is a schematic view ofc is a marker protein and TAMRA-N3And (4) carrying out fluorescence imaging on the SDS-PAGE gel after coupling.
Fig. 10 a is an ESI-TOF spectrum of unlabeled myoglobin and myoglobin labeled with compound 1F.
FIG. 10 b is an ESI-MS/MS spectrum of the cleaved peptide fragment after myoglobin was labeled with Compound 1F.
FIG. 10 c is the marker protein and TAMRA-N3And (4) carrying out fluorescence imaging on the SDS-PAGE gel after coupling.
FIG. 11 shows the selective labeling of amino groups in the Nanobody-anti-human epidermal growth factor receptor antibody by Compound 1F, and the preparation of antibody-fluorescent conjugates.
Fig. 12 a is ESI-TOF spectrum of unlabeled nanobody-anti-human epidermal growth factor receptor and antibody labeled by compound 1F.
FIG. 12 b is ESI-MS/MS spectrum of enzyme digestion peptide fragment after nanometer antibody-anti-human epidermal growth factor receptor is labeled by compound 1F.
FIG. 12 c is a graph showing the binding of labeled antibody to TAMRA-N3And (4) carrying out fluorescence imaging on the SDS-PAGE gel after coupling.
Detailed Description
In all examples, 1H NMR was recorded by a Bruker Avance III-300 or Avance III-400 model nuclear magnetic resonance apparatus, chemical shifts being expressed in delta (ppm); mass spectra were determined by MS mass spectra UPLC-MS (esi); wherein the UPLC model is Waters HPLC H-CLASS, and the MS (ESI) model is Waters SQ Detector 2; the anhydrous tetrahydrofuran is prepared by benzophenone/metallic sodium reflux drying and deoxidization, and the anhydrous toluene and the anhydrous dichloromethane are prepared by calcium chloride reflux drying; solvents such as petroleum ether, ethyl acetate and dichloromethane for column chromatography mobile phase are all purchased from chemical reagents of national medicine group; the thin layer chromatography silica gel plate (HSGF254) used in the reaction detection is from chemical reagents of national drug group, Inc.; the compound separation is performed by using 200-300 mesh silica gel of national drug group chemical reagent, Inc. The starting materials of the present invention can be obtained commercially, such as the main reagents purchased from the national pharmaceutical group chemical agents limited, or prepared by methods known in the art, or prepared according to the methods described in the present invention.
Example 1: n- (3-azidopropyl) -4- (hydroxymethyl) -3-nitrobenzamide
Figure BDA0002748074610000091
Step 1-1: compound S1(4- (bromomethyl) -3-nitrobenzoic acid) (5.00g, 19.2mmol) was dissolved in acetone/H2To the O (1: 1, 150mL) mixed solution, Na was added2CO3(7.13g, 67.3mmol) was refluxed for 3 hours. Acetone was removed in vacuo and the aqueous phase was taken up in Et2And O is extracted twice. Adding concentrated HCl into the water phase until the pH value is 2-3, and extracting for 3 times by using ethyl acetate. The organic layer was washed with water and brine, dried over magnesium sulfate, and concentrated in vacuo without further purification to give compound 1A as a brown oil (3.78g, 98%).1H NMR(500MHz,MeOD)δ8.60(s,1H),8.29(d,J＝8.0Hz,1H),7.97(d,J＝8.1Hz,1H),5.00(s,2H).
Step 1-2: 1A (1.0eq), HATU (1.2eq) and propylamine (1B, 3.0eq) or propargylamine (1C, 3.0eq) or 3-azidopropan-1-amine (1D, 1.0eq) were dissolved in anhydrous DMF and DIPEA (3.0eq) was added dropwise at 0 ℃ and the mixture was stirred at room temperature overnight. Adding H to the mixture2O, and extracted 3 times with ethyl acetate. The organic layer was washed with saturated NaHCO3、0.1M HCl、H2O, brine, over Na2SO4Dried and concentrated in vacuo and the residue purified by silica chromatography to give the products 1E to G. Compound 1E, yield: and 64 percent.1H NMR(400MHz,MeOD)δ8.50(d,J＝1.7Hz,1H),8.14(dd,J＝8.1,1.7Hz,1H),7.96(d,J＝8.2Hz,1H),4.99(s,2H),3.39–3.33(m,2H),1.66(dq,J＝14.7,7.4Hz,2H),1.02–0.94(m,3H).13C NMR(101MHz,MeOD)δ167.5,148.4,142.5,135.6,132.9,129.7,124.5,61.8,42.9,23.6,11.8.HRMS(ESI-Q-TOF):m/z[M+H]+Calcd for C11H15N2O4 +239.1026; found 239.1040 compound 1F, yield:58 percent of Yield.1H NMR(400MHz,MeOD)δ8.51(d,J＝1.7Hz,1H),8.15(dd,J＝8.2,1.8Hz,1H),7.97(d,J＝8.2Hz,1H),4.99(s,2H),4.18(d,J＝2.5Hz,2H),2.64–2.62(m,1H).13C NMR(101MHz,MeOD)δ167.1,148.3,142.8,134.9,133.0,129.8,124.6,80.4,72.3,61.8,30.1.HRMS(ESI-Q-TOF):m/z[M-H]-Calcd for C11H9N2O4 -233.0568; found 233.0566 compound 1G, yield: 51 percent.1H NMR(500MHz,MeOD)δ8.52(d,J＝1.8Hz,1H),8.15(dd,J＝8.1,1.8Hz,1H),7.98(d,J＝8.1Hz,1H),4.99(s,2H),3.49(t,J＝6.9Hz,2H),3.43(t,J＝6.7Hz,2H),1.90(p,J＝6.8Hz,2H).13C NMR(126MHz,MeOD)δ167.7,148.5,142.6,135.5,133.0,129.8,124.5,61.8,50.2,38.6,29.7.HRMS(ESI-Q-TOF):m/z[M+Na]+Calcd for C11H13N5NaO4 +:302.0860；found:302.0853.
Example 2: 4- (hydroxymethyl) -3-nitro-N- (2- (5- ((3aS, 4S, 6aR) -2-oxocyclohexane-1H-thieno [3,4-d ] imidazol-4-yl) pentyl) ethyl) benzamide
Figure BDA0002748074610000101
Step 2-1: a solution of D-biotin (3.00g, 12.3mmol), EDCI. HCl (2.82g, 14.7mmol) and NHS (1.70g, 14.7mmol) in DMF (100mL) was stirred at room temperature overnight. The reaction mixture was concentrated in vacuo and the residue was filtered, washed with EtOH/AcOH/H2O (95: 1: 4) and dried in vacuo. The product, compound 2A, was obtained as a yellow oil without further purification (3.74g, 89%).1H NMR(500MHz,DMSO)δ6.45(s,1H),6.38(s,1H),4.30(dd,J＝7.5,5.2Hz,1H),4.16–4.12(m,1H),3.13–3.07(m,1H),2.84(d,J＝5.1Hz,1H),2.81(s,4H),2.67(t,J＝7.4Hz,2H),2.58(d,J＝13.3Hz,1H),1.74–1.57(m,6H).
Step 2-2: to a stirred solution of ethylenediamine (3.91mL, 58.6mmol) in anhydrous DMF (20mL) was added a solution of compound 2A (1.00g, 2.93mmol) in anhydrous DMF (10mL) dropwise and stirred overnight. Et was added to the mixture2O, the precipitated product was filtered, washed with ethyl acetate and dried in vacuo without further purification (753mg, 90%). The product from the previous step (400mg, 1.40mmol), Compound 1A (276mg, 1.40 m)mol) and HATU (637mg, 1.68mmol) were dissolved in anhydrous DMF (15mL), DIPEA (0.69mL, 4.19mmol) was added dropwise at 0 deg.C, and the mixture was stirred at room temperature overnight. Adding H to the mixture2O, and extracted 3 times with ethyl acetate. The organic layer was washed with saturated NaHCO3、1M HCl、H2O, brine, over Na2SO4Dried and concentrated in vacuo, and purified by silica gel chromatography to give compound 2B as a yellow oil (331mg, 51%).1H NMR(500MHz,MeOD)δ8.52(d,J＝1.7Hz,1H),8.15(dd,J＝8.1,1.7Hz,1H),7.99(d,J＝8.2Hz,1H),5.00(s,2H),4.46(dd,J＝7.9,4.9Hz,1H),4.24(dd,J＝7.9,4.5Hz,1H),3.53(t,J＝5.9Hz,2H),3.44(t,J＝5.8Hz,2H),3.14–3.09(m,1H),2.89(dd,J＝12.8,5.0Hz,1H),2.68(d,J＝12.7Hz,1H),2.21(t,J＝7.4Hz,2H),1.66(m,4H),1.59–1.49(m,2H).13C NMR(126MHz,MeOD)δ176.7,167.8,166.1,148.5,142.6,135.5,133.0,129.9,124.6,63.3,61.8,61.6,56.9,41.1,41.0,39.9,36.8,29.7,29.4,26.8.HRMS(ESI-Q-TOF):m/z[M+H]+Calcd for C20H28N5O6S+:466.1755；found:466.1744.
Example 3: n- (6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) hexyl) -4- (hydroxymethyl) -3-nitrobenzamide
Figure BDA0002748074610000111
Step 3-1: compound S2(74mg, 0.25mmol) was added to 2: 1CH2Cl2And trifluoroacetic acid (1.5mL) at room temperature for 1h, and then the solvent was removed in vacuo and directly fed to the next step.
Step 3-2: compound 1A (54mg, 0.275mmol), HATU (104mg, 0.275mmol) and the resulting oil 3A were dissolved in anhydrous DMF and DIPEA (227. mu.L, 1.38mmol) was added to the solution. The reaction was stirred at room temperature for 1 hour. The mixture was diluted with ethyl acetate, washed with water and brine, over Na2SO4After drying and removal of the solvent in vacuo, the resulting residue was purified by flash chromatography to give compound 3B as a yellow oil (30mg, 32%).1HNMR(600MHz,DMSO)δ8.74(t,J＝5.5Hz,1H),8.49(d,J＝1.7Hz,1H),8.20(dd,J＝8.1,1.7Hz,1H),7.92(d,J＝8.1Hz,1H),6.99(s,2H),5.65(t,J＝5.5Hz,1H),4.87(d,J＝5.5Hz,2H),3.39(t,J＝7.1Hz,2H),3.25(dd,J＝12.8,6.9Hz,2H),1.53–1.46(m,4H),1.34–1.29(m,2H),1.25–1.22(m,2H).13C NMR(126MHz,DMSO)δ171.1,163.8,146.6,141.1,134.4,134.0,132.0,128.4,123.0,59.9,37.0,28.8,27.9,25.9,25.8.HRMS(ESI-Q-TOF):m/z[M+H]+Calcd for C18H22N3O6 +:376.1503；found:376.1496.
Example 4
Compound 1G on-NH of polypeptide2Selective labelling assay of (1). FIG. 1 shows the selective reaction of compound 1G for-NH 2 on the polypeptide. Compound 1G (1.25mM) and polypeptide (AcSRKYDH in FIG. 1) (0.5mM) in 100mM PBS/MeOH (9: 1, pH 7.4) were treated with 365nm UV light and shaken for 30 min at 25 deg.C, samples were collected with MeOH/H2O was diluted and analyzed by UPLC-MS. FIG. 2 shows the molecular weight spectra of the polypeptide product labeled with Compound 1G, indicating that the light-induced labeling of the polypeptide is very rapid and that the desired product can be obtained by UPLC-MS analysis of the reaction mixture.
Example 5
Compound 1F on-NH of polypeptide2Selective labelling assay of (1). FIG. 3 shows the selective reaction of Compound 1F to-NH 2 on the polypeptide. Compound 1F (1.25mM) and polypeptide (AcRCYMNK, 0.5mM in FIG. 3) in 100mM PBS/MeOH (9: 1, pH 7.4) were treated with 365nm UV light and shaken for 30 min at 25 deg.C, samples were collected with MeOH/H2O was diluted and analyzed by UPLC-MS. FIG. 4 shows the molecular weight spectra of the polypeptide product labeled with Compound 1F, indicating that the light-induced labeling of the polypeptide is very rapid and that UPLC-MS analysis of the reaction mixture can yield the desired product.
Example 6
And (3) under a light induction condition, marking Affiniody protein by using the compound 1F.
Compound 1F (2.5mM) was exposed to 365nm UV light in methanol for 7min, and Affinibody (55. mu.M) was added to bring the final concentration of o-nitrobenzyl alcohol compound 1F to 125. mu.M in PBS. After mixing, shaking for 1h at 25 ℃. Collecting samples, adding water for dilution, and carrying out ESI-TOF protein molecular weight analysis. FIG. 5 shows ESI-TOF spectra of unlabeled Affiniody and 1F-labeled Affiniody, indicating that their covalent modifications were almost quantitatively modified (FIG. 5). The molecular weight of the unlabeled protein is 7594.61 or 7725.50, and the molecular weight of the labeled protein is 7792.74, 7923.52; and the molecular weights of the two labeled molecules are 7990.46, 8121.74.
Affinibody amino acid sequence (SEQ ID NO: 1):
MTSVDNKFNKELSVAGREIVTLPNLNDPQKKAFIFSLWDDPSQSANLLAEAKKLNDAQAPKGSHHHHHH
example 7
Compound 1F was used for Nanobody-EGFR labeling under light induction.
Compound 1F (2.5mM) was exposed to 365nm UV light in methanol for 7min, and Nanobody-EGFR (55. mu.M) was added to make the final concentration of o-nitrobenzyl alcohol compound 1F 250. mu.M in PBS solution. After mixing, shaking for 1h at 25 ℃. Collecting samples, adding water for dilution, and carrying out ESI-TOF protein molecular weight analysis. FIG. 6 shows ESI-TOF spectra of unlabeled Nanobody-EGFR and 1F-labeled Nanobody-EGFR, indicating that its covalent modification is almost quantitatively modified. The molecular weights of the proteins before labeling are 14538.30 and 1455.12, and the molecular weight distributions after labeling are 14736.39, 14753.30, 14934.22, 14951.20, 15149.95, 15132.70 and 15347.14 respectively.
Nanobody-EGFR amino acid sequence (SEQ ID NO: 2):
QVKLEESGGGSVQTGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSAWYGTLYEYDYWGQGTQVTVSSALEHHHHHH
example 8
Experiment for labeling Ubiquitin protein by compound 1F under photoinduction condition
Compound 1F (2.5mM) was exposed to 365nm UV light in methanol for 7min, and Ubiquitin (55. mu.M) was added to make the final concentration of o-nitrobenzyl alcohol compound 1F 250. mu.M in PBS solution. After mixing, shaking for 1h at 25 ℃. Collecting samples, adding water for dilution, and carrying out ESI-TOF protein molecular weight analysis. FIG. 7 shows ESI-TOF spectra of unlabeled Ubiquitin and 1F-labeled modified Ubiquitin, indicating that its covalent modification is almost quantitatively modified. Molecular weight 10035.19, 10166.39 before labeling; the molecular weight distributions after labeling were 10233.29, 10364.50, 10431.40, 10562.58, 10629.27, 10760.60, 10827.36 and 10958.34, respectively.
Ubiquitin amino acid sequence (SEQ ID NO: 3):
MTSMQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGLEHHHHHHHH
example 9
Compound 1F was tested for Chymotrypsisinogen protein A labeling under light induction.
Compound 1F was prepared as a 2.5mM MeOH solution and chymotrypsinogen A was prepared as a 27.5 μ M solution in PBS. Compound 1F (2.5mM) in MeOH was treated with 365nm UV light for 7min, then added to chymotrypsinogen A (27.5. mu.M) in PBS to a final concentration of 125. mu.M and mixed. The mixture was shaken at 25 ℃ for 1 hour, samples were collected, diluted with water and analyzed by ESI-TOF to achieve labeling of the chromophorinogen a protein. Mixing TAMRA-N3Preparing into 1.25mM DMSO solution, and preparing CuSO4 and THPTA into 50mM H solution2O solution, and mixing at a ratio of 1: volume ratio of 5. Making sodium ascorbate into 100mM H2Stock solution of O solution. 100 microliters of o-nitrobenzyl alcohol compound 1F-labeled protein (25. mu.M or 50. mu.M), TAMRA-N3 (100. mu.M), premixed CuSO4 (100. mu.M), THPTA (500. mu.M) and sodium ascorbate (5mM) were added, and the mixture was reacted at 25 ℃ for 1h and analyzed by SDS-PAGE. FIG. 8a is an ESI-TOF spectrum of unlabeled Chymotrypsisinogen A and Chymotrypsinogen labeled with 1F, indicating that its covalent modification is almost quantitatively modified (FIG. 8 a). The molecular weight before labeling is 26655.97, and the molecular weight distribution after labeling is 25854.10, 26052.25, 26250.34, 26488.34 and 26646.27. Trypsin digestion and subsequent ESI-MS/MS analysis of the fragments also confirmed lysine (K) specific labeling (b of FIG. 8) and subsequent conjugation with tetramethylrhodamine, with good biocompatibility (c of FIG. 8).
Chymotrypsisinogen A amino acid sequence (SEQ ID NO: 4):
CGVPAIQPVLSGLSRIVNGEEAVPGSWPWQVSLQDKTGFHFCGGSLINENWVVTAAHCGVTTSDVVVAGEFDQGSSSEKIQKLKIAKVFKNSKYNSLTINNDITLLKLSTAASFSQTVSAVCLPSASDDFAAGTTCVTTGWGLTRYTNANTPDRLQQASLPLLSNTNCKKYWGTKIKDAMICAGASGVSSCMGDSGGPLVCKKNGAWTLVGIVSWGSSTCSTSTPGVYARVTALVNWVQQTLAAN
example 10
Compound 1F was tested for Lysozyme protein labeling under light-induced conditions.
Compound 1F was prepared as a 2.5mM MeOH solution and lysozyme was prepared as a 27.5. mu.M solution in PBS. Compound 1F (2.5mM) in MeOH was treated with 365nm UV light for 7min, then added to lysozyme (27.5. mu.M) in PBS to a final concentration of 125. mu.M and mixed. The mixture was shaken at 25 ℃ for 1 hour, and samples were collected, diluted with water and analyzed by ESI-TOF to effect labeling of Lysozyme protein. Mixing TAMRA-N3Preparing into 1.25mM DMSO solution, and preparing CuSO4 and THPTA into 50mM H solution2O solution, and mixing at a ratio of 1: volume ratio of 5. Making sodium ascorbate into 100mM H2Stock solution of O solution. Add 100. mu.l o-nitrobenzyl alcohol Compound 1F-labeled protein (25. mu.M or 50. mu.M), TAMRA-N3(100. mu.M), premixed CuSO4 (100. mu.M), THPTA (500. mu.M) and sodium ascorbate (5mM), the mixture was reacted at 25 ℃ for 1h and analyzed by SDS-PAGE. FIG. 9 a is ESI-TOF spectrum of unlabeled Lysozyme protein and Lysozyme labeled with Compound 1F, indicating that its covalent modification is almost quantitatively modified (FIG. 9 a). Molecular weight was 14304.07 before labeling and 14502.21, 14700.33, 14898.26 after labeling. Trypsin digestion and subsequent ESI-MS/MS analysis of the fragments also confirmed lysine-specific labeling (b of FIG. 9). FIG. 9 c is the marker protein with TAMRA-N3The fluorescence imaging picture of the coupled SDS-PAGE gel shows that the coupled SDS-PAGE gel can also be coupled with tetramethyl rhodamine and has good biocompatibility.
Lysozyme amino acid sequence (SEQ ID NO: 7):
KVFGRCELAAAMKRHGLDNYRGYSLGNWVCAAKFESNFNTQATNRNTDGSTDYGILQINSRWWCNDGRTPGSRNLCNIPCSALLSSDITASVNCAKKIVSDGNGMNAWVAWRNRCKGTDVQAWIRGCRL
example 11
Compound 1F was tested for Myoglobin protein labeling under light induction conditions.
Compound 1F was prepared as a 2.5mM MeOH solution and myoglobin was prepared as a 27.5. mu.M solution in PBS. Compound 1F (2.5mM) in MeOH was treated with 365nm UV light for 7min, then added to myoglobin (27.5. mu.M) in PBS to a final concentration of 125. mu.M and mixed. The mixture was shaken at 25 ℃ for 1 hour, samples were collected, diluted with water and analyzed by ESI-TOF to achieve labeling of the Myoglobin protein. Mixing TAMRA-N3Preparing into 1.25mM DMSO solution, and preparing CuSO4 and THPTA into 50mM H solution2O solution, and mixing at a ratio of 1: volume ratio of 5. Making sodium ascorbate into 100mM H2Stock solution of O solution. Add 100. mu.l o-nitrobenzyl alcohol Compound 1F-labeled protein (25. mu.M or 50. mu.M), TAMRA-N3(100. mu.M), premixed CuSO4 (100. mu.M), THPTA (500. mu.M) and sodium ascorbate (5mM), the mixture was reacted at 25 ℃ for 1h and analyzed by SDS-PAGE. Fig. 10 a is an ESI-TOF spectrum of unlabeled Myoglobin protein and Myoglobin labeled with compound 1F, indicating that its covalent modification is almost quantitatively modified (fig. 10 a). The molecular weight before labeling is 16951.38, and the molecular weight distribution after labeling is 17149.40, 17347.63, 17545.73 and 17743.63. Trypsin digestion and subsequent ESI-MS/MS analysis of the fragments also confirmed lysine-specific labeling (b of FIG. 10). FIG. 10 c is the marker protein and TAMRA-N3The fluorescence imaging picture of the coupled SDS-PAGE gel shows that the coupled tetramethylrhodamine has good biocompatibility (figure 10 c).
Myoglobin amino acid sequence (SEQ ID NO: 5):
GLSDGEWQQVLNVWGKVEADIAGHGQEVLIRLFTGHPETLEKFDKFKHLKTEAEMKASEDLKKHGTVVLTALGGILKKKGHHEAELKPLAQSHATKHKIPIKYLEFISDAIIHVLHSKHPGDFGADAQGAMTKALELFRNDIAAKYKELGFQG
example 12
Compound 1F was tested for Nanobody-HER2 protein labeling under light-induced conditions. FIG. 11 shows the selective labeling of the amino group in the Nanobody-HER2 antibody by Compound 1F, and the preparation of antibody-fluorescent conjugates.
Preparation of Compounds in MeOH solutionSubstance 1F in 2.5 mM. Compound 1F (2.5mM) in MeOH was treated with 365nm UV light for 7min, then added to a solution of Nanobody-HER2 (55. mu.M, 0.77mg/mL) in PBS to a final concentration of 125. mu.M and mixed. The mixture was incubated at 25 ℃ for 1 h. Samples were collected, diluted with H2O, and analyzed by ESI-TOF. The obtained compound 1F-labeled Nanobody-HER2 protein. Mixing TAMRA-N3Preparing into 1.25mM DMSO solution, and preparing CuSO4 and THPTA into 50mM H solution2O solution, and mixing at a ratio of 1: volume ratio of 5. Making sodium ascorbate into 100mM H2Stock solution of O solution. Add 100. mu.l o-nitrobenzyl alcohol Compound 1F-labeled protein (25. mu.M or 50. mu.M), TAMRA-N3(100. mu.M), premixed CuSO4 (100. mu.M), THPTA (500. mu.M) and sodium ascorbate (5mM), the mixture was reacted at 25 ℃ for 1h and analyzed by SDS-PAGE. FIG. 12 a is the ESI-TOF spectrum of unlabeled Nanobody-HER2 antibody and the antibody labeled with Compound 1F, indicating that its covalent modification is almost quantitatively modified (FIG. 12 a). The molecular weight before labeling is 13694.56, and the molecular weight distribution after labeling is 13892.63, 14090.77, 14288.56 and 14485.93. Trypsin digestion and subsequent ESI-MS/MS analysis of the fragments also confirmed lysine-specific labeling (b of FIG. 12). FIG. 12 c is a graph showing the binding of labeled antibody to TAMRA-N3The fluorescence imaging picture of SDS-PAGE gel after coupling shows that the conjugate can also be coupled with tetramethyl rhodamine to prepare the antibody-fluorescence conjugate.
Nanobody-HER2-wt-His6 amino acid sequence (SEQ ID NO: 6):
MQVQLQESGGGSVQAGGSLKLTCAASGYIFNSCGMGWYRQSPGRERELVSRISGDGDTWHKESVKGRFTISQDNVKKTLYLQMNSLKPEDTAVYFCAVCYNLETYWGQGTQVTVSSGGHHHHHH
SEQUENCE LISTING
<110> Shanghai pharmaceutical research institute of Chinese academy of sciences
<120> linker of the same kind used for protein labeling and application thereof in biological medicine
<130> DI20-1875-XC91
<160> 7
<170> PatentIn version 3.5
<210> 1
<211> 69
<212> PRT
<213> Artificial Sequence
<220>
<223> Affibody
<400> 1
Met Thr Ser Val Asp Asn Lys Phe Asn Lys Glu Leu Ser Val Ala Gly
1 5 10 15
Arg Glu Ile Val Thr Leu Pro Asn Leu Asn Asp Pro Gln Lys Lys Ala
20 25 30
Phe Ile Phe Ser Leu Trp Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu
35 40 45
Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Gly Ser His
50 55 60
His His His His His
65
<210> 2
<211> 133
<212> PRT
<213> Artificial Sequence
<220>
<223> Nanobody-EGFR
<400> 2
Gln Val Lys Leu Glu Glu Ser Gly Gly Gly Ser Val Gln Thr Gly Gly
1 5 10 15
Ser Leu Arg Leu Thr Cys Ala Ala Ser Gly Arg Thr Ser Arg Ser Tyr
20 25 30
Gly Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ser Gly Ile Ser Trp Arg Gly Asp Ser Thr Gly Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Asp
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr Cys
85 90 95
Ala Ala Ala Ala Gly Ser Ala Trp Tyr Gly Thr Leu Tyr Glu Tyr Asp
100 105 110
Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Leu Glu His
115 120 125
His His His His His
130
<210> 3
<211> 88
<212> PRT
<213> Artificial Sequence
<220>
<223> Ubiquitin
<400> 3
Met Thr Ser Met Gln Ile Phe Val Lys Thr Leu Thr Gly Lys Thr Ile
1 5 10 15
Thr Leu Glu Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys
20 25 30
Ile Gln Asp Lys Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile Phe
35 40 45
Ala Gly Lys Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile
50 55 60
Gln Lys Glu Ser Thr Leu His Leu Val Leu Arg Leu Arg Gly Leu Glu
65 70 75 80
His His His His His His His His
85
<210> 4
<211> 245
<212> PRT
<213> Artificial Sequence
<220>
<223> Chymotrypsinogen A
<400> 4
Cys Gly Val Pro Ala Ile Gln Pro Val Leu Ser Gly Leu Ser Arg Ile
1 5 10 15
Val Asn Gly Glu Glu Ala Val Pro Gly Ser Trp Pro Trp Gln Val Ser
20 25 30
Leu Gln Asp Lys Thr Gly Phe His Phe Cys Gly Gly Ser Leu Ile Asn
35 40 45
Glu Asn Trp Val Val Thr Ala Ala His Cys Gly Val Thr Thr Ser Asp
50 55 60
Val Val Val Ala Gly Glu Phe Asp Gln Gly Ser Ser Ser Glu Lys Ile
65 70 75 80
Gln Lys Leu Lys Ile Ala Lys Val Phe Lys Asn Ser Lys Tyr Asn Ser
85 90 95
Leu Thr Ile Asn Asn Asp Ile Thr Leu Leu Lys Leu Ser Thr Ala Ala
100 105 110
Ser Phe Ser Gln Thr Val Ser Ala Val Cys Leu Pro Ser Ala Ser Asp
115 120 125
Asp Phe Ala Ala Gly Thr Thr Cys Val Thr Thr Gly Trp Gly Leu Thr
130 135 140
Arg Tyr Thr Asn Ala Asn Thr Pro Asp Arg Leu Gln Gln Ala Ser Leu
145 150 155 160
Pro Leu Leu Ser Asn Thr Asn Cys Lys Lys Tyr Trp Gly Thr Lys Ile
165 170 175
Lys Asp Ala Met Ile Cys Ala Gly Ala Ser Gly Val Ser Ser Cys Met
180 185 190
Gly Asp Ser Gly Gly Pro Leu Val Cys Lys Lys Asn Gly Ala Trp Thr
195 200 205
Leu Val Gly Ile Val Ser Trp Gly Ser Ser Thr Cys Ser Thr Ser Thr
210 215 220
Pro Gly Val Tyr Ala Arg Val Thr Ala Leu Val Asn Trp Val Gln Gln
225 230 235 240
Thr Leu Ala Ala Asn
245
<210> 5
<211> 153
<212> PRT
<213> Artificial Sequence
<220>
<223> Myoglobin
<400> 5
Gly Leu Ser Asp Gly Glu Trp Gln Gln Val Leu Asn Val Trp Gly Lys
1 5 10 15
Val Glu Ala Asp Ile Ala Gly His Gly Gln Glu Val Leu Ile Arg Leu
20 25 30
Phe Thr Gly His Pro Glu Thr Leu Glu Lys Phe Asp Lys Phe Lys His
35 40 45
Leu Lys Thr Glu Ala Glu Met Lys Ala Ser Glu Asp Leu Lys Lys His
50 55 60
Gly Thr Val Val Leu Thr Ala Leu Gly Gly Ile Leu Lys Lys Lys Gly
65 70 75 80
His His Glu Ala Glu Leu Lys Pro Leu Ala Gln Ser His Ala Thr Lys
85 90 95
His Lys Ile Pro Ile Lys Tyr Leu Glu Phe Ile Ser Asp Ala Ile Ile
100 105 110
His Val Leu His Ser Lys His Pro Gly Asp Phe Gly Ala Asp Ala Gln
115 120 125
Gly Ala Met Thr Lys Ala Leu Glu Leu Phe Arg Asn Asp Ile Ala Ala
130 135 140
Lys Tyr Lys Glu Leu Gly Phe Gln Gly
145 150
<210> 6
<211> 124
<212> PRT
<213> Artificial Sequence
<220>
<223> Nanobody-HER2-wt-His6
<400> 6
Met Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly
1 5 10 15
Gly Ser Leu Lys Leu Thr Cys Ala Ala Ser Gly Tyr Ile Phe Asn Ser
20 25 30
Cys Gly Met Gly Trp Tyr Arg Gln Ser Pro Gly Arg Glu Arg Glu Leu
35 40 45
Val Ser Arg Ile Ser Gly Asp Gly Asp Thr Trp His Lys Glu Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Val Lys Lys Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys
85 90 95
Ala Val Cys Tyr Asn Leu Glu Thr Tyr Trp Gly Gln Gly Thr Gln Val
100 105 110
Thr Val Ser Ser Gly Gly His His His His His His
115 120
<210> 7
<211> 129
<212> PRT
<213> Artificial Sequence
<220>
<223> Lysozyme
<400> 7
Lys Val Phe Gly Arg Cys Glu Leu Ala Ala Ala Met Lys Arg His Gly
1 5 10 15
Leu Asp Asn Tyr Arg Gly Tyr Ser Leu Gly Asn Trp Val Cys Ala Ala
20 25 30
Lys Phe Glu Ser Asn Phe Asn Thr Gln Ala Thr Asn Arg Asn Thr Asp
35 40 45
Gly Ser Thr Asp Tyr Gly Ile Leu Gln Ile Asn Ser Arg Trp Trp Cys
50 55 60
Asn Asp Gly Arg Thr Pro Gly Ser Arg Asn Leu Cys Asn Ile Pro Cys
65 70 75 80
Ser Ala Leu Leu Ser Ser Asp Ile Thr Ala Ser Val Asn Cys Ala Lys
85 90 95
Lys Ile Val Ser Asp Gly Asn Gly Met Asn Ala Trp Val Ala Trp Arg
100 105 110
Asn Arg Cys Lys Gly Thr Asp Val Gln Ala Trp Ile Arg Gly Cys Arg
115 120 125
Leu

Claims (8)

1. A compound represented by the general formula (I), tautomers, enantiomers, diastereomers, racemates, isotopic compounds thereof, and salts in various forms or hydrates thereof:
Figure FDA0002748074600000011
wherein Y is selected from: -CO-, -NH-CH2-、-O-CO-CH2-、-NH-COO-CH2-、-NH-CO-NH-CH2-、-COOCH2-、-CO-NH-、-O-CH2-、-CH2-、-COO-、-OCO-、-O-、-S-、-SO2-、-C≡C-、-C＝C-、-SO2NH-、-NHCONH-、-NHCSNH-、-NH-、-CONH-CH2-or absent, wherein one end of Y may be attached to the 3,4, 5 or 6 position of the phenyl ring in the ortho-nitrobenzyl alcohol structure;
wherein R is1Is one or more than one substitution of any position except Y substitution position on 3,4, 5 or 6 position in the structure of o-nitrobenzyl alcohol, and when R is polysubstituted1Identical or different, R1Each independently selected from hydrogen, deuterium, amino, halogen, nitro, cyano, C1-6Alkyl radical, C3-10Cycloalkyl radical, C1-5Alkoxy radical, C1-6Alkylamino or aminoalkyl radical, C1-C6Alkylcarbonyl group, C2-C6Alkoxycarbonyl group, C2-C6Alkylamino carbonyl, C5-8Heterocyclic group, C6-10Aryl radical, C5-6A heteroaryl group,
Figure FDA0002748074600000012
Wherein n is0And n1Is 1, 2, 3,4 or 5, wherein said alkyl, cycloalkyl, alkoxy, alkylamino or aminoalkyl, alkylcarbonyl, alkoxycarbonyl, alkylaminocarbonyl, heterocyclyl, aryl, heteroaryl is optionally further substituted with one or more substituents selected from the group consisting of halogen, hydroxy, amino, C1-C6Alkoxy, cyano, nitro;
r is selected from hydrogen, deuterium, halogen, nitro, cyano, hydroxyl, alkyl hydroxyl, aryl hydroxyl, alkyl amino, aryl amino, sulfydryl, alkyl sulfydryl, aryl sulfydryl, carboxylic acid, alkyl carboxylic acid, aryl carboxylic acid, alkynyl, alkyl alkynyl, aryl alkynyl, azide, alkyl azide, aryl azide, carbonyl, alkyl carbonyl, aryl carbonyl, aldehyde group, alkyl aldehyde group, aryl aldehyde group, alkyl, cycloalkyl, alkoxy, heterocyclic group, aryl, heteroaryl or any combination thereof, wherein the above groups are optionally further substituted by one or more groups selected from halogen, hydroxyl, amino, C1-C6Alkoxy, cyano, nitro.
2. A compound according to claim 1, its tautomers, enantiomers, diastereomers, racemates, isotopic compounds and salts in various forms or hydrates thereof, wherein
Y is selected from: -CO-, -NH-CH2-、-O-CO-CH2-、-NH-COO-CH2-、-NH-CO-NH-CH2-、-COOCH2-、-CO-NH-、-O-CH2-、-CH2-、-COO-、-OCO-、-O-、-S-、-SO2-、-C≡C-、-C＝C-、-SO2NH-、-NHCONH-、-NHCSNH-、-NH-、-CONH-CH2-or absent, wherein one end of Y may be attached to the 3,4, 5 or 6 position of the phenyl ring in the ortho-nitrobenzyl alcohol structure;
r is selected from hydrogen, deuterium, halogen, nitro, cyano, hydroxyl, alkyl hydroxyl, aryl hydroxyl, alkyl amino, aryl amino, sulfydryl, alkyl sulfydryl, aryl sulfydryl, carboxylic acid, alkyl carboxylic acid, aryl carboxylic acid, alkynyl, alkyl alkynyl, aryl alkynyl, azide, alkyl azide, aryl azide, carbonyl, alkyl carbonyl, aryl carbonyl, aldehyde group, alkyl aldehyde group, aryl aldehyde group, alkyl, cycloalkyl, alkoxy, heterocyclic group, aryl, heteroaryl or any combination thereof, wherein the above groups are optionally further substituted by one or more groups selected from halogen, hydroxyl, amino, C1-C6Alkoxy, cyano, nitro;
wherein R is1Is one or more than one substitution of any position except Y substitution position on 3,4, 5 or 6 position in the structure of o-nitrobenzyl alcohol, and when R is polysubstituted1Identical or different, R1Each independently selected from hydrogen, deuterium, amino, halogen, C1-3Alkoxy, nitro or
Figure FDA0002748074600000021
Wherein n is0And n11, 2, 3,4 and 5.
3. A compound according to claim 1 or 2, its tautomers, enantiomers, diastereomers, racemates, precursor compounds, isotopic compounds and salts in various forms or hydrates thereof, wherein the compound of formula (i) is selected from the group consisting of the following formulae:
Figure FDA0002748074600000022
wherein R, R1Is defined and corresponds to the claimsThe requirements are the same.
4. Use of a compound according to any one of claims 1 to 3, or a tautomer, enantiomer, diastereomer, racemate, precursor compound, isotopic compound, salt in various forms, or hydrate thereof, for the preparation of a label for selectively labeling a polypeptide or a lysine free amino group in a protein side chain.
5. Use of the compound of any one of claims 1 to 3, or a tautomer, enantiomer, diastereomer, racemate, precursor compound, isotopic compound, salt or hydrate thereof in various forms, for producing an antibody-drug conjugate, an antibody-affinity label, and an antibody-fluorescent substance.
6. The use according to claim 5, wherein the conjugate has the characteristic structural formula (II):
Figure FDA0002748074600000031
wherein, Y, R1Are as defined in the corresponding claims,
a is a polypeptide or protein;
z is an integer of L-X,
x includes one or none of affinity tags, labeled fluorescent substances including rhodamine, fluorescein, pigment, coumarin, and active drugs or their derivatives, particularly, affinity tags such as biotin and folic acid, wherein the active drugs include Maytansinoids (Maytansinoids), Auristatins (Auristatins), Calicheamicins (Calichemicins), adriamycin (Doxorubicin), pyrrolobenzodiazepine dimer (PBDs), Triptolide (Triptolide), Colchicine (Colchicine), Combretastatin (Combretastatin), Homoharringtonine (Homoharringtonine), Camptothecin (Camptothecin), Paclitaxel (Paclitaxel), and also includes all drugs that can be used for antibody drug conjugates,
l is: C1-C9 alkyl, C2-C9 alkenyl, C2-C9 alkynyl, aryl, heteroaryl, C3-C9 cycloalkyl, C3-C9 heterocyclyl, -NR1-, -O-, -S-, -CO-, -OCO-, -COO-, -NHCO-, -CONR1-, -C ═ NR1-, -C ═ S-O-, -C ═ S-NR1-, -CS2-, -NR1CO-, -NR1CSNR2-, -OCONR1-, -OSO-, Val-Val-PAB, Val-Cit-PAB, Val-Ala-PAB, Val-Lys (Ac) -PAB, Phe-Lys (Ac) -PAB, D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn-PAB, Ala-PAB, PAB and any combination or null thereof, wherein R1 and R2 are independently selected from H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, aryl, heteroaryl, C3-C9 cycloalkyl and C3-C9 heterocyclic group, and n3 is 0-23; or L is absent.
7. The antibody-drug conjugate of claim 6, wherein L is selected from the group consisting of the following structures and any combination thereof:
Figure FDA0002748074600000041
wherein n is4Is an integer of 0 to 23.
8. The use of claim 7, wherein the antibody-drug conjugate is selected from the following structures;
Figure FDA0002748074600000042
wherein, Y, L, R1Are as defined in the corresponding claims,
Figure FDA0002748074600000043
refers to an antibody, which refers to a unit capable of binding, reactively associating or complexing a receptor or antigen, such as a chimeric antibody, a humanized antibody, a human antibody or an antibody fragment.
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